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1.
Acta cir. bras ; 38: e380923, 2023. graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1429538

ABSTRACT

Purpose: To investigate the role and mechanism of ß1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) in esophageal cancer (ESCA). Methods: The starBase database was used to evaluate the expression of B3GNT3. B3GNT3 function was measured using KYSE-30 and KYSE-410 cells of esophageal squamous cell carcinoma (ESCC) cell lines. The mRNA levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8, clone formation assay and transwell assay were used to detect the changes of proliferation, invasion and migration. Results: B3GNT3 expression was higher in ESCA tissues than in normal tissues. The overall survival rate of ESCA patients with high B3GNT3 expression was lower than that of ESCA patients with low B3GNT3 expression. In vitro functional experiments showed that the proliferation ability, migration and invasion ability of KYSE-30 and KYSE-410 cells with B3GNT3 interference were lower than those of the control, and the overexpression of B3GNT3 had the opposite effect. After silencing B3GNT3 expression in ESCC cell lines, the growth of both cell lines was inhibited and the invasiveness was decreased. Knockdown of B3GNT3 reduced the growth rate and Ki-67 expression level. Conclusion: B3GNT3, as an oncogene, may promote the growth, invasion and migration of ESCC cell.


Subject(s)
Oncogenes , N-Acetylglucosaminyltransferases/analysis , Cell Migration Assays , Transcriptome , Esophageal Squamous Cell Carcinoma , Esophageal Neoplasms/physiopathology
2.
Indian J Exp Biol ; 2022 Jun; 60(6): 386-396
Article | IMSEAR | ID: sea-222497

ABSTRACT

The Grainy head like-2 (Grhl2) transcription factor plays a major role in embryonic and cancer development. The role of Grhl2 has been intensively studied in various cancers but not for brain cancer. Hence, in this study, we provide a preliminary understanding on the role of Grhl2 that regulate the transition of astrocytoma cells. The human A172 astrocytoma cell line, a mesenchymal cell characterized by mild overexpression of Grhl2 transcription factor, was used in this study. At first, the Grhl2 stably overexpressing A172 clones into three types i.e., Grhl2+ (mild), Grhl2++ (moderate) and Grhl2+++ (high) based on mRNA and protein expression levels of Grhl2 were characterized. Phenotypic characteristics of vector and Grhl2+ cells were observed using phase contrast microscopy. Quantitative PCR (qPCR), Western blot and immunofluorescence were used to detect the level of mesenchymal markers (N-cadherin/vimentin) and also epithelial markers (E-cadherin/ ?- catenin) in vector and Grhl2+ cells. The migration and invasion characteristics of vector and Grhl2+ cells were determined by scratch assay and Boyden chamber assay. Further, the Grhl2+ cells were characterized to determine the effect of temozolomide chemotherapy drug which were widely used in treating brain cancer. As expected, in phase contrast image, we observed the mesenchymal characteristic of A172 cells becomes hybrid phenotype i.e., mixture of mesenchymal (spindle-like fibroblast morphology) and epithelial (cobblestone like appearance) cells upon Grhl2 mild expression (Grhl2+) when compared to vector cells. Further, we found that there was a significant upregulation of E-cadherin at both mRNA and protein levels in Grhl2+ cells when compared to vector cells. There was a significant upregulation of ?-catenin, N-cadherin and vimentin at mRNA levels, but there was no significant upregulation at the protein levels in Grhl2+ cells compared to the vector cells. The migration and invasion were diminished in Grhl2+ cells when compared to the vector control cells. We observed that the Grhl2+ were sensitive to the temozolomide compared to the vector cells. This infers that the Grhl2+ cells were unable to attain complete transition of mesenchymal to epithelial state, and hence we categorized the Grhl2+ cells as hybrid phenotype. The results provide a better understanding of the largely unknown function of Grhl2 in human astrocytoma cells as tumor progression or suppression.

3.
Journal of Medical Biomechanics ; (6): E162-E168, 2022.
Article in Chinese | WPRIM | ID: wpr-920685

ABSTRACT

Objective To investigate the effects of cyclic stretch on migration of MC3T3-E1 cells and its related mechanism. Methods The strain loading system was used to stretch MC3T3-E1 cells cultured in vitro with 15% amplitude, to simulate the mechanical condition in vivo. The wound healing assay was used to detect the migration of MC3T3-E1 cells. Western blotting was used to test Runx2 expression. RNA interfering was used to decrease Runx2 expression. Results Cyclic mechanical stretch with 15% amplitude, 1.25 Hz frequency and lasting for 24 hours could promote the migration of MC3T3-E1 cells and increase the expression level of Runx2. Runx2 interference inhibited the migration of MC3T3-E1 cells in static culture condition. Interference with Runx2 expression in MC3T3-E1 cells could partially reduce the positive effect of cyclic mechanical stretch on cell migration. Conclusions Cyclic stretch can promote the migration of MC3T3-E1 cells, and Runx2 may play an important role in this process. This study provides experimental basis for finding innovative clinical treatment method to promote fracture healing.

4.
Journal of Chinese Physician ; (12): 377-382, 2022.
Article in Chinese | WPRIM | ID: wpr-932073

ABSTRACT

Objective:To investigate the methylation status of O6-methylguanine-DNA methyltransferase (MGMT) in meningioma tissue and its effect on the growth and metastasis of human meningioma cell line IOMM-Lee cells.Methods:The specimens of 34 patients with meningioma were collected. Methylation-specific polymerase chain reaction (MSP) method was used to detect MGMT methylation in meningioma tissue and normal brain tissue; immunohistochemical staining was used to detect the expression of MGMT in meningioma tissue and normal brain tissue. The cultured human glioma cell line IOMM-Lee cells were divided into blank control group (normal cultured IOMM-Lee cells), negative control group (empty vector virus transfected IOMM-Lee cells) and RNAi lentivirus transfection group (transfected with RNAi lentivirus vector to down-regulate the expression of MGMT). The expression of MGMT in IOMM-Lee cells was silenced by RNAi technology. The expression levels of MGMT mRNA and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot. The proliferation activity of cells was detected by cell counting kit-8 (CCK-8) test, the colony forming ability was detected by cell clone formation test, and the invasion and migration ability of cells in each group were detected by Transwell test and cell scratch test.Results:The methylation of MGMT in meningioma tissue reached 88.23% (30/34). MGMT methylation was not detected in normal brain tissue; the staining intensity of MGMT in meningioma tissue was significantly higher than that in normal brain tissue. Compared with the blank control group and the negative control group, the relative expression of MGMT mRNA and protein in IOMM-Lee cells of the RNAi lentiviral transfection group were significantly decreased (all P<0.05). After 24, 48 and 72 hours of transfection, the proliferation activity of IOMM-Lee cells decreased significantly (all P<0.05), with reduced number of cell clone formation and cell invasion (all P<0.05). The rate of scar healing decreased significantly ( P<0.05). Conclusions:MGMT is mostly hypermethylated in human meningiomas. Silencing the expression of MGMT in meningiomas can inhibit the growth and metastasis of meningiomas.

5.
Journal of Chinese Physician ; (12): 216-220, 2022.
Article in Chinese | WPRIM | ID: wpr-932046

ABSTRACT

Objective:To observe the expression of microRNA (miRNA)-7159-5p in gastric cancer tissues, and explore its effect on the proliferation and invasion of gastric cancer cells and its molecular mechanism.Methods:The cancer tissues and adjacent tissues of 29 patients with gastric cancer who underwent surgical resection in Hubei 672 Orthopedic Hospital of Integrated Traditional Chinese and Western Medicine from March 2018 to November 2019 were collected. Fluorescence real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-7159-5p in gastric cancer tissues and adjacent tissues, gastric cancer cell lines and normal gastric epithelial cell lines. Select the cell line with the lowest expression of miR-7159-5p, set the control group and the experimental group, and be transfected with control and miR-7159-5p mimics respectively. qRT-PCR was used to detect the expression of miR-7159-5p in transfected cells. The lymphocyte proliferation test (MTS method) was used to detect the proliferation of transfected cells, and the Transwell chamber method was used to detect the invasion activity of transfected cells. The miRNAMap database and dual luciferase reporter gene experiment were used to predict and verify the target genes of miR-7159-5p. qRT-PCR and Western blot were used to detect the expression of target genes in transfected cells.Results:The expression of miR-7159-5p in gastric cancer tissues was lower than that in adjacent tissues ( P<0.01), the expression of miR-7159-5p in gastric cancer cell lines was lower than that of normal gastric epithelial cells (all P<0.01), and the expression of miR-7159-5p was the lowest in SGC7901 cells ( P<0.01). After transfection, the expression of miR-7159-5p in SGC7901 cells of the experimental group was significantly higher than that in the control group ( P<0.01). After transfection, the proliferation activity and the cell invasion activity of the experimental group was significantly lower than that of the control group (all P<0.01). The target gene of miR-7159-5p was tripartite motif 26 (TRIM26). After transfection, the expression of TRIM26 mRNA in SGC7901 cells of the experimental group was significantly lower than that in the control group ( P<0.01). Western blot showed that after transfection, the expression of TRIM26, c-Myc, cyclin D1, β-catenin protein were lower than those in the control group (all P<0.05). Conclusions:The expression of miR-7159-5p is low in gastric cancer tissues, and miR-7159-5p can inhibit the proliferation and invasion of SGC7901 cells by down-regulating the expression of TRIM26.

6.
Article in Chinese | WPRIM | ID: wpr-955846

ABSTRACT

Objective:To investigate the effects of miR-5011-5p on apoptosis and migration of bladder cancer cell line J82 and the underlying mechanism.Methods:J82 cells were transfected with random sequence molecules (NC group) and miR-5011-5p sequence molecules (miR-5011-5p group). Flow cytometry and scratch experiment were performed to analyze the effects of miR-5011-5p on apoptosis and migration of J82 cells. The target gene of miR-5011-5p was predicted by bioinformatics. Real-time fluorescent quantitative polymerase chain reaction and western blot assay were performed to investigate the effects of miR-5011-5p on target gene expression.Results:The relative expression of miR-5011-5p in J82 cells in the miR-5011-5p group was significantly higher than that in the NC group (10.73 ± 1.67 vs. 1.04 ± 0.16, t = 5.81, P < 0.01). There was significant difference in the apoptosis rate of J82 cells between NC and miR-5011-5p groups [(8.83 ± 1.67)% vs. (34.96 ± 3.80)%, t = 6.30, P < 0.01]. The migration rate of J82 cells differed significantly between NC and miR-5011-5p groups [(71.31 ± 7.69)% vs. (37.43 ± 5.01)%, t = 3.69, P < 0.05]. The target gene of miR-5011-5p may be Yes-related protein 1 (YAP1). Compared with the NC group, miR-5011-5p exhibited an obvious inhibitory effect on the YAP1 expression in J82 cells ( P < 0.01). Conclusion:miR-5011-5p may promote the apoptosis of J82 cells and inhibit their migration in bladder cancer through targeted inhibition of YAP1 gene expression.

7.
International Journal of Surgery ; (12): 658-662,C2, 2022.
Article in Chinese | WPRIM | ID: wpr-954271

ABSTRACT

Objective:To explore the expression level of miR-769-3p in bladder cancer tissues, and observe the effect of silencing miR-769-3p on the migration ability and cell cycle of J82 cells by down-regulating the expression level of miR-769-3p in bladder cancer J82 cells.Methods:The OncomiR database was used to analyze the expression differences of miR-769-3p in bladder cancer tissues and adjacent tissues. J82 cells were transfected with Lipofectamine 2000 transfection reagent and divided into si-miR-769-3p group (transfected with miR-769-3p small molecule interference fragments) and control group (transfected with meaningless sequences). quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative expression level of miR-769-3p after transfection. The cell scratch test and flow cytometry were used to compare the migration ability and cell cycle differences between the two groups of J82 cells. The bioinformatics software MicroRNAdb was used to predict the target gene of miR-769-3p. The dual-luciferase reporter gene assay was used to verify the complementary binding of miR-769-3p to the target gene. qRT-PCR and Western blotting were used to detect the expression levels of miR-769-3p target gene. Measurement data were expressed as mean ± standard deviation ( ± s), and t-test was used for comparison between two groups. Results:The expression of miR-769-3p was significantly increased in bladder cancer tissues compared with adjacent tissues, the difference was statistically significant ( P<0.01). The relative expression of miR-769-3p in the si-miR-769-3p group (1.02 ± 0.16) was significantly lower than that of the control group (4.50 ± 0.60), the difference was statistically significant ( P<0.01). The cell migration rate of the si-miR-769-3p group [(26.67±3.98)%] was significantly lower than that of the control group [(61.86±4.70)%], the difference was statistically significant ( P<0.01). The proportion of cells in the G 0-G 1 phase in the si-miR-769-3p group [(57.66±5.74)%] was significantly higher than that in the control group [(31.26±3.24)%], the difference was statistically significant ( P<0.01). Dual-luciferase reporter gene assay confirmed that endothelin 3 ( EDN3) was the target gene of miR-769-3p. The relative expression of EDN3 mRNA in J82 cells in control group and si-miR-769-3p group was 1.99 ± 0.66 and 6.98 ± 0.76, compared with the control group, the EDN3 mRNA relative expression level of the si-miR-769-3p group was significantly higher than that of the control group, the difference was statistically significant ( P<0.01). Conclusion:Low expression of miR-769-3p can inhibit the migration of bladder cancer J82 cells and block the J82 cell cycle by promoting the expression of EDN3 gene.

8.
International Journal of Surgery ; (12): 654-658,C1, 2022.
Article in Chinese | WPRIM | ID: wpr-954270

ABSTRACT

Objective:To investigate the expression of long non-coding RNA (lncRNA) CALCOCO1 in bladder cancer tissue and its effect on the proliferation and migration of bladder cancer cells by regulating miR-200a-3p.Methods:The relative expression levels of CALCOCO1 in bladder cancer tissues and adjacent tissues were analyzed by TCGA database. Human bladder cancer cells UM-UC-3 were selected, and the cells were divided into negative control group and CALCOCO1 group, and NC plasmid and CALCOCO1 plasmid were transfected into UM-UC-3 cells respectively. The expression level of CALCOCO1 in each group was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation and migration ability of UM-UC-3 cells were detected by MTT assay and Transwell migration assay. Bioinformatics technology was used to predict and dual-luciferase reporter gene experiments to verify the targeting relationship between CALCOCO1 and miR-200a-3p. The expression levels of miR-200a-3p in UM-UC-3 cells in each group were detected by qRT-PCR. Western blotting was used to detect the expression of UM-UC-3 cells proliferation and migration phenotype in each group. Measurement data were expressed as mean ± standard deviation ( ± s), t-test was used for comparison between two groups, and repeated measurement analysis of variance was used for comparison at different time. Results:Compared with adjacent tissues, the relative expression level of CALCOCO1 in bladder cancer tissues was significantly lower, the difference was statistically significant( P<0.01). The relative expression of CALCOCO1 in UM-UC-3 cells in CALCOCO1 group and negative control group was 9.66±2.51 and 1.07±0.59, respectively. The relative expression level of CALCOCO1 in CALCOCO1 group was significantly higher than that in negative control group, the difference was statistically significant ( P<0.01). Compared with the negative control group, the proliferation activity of UM-UC-3 cells in the CALCOCO1 group was decreased ( P<0.05), and the migration number of UM-UC-3 cells was significantly decreased ( P<0.01). CALCOCO1 had a binding site with miR-200a-3p ( P<0.01). The relative expression of miR-200a-3p in UM-UC-3 cells in CALCOCO1 group and negative control group was 1.02 ± 0.31 and 5.79 ± 1.68, respectively, the difference was statistically significant ( P<0.01). Compared with the negative control group, the expression levels of proliferation phenotype proteins CCNB1, CCNE1 and CCND2 in UM-UC-3 cells in CALCOCO1 group decreased, and the expression levels of migration phenotype proteins FOXC2 and Fibronectin decreased. Conclusion:The expression of CALCOCO1 is down-regulated in bladder cancer tissue, promoting the expression of CALCOCO1 can inhibit the proliferation and migration of bladder cancer UM-UC-3 cells through targeted down-regulation of miR-200a-3p expression.

9.
Journal of Medical Biomechanics ; (6): E433-E440, 2022.
Article in Chinese | WPRIM | ID: wpr-961747

ABSTRACT

Objective From the perspective of biophysics and immunology, the effects of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on biophysical characteristics, cytoskeleton and migration ability of mouse derived dendritic cells (DCs) were analyzed, so as to explore the effect of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) on immune function of DCs and its potential mechanism. Methods The bone marrow-derived monocytes from C57BL/6J mice were isolated and induced to differentiate into immature dendritic cells (imDCs) by 20 ng/mL recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and 10 ng/mL recombinant mouse interleukin-4 (rmIL-4), After 6 days, 100 ng/mL lipopolysaccharide was added to induce mature dendritic cells (mDCs). Further the morphological observation and positive rate of CD11c in imDCs and mDCs were analyzed, Then at different concentrations of EPA and DHA (0-60 μmol/L), the cell viability and apoptosis of DCs were detected by cell counting kit-8 (CCK-8) kit and flow cytometry. After the optimal concentration of EPA and DHA were determined, the changes of the membrane fluidity, electrophoretic mobility (EPM) and osmotic fragility of DCs were separately detected by the fluorescence polarization, cell electrophoresis and concentration gradient. The expression of filamentous actin (F-actin) was detected by the immunofluorescence. Finally, the migration ability of DCs was detected by the Transwell system. Results The positive rate of CD11c in DCs was about 80%. The viability of DCs decreased in a dose-dependent manner under the action of EPA and DHA of different concentrations, which didn’t induce the apoptosis of DCs. Under the action of 50 μmol/L EPA and DHA, the osmotic fragility and EPM of DCs significantly decreased, and the membrane fluidity significantly increased. The expression of F-actin in DCs obviously increased, and the migration rate of cells obviously decreased. Conclusions ω-3 PUFAs may affect the cytoskeleton structure and biophysical characteristics of DCs, inhibit the migration, and then affect its immune function.

10.
Journal of Medical Biomechanics ; (6): E395-E402, 2022.
Article in Chinese | WPRIM | ID: wpr-961742

ABSTRACT

Objective To investigate the effect of cyclic stretch on Src and Runt-related transcription factor 2 (RUNX2), and their pivotal roles in migration of vascular smooth muscle cells (VSMCs). Methods The 5% cyclic stretch (to simulate normotensive physiological condition) or 15% cyclic stretch (to simulate hypertensive pathological condition) was applied to VSMCs by FX-5000T system. Western blotting was used to detect the expression of RUNX2 and phosphorylation of Src in VSMCs. The Ingenuity Pathway Analysis (IPA) bioinformatic software was used to analyze the potential regulatory effect of Src on RUNX2. Small interfering RNA (siRNA) was transfected to decrease the expression of RUNX2. Src inhibitor-1 was used to repress Src kinase activity; Wound-healing assay was applied to detect VSMC migration. Results Compared with 5% cyclic stretch, 15% cyclic stretch significantly increased RUNX2 expression in VSMCs. Under both static and 15% cyclic stretch conditions, VSMC migration was significantly inhibited after reducing RUNX2 expression with siRNA transfection. IPA indicated that Src kinase might be the upstream modulator of RUNX2, and Western blotting validated that RUNX2 expression was significantly decreased after inhibiting Src. Furthermore, under 15% cyclic stretch, Src inhibitor-1 markedly repressed RUNX2 expression and VSMC migration.Conclusions High cyclic stretch increased phosphorylation of Src kinase and expression of RUNX2, which subsequently induced VSMC abnormal migration. Exploring the mechanobiological mechanism of VSMC migration regulated by cyclic stretch may contribute to further revealing the mechanism of vascular physiological homeostasis and vascular pathological remodeling, as well as providing new perspective for the translational research of vascular remodeling upon hypertension.

11.
Journal of Medical Biomechanics ; (6): E335-E341, 2022.
Article in Chinese | WPRIM | ID: wpr-961733

ABSTRACT

Objective To explore the role of adenosine monophosphate-activated protein kinase (AMPK), a key regulator of cellular energy metabolism, in vascular smooth muscle cell (VSMC) migration in response to physiological cyclic stretch. Methods The Flexcell-5000T mechanical loading system was applied with a physiological cyclic stretch at 10% amplitude and 1.25 Hz frequency to primary rat VSMCs, to simulate mechanical stimulation of VSMCs in vivo. The protein expression of p-AMPK in VSMCs was detected by Western blotting, and VSMC migration was detected by wound healing test. Results Compared with the static group, physiological cyclic stretch loading for 24 h significantly decreased the area of wound healing, indicating that physiological cyclic stretch inhibited VSMC migration. The protein expression of p-AMPK in VSMCs was increased significantly after physiological cyclic stretch loading for 3 h, and was decreased significantly after 24 h. Under physiological cyclic stretch loading conditions, incubating AMPK inhibitor could significantly reduce the protein expression of p-AMPK after 3 h, and promote VSMC migration after 24 h; incubating AMPK activator AICAR under static conditions significantly increased the protein expression of p-AMPK after 3 h, and weakened VSMC migration after 24 h. Conclusions Physiological cyclic stretch inhibits VSMC migration by increasing the protein expression of p-AMPK, indicating that VSMC migration regulated by physiological cyclic stretch is of great significance for maintaining vascular homeostasis.

12.
Cancer Research and Clinic ; (6): 648-653, 2022.
Article in Chinese | WPRIM | ID: wpr-958909

ABSTRACT

Objective:To investigate the expression of miRNA-522-3p (miR-522-3p) in gastric cancer tissues/cells and its regulation of RSU1 expression and to analyze the effect of miR-522-3p on biological function of gastric cancer cells in vitro.Methods:miR-522-3p relative expression levels in tumor tissues and adjacent tissues of 50 patients clinically diagnosed as gastric cancer in Shanxi Bethune Hospital from May 2019 to June 2019 were detected by using real-time quantitative polymerase chain reaction (qRT-PCR). MGC-803 cells and BGC-823 cells in gastric cancer were divided into miR-522-3p inhibitor group (transfected with miR-522-3p inhibitor) and empty vector group (transfected with empty vector). The cell proliferation was detected by using CCK-8 assay, cell scratch assay was used to detect the scratch healing ability of cells, and flow cytometry was used to detect the apoptosis. The target correlation between miR-522-3p and RSU1 was detected by using double luciferase reporter gene assay, the change of RSU1 protein was detected by using Western blot.Results:qRT-PCR showed that compared with adjacent cancer tissues, the relative expression level of miR-522-3p was up-regulated, and the difference was statistically significant (0.84±0.31 vs. 0.48±0.22, t = 2.93, P < 0.05). There were no statistically significant differences in the relative expression level of miR-522-3p in gastric cancer tissues with different tumor diameter and pathological grade (all P < 0.05). In vitro experimental assay showed that the cell proliferation rates of MGC-803 and BGC-823 cells in miR-522-3p inhibitor group at 48 h and 72 h were decreased (all P < 0.05). Furthermore, MGC-803 and BGC-823 tranfected with miR-522-3p inhibitor could effectively inhibit the scratch healing ability of MGC-803 and BGC-823 cells. Dual-luciferase reporter gene assay verified that miR-522-3p targeted to RSU1 3'UTR and affected its fluorescence activity. Western blot results showed that miR-522-3p could promote RSU1 protein expression. Conclusions:miR-522-3p is involved in the progression of gastric cancer probably via the regulation of RSU1 expression, and it may be a potential therapeutic target.

13.
Cancer Research and Clinic ; (6): 521-524, 2022.
Article in Chinese | WPRIM | ID: wpr-958886

ABSTRACT

Objective:To investigate the effects of solute carrier family 39 (SLC39) A14 on proliferation, migration and invasion of diffuse large B-cell lymphoma (DLBCL) OCI-LY3 cells.Methods:The human DLBCL cell line OCI-LY3 was divided into Vector group (transfected with empty control plasmid) and SLC39A14 group (transfected with SLC39A14 plasmid). The proliferation of OCI-LY3 cells in the two groups was detected by CCK-8 method, the migration and invasion of cells were detected by Transwell method, and the expression level of SLC39A14 protein and the expressions of PI3K-AKT-mTOR signaling pathway-related proteins in OCI-LY3 cells were detected by Western blotting.Results:Compared with the Vector group, the cell proliferation ability in the SLC39A14 group was increased from day 3 to day 5 (all P < 0.05).The results of Transwell cell migration assay showed that the number of migrating cells after 36 h in the Vector group was (64±4) cells, and that in the SLC39A14 group was (236±25) cells. The cell migration ability in the SLC39A14 group was increased, and the difference was statistically significant ( t = 15.02, P < 0.05). The results of Transwell cell invasion assay showed that the number of invasive cells in the Vector group was (32±2) cells, and that in the SLC39A14 group was (127±17) cells. The cell invasion ability in the SLC39A14 group was increased, and the difference was statistically significant ( t = 8.33, P < 0.05).The results of Western blotting showed that the expression levels of pmTOR, pAKT and pPI3K proteins in the SLC39A14 group were all increased. Conclusions:SLC39A14 may be involved in the occurrence and development of DLBCL through PI3K-AKT-mTOR signaling pathway.

14.
Chinese Journal of Digestion ; (12): 667-674, 2022.
Article in Chinese | WPRIM | ID: wpr-958350

ABSTRACT

Objective:To examine the expression of transmembrane emp24 domain-containing protein 4(TMED4) in liver tissue of patients with hepatocellular carcinoma, and to investigate the effects of TMED4 gene on the proliferation and migration of hepatocellular carcinoma cells and related molecular mechanisms. Methods:The expression of TMED4 at protein level in liver cancer tissue and paracancerous tissue of patients with hepatocellular carcinoma were detected by Western blotting and immunohistochemical stainning, and the correlation between its expression and clinicopathological features was analyzed. The effects of TMED4 overexpression or knockdown on proliferation, migration and healing ability of hepatocellular carcinoma cells in vitro and in vivo were determined by cell proliferation test, Transwell test, wound healing test and subcutaneous tumor formation in nude mice. The molecular mechanism of TMED4 in regulating the biological behavior of hepatocellular carcinoma cells was preliminarily explored by pathway analysis. Independent sample t test, Mann-Whitney U test and chi-square test were used for statistical analysis. Results:The results of Western blotting showed that the expression of TMED4 at protein level in hepatocellular carcinoma tissue was lower than that in paracancerous tissue(0.52±0.29 vs. 0.83±0.22), and the difference was statistically significant( t=2.54, P=0.022). The results of immunohistochemical examination indicated that the expression of TMED4 at protein level in liver cancer tissue was lower than that in paracancerous tissue(5.46±3.37 vs. 7.58±3.08), and the difference was statistically significant( t=3.49, P<0.001). The expression of TMED4 at protein level was significantly correlated with vascular invasion and Barcelona clinic liver cancer stage( χ2=6.83 and 4.20, P=0.009 and 0.040). The results of cell proliferation assay showed that the absorbance value of SMMC-7721 cells in TMED4 overexpression group was lower than that in control group(1.38±0.05 vs. 2.37±0.08), while the optical density value of HepG2 in TMED4 knockdown group was higher than that in control group(0.76±0.04 vs. 0.54±0.01), and the differences were statistically significant( t=18.23 and 8.85, both P<0.001). The results of Transwell test showed that the number of migrated SMMC-7721 cells in TMED4 overexpression group was less than that in control group(286.30±13.01 vs. 439.70±12.34), while the number of migrated HepG2 cells in TMED4 knockdown group was higher than that in control group(249.00±6.00 vs. 160.00±6.56), and the differences were statistically significant( t=14.81 and 17.34, both P<0.001). The wound healing test showed that the healing rate of SMMC-7721 cells in TMED4 overexpression group was lower than that in control group((0.21±0.01)% vs.(0.45±0.01)%), the healing rate of HepG2 cells in TMED4 knockdown group was higher than that in control group((0.46±0.01)% vs.(0.20±0.01)%), and the differences were statistically significant( t=200.10 and 30.46, both P<0.001). The results of subcutaneous tumor formation assay in nude mice showed that the growth rate of cells in TMED4 overexpression group was slower than that in control group. After cell inoculation for 6 weeks, the subcutaneous tumor volume of mice in TMED4 overexpression group was smaller than that in control group(27.36 mm 3(138.70 mm 3) vs. 1 741.62 mm 3(1 783.39 mm 3)), the tumor weight was lower than that in control group(0.06 g(0.14 g) vs. 1.46 g(1.09 g)), and the differences were statistically significant(both Z=-2.31, both P<0.001). The results of Western blotting showed that the expression of Snail at protein level in SMMC-7721 cells of the TMED4 overexpression group was lower than that of the control group(0.32±0.01 vs. 0.90±0.03), the protein level of Snail in HepG2 cells of TMED4 knockdown group was higher than that of control group(1.03±0.01 vs. 0.97±0.01), and the differences were statistically significant( t=28.49 and 12.31, both P<0.001). The results of real time fluorescent quantitative polymerase chain reaction showed that the expression of Snail at mRNA level in SMMC-7721 cells of TMED4 overexpression group was lower than that of control group(0.13±0.05 vs. 1.00±0.15), the expression of Snail at mRNA level in HepG2 cells of TMED4 knockdown group was higher than that of control group(1.25±0.32 vs. 0.21±0.14), and the differences were statistically significant( t=9.62 and 5.10, P<0.001 and P=0.007). Conclusion:TMED4 may affect the proliferation and migration of hepatocarcinoma cells by regulating the expression of Snail, and which is expected to become a potentially therapeutic target for hepatocellular carcinoma.

15.
Article in Chinese | WPRIM | ID: wpr-957042

ABSTRACT

Objective:To investigate the effect of cell migration-inducing hyaluronidase 1 (CEMIP) on biological function of gallbladder cancer GBC-SD cells and its possible mechanism.Methods:The expression of CEMIP in biliary epithelial cell line HIBEC and gallbladder cancer cell line NOZ and GBC-SD was detected by Western blot. GBC-SD cells in logarithmic growth phase were divided into blank control group, negative control group (transfection with nonsense siRNA), siCEMIP-1 group (transfection with siCEMIP-1) and siCEMIP-2 group (transfection with siCEMIP-2). The expression of CEMIP and binding immunoglobulin protein (Bip) and calreticulin (CRT) in GBC-SD cells was detected by Western blot after culturing for 48h in blank control group, negative control group, siCEMIP-1 and siCEMIP-2 group. The relative survival rate was determined by CCK-8 assay. The wound healing rate and apoptotic rate was detected by wound healing assay and flow cytometry. The migration and invasion abilities were evaluated by Transwell chamber assay. Twelve 5-week-old BALB/c-nude mice were selected and divided into control group and experimental group (6 mice in each group). GBC-SD cells and GBC-SD cells with silenced CEMIP were subcutaneously injected into the right armpit (forelimb) of the two groups of mice, respectively. The volume and weight of transplanted tumor were compared 33 days later.Results:Compared with HIBEC cells, the relative protein level of CEMIP in gallbladder cancer GBC-SD cells [(0.750±0.034) vs. (0.120±0.002)] and NOZ cells [(0.690±0.013) vs. (0.120±0.002)] was significantly higher ( P<0.05). Compared with blank control group and negative control group, the relative protein level of CEMIP, Bip and CRT in siCEMIP-1 group and siCEMIP-2 group was significantly lower ( P<0.05). Compared with blank control group and negative control group, the relative survival rate and wound healing rate and number of migration cells and invading cells in siCEMIP-1 group and siCEMIP-2 group were also significantly lower ( P<0.05). While the apoptotic rate in siCEMIP-1 group and siCEMIP-2 group were higher than that in blank control group and negative control group ( P<0.05). Compared with control group, the average tumor volume [(543.6±114.7) vs. (801.5±256.3) mm 3] and tumor weight [(0.453±0.093) vs. (0.728±0.278) g ] of the experimental group was significantly decreased ( P<0.05). Conclusions:CEMIP was up-regulated in gallbladder cancer cell line GBC-SD and NOZ. Silencing CEMIP inhibited cell proliferation, wound healing rate, migration and invasion, and promoted apoptosis in gallbladder cancer GBC-SD cells, which may be related to the inhibition of endoplasmic reticulum chaperone Bip and CRT expression.

16.
Journal of Chinese Physician ; (12): 1210-1214,1219, 2022.
Article in Chinese | WPRIM | ID: wpr-956286

ABSTRACT

Objective:To explore the expression of miR-6883-5p in the serum of bladder cancer patients and its effect on the migration and proliferation ability of bladder cancer cells.Methods:The real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) method was used to detect the expression level of miR-6883-5p in the serum of 39 patients with bladder cancer and 39 healthy subjects, as well as the expression level of miR-6883-5p in normal bladder mucosal epithelial cells and bladder cancer cells. The bladder cancer cells with the lowest expression of miR-6883-5p were transfected with miR-6883-5p mimics or negative control (NC), namely miR-6883-5p group and NC group. Transwell migration experiment and cell counting kit-8 (CCK-8) colorimetric method were used to detect the migration and proliferation ability of cells transfected with miR-6883-5p. Bioinformatics software and dual luciferase reporter gene were used to predict and test the target genes of miR-6883-5p. qRT-PCR and Western blot were used to detect the expression levels of target genes in cells transfected with miR-6883-5p.Results:The expression level of miR-6883-5p in the serum of bladder cancer patients was significantly lower than that of healthy persons ( P<0.01). The expression level of miR-6883-5p in bladder cancer cells was significantly lower than that in normal bladder mucosal epithelial cells (all P<0.05), and the lowest expression of miR-6883-5p was in 5637 cells ( P<0.01). After transfection with miR-6883-5p, the migration and proliferation of cells in miR-6883-5p group were significantly lower than those in NC group (all P<0.05). Phosphatidyl alcohol proteoglycan-6 (GPC6) was the target gene of miR-6883-5p. After transfection with miR-6883-5p, the expression levels of GPC6 mRNA and protein in miR-6883-5p group significantly decreased ( P<0.01). Conclusions:miR-6883-5p is significantly low expressed in the serum of bladder cancer patients, and miR-6883-5p is related to the occurrence and development of bladder cancer; overexpression of miR-6883-5p can inhibit the migration and proliferation of bladder cancer cells; miR-6883-5p can inhibit the occurrence and development of bladder cancer by down-regulating the expression of GPC6.

17.
Journal of Chinese Physician ; (12): 1193-1197,1203, 2022.
Article in Chinese | WPRIM | ID: wpr-956283

ABSTRACT

Objective:To explore the effect of miR-3607-3p on the malignant phenotype of cervical cancer cells and related mechanisms.Methods:The OncoLnc bioinformatics website was used to analyze the relationship between the expression of miR-3607-3p and the prognosis of cervical cancer patients. Real time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-3607-3p in human normal cervical epithelial cells (H8) and cervical cancer cell lines (Hela, HCC94, SiHa, C33A). SiHa cells transfected with negative control nucleotide sequence in vitro were defined as negative control (NC) group, and SiHa cells transfected with miR-3607-3p mimicked sequence was defined as miR-3607-3p group. qRT-PCR was used to detect the expression changes of miR-3607-3p in SiHa cells. Cell counting kit-8 (CCK-8) method and scratch test were used to detect the malignant biological behavior changes of SiHa cells. qRT-PCR and Western blot were used to detect the expression changes of profilin 2 (PFN2) gene, and the dual luciferase reporter gene experiment was used to detect the targeting relationship between miR-3607-3p and PFN2. Results:The survival time of patients with high expression of miR-3607-3p was significantly higher than that of patients with low expression of miR-3607-3p ( P<0.01). Compared with H8 cells, the expression of miR-3607-3p was abnormally low in human cervical cancer cell lines ( P<0.05), and the expression of miR-3607-3p was the lowest in the SiHa cell line ( P<0.01). The expression of miR-3607-3p in the NC group and miR-3607-3p group was (1.04±0.31) and (9.28±1.76), respectively. The expression level of miR-3607-3p in SiHa cells transfected with miR-3607-3p mimic sequence was significantly higher than that in NC group, and the proliferation activity and scratch healing rate were significantly lower than that in NC group (all P<0.01). Dual-luciferase reporter gene assay confirmed that miR-3607-3p directly targeted and binded to PFN2 ( P<0.01). qRT-PCR and Western blot results showed that the expression of PFN2 mRNA and protein in miR-3607-3p group was lower than that in NC group; Western blot results showed the expression of proliferating proteins CDK3 and CDK2 in miR-3607-3p group was lower than that in NC group (all P<0.05), and the expression of epithelial phenotype related proteins Claudin-1 and ZO-1 in miR-3607-3p group was higher than that in NC group (all P<0.05). Conclusions:miR-3607-3p is positively correlated with the survival of cervical cancer patients. Up-regulating the expression of miR-3607-3p can inhibit the proliferation and migration of cervical cancer SiHa cells. The mechanism may be related to the targeted inhibition of PFN2.

18.
Article in Chinese | WPRIM | ID: wpr-936325

ABSTRACT

OBJECTIVE@#To investigate the effect of triptolide (TPL) on inflammatory response and migration of fibroblast like synovial cells (FLS) in rheumatoid arthritis (RA-FLS) and the mechanism of circular noncoding RNA (circRNA) 0003353 for mediating this effect.@*METHODS@#We collected peripheral blood mononuclear cells (PBMCs) and serum samples from 50 hospitalized RA patients and 30 healthy individuals for detecting the expression of circRNA 0003353, immune and inflammatory indexes (ESR, CRP, RF, anti-CCP, IgA, IgG, IgM, C3, and C4) and DAS28 score. Cultured RA-FLS was treated with 10 ng/mL TPL and transfected with a circRNA 0003353 overexpression plasmid, and cell counting kit-8 (CCK-8) assay and Transwell assay were used to detect the changes in the viability and migration of the cells. Enzyme-linked immunosorbent assay (ELISA) was used to examine the cytokines IL-4, IL-6, and IL-17, and real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect the expression of circRNA 003353; Western blotting was used to detect the expressions of p-JAK2, pSTAT3, JAK2 and STAT3 proteins in the treated cells.@*RESULTS@#The expression of circRNA 0003353 was significantly increased in PBMCs from RA patients and showed a good performance in assisting the diagnosis of RA (AUC=90.5%, P < 0.001, 95% CI: 0.83-0.98). CircRNA 0003353 expression was positively correlated with ESR, RF and DAS28 (P < 0.05). Treatment with TPL significantly decreased the expression of circRNA 0003353, suppressed the viability and migration ability, decreased the expressions of IL-6 and IL-17, and increased the expression IL-4 in cultured RA-FLS in a time-dependent manner (P < 0.01). TNF-α stimulation of RA-FLS significantly increased the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, which were obviously lowered by TPL treatment (P < 0.01). TPL-treated RA-FLS overexpressing circRNA 0003353 showed significantly increased cell viability and migration ability with decreased IL-4 expression and increased IL-6 and IL-17 expressions and ratios of p-JAK2/ JAK2 and p-STAT3/STAT3 (P < 0.01).@*CONCLUSION@#The expression of circRNA 0003353 is increased in PBMCs in RA patients and in RA-FLS. TPL treatment can regulate JAK2/STAT3 signal pathway and inhibit the inflammatory response and migration of RA-FLS through circRNA 0003353.


Subject(s)
Humans , Arthritis, Rheumatoid/pathology , Cells, Cultured , Diterpenes/pharmacology , Epoxy Compounds/pharmacology , Fibroblasts/pathology , Interleukin-17/metabolism , Interleukin-4/metabolism , Interleukin-6/metabolism , Janus Kinase 2/metabolism , Leukocytes, Mononuclear/metabolism , Phenanthrenes/pharmacology , RNA, Circular/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Synovial Membrane/pathology
19.
International Eye Science ; (12): 1252-1256, 2022.
Article in Chinese | WPRIM | ID: wpr-934993

ABSTRACT

AIM: To investigate the effect and mechanism of matrix metalloproteinase(MMPs)inhibitor AG3340 on the migration and invasion ability of retinal pigment epithelial cells-19(ARPE-19)cultured in high glucose(CHG). METHODS: ARPE-19 cells cultured in vitro were divided into four groups: Control group, the glucose at the concentration of 5.6mmol/L in DMEM/F12 medium; HG group, the glucose at the concentration of 30mmol/L was cultured with DMEM/F12 medium; HG+AG3340 group, the cells were pretreated with AG3340 for 12h, and then cultured in DMEM/F12 medium containing 30mmol/L glucose; The mannitol(MA)group, cultured with DMEM/F12 medium of 5.6mmol/L glucose and 24.4mmol/L mannitol, which used as hypertonic control group. The migration ability of cells was detected by wound healing assay, the invasion ability of cells was detected by Transwell assay, and the relative expression levels of MMP-9, MMP-2, fibronectin and collagen Ⅳ were detected by Western blot.RESULTS: The results of wound healing assay showed that compared with the Control group, the cell migration rate of scratching after 24h and 48h in the HG group was significantly increased(all P<0.001).After pretreated by AG3340, the cell migration rate was significantly lower than that in the HG group(all P<0.01).Transwell assay showed that compared with the Control group, the number of cell invasion in the HG group was significantly higher than that in the Control group(all P<0.001). After pretreated by AG3340, the number of cell invasion was decreased than the HG group(all P<0.01). Western blot results showed that compared with the Control group, the relative expression levels of MMP-9 and MMP-2 of the cells in the HG group were increased, and the relative expression levels of Fibronectin and Collagen Ⅳ were decreased(all P<0.001). Compared with the HG group, the relative expression levels of MMP-9 and MMP-2 protein in AG3340 pretreatment group were decreased, and the relative expression levels of Fibronectin and Collagen Ⅳ were increased(all P<0.05). CONCLUSION: High glucose induced ARPE-19 cells with enhanced migration and invasion ability, and AG3340 partially reversed this effect, which was related to the inhibition of MMP-9 and MMP-2 expression and the stability of extra-cellular matrix components.

20.
Cancer Research and Clinic ; (6): 171-175, 2022.
Article in Chinese | WPRIM | ID: wpr-934651

ABSTRACT

Objective:To explore the effects of long non-coding RNA (lncRNA) FAM224A on the proliferation and migration of ovarian cancer cells by regulating the expression of miRNA-590-3p (miR-590-3p).Methods:Human ovarian cancer cell lines OC3, SKOV-3, HO-8910, A2780 and human normal ovarian epithelial cell line IOSE80 were selected, and the relative expression of FAM224A in each cell line was detected by real-time quantitative polymerase chain reaction (qRT-PCR). The cell line with the lowest relative expression level of FAM224A was screened for follow-up experiment. The cells were divided into FAM224A group (transfected with FAM224A mimic gene) and control group (transfected with control mimic gene). CCK-8 method and cell scratch test were used to detect the cell proliferation and migration ability of the two groups. The bioinformatics website LncBase v.2 predicted that the target gene that FAM224A might complementarily bind to was miR-590-3p. qRT-PCR was used to detect the relative expression levels of miR-590-3p and forkhead box protein A2 (FOXA2) mRNA, and the expressions of related proteins were detected by Western blot.Results:The relative expression levels of FAM224A in ovarian cancer cell lines OC3, SKOV-3, HO-8910, A2780 and normal ovarian epithelial cell line IOSE80 were 0.23±0.04, 0.65±0.05, 0.45±0.03, 0.63±0.08 and 1.02±0.11, respectively, and the difference was statistically significant ( F = 14.78, P < 0.01), and the cell line with the lowest relative expression level of FAM224A was OC3. The results of CCK-8 method showed that the proliferation ability of OC3 cells in the FAM224A group was lower than that in the control group on the 2nd, 3rd, 4th and 5th day of culture (all P < 0.05). The scratch healing rates of OC3 cells in the FAM224A group and the control group were (18.6±2.3)% and (71.7±7.2)%, respectively, and the difference was statistically significant ( t = 6.99, P < 0.01). The relative expression levels of FAM224A in OC3 cells in the FAM224A group and the control group was 12.36±1.45 and 1.14±0.24, respectively ( t = 13.08, P < 0.01); the relative expression levels of miR-590-3p were 0.19±0.06 and 1.04±0.20, respectively ( t = 4.01, P < 0.01); the relative expression levels of FOXA2 mRNA were 6.37±1.37 and 1.05±0.08, respectively ( t = 3.86, P < 0.01). Compared with the control group, the expression of FOXA2 protein in OC3 cells in the FAM224A group was increased, the expressions of cell proliferation protein cyclin-dependent kinase 2 (CDK2) and cyclin D3 were decreased, and the expression of cell migration protein Snail was decreased. Conclusions:FAM224A is low expressed in ovarian cancer cell lines. FAM224A reduces the proliferation and migration ability of ovarian cancer OC3 cells by inhibiting the expression of miR-590-3p.

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