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Objective To investigate the effects of chronic starvation stress on the proliferation and migration of colorectal cancer cells, as well as the underlying mechanisms. Methods By using prolonged serum starvation to simulate chronic starvation stress in tumor cells, we established enduring serum-deprived models of SW480 and DLD-1 cells and observed cellular morphological change. Effects of prolonged serum starvation on SW480 and DLD-1 proliferative and migratory capabilities were assessed using CCK-8 and Transwell assays. Differential gene-expression analysis on SW480 cultured with 1% FBS or 10% FBS medium was followed by GO and KEGG pathway assessments. Migration-related protein interactions were explored using String database and Metascape software, leading to 16 genes being selected for RT-qPCR validation. Protein levels of ITGB1 and key molecules in the relevant pathways were measured. Mobility changes in SW480 were observed through Transwell assay after ITGB1 knockdown or STAT3 inhibition. Results Prolonged serum starvation significantly inhibited the proliferation of SW480 and DLD-1 cells, and DLD-1 mobility, while enhanced SW480 migration. Transcriptome analysis revealed that prolonged serum deprivation caused the upregulation of 3016 genes, among which 283 were involved in cell migration. Metascape analysis identified the correlations among potential core genes ITGB1, CD44, TNS1, STAT3, etc. Prolonged serum deprivation increased the mRNA levels of VTN, TNS1, VEGFA, STAT3, and ITGB1 while also increasing the protein levels of ITGB1 and MMP2 and the phosphorylation levels of JAK2 and STAT3. Mobility reduction in prolonged serum-starved SW480 cells was achieved through ITGB1 knockdown or a STAT3 inhibitor. Conclusion Colorectal cancer cells can endure chronic starvation stress which enhances migration capability by upregulating ITGB1 expression.
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Endometriosis is a common chronic gynecological disease,and its pathogenesis has not been fully elucidated.Mesenchymal stem cells are a kind of pluripotent stem cells with multi-directional differentiation potential derived from mesoderm,which can differentiate into a variety of tissues and organs.Endometrial mesenchymal stem cells,menstrual blood-derived mesenchymal stem cells,adipose mesenchymal stem cells,bone marrow mesenchymal stem cells and umbilical cord blood mesenchymal stem cells can participate in the pathogenesis of endometriosis from cell proliferation and differentiation,ectopic migration,angiogenesis,inflammatory response and fibrosis formation,and play a certain role in progression of the disease.Mesenchymal stem cells provide new ideas for elucidating the pathogenesis of endometriosis,and may also become a potential method for the treatment of endometriosis.
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Objective:To observe the expression of CD36 in hepatocellular carcinoma tissues and cell lines, and to investigate the effects of CD36 on the proliferation and migration abilities of human hepatocellular carcinoma cell lines and human hepatocellular carcinoma cell xenograft models in nude mice.Methods:Differences in the expression levels of CD36 transcripts in 371 hepatocellular carcinoma and paracancerous tissues were analyzed based on information from The Cancer Genome Atlas (TCGA) database. Cancer tissues and corresponding paracancerous tissues of 48 hepatocellular carcinoma patients who were diagnosed and underwent surgical treatment at the Affiliated Hospital of Yangzhou University from January 2019 to February 2021 were prospectively collected, and the levels of CD36 mRNA in the tissues were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) method. Western blotting was used to detect CD36 protein levels in human hepatocellular carcinoma cell lines Huh7 and HCCLM3 and human normal liver cell line LO2. Plasmids containing CD36 interfering sequences and empty plasmids were transfected into Huh7 cells or HCCLM3 cells for sh-CD36 group and control group, respectively. The CCK-8 assay was used to detect the proliferation ability (expressed as absorbance value) of cells in each group at 0, 12, 24, 36, 48 and 60 h of culture, and the scratch healing assay and Transwell assay were used to detect the migration ability of cells in each group. The Huh7 cells of sh-CD36 group or control group were injected into the axillary subcutis of BALB/c nude mice, with 4 mice in each group, to construct nude mice models of human hepatocellular carcinoma xenografts; the long and short diameters of tumor were measured weekly after 1 week of inoculation, and the tumor volume was calculated. The nude mice were put to death after 5 weeks of inoculation, and the tumor specimens were collected and weighed; the tumor cell morphology was observed under the microscope, and the expressions of CD36 and Ki-67 proteins in the tumor tissues was detected by immunohistochemistry (IHC).Results:Analysis of the data from the TCGA database showed that the level of CD36 transcripts was higher in hepatocellular carcinoma tissues compared with that in paracancerous tissues (4.2±1.8 vs. 3.2±1.5, t = 2.28, P = 0.035). Tissues detection using qRT-PCR in 48 patients with hepatocellular carcinoma showed that the relative expression of CD36 mRNA in hepatocellular carcinoma tissues was higher than that in paracancerous tissues (0.76±0.26 vs. 0.48±0.23, t = 3.52, P < 0.001). Western blotting assay showed that CD36 protein level in Huh7 and HCCLM3 cells was higher than that in LO2 cells, which were (1.42±0.11) times and (1.68±0.16) times higher than LO2 cells, respectively (both P < 0.001). At the mRNA and protein levels, the CD36 of Huh7 and HCCLM3 cells in the sh-CD36 group was lower than that in the corresponding control group (both P < 0.001). CCK-8 assay showed that the proliferative ability of Huh7 cells and HCCLM3 cells in the sh-CD36 group was lower than that in the corresponding control group after 36 and 24 h of culture (both P < 0.01). Scratch healing assay showed that the scratch healing rates of Huh7 cells [(12±3)% vs. (30±5)%, t = 4.01, P < 0.001] and HCCLM3 cells [(15±4)% vs. (29±5)%, t = 4.16, P < 0.001] in the sh-CD36 group were lower than those in the corresponding control group at 48 h of culture; Transwell assay showed that the number of Huh7 cells [(46±6) cells/field of view vs. (88± 6) cells/field of view, t = 5.56, P < 0.001] and HCCLM3 cells [(42±5) cells/field of view vs. (82±7) cells/field of view, t = 5.34, P < 0.001] penetrating into the membrane in 24 h in the sh-CD36 group was less than that in the corresponding control group. Five weeks after subcutaneous injection, the tumor volume [(682±268) mm 3vs. (1 375±512) mm 3, t = 4.73, P = 0.006] and tumor mass [(432±95) mg/mouse vs. (871±109) mg/mouse, t = 6.57, P < 0.001] of nude mice injected with Huh7 cells of the sh-CD36 group were lower than those of nude mice injected with Huh7 cells of the control group; under the microscope, the density of tumor cells in transplanted tumor specimens of nude mice injected with Huh7 cells of the sh-CD36 group was lower than that in nude mice injected with Huh7 cells of the control group, and the expression levels of both CD36 and Ki-67 proteins were also low. Conclusions:CD36 expression is up-regulated in cancer tissues of hepatocellular carcinoma patients and human hepatocellular carcinoma cell lines Huh7 and HCCLM3, and it may associate with cell proliferation and migration of hepatocellular carcinoma. Knockdown of CD36 expression significantly inhibits the proliferation and migration abilities of hepatocellular carcinoma cells in vitro, and inhibits the tumors of human hepatocellular carcinoma cell xenograft models in nude mice.
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Objective:To investigate the level of the transporter RNA (tRNA) derivative tRF-5026a in the serum of breast cancer patients and its value for the diagnosis of breast cancer, and to investigate its effect on the biological functions of breast cancer cells in vitro and the possible mechanisms.Methods:Sixty female breast cancer patients (breast cancer group) hospitalized in Jiangsu Cancer Hospital from January 2016 to February 2019 and 20 healthy women undergoing physical examination during the same period (healthy control group) were retrospectively selected. The relative expression of serum tRF-5026a in the study subjects was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The receiver operating characteristic (ROC) curve of serum tRF-5026a level for the diagnosis of breast cancer was drawn with pathological diagnosis as the gold standard. tRF-5026a mimics (tRF-5026a group) and negative control sequences (negative control group) were transiently transfected into MCF-7 and BT549 cells by lipofectamine method; CCK-8 assay and 5-ethynyl-2-deoxyuridine (EdU) assay were used to detect the ability of cell proliferation in cells of each group; cell apoptosis in cells of each group was detected by flow cytometry; the abilities of cell invasion and migration in cells of each group were detected by Transwell assay; the expressions of epithelial mesenchymal transition-related proteins in cells of each group were detected by Western blotting.Results:The relative expressions of tRF-5026a [ M ( Q1, Q3)] in serum of healthy control group and breast cancer group were 16.58 (6.37, 26.31) and 3.46 (0.32, 9.01), with a statistically significant difference ( Z = -4.27, P < 0.001). ROC curve analysis showed that the area under the curve (AUC) for diagnosis of breast cancer by the relative expression of serum tRF-5026a was 0.820 (95% CI: 0.722-0.918), with an optimal cut-off value of 9.082, and the corresponding sensitivity and specificity were 75.0 % and 76.7%, respectively. The apoptosis rates of MCF-7 cells in the tRF-5026a group and the corresponding negative control group were (16.52±0.51)% and (12.28±1.75)%, and the BT549 cells were (13.27±2.18)% and (8.86±0.29)%, the differences were not statistically significant (both P > 0.05). MCF-7 and BT549 cells in the tRF-5026a group had lower proliferative, invasive and migratory abilities than cells in the corresponding negative control group (all P < 0.05). MCF-7 and BT549 cells in the tRF-5026a group had lower protein expressions of N-cadherin, matrix metalloproteinase (MMP)-9 and MMP-3 than cells in the corresponding negative control group. Conclusions:tRF-5026a has low level in the serum of breast cancer patients and it may be an indicator for breast cancer diagnosis. tRF-5026a can inhibit the proliferation, invasion and migration of breast cancer MCF-7 and BT549 cells in vitro, which may be related to the regulation of epithelial mesenchymal transition.
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Objective:To explore the expression of long non-coding RNA (lncRNA) RP13-349O20.2 in cervical cancer tissues and its impact on the migration, invasion abilities and chemotherapy sensitivity of cervical cancer cells in vitro and the possible mechanisms.Methods:The GEPIA.CANCER website (the data was updated in June 2023) was used to analyze the relationship between the expression level of RP13-349O20.2 and the overall survival of 253 cervical cancer patients. From January 2020 to August 2022, cancer tissues and paracancerous tissues (>2 cm from the tumor edge) from 40 cervical cancer patients in the Affiliated Tengzhou Central People's Hospital of Xuzhou Medical University were retrospectively collected. Human normal cervical epithelial cells H8 and human cervical cancer cell lines HCC94, C33A, Hela, HCC1106 and SiHa were used for cell experiments in vitro. Real-time fluorescence polymerase chain reaction (qRT-PCR) was used to detect the relative expression of RP13-349O20.2 in cervical cancer tissues, paracancerous tissues and each cell line. The C33A cells with the highest relative expression level of RP13-349O20.2 were transfected with small interfering RNA (siRNA) of RP13-349O20.2 and siRNA of its negative control sequence, and they were si-RP13-349O20.2 group and si-Con group, respectively. The scratch healing assay was used to detect the migration ability of C33A cells in the two groups, the Transwell assay was used to detect the invasion ability of C33A cells, and the CCK-8 method was used to detect the sensitivity of C33A cells to 5-fluorouracil. The absorbance value indicated the cell proliferation ability, the lower the absorbance value, the weaker the proliferation ability, the more sensitive to the drug. Dual-luciferase reporter gene assay was used to verify the targeting relationship between RP13-349O20.2 and miRNA-493-5p (miR-493-5p), miR-493-5p and Nectin-4. qRT-PCR was used to detect the relative expression of miR-493-5p and Nectin-4 mRNA in two groups of C33A cells, and Western blotting was used to detect the expressions of Nectin-4 protein and PI3K-AKT signaling pathway proteins in two groups of cells.Results:Analysis based on data from GEPIA.CANCER website shows that patients with low expression of RP13-349O20.2 had better overall survival than patients with high expression ( P < 0.01). The relative expression levels of RP13-349O20.2 in cervical cancer tissues and paracancerous tissues of 40 patients were 4.04±0.32 and 1.18±0.14, and the difference was statistically significant ( t = 8.29, P < 0.01). Compared with H8 cells, the expressions of RP13-349O20.2 in human cervical cancer cell lines HCC94, C33A, Hela, HCC1106 and SiHa were higher (all P < 0.01). The relative expression levels of RP13-349O20.2 in C33A cells in the si-Con group and si-RP13-349O20.2 group were 7.30±0.30 and 1.01±0.27, and the difference was statistically significant ( t = 15.62, P < 0.01). The scratch healing rates of C33A cells in the si-Con group and si-RP13-349O20.2 group were (32±9)% and (75±6)% ( t = 3.97, P < 0.01), and the numbers of invasive cells were (106±12) cells and (36±8) cells ( t = 4.79, P < 0.01). After the action of 5, 10, 20, 40 and 80 μmol/L 5-fluorouracil for 24 h, the absorbance value of C33A cells in the si-RP13-349O20.2 group was lower than that in the si-Con group. Dual-luciferase reporter gene assay confirmed that there was a targeting relationship between P13-349O20.2 and miR-493-5p ( P < 0.01), and there was a targeting relationship between miR-493-5p and Nectin-4 ( P < 0.01) . The relative expression levels of miR-493-5p in C33A cells in the si-Con group and si-RP13-349O20.2 group was 1.02±0.13 and 5.48±0.85 ( t = 5.21, P < 0.01). The relative expression levels of Nectin-4 mRNA were 5.65±0.33 and 0.99±0.34 ( t = 9.87, P < 0.01). The expression of Nectin-4 protein in C33A cells in the si-RP13-349O20.2 group was lower than that in the si-Con group ( t = 9.21, P = 0.001), and the expressions of PI3K-AKT signaling pathway proteins p-STAT3, p-PI3K, p-AKT and p-mTOR were lower than those in the si-Con group (all P < 0.01). Conclusions:The level of RP13-349O20.2 in cervical cancer tissues is high, and its high expression may indicate the poor prognosis of patients. Interfering with the expression of RP13-349O20.2 in vitro can inhibit the migration and invasion abilities of cervical cancer cells and promote the sensitivity of cervical cancer cells to 5-fluorouracil. The mechanism may be related to the miR-493-5p/Nectin-4 signaling pathway and the PI3K-AKT signaling pathway.
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ObjectiveTo investigate the effect of ankyrin-repeat domain-containing protein 22 （ANKRD22） on the proliferation， invasion， and migration of human hepatoma cells and its molecular mechanism. MethodsThe TCGA database was used to analyze the expression level of ANKRD22 in normal liver tissue and hepatocellular carcinoma tissue and its association with prognosis. Western Blot and qRT-PCR were used to measure the expression of ANKRD22 in human normal liver cells （L-02） and human hepatoma cells （Huh7， HepG2， MHCC-97H， SK-HEP-1， and SMMC-7721）； CCK-8 assay， EdU， wound healing assay， and Transwell assay were used to observe the effect of ANKRD22 on the proliferation， invasion， and migration of hepatoma cells； Western Blot was used to investigate the association of ANKRD22 with cyclins and EMT-related proteins； KEGG and ssGSEA analyses were performed to investigate the mechanism of action of ANKRD22 in hepatoma cells， and related experiments were conducted for validation. The independent-samples t-test was used for comparison of continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsIn the TCGA database， the expression level of ANKRD22 in hepatoma tissue was significantly higher than that in normal liver tissue （t=5.083， P<0.05）， and the patients with a high expression level of ANKRD22 had longer overall survival and disease-related survival than those with a low expression level of ANKRD22 （P<0.05）. The expression level of ANKRD22 in various human hepatoma cell lines was higher than that in human normal liver cells （all P<0.05）. Cell proliferation assay showed that the ANKRD22 overexpression group had significantly higher EdU positive rate and proliferation rate than the Vector group （t=19.60 and 6.72， both P<0.001）， and compared with the si-NC group， the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower EdU positive rate and proliferation rate （all P<0.001）. Compared with the Vector group， the overexpression group had significantly higher expression levels of Cyclin E1， Cyclin D1， CDK7， and CDK4 （t=3.54， 4.95， 6.34， and 5.19， all P<0.01）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group （all P<0.001）. The overexpression group had a significantly lower expression level of P27 than the Vector group （t=6.12， P<0.001）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had a significantly higher expression level than the si-NC group （both P<0.001）. Invasion and migration experiments showed that compared with the Vector group， the ANKRD22 overexpression group had significantly higher migration rate and number of crossings through the membrane （migration group and invasion group） （t=5.01， 25.60， and 3.67， all P<0.05）， and compared with the si-NC group， thesi-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower migration rate and number of crossings through the membrane （migration group and invasion group） （all P<0.01）. The overexpression group had significantly higher expression levels of N-cadherin， Vimentin， and Snail than the Vector group （t=12.13， 8.85， and 13.97， all P<0.001）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group （all P<0.001）； the overexpression group had a significantly lower expression level of E-cadherin than the Vector group （t=4.98， P<0.01）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had a significantly higher expression level than the si-NC group （both P<0.001）. The KEGG enrichment analysis and the ssGSEA analysis showed that ANKRD22 was associated with the PI3K/AKT/mTOR signaling pathway in hepatocellular carcinoma， and the overexpression group had significantly higher expression levels of p-AKT/AKT， p-PI3K/PI3K， and p-mTOR/mTOR than the Vector group （t=12.21， 3.43， and 9.75， all P<0.01）， and the si-ANKRD22#2 group and the si-ANKRD22#3 group had significantly lower expression levels than the si-NC group （all P<0.001）. ConclusionANKRD22 is highly expressed in hepatoma cells and can promote the proliferation， invasion， and migration of hepatoma cells and the activation of the PI3K/AKT/mTOR signaling pathway.
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ObjectiveTo investigate the role and prognostic value of matrix metalloproteinase-7 （MMP7） in the migration and immune cell infiltration of hepatocellular carcinoma. MethodsMMP7_siRNA for downregulating the target gene MMP7 and pMMP7 for upregulating MMP7 were constructed and were used to transfect hepatocellular carcinoma cell line （MHCC97H）. RT-qPCR and Western Blot were used to measure the mRNA and protein expression levels of the target gene in cells. Scanning electron microscopy and Transwell assay were used to observe the changes in cell pseudopodia and migration ability， and bioinformatics methods were used to investigate the correlation of MMP7 with immune cells and immune infiltration score in TCGA and TIMER databases in patients with hepatocellular carcinoma， as well as the association between MMP7 and the prognosis of patients with hepatocellular carcinoma. The Spearman method was used for correlation analysis. Sanger Box online tool was used to assess the value of MMP7 in the overall survival curve and disease-specific survival of hepatocellular carcinoma. The Kaplan-Meier method was used to plot survival curves， and the Log-rank test was used for comparison of prognosis between different samples. ResultsAfter MHCC97H cells were transfected with MMP7_siRNA or pMMP7， there was a significant reduction or increase in the expression of the target gene MMP7； after downregulation of MMP7， there were significant reductions in the number and length of the pseudopodia， while after MMP7 overexpression， there were significant increases in the number and length of filopodia with radial arrangement. The Transwell chamber assay showed that MMP7_siRNA2 significantly reduced the migration ability of cells （P<0.05）， and there was a significant increase in migration ability after pMMP7 transfection. The expression of MMP7 was significantly correlated with B lymphocytes （r=0.37， P<0.05）， CD4+ T lymphocytes （r=0.40， P<0.05）， neutrophils （r=0.49， P<0.05）， macrophages （r=0.49， P<0.05）， and dendritic cells （r=0.47， P<0.05）. In the TCGA database， the patients with hepatocellular carcinoma were divided into MMP7 high expression group with 267 patients and MMP7 low expression group with 146 patients based on overall survival， and the results showed that the MMP7 high expression group had a significantly shorter overall survival time than the MMP7 low expression group （P<0.05）； based on the disease-specific survival time， the patients were divided into MMP7 high expression group with 257 patients and MMP7 low expression group with 145 patients， and the analysis showed that the MMP7 high expression group also had a significantly shorter disease-specific survival time than the MMP7 low expression group （P<0.05）. ConclusionMMP7 promotes the migration of hepatocellular carcinoma cells and plays a major role in immune cell infiltration， and the expression of MMP7 is also significantly associated with the prognosis of hepatocellular carcinoma.
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Objective:To investigate the characteristics related to proliferation, migration and invasion of radiation-induced polyploid colon cancer SW1116 cells and their progeny.Methods:Colon cancer SW1116 cells were conventionally cultured in Leibovitz's L-15 medium containing 10% fetal bovine serum. SW1116 cells at logarithmic growth stage were irradiated with 7 Gy X-ray, and the morphological changes of the cells were observed by inverted microscope on days 3, 5, 10 and 19 after radiation induction. According to the morphological changes of the cells, the cells at day 3 after radiation induction were labeled as polyploid giant cancer cell (PGCC) group, and the cells at day 19 were recorded as PGCC progeny group. SW1116 cells without radiation induction were used as control group. Flow cytometry was used to detect cell ploidy in the control, PGCC and PGCC progeny groups, CCK-8 assay was used to detect the proliferation ability of the three groups, cell migration and invasion abilities of the three groups were detected by cell scratch assay and Transwell assay, and Western blotting was used to detect the expressions of cell cycle and proliferation-related proteins and epithelial-mesenchymal transition (EMT) marker N-cadherin (N-cad) in the three groups.Results:The volume of SW1116 cells gradually became larger on days 3, 5 and 10 after radiation induction, and returned to normal on day 19. The proportions of polyploid (DNA content >4N) cell subsets in the control group, PGCC group and PGCC progeny group were (2.3±1.1)%, (23.1±8.1)% and (3.2±0.5)%, the difference was statistically significant ( F = 18.52, P < 0.05), and the proportion of polyploid cell subpopulations in the PGCC group was higher than that in the control group ( t = 5.38, P < 0.01), but the differences between the PGCC progeny group and the control group were not statistically significant ( t = 0.22, P > 0.05). After 72 h of culture, the cell proliferation rates of the control, PGCC and PGCC progeny groups were (100.0±4.1)%, (73.5±0.7)% and (123.9±3.5)%, and the difference was statistically significant ( F = 190.27, P < 0.001). After 48 h of cell scratching, the scratch healing rates in the control, PGCC and PGCC progeny groups were (38.0±2.7)%, (41.5±4.0)% and (63.7±4.2)%, and the difference was statistically significant ( F = 43.05, P < 0.001). After 24 h of culture, the number of invasive cells in the control, PGCC and PGCC progeny groups was 12.9±1.2, 3.4±0.6 and 23.7±1.5, and the difference was statistically significant ( F = 63.64, P < 0.001). The expression levels of cell cycle-related proteins P-cdc25c, cdc25c and cdc2 in the PGCC group were lower than those in the control group (all P < 0.05), and the expression levels of transcription factor-related proteins E2F-2, E2F-3 and EMT marker N-cad were downregulated compared with the control group (all P < 0.05); the expression levels of P-cdc25c, cdc25c, cdc2, E2F-2, E2F-3 and N-cad proteins in the PGCC progeny group were higher than those in the control group (all P < 0.05). Conclusions:Radiation can induce colon cancer SW1116 cells to produce polyploid, which may then generate daughter cells through asymmetric mitosis and gain new life, and then promote the recurrence and metastasis of colon cancer.
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Objective:To investigate the relationship between long non-coding RNA (lncRNA) DHRS4-AS1 and disease-free survival in osteosarcoma patients and the mechanisms of its effect on proliferation and migration of osteosarcoma cells in vitro.Methods:The data of DHRS4-AS1 transcriptome levels and survival status of osteosarcoma patients in GEPIA database were collected since the database was established, and the patients were divided into high DHRS4-AS1 expression group and low DHRS4-AS1 expression group based on the median DHRS4-AS1 transcriptome level, with 59 cases in each group, and the Kaplan-Meier method was used to analyze the disease-free survival of the two groups. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of DHRS4-AS1 in osteosarcoma cell lines MG-63, HOS, 143B, U-2OS, Saos2 and normal osteoblast cell line hFOB1.19, and the osteosarcoma cell line with the lowest DHRS4-AS1 expression level was selected for subsequent experiments. The plasmid carrying DHRS4-AS1 sequence and the plasmid carrying negative control sequence were transfected into the selected osteosarcoma cells as DHRS4-AS1 group and control group. CCK-8 method was used to detect the proliferation of each group of cells, and the absorbance value was used as the cell proliferation ability; cell scratch assay was used to detect the migration of each group of cells. The bioinformatics website starBase V2.0 was used to predict the target genes of DHRS4-AS1, and the dual luciferase reporter gene assay was used to verify the targeting relationship between DHRS4-AS1 and the target genes. The expression levels of target genes and downstream genes of osteosarcoma cells in control group and DHRS4-AS1 group were detected by qRT-PCR and Western blotting.Results:Survival analysis showed that the disease-free survival of osteosarcoma patients in the high DHRS4-AS1 expression group in GEPIA database was superior to that of the low DHRS4-AS1 expression group ( P < 0.001). Compared with normal osteoblastic hFOB1.19 cells, the expression level of DHRS4-AS1 was low in all osteosarcoma cells (all P < 0.01), with the lowest expression level of DHRS4-AS1 in U-2OS cells ( P < 0.001). Cell proliferation ability was reduced in U-2OS cells of the DHRS4-AS1 group after 1, 2, 3 and 4 d of culture compared with the control group (all P < 0.05). The migration rate of U-2OS cells in the DHRS4-AS1 group was lower than that in the control group [(31±6)% vs. (63±4)%, t = 4.38, P = 0.005]. starBase V2.0 website predicted that DHRS4-AS1 complementarily bound to miRNA-411-3p (miR-411-3p); dual luciferase reporter gene assay showed that miR-411-3p overexpression reduced the luciferase activity of the wild-type DHRS4-AS1 reporter gene ( P < 0.001), but had no effect on the luciferase activity of the mutant DHRS4-AS1 reporter gene ( P > 0.05). qRT-PCR showed that the relative expression of miR-411-3p in U-2OS cells of the DHRS4-AS1 group was low (0.22±0.06 vs. 1.06±0.23, t = 3.55, P = 0.012) and the relative expression of metastasis suppressor MTSS1 mRNA was high (5.58±1.03 vs. 1.06±0.22, t = 4.28, P = 0.005) compared with the control group; Western blotting showed that MTSS1 expression was elevated, and the expression levels of cell proliferation phenotype proteins CDK3 and cyclin C and cell migration phenotype proteins ZEB2 and KLF8 were low. Conclusions:Osteosarcoma patients with high expression of lncRNA DHRS4-AS1 have better disease-free survival, and its expression is low in osteosarcoma cell lines. DHRS4-AS1 may promote MTSS1 gene expression and inhibit cell proliferation and migration by targeting and down-regulating miR-411-3p expression in osteosarcoma cells.
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Objective:To investigate the effects of miRNA-30a-5p (miR-30a-5p) and metadherin (MTDH) on the proliferation, invasion and migration abilities of human breast cancer cells in vitro.Methods:The expression of MTDH in cancer and paracancerous tissues of 112 breast cancer patients in the database and miR-30a-5p in cancer and paracancerous tissues of 103 breast cancer patients in the database were analyzed using data from The Cancer Genome Atlas (TCGA) database. Pearson correlation analysis was used to analyze the correlation between miR-30a-5p and MTDH in 1 222 breast cancer patients in the database; the data were updated to August 2022. Breast cancer MDA-MB-231 cells were divided into negative control group (transfected with negative control sequence), miR-30a-5p overexpression group (transfected with miR-30a-5p mimics), siMTDH group [transfected with small interfering RNA against MTDH (siMTDH)], siMTDH+miR-30a-5p overexpression group (transfected with both siMTDH and miR-30a-5p mimics); cell proliferation ability was detected by methyl thiazol tetrazolium (MTT) assay, cell migration ability was detected by cell scratch assay, cell invasion ability was detected by Transwell assay. The relative expressions of miR-30a-5p, MTDH, matrix metalloproteinase (MMP)-2, MMP-9, vimentin and β-catenin mRNA in cells were detected by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR), and the expressions of MTDH, N-cadherin (N-cad), β-catenin, Snail and MMP-9 proteins were detected by Western blotting.Results:In the TCGA database, MTDH expression level was higher and miR-30a-5p expression level was lower in breast cancer tissues compared with paracancerous tissues, and the differences were statistically significant (both P < 0.001). There was a negative correlation between MTDH and miR-30a-5p expressions in 1 222 patients with breast cancer ( r=-0.134, P < 0.001). Compared with the negative control group, the cell proliferation ability was reduced in both siMTDH group and miR-30a-5p overexpression group at 24, 48 and 72 h (all P < 0.001). The cell scratch healing rate in miR-30a-5p overexpression group and siMTDH group was lower than that in negative control group [(61.6±1.6)%, (54.7±5.9)% vs. (80.3±3.0)%] (both P < 0.05). Compared with the negative control group, The number of migrated cells in miR-30a-5p overexpression group and siMTDH group was less than that in negative control group (881±50, 725±63 vs. 1 172±66) (both P < 0.05). Compared with the negative control group, the relative expressions of MMP-2, MMP-9, vimentin and β-catenin mRNA were all down-regulated in MDA-MB-231 cells of miR-30a-5p overexpression group and siMTDH group (all P < 0.05). Compared with the negative control group, the relative expressions of N-cad, β-catenin, Snail and MMP-9 proteins were down-regulated in MDA-MB-231 cells of miR-30a-5p overexpression group and siMTDH group (all P < 0.05). There was no statistical difference in the number of migrated MDA-MB-231 cells between siMTDH+miR-30a-5p overexpression group and siMTDH group (476±5 vs. 389±46, t = 3.37, P = 0.078). There was no statistical difference in the number of migrated cells between siMTDH+miR-30a-5p overexpression group and miR-30a-5p overexpression group (476±5 vs. 477±22, t = 0.02, P = 0.983). Conclusions:The expression of miR-30a-5p is negatively correlated with the expression of MTDH in breast cancer tissues, and either overexpression of miR-30a-5p or silence of MTDH in breast cancer MDA-MB-231 cells in vitro can inhibit cell proliferation, invasion and migration, but MTDH may not be a target gene of miR-30a-5p.
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Objective:To investigate the effects of Anhydroicaritin (AHI) , an isopentenylated flavo-noid compound, on proliferation, migration and apoptosis of human hepatocarcinoma cell line MHCC-97H.Methods:Human hepatocarcinoma cell line MHCC-97H and human normal liver cell line L02 were cultured in vitro. MHCC-97H cells were treated with 0, 20, 40, 80, 120, 160, 200 μg/ml of AHI respectively and L02 cells were treated with 0, 25, 50, 100, 150, 200, 400, 500 μg/ml of AHI respectively. CCK-8 and clone formation assay were used to detect cell proliferation. Scratch test was used to explore cell migration ability. Hoechst33342 assay and flow cytometer were used to detect cell apoptosis. The expressions of apoptosis-related proteins were detected by Western blotting. Results:The cell viabilities of MHCC-97H cells treated with 0, 20, 40, 80, 120, 160, 200 μg/ml of AHI for 24 h were (100.00±0.00) %, (97.41±2.10) %, (96.58±3.23) %, (87.72±4.85) %, (78.33±3.76) %, (56.97±2.61) % and (15.25±2.51) % respectively, and there was a statistically significant difference ( F=429.20, P<0.001) . There were statistically significant differences between 0 μg/ml and 80, 120, 160, 200 μg/ml of AHI treatment (all P<0.001) . The cell viabilities of L02 cells treated with 0, 25, 50, 100, 150, 200, 400, 500 μg/ml of AHI for 24 h were (100.00±0.00) %, (96.82±3.79) %, (95.36±3.43) %, (90.79±5.75) %, (77.67±5.66) %, (63.98±5.22) %, (34.22±4.01) % and (33.84±4.41) % respectively, and there was a statistically significant difference ( F=233.20, P<0.001) . There were statistically significant differences between 0 μg/ml and 100, 150, 200, 400, 500 μg/ml of AHI treatment (all P<0.05) . The 24 h half maximal inhibitory concentration (IC 50) value of AHI treated L02 cells was (300.20±17.10) μg/ml, which was significantly higher than that of MHCC-97H cells [ (158.60±5.50) μg/ml], and there was a statistically significant difference ( t=13.65, P<0.001) . The cell clone numbers of MHCC-97H cells treated with 0, 120, 160 and 200 μg/ml of AHI for 24 h were 1 993.00±46.29, 1 355.00±54.84, 998.33±21.03 and 218.33±35.95 respectively, and there was a statistically significant difference ( F=954.80, P<0.001) . There were statistically significant differences between 0 μg/ml and 120, 160, 200 μg/ml of AHI treatment (all P<0.001) . The healing rates of MHCC-97H cells treated with 0, 120, 160 and 200 μg/ml of AHI for 24 h were (51.68±1.93) %, (16.04±0.73) %, (8.88±0.31) % and (-6.94±0.46) % respectively, and there was a statistically significant difference ( F=1 616.00, P<0.001) . There were statistically significant differences between 0 μg/ml and 120, 160, 200 μg/ml of AHI treatment (all P<0.001) . Hoechst33342 experiment showed that MHCC-97H cells treated with 0 μg/ml AHI showed uniform dark blue with a complete nuclear state under inverted microscope. Compared with 0 μg/ml AHI treated cells, cells in the 120, 160, 200 μg/ml AHI treatment groups wrinkled and broken, and nuclei were also morphologically abnormal, with some nuclei stained bright blue, and the situation became more obvious with increasing dose. The apoptosis rates of MHCC-97H cells treated with 0, 120, 160 and 200 μg/ml AHI for 24 h were (10.51±0.56) %, (42.23±0.87) %, (61.92±0.52) % and (72.05±0.74) % respectively, and there was a statistically significant difference ( F=4 677.00, P<0.001) . There were statistically significant differences between 0 μg/ml and 120, 160, 200 μg/ml of AHI treatment (all P<0.001) . There were statistically significant differences among the different expression levels of Bax, Cleaved Caspase-3/Caspase-3, Cleaved Caspase-9/Caspase-9, and Bcl-2 proteins in MHCC-97H cells of 0, 120, 160, and 200 μg/ml of AHI treatment ( F=30.43, P<0.001; F=212.80, P<0.001; F=475.30, P<0.001; F=10.75, P=0.004) . The Bax protein expression of 160 and 200 μg/ml was significantly increased than that of 0 μg/ml AHI (both P<0.001) . The Cleaved Caspase-3/Caspase-3, Cleaved Caspase-9/Caspase-9 protein expressions of 120, 160 and 200 μg/ml were significantly increased than those of 0 μg/ml AHI (all P<0.001) . The Bcl-2 protein expression of 120, 160, 200 μg/ml was significantly decreased compared with that of 0 μg/ml AHI (all P<0.05) . Conclusion:AHI can inhibit the proliferation and migration of hepatocellular carcinoma cell line MHCC-97H, and promote its apoptosis.
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Objective To investigate the effect of genistein on the proliferation,migration and invasion of prostate cancer cells and its molecular mechanism.Methods Prostate cancer LNCaP and CWR22RV1 cells were divided into the control group(conventional culture)and the experimental group(50μmol/L genistein treatment).The effect of genistein on the proliferation of prostate cancer cells were analyzed by MTT assay.The effect of genistein on the migration and invasion of prostate cancer cells were analyzed by cell scratch assay and Transwell assay.The protein levels of epithelial interstital transformation(EMT)intermediate markers E-Cadherin,N-Cadherin,Vimentin,and tumor stem cell markers CD44 and Oct-4 were detected by Western blot assay.Results MTT assay showed that genistein could inhibit the proliferation of prostate cancer cells.The scratch closure rates of LNCaP and CWR22RV1 cells were significantly reduced in the experimental group compared with those in the control group,and the number of cells passing through the Transwell membrane was significantly reduced(P＜0.05).Western blot assay showed that genistein could down-regulate the expression levels of N-Cadherin,Vimentin,CD44 and Oct4 in prostate cancer cells,and up-regulate the expression of E-Cadherin in epithelial cells(P＜0.01).Conclusion Genistein reduces the dryness of prostate cancer cells by inhibiting the EMT process,thus reducing the proliferation,migration and invasion of prostate cancer cells.
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Objective To investigate the impact of shikonin(SHI)on the malignant biological activity of liver cancer cells by regulating Notch signaling pathway.Methods Western blot assay was used to detect the expression of Notch,Hairy mitosis-related enhancer-1(Hes1),hairy-related transcription factor-1(HEY1)protein in liver cancer tissue,paracancerous tissue,hepatoma cells(HepG2 cells,Hep3B cells,HCCLM3 cells,Huh-7 cells and SMMC-7721 cells)and normal liver cells(HL-7702 cells).Huh-7 cells were divided into the control group,the L-SHI group(1μmol/L SHI),the M-SHI group(2μmol/L SHI),the H-SHI group(4μmol/L SHI),the DAPT group(50μmol/L Notch signal inhibitor DAPT)and the H-SHI+VPA group[4μmol/L SHI and 8 mmol/L Notch pathway activator Valproic acid(VPA)].The proliferation of Huh-7 cells was detected by CCK-8 method and plate cloning test.The apoptosis and cell cycle of Huh-7 cells were detected by flow cytometry.Cell scratch test and Transwell invasion test were used to detect migration and invasion of Huh-7 cells.Western blot assay was used to detect the expression of epithelial-mesenchymal transformation(EMT)and apoptosis related proteins.Results The expression levels of Notch,HES1 and HEY1 were obviously increased in liver cancer tissue and cells,and Huh-7 cells showed the most obvious difference,therefore,Huh-7 cells were taken as the research object.Compared with the control group,the protein levels of Notch,HES1,HEY1 and Bcl-2 decreased,and the proportions of S phase and G2 phase cells,OD450 value,number of clones,migration rate,number of invasive cells and levels of N-cadherin and Vimentin decreased significantly in the L-SHI group,the M-SHI group,the H-SHI group and the DAPT group(P＜0.05).The proportion of G1/G0 phase cells,apoptosis rate and levels of Bax,cleaved Casase-3,and E-cadherin increased obviously(P＜0.05).The effect of SHI was dose-dependent.Compared with the H-SHI group,the above indexes showed the opposite trend in the H-SHI+VPA group.VPA attenuated the effect of SHI on reducing the malignant biological activity of liver cancer cells.Conclusion SHI may inhibit the proliferation,migration and invasion of Huh-7 cells and promote apoptosis of Huh-7 cells by inhibiting Notch signal pathway.
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Objective:To evaluate the effect of dexmedetomidine (Dex) on the proliferation, migration and invasion ability of renal carcinoma cells and the relationship with ferroptosis.Methods:Experiment Ⅰ GRC-1 cells at the logarithmic growth phase were selected and divided into 5 groups ( n=6 each) using a random number table method: control group (group C) and different concentrations of dexmedetomidine groups(D1, D2, D3, and D4 groups). Group C was routinely incubated for 24 h. D1, D2, D3, and D4 groups were incubated with dexmedetomidine at 0.1, 1.0, 10.0 and 100.0 μmol/L respectively, for 24 h. The cell proliferation ability was assessed by CCK-8 assay.The cell migration and invasion ability was was evaluated by Transwell chamber assay. Experiment Ⅱ GRC-1 cells at the logarithmic growth phasewere selected and divided into 3 groups ( n=6 each) using a random number table method: control group (group C), dexmedetomidine group (group D), and dexmedetomidine+ Ferrostatin-1 group (group D+ F). Group C was routinely cultured for 24 h. Dexmedetomidine 10 μmol/L was added and cells were incubated for 24 h in group D. Dexmedetomidine 10 μmol/L was added, Ferrostatin-1 1 μmol/L was simultaneously added, and then cells were incubated for 24 h in group D+ F. The proliferation ability of the cells was tested by CCK-8 assay, and the migration and invasion ability of the cells was detected by Transwell assay. The contents of glutathione (GSH), malondialdehyde (MDA) and Fe 2+ were measured by the colorimetric method. The expression of glutathione peroxidase 4(GPX4) and ATF4-induced solute carrier family 7a member 11 (SLC7A11) was detected by Western blot. Results:Experiment I Compared with group C, the cell proliferation and the number of migrating and invading cells were significantly decreased in D3 and D4 groups ( P<0.05), and no significant change was found in aforementioned indexes in D1 and D2 groups ( P>0.05). Experiment Ⅱ Compared with group C, the cell proliferation and the number of migrating and invading cells were significantly decreased, the content of Fe 2+ was increased, the content of GSH was decreased, the expression of GPX4 and SLC7A11 was down-regulated ( P<0.05), and no significant change was found in MDA content in group D( P>0.05). Compared with group D, the cell proliferation and the number of migrating and invading cells were significantly increased, the content of Fe 2+ was decreased, the content of GSH was increased, the expression of GPX4 and SLC7A11 was up-regulated ( P<0.05), and no significant change was found in the MDA content in group D+ F( P>0.05). Conclusions:Dexmedetomidine can inhibit the proliferation, migration and invision ability of renal carcinoma cells, and the mechanism is related to promotion of ferroptosis.
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Objective:To evaluate the effects of different concentrations of ropivacaine on the growth and migration of lung cancer cells.Methods:Human lung adenocarcinoma cell strain A549 cells and human lung squamous cell strain H520 cells were divided into 4 groups ( n=24 each) using a random number table method: control group (group C) and different concentrations of ropivacaine groups (Ⅰ-Ⅲ groups). Cells were commonly cultured in group C. Ropivacaine 3, 5 and 7 mmol/L were added and then the cells were cultured in Ⅰ-Ⅲ groups, respectively. The cell survival rate was determined using the CCK-8 method at 24, 48 and 72 h of treatment (T 1-3). The cell cycle and apoptosis were detected at T 1 using flow cytometry. The expression of Cyclin D1, cyclin-dependent kinase 4 (CDK4), cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and cleaved caspase-3 was detected using Western blot. Wound healing assay was used to measure cell migration distance. The activities of RhoA and Rac1 were detected by microplate spectrophotometry. Results:The cell viability of A549 and H520 cells sequentially decreased at T 1-3, the proportion of G0/G1 phase and apoptosis sequentially increased, the expression of Cyclin D1 and CDK4 was down-regulated sequentially at T 1, the expression of cleaved PARP-1 and cleaved caspase-3 was up-regulated sequentially, and the cell migration distance, RhoA, and Rac1 activity decreased sequentially in C, Ⅰ, Ⅱ and Ⅲ groups ( P<0.05). Conclusions:Ropivacaine can inhibit the growth and migration ability of lung cancer cells in a concentration-dependent manner, which is related to induction of cell cycle arrest and apoptosis.
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Objective:To investigate effects of the ROCK1 gene on proliferation and migration of and related molecular expression in keloid fibroblasts.Methods:Immunohistochemical technique was used to detect ROCK1 protein expression in human keloids and normal skin tissues, and Western blot analysis was performed to detect the expression of ROCK1, transforming growth factor β1 (TGF-β1) and E-cadherin in keloid tissues. In vitro cultured human keloid fibroblasts (HKFs) were divided into 4 groups: ROCK1 gene overexpression control group (ROCK1 NC group) transfected with ROCK1 gene overexpression control vectors, ROCK1 gene overexpression group (ROCK1 OE group) transfected with ROCK1 gene overexpression vectors, ROCK1 gene knockdown control group (sh NC group) transfected with ROCK1 gene knockdown control vectors, and ROCK1 gene knockdown group (shROCK1 group) transfected with ROCK1 gene knockdown vectors. Cell counting kit-8 (CCK8) assay was performed to evaluate the effect of ROCK1 gene on the survival rate of HKFs, Transwell assay to evaluate the effect on the migration of HKFs, and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were conducted to determine the mRNA and protein expression of ROCK1, TGF-β1 and E-cadherin, respectively. Results:Immunohistochemical study showed that ROCK1 protein expression decreased significantly in the human keloid tissues compared with the normal tissues ( t = 6.47, P = 0.003) ; Western blot analysis showed that the expression levels of ROCK1 and E-cadherin significantly decreased ( t = 14.02, 162.20, respectively, both P < 0.001), while TGF-β1 expression significantly increased ( t = 76.01, P < 0.001) in the keloid tissues compared with the expression levels of corresponding proteins in the normal tissues. CCK8 assay showed that the cell activity was significantly lower in the ROCK1 OE group than in the ROCK1 NC group after 24-hour transfection ( t = 3.25, 3.78, P = 0.031, 0.019, respectively), and significantly higher in the shROCK1 group than in the sh NC group ( t = 3.12, 2.79, P = 0.036, 0.049, respectively). Transwell assay showed that the number of migratory cells was significantly lower in the ROCK1 OE group than in the ROCK1 NC group ( t = 5.17, P = 0.004), and significantly higher in the shROCK1 group than in the sh NC group ( t = 9.28, P < 0.001). Compared with the ROCK1 NC group, the ROCK1 OE group showed significantly increased mRNA and protein expression levels of ROCK1 and E-cadherin ( P < 0.05 or < 0.001), but decreased mRNA and protein expression levels of TGF-β1 (both P < 0.001) ; compared with the sh NC group, the shROCK1 group showed significantly decreased mRNA and protein expression levels of ROCK1 and E-cadherin ( P < 0.05 or < 0.001), but significantly increased mRNA and protein expression levels of TGF-β1 ( P = 0.005 or < 0.001) . Conclusions:The ROCK1 gene can inhibit the proliferation and migration of HKFs. Overexpression of the ROCK1 gene can down-regulate the TGF-β1 gene expression and up-regulate the E-cadherin gene expression in HKFs.
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Abstract Objectives Metastasis is one of the biggest challenges in the management of Esophageal Squamous Cell Carcinoma (ESCC), of which molecular mechanisms remain elusive. The present study aimed to explore the roles and underlying mechanisms of Transmembrane protein 26 (TMEM26) in ESCC. Method TMEM26 expressions in tumorous and adjacent tissues from patients with ESCC and in normal esophageal epithelial and ESCC cell lines were detected by immunostaining and western blotting, respectively. The Epithelial-Mesenchymal Transition (EMT), a critical process during metastasis, was investigated by wound healing and Transwell assays, and EMT-related proteins were examined after the TMEM26 alteration in ESCC cell lines. NF-κB signaling activation and Tight Junction (TJ) protein expression were analyzed by western blotting and immunofluorescence, respectively. In vivo verification was performed on the liver metastatic murine model. Results Compared with non-cancerous esophageal tissues and cells, the TMEM26 expression level was higher in ESCC samples and cell lines, where the plasma membrane localization of TMEM26 was observed. The EMT-related processes of ESCC cells were suppressed by RNAi depletion of TMEM26 but aggravated by TMEM26 overexpression. Mechanistically, TMEM26 promoted NF-κB signaling to accelerate EMT in ESCC cells. The plasma membrane presentation and assembly of TJ proteins were impaired by TMEM26. Conclusion Overall, TMEM26 acts as a critical determinant for EMT in ESCC cells by disrupting TJ formation and promoting NF-κB signaling, which may be a potential therapeutic target for treating metastatic ESCC.
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Objective:To explore the effect of bone morphogenetic protein 4 (BMP4) on the glycolysis level of human retinal microvascular endothelial cells (hRMECs).Methods:A experimental study. hRMECs cultured in vitro were divided into normal group, 4-hydroxynonenal (HNE) group (4-HNE group) and 4-HNE+BMP4 treatment group (BMP4 group). 4-HNE group cell culture medium was added with 10 μmmol/L 4-HNE; BMP4 group cell culture medium was added with recombinant human BMP4 100 ng/ml after 6 h stimulation with 10 μmol/L 4-HNE. The levels of intracellular reactive oxygen species (ROS) were detected by flow cytometry. The effect of 4-HNE on the viability of cells was detected by thiazole blue colorimetry. Cell scratch test and Transwell cell method were used to determine the effect of 4-HNE on cell migration. The relative expression of BMP4 and SMAD9 mRNA and protein in normal group and 4-HNE group were detected by realtime quantitative polymerase chain reaction and Western blot. Seahorse XFe96 cell energy metabolism analyzer was used to determine the level of intracellular glycolysis metabolism in normal group, 4-HNE group and BMP4 group. One-way analysis of variance was used for comparison between groups.Results:The ROS levels in hRMECs of normal group, 4-HNE group and BMP4 group were 21±1, 815±5, 810±7, respectively. Compared with the normal group, the levels of ROS in the 4-HNE group and the BMP4 group were significantly increased, and the difference was statistically significant ( F=53.40, 50.30; P<0.001). The cell viability in the normal group and 4-HNE group was 1.05±0.05 and 1.28±0.05, respectively; the migration rates were (0.148±0.005)%, (0.376±0.015)%; the number of cells passing through the pores were 109.0±9.6, 318.0±6.4, respectively. Compared with the normal group, the 4-HNE group had significantly higher cell viability, cell migration rate, and the number of cells passing through the pores, and the differences were statistically significant ( F=54.35, 52.84, 84.35; P<0.05). The relative expression levels of BMP4 and SMAD9 mRNA in the cells of the 4-HEN group were 1.680±0.039 and 1.760±0.011, respectively; compared with the normal group, the difference was statistically significant ( F=53.66, 83.54; P<0.05). The relative expression levels of BMP4 and SMAD9 proteins in the cells of the normal group and 4-HEN group were 0.620±0.045, 0.860±0.190, 0.166±0.049, 0.309±0.038, respectively; compared with the normal group, the differences were statistically significant ( F=24.87, 53.84; P<0.05). The levels of intracellular glycolysis, glycolytic capacity and glycolytic reserve in normal group, 4-HNE group and BMP4 group were 1.21±0.12, 2.84±0.24, 1.78±0.36, 2.59±0.11, 5.34±0.32, 2.78±0.45 and 2.64±0.13, 5.20±0.28, 2.66±0.33. Compared with the normal group, the differences were statistically significant (4-HNE group: F=86.34, 69.75, 58.45; P<0.001; BMP4 group: F=56.87, 59.35, 58.35; P<0.05). There was no significant difference in intracellular glycolysis, glycolysis capacity and glycolysis reserve level between 4-HNE group and BMP4 group ( F=48.32, 56.33, 55.01; P>0.05). Conclusion:BMP4 induces the proliferation and migration of hRMECs through glycolysis.
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Objective:To observe the effect of bone morphogenetic protein 4 (BMP4) on the proliferation and migration of human retinal microvascular endothelial cells (hRMEC) under oxidative stress.Methods:The hRMEC cultured in vitro were divided into control group, 4-hydroxynonenal (HNE) treatment group (4-HNE group), 4-HNE+BMP4 group (BMP4 group). Cell culture medium of 4-HNE treatment group was added with 10 μmmol/L 4-HNE; cell culture of BMP4 group was cultured with 10 μmmol/L 4-HNE, and after stimulation for 6 h, 100 ng/ml recombinant human BMP4 was added. The effects of 4-HNE and BMP4 on hRMEC viability was detected by thiazole blue colorimetric method. The effects of 4-HNE and BMP4 on cell migration was determined by cell scratch test. The relative expression of BMP4 mRNA in the cells of the control group and 4-HNE treatment group and the mRNA expression of the control group, the fibronectin (FN) of BMP4 group, laminin (Laminin), α-smooth muscle contractile protein (α-SMA), and collagen type Ⅰ (Collagen Ⅰ), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) were detected by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the relative expression of BMP4 protein in the control group and 4-HNE group. The control group and 4-HNE group were compared by t test. Results:Compared with the control group, cell viability ( t=12.73, 16.26, P=0.000 2, <0.000 1), cell migration rate ( t=28.17, 37.48, P<0.000 1, <0.000 1) in 4-HNE group and BMP4 group were significantly increased, and the difference was statistically significant; the relative expression of BMP4 mRNA and protein in the 4-HNE group was significantly increased, and the difference was statistically significant ( t=16.36, 69.35, P=0.000 1, <0.000 1). The qRT-PCR test results showed that compared with the control group, the relative expression of VEGF, FN, Laminin, α-SMA, Collagen Ⅰ, and CTGF mRNA in the cells of the BMP4 group was significantly increased, and the difference was statistically significant ( t=10.61, 17.00, 14.85, 7.78, 12.02, 10.61, P=0.0004, <0.000 1, 0.000 1, 0.001 5, 0.000 1, 0.000 4). Conclusion:BMP4 can induce the proliferation and migration of hRMEC; it can also regulate the expression of angiogenesis factors and fibrosis-related factors in hRMEC.
الملخص
Objective:To investigate the effect of ring finger protein 187 (RNF187) on cell pro-liferation, migration and invasion of hepatocellular carcinoma (HCC).Methods:Messenger RNA (mRNA) level of RNF187 in HCC was analyzed by bioinformatics. Huh7 cells transfected with small interfering RNA (siRNA) of negative control or target gene respectively were classified as non-transfection (NC) group and RNF187 knockdown group. After 24 hours of transfection, the above two groups dimethyl sulfoxide (DMSO) were used as NC+ DMSO group and RNF187 knockdown + DMSO group. 24 hours after transfection with siRNA of target gene, the cells dealt with bafliomycin A1 (BFA) were set as RNF187 knockdown + BFA group. The regulation of RNF187 on malignant biological behavior and autophagy level of HCC cells were explored by cell counting kit-8 (CCK8) proliferation assay, cell scratch assay, transwell assay and western blot.Results:Compared with normal liver tissue, the mRNA level of RNF187 was higher in HCC tissue ( P<0.05). Compared with NC group, the absorbance at 48 h and 72 h and the scratch healing rate at 12 h and 24 h of RNF187 knockdown group were all lower, the differences were statistically significant (all P<0.001). The number of transmembrane cells in RNF187 knockdown group (39.50±5.57) at 24 h was lower than that in NC group (128.25±17.35), the differences were statistically significant ( t=9.74, P<0.001). Compared with NC group, the relative expression of total LC3 and Beclin-1 in RNF187 knockdown group all increased, while the relative expression of phosphorylated mammalian target of rapamycin decreased, the difference were statistically significant (all P<0.05). Compared with RNF187 knockdown+ DMSO group, the autophagy flow level, the 48 h and 72 h absorbance, the scratch healing rate at 24 h in RNF187 knockdown + BFA group were higher, the differences were statistically significant (all P<0.001). The number of transmembrane cells in RNF187 knockdown + BFA group (119.00±2.65) was more than that in RNF187 knockdown + DMSO group (57.67±2.52), the differences were statistically significant ( t=29.09, P<0.001). Conclusion:RNF187 is highly expressed in HCC tissue and knockdown of RNF187 inhibits the malignant biological behavior of HCC by enhancing the level of autophagy.