The role of B3GNT3 as an oncogene in the growth, invasion and migration of esophageal cancer cells

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.

Expression of miR-4783-3p in hepatocellular carcinoma tissue and its effect on the proliferation and migration of hepatocellular carcinoma cells / 国际外科学杂志

Objective:To study the expression of microRNA (miRNA)-4783-3p in liver cancer tissue and its effect on the proliferation and migration of liver cancer Huh-7 cells.Methods:The cBioPortal database was used to analyze the expression of miR-4783-3p in liver cancer tissues and adjacent tissues. In strict accordance with the instructions of Lipofectamine? 2000 transfection kit, miR-4783-3p overexpression mimics or overexpression control mimics were transfected into Huh-7 cells respectively, namely overexpression group and control group. The proliferation of Huh-7 cells was analyzed by CCK-8 assay, and the migration of Huh-7 cells was analyzed by cell scratch method. The targeting relationship between miR-4783-3p and insulin-like growth factor binding protein 2 ( IGFBP2) mRNA was detected by dual-luciferase reporter gene assay. RT-qPCR was used to detect the expression of IGFBP2 mRNA. Western-blotting was used to detect the expression of IGFBP2 protein and EGFR-STAT3 molecular pathway proteins. Results:The expression of miR-4783-3p in liver cancer tissues was significantly lower than that in adjacent tissues ( P<0.01). Compared with the control group, the proliferation ability of Huh-7 cells in the overexpression group was significantly decreased ( P<0.05). The scratch healing rates of Huh-7 cells in the control group and the overexpression group were (67.71±9.04)% and (29.58±10.51)%, respectively, and the scratch healing rate in the overexpression group was significantly lower ( P<0.01). miR-4783-3p targeted and bound to IGFBP2 mRNA ( P<0.01). The expression of IGFBP2 mRNA in the control and overexpression groups was 5.76±1.44 and 0.99±0.47, respectively, and miR-4783-3p negatively regulated the expression of IGFBP2 mRNA ( P<0.01). Compared with the control group, the expressions of IGFBP2 protein and EGFR-STAT3 molecular pathway protein were decreased in the overexpression group. Conclusions:miR-4783-3p is lowly expressed in liver cancer tissues. miR-4783-3p can attenuate the proliferation and invasion ability of liver cancer Huh-7 cells by inducing the low expression of IGFBP2 gene.

Expression of glucose transporter 3 in cutaneous squamous cell carcinoma and its effect on the proliferation, invasion and migration of A431 cells / 中华皮肤科杂志

Objective:To determine the expression of glucose transporter 3 (GLUT3) in cutaneous squamous cell carcinoma (cSCC), and to evaluate its effect on the cSCC cell line A431.Methods:From June 2016 to December 2020, 22 paraffin-embedded tissue specimens were collected from patients with pathologically confirmed cSCC in the Department of Dermatology, Peking University Third Hospital, and 20 discarded normal skin tissues after dermatological surgeries served as controls. Immunohistochemical assay was performed to determine the GLUT3 expression in cSCC tissues and normal skin tissues. Cultured A431 cells were divided into two groups: GLUT3 overexpression group transfected with a lentiviral vector carrying the SLC2A3 gene, and negative control group transfected with an empty lentiviral vector. Real-time fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of GLUT3 in A431 cells in different groups, the cell proliferation assay (MTS assay) was performed to estimate the cell proliferative activity, and the live-cell analysis system Incucyte S3 was used for real-time evaluation of the migratory and invasive abilities of A431 cells in different groups. The relative glucose consumption and lactic acid production in A431 cells at 48 hours were measured by using glucose and lactic acid assay kits, retrospectively. Two independent samples t-test was used for comparisons between two groups, and one-way analysis of variance was used for comparisons among multiple groups. Results:The GLUT3 expression was significantly higher in the cSCC tissues than in the normal skin tissues (immunohistochemical assay score: 9.39 ± 2.56 points vs. 2.30 ± 2.60 points; t = 8.91, P < 0.05). Compared with the negative control group, the mRNA and protein expression of GLUT3 markedly increased in the GLUT3 overexpression group. MTS assay showed significantly increased proliferative activity of A431 cells in the GLUT3 overexpression group compared with the negative control group after 24- and 96-hour treatment ( t = 2.49, 3.54, P = 0.048, 0.012, respectively); cell fusion rates in the scratched area were significantly higher in the GLUT3 overexpression group than in the negative control group in the cell migration assay at 6, 12 18, and 24 hours and cell invasion assay at 12, 18, and 24 hours (all P < 0.05). At 48 hours, the relative glucose consumption and lactic acid production in A431 cells were significantly higher in the GLUT3 overexpression group than in the negative control group ( t = 2.98, 2.20, P = 0.011, 0.038, respectively) . Conclusion:GLUT3 was highly expressed in the cSCC tissues, and may participate in the occurrence and development of cSCC by providing energy to cSCC cells via promoting glucose uptake in cells to enhance their proliferative, migratory and invasive abilities.

lncRNA CALCOCO1 inhibits the proliferation and migration of bladder cancer cells by regulating miR-200a-3p / 国际外科学杂志

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.

Expression of miR-128-3p in gastric cancer and its effect on migration and invasion of gastric cancer cells / 中华普通外科杂志

Objective:To study the effects of miR-128-3p on the migration and invasion of the gastric cancer cells.Methods:qRT-PCR was used to detect the expression of miR-128-3p in 126 gastric cancer tissues and adjacent tissues from Jan 2014 to Jan 2016 at He'nan Cancer Hospital. The effect of miR-128-3p on the invasion and migration of gastric cancer cell line was detected.The expression of miR-128-3p related proteins was detected by Western blotting, miRNA on-line target prediction tool for the prediction of miR-128-3p directly regulated downstream target genes.Results:the expression of miR-128-3p in gastric cancer was significantly higher than that in adjacent non-tumor tissues ( P<0.05). The expression of miR-128-3p was correlated with the vascular tumor thrombus, pN staging and pTNM staging, the prognosis of patients with high expression of miR-128-3p was poor (all P<0.05). MiR-128-3p expression was significantly higher in gastric cancer cell lines ( P<0.05). Online target prediction tool and double luciferase reporter gene showed that CLDN18 was a downstream target gene directly regulated by mir-128-3p. Conclusion:The high expression of miR-128-3p is related to the poor prognosis of gastric cancer patients.

Role of long non-coding RNA 068 in the migration of melanoma cells / 中华皮肤科杂志

Objective:To investigate the effect of long non-coding RNA 068 (lncRNA 068) on the migration of a melanoma cell line A375, and to explore its mechanism of action.Methods:From December 2015 to November 2020, 21 patients with pathologically confirmed cutaneous melanoma were collected from Department of Dermatology, Affiliated Hospital of Nantong University, and quantitative PCR (qPCR) was performed to determine the expression of lncRNA 068 in melanoma and paracancerous tissues. LncRNA 068 was overexpressed or knocked down via lentiviral transfection in A375 human melanoma cells in the following experiments. Specifically, A375 cells were divided into lentiviral vector (LV) -green fluorescent protein (GFP) group and LV-lncRNA 068 group to be transfected with a GFP-expressing LV and a LV containing lncRNA 068 respectively in the overexpression experiment, and were divided into LV-LacZ short hairpin RNA (shRNA) group and LV-lncRNA 068 shRNA group to be transfected with a LV containing the reporter gene LacZ-specific shRNA and a LV containing the lncRNA 068-targeting shRNA respectively in the low-expression experiment, with the LV-GFP group and LV-LacZ shRNA group serving as the control groups. Transwell and scratch assays were performed to evaluate cell migration, EdU cell proliferation assay and cell counting kit-8 (CCK8) assay to determine the proportion of proliferative cells and cell viability respectively, and immunofluorescence staining was conducted to evaluate epithelial-mesenchymal transformation in the above groups. Lentivirus-transfected A375 cells from the above groups were inoculated into the axillae of BALB/c nude mice, and tumor volume was measured and calculated every 3 days. After 30 days, all mice were sacrificed, and tumor tissues were resected to measure the tumor volume and weight. Cultured B16F10 cells were subcutaneously inoculated into the back and foot of BALB/c nude mice to construct mouse models of subcutaneously transplanted B16F10 melanoma. After 2 weeks, the mice were sacrificed, and qPCR and Western blot analysis were performed to determine the mRNA expression of inflammatory factors in transplanted B16F10 melanoma and paracancerous tissues, and expression of IκB kinase (IKK) /P65 signaling pathway-related proteins, respectively. Comparisons between 2 groups were done by t test, and comparisons of tumor volume and weight at different time points among groups were done by repeated measures analysis of variance. Results:qPCR showed that the relative expression of lncRNA 068 was significantly lower in human melanoma tissues and transplanted B16F10 murine melanoma tissues (0.414 ± 0.109, 0.717 ± 0.041, respectively) than in the corresponding paracancerous tissues (1.050 ± 0.103, 1.011 ± 0.023, t = 19.48, 10.83, respectively, both P < 0.001). Transwell and scratch assays both showed that the cellular migratory ability was significantly lower in the LV-lncRNA 068 group than in the LV-GFP group (both P < 0.01), and significantly higher in the LV-lncRNA 068 shRNA group than in the LV-LacZ shRNA group (both P < 0.05). Immunofluorescence assay showed significantly increased fluorescence intensity of E-cadherin and decreased fluorescence intensity of N-cadherin in the LV-lncRNA 068 group compared with the LV-GFP group (both P < 0.001), but significantly decreased fluorescence intensity of E-cadherin and increased fluorescence intensity of N-cadherin in the LV-lncRNA 068 shRNA group compared with the LV-LacZ shRNA group (both P < 0.05). In vivo tumor formation experiment in nude mice showed that there were no significant differences in the volume or weight of melanoma between the LV-lncRNA 068 group and LV-GFP group (both P > 0.05), as well as between the LV-lncRNA 068 shRNA group and LV-LacZ shRNA group (both P > 0.05). As qPCR and Western blot analysis showed, the mRNA and protein expression of interleukin-10 (IL-10) and claudin-1 in A375 cells were significantly higher in the LV-lncRNA 068 group than in the LV-GFP group (both P < 0.05), but significantly lower in the LV-lncRNA 068 shRNA group than in the LV-LacZ shRNA group (both P < 0.05). Compared with the paracancerous tissues, B16F10 melanoma tissues showed significantly decreased mRNA expression of IL-10 ( P < 0.01), but significantly increased mRNA expression of IL-6 and tumor necrosis factor-α, as well as protein expression of phosphorylated P65 and phosphorylated IKK ( P < 0.01) . Conclusion:Overexpression of lncRNA 068 can inhibit the migration of A375 melanoma cells, and may affect the development of inflammation and inhibit the epithelial-mesenchymal transformation by inhibiting the IKK/P65 signaling pathway.

miRNA-522-3p expression in gastric cancer and the effect of its targeted inhibition of RSU1 expression on the biological function of gastric cancer cells / 肿瘤研究与临床

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.

Mechanism of silencing miR-4320 expression in inhibiting proliferation and migration of gastric cancer cells / 国际外科学杂志

Objective:To explore the effect and mechanism of microRNA (miRNA)-4320 on the proliferation and migration of gastric cancer MGC803 cells.Methods:Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-4320 in four gastric cancer cell lines(MGC803, HS-746T, SGC7901, BGC823) MGC803 cells were infected with recombinant lentivirus carrying miR-4320 interference fragments or blank lentivirus, and set as si-miR-4320 group and NC group. Thiazole blue colorimetry and Transwell small box experiment were used to detect the proliferation and migration of MGC803 cells after miR-4320 was down-regulated. The bioinformatics software RNAhybrid was used to predict the target gene of miR-4320. The targeting relationship between miR-4320 and target gene was verified by dual-luciferase reporter gene experiment. qRT-PCR and Western blot were used to detect the expression of miR-4320 target gene. Measurement data were expressed as mean ± standard deviation ( ± s), and t-test or one-way ANOVA was used for comparison between groups. Results:The expression of miR-4320 in the four gastric cancer cell lines was significantly higher than that of normal gastric mucosal epithelial cells ( P<0.01). The expression of miR-4320 in MGC803 cells in the NC group and the si-miR-4320 group were 8.19±1.00 and 1.09±0.31, respectively. The miR-4320 interference fragment significantly reduced the expression of miR-4320 ( P<0.01). The absorbance of MGC803 cells in the si-miR-4320 group was significantly lower than that of the NC group ( P<0.05), and the migration ability was significantly lower than that of the NC group ( P<0.01). Suppressor of cytokine signaling1 ( SOCSI) is the target gene of miR-4320. Compared with the NC group, the SOCS1 gene expression in the si-miR-4320 group was significantly up-regulated ( P<0.01). Conclusions:The expression of miR-4320 is increased in gastric cancer cell lines. Down-regulating the expression of miR-4320 can inhibit the proliferation and migration of gastric cancer MGC803 cells by inducing the expression of SOCS1 gene.

Effects of microRNA-26a targeted high-mobility group A2 protein antibody on the proliferation and migration of hepatoma cells / 中国基层医药

Objective:To investigate the effects of miRNA-26a (miR-26a) on the target gene HMGA2 on the proliferation and migration of hepatoma cells and the underlying mechanism. Methods:Liver cancer tissue samples ( n = 30) and adjacent normal tissue samples ( n = 30) pathologically confirmed by Wenzhou Hospital of Traditional Chinese Medicine between September 2018 and September 2019 were collected. MiR-26a mimics, control mimics (miR-Control), high-mobility group A2 protein (HMGA2) siRNA or negative control siRNA (Control) were transfected into human hepatoma cell lines HepG2 or Huh-7 cells. The expression of miR-26a in hepatocellular carcinoma tissue was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). MTT assay and scratch test were performed to determine the ability of cell proliferation and migration. RT-qPCR and western blotting were performed to detect miR-26a and HMGA2 mRNA expression. The relationship between miR-26a and HMGA2 mRNA was analyzed using Bioinformatics and luciferase reporter gene assay. Results:RT-qPCR results showed that the expression level of miR-26a in hepatocellular carcinoma tissue was 0.11 ± 0.02, which was significantly lower than that in normal tissues (0.25 ± 0.03, t = 21.268, P < 0.05). The expression level of miR-26a in stage III + IV was 0.05 ± 0.01, which was significantly lower than that in stage I + II (0.09 ± 0.01, t = 15.491, P < 0.05). Cell experiment showed that in the miR-26a group, the proliferation ability of Huh-7 cells was (3.10 ± 0.30) and (4.10 ± 0.40), and the proliferation ability of HepG2 cells was (3.08 ± 0.31) and (4.11 ± 0.40), which was significantly lower than that in the control group [(3.90 ± 0.40), (5.50 ± 0.60), (3.92 ± 0.41), (5.49 ± 0.58), t = 8.764, 10.634, 11.148, 10.728, all P < 0.05]. In the miR-26a group, the migration ability was (0.50 ± 0.06), (0.65 ± 0.07), which was significantly lower than that in the control group [(1.00 ± 0.10), (0.96 ± 0.10), t = 23.483, 13.910, both P < 0.05]. Bioinformatics and in vitro experiments showed that HMGA2 was a direct target of miR-26a. Restoring the expression of HMGA2 in miR-26a mimics-transfected cells, compared with that in the miR-26a group [(0.24 ± 0.02), (0.31 ± 0.03);(0.45 ± 0.05)], could significantly reverse the inhibitory effect of miR-26a on tumor cell proliferation and migration [(0.31 ± 0.03), (0.40 ± 0.04);(0.93 ± 0.08), t = 10.634, 9.859, 27.868, all P < 0.05). Conclusion:miR-26a inhibits the proliferation and migration of hepatoma cells by directly targeting HMGA2. The abnormal decrease of miR-26a and the increase of HMGA2 may be the important factors that participate in the occurrence and development of liver cancer.

Effect of Beclin1 overexpression on biological behaviors of SK-MEL-2 human malignant melanoma cells / 中华皮肤科杂志

Objective@#To evaluate the effect of overexpression of the autophagy marker gene Beclin1 on biological behaviors of SK-MEL-2 human malignant melanoma cells.@*Methods@#Western blot analysis was performed to determine the protein expression of Beclin1 in melanoma cell lines A375 and SK-MEL-2. SK-MEL-2 cells with low Beclin1 protein expression were selected as research objects, and divided into 3 groups: blank group receiving no treatment, negative control group transfected with pcDNA.3.1/myc-His (-) A, and experimental group transfected with pcDNA3.1-Beclin1 plasmid. After 2-week culture, cell counting kit-8 (CCK-8) assay was conducted to evaluate the effect of Beclin1 on cell proliferation at 24, 48 and 72 hours, and Transwell assay and wound-healing assay were performed to assess the effect of Beclin1 overexpression on the invasion and migration abilities of SK-MEL-2 cells. Repeated measures analysis of variance and completely randomized analysis of variance were used to analyze differences in indices among groups, and least significant difference (LSD) -t test was used for multiple comparisons.@*Results@#The protein expression of Beclin1 was significantly lower in the SK-MEL-2 cells (0.037 ± 0.010) than in the A375 cells (0.670 ± 0.150, F = 46.62, P<0.05) . The experimental group showed significantly increased protein expression of Beclin1 (0.32 ± 0.04) compared with the negative control group (0.06 ± 0.02, P < 0.05) and blank group (0.07 ± 0.02, P < 0.05) . CCK-8 assay revealed a significant difference in the cell proliferation rate among different groups and different time points (F = 1 077.36, 4 903.04 respectively, both P<0.05) , and there was a significant interaction between the transfection treatment and time (F= 205.20, P<0.05) . Transwell assay showed that the number of SK-MEL-2 cells crossing the chamber per high-power field (× 200) after 24-hour treatment was significantly lower in the experimental group (18.67 ± 1.19) than in the negative control group (87.89 ± 6.05, P<0.05) and blank group (86.78 ± 5.93, P<0.05) . In the wound-healing assay, the cell migration distance was significantly shorter in the experimental group than in the blank group and negative control group at 24 and 48 hours (all P < 0.05) .@*Conclusion@#Beclin1 overexpression can markedly inhibit the proliferation, invasion and migration of SK-MEL-2 cells.

Effect of Beclin1 overexpression on biological behaviors of SK-MEL-2 human malignant melanoma cells / 中华皮肤科杂志

Objective To evaluate the effect of overexpression of the autophagy marker gene Beclin I on biological behaviors of SK-MEL-2 human malignant melanoma cells.Methods Western blot analysis was performed to determine the protein expression of Beclin 1 in melanoma cell lines A375 and SK-MEL-2.SK-MEL-2 cells with low Beclin1 protein expression were selected as research objects,and divided into 3 groups:blank group receiving no treatment,negative control group transfected with pcDNA.3.1/myc-His (-) A,and experimental group transfected with pcDNA3.1-Beclin1 plasmid.After 2-week culture,cell counting kit-8 (CCK-8) assay was conducted to evaluate the effect of Beclin1 on cell proliferation at 24,48 and 72 hours,and Transwell assay and wound-healing assay were performed to assess the effect of Beclin 1 overexpression on the invasion and migration abilities of SK-MEL-2 cells.Repeated measures analysis of variance and completely randomized analysis of variance were used to analyze differences in indices among groups,and least significant difference (LSD)-t test was used for multiple comparisons.Results The protein expression of Beclin1 was significantly lower in the SK-MEL-2 cells (0.037 ± 0.010) than in the A375 cells (0.670 ± 0.150,F =46.62,P ＜ 0.05).The experimental group showed significantly increased protein expression of Beclin1 (0.32 ± 0.04) compared with the negative control group (0.06 ± 0.02,P ＜0.05) and blank group (0.07 ± 0.02,P ＜ 0.05).CCK-8 assay revealed a significant difference in the cell proliferation rate among different groups and different time points (F =1 077.36,4 903.04 respectively,both P＜ 0.05),and there was a significant interaction between the transfection treatment and time (F =205.20,P ＜ 0.05).Transwell assay showed that the number of SK-MEL-2 cells crossing the chamber per high-power field (× 200) after 24-hour treatment was significantly lower in the experimental group (18.67 ±1.19) than in the negative control group (87.89 ± 6.05,P＜ 0.05) and blank group (86.78 ± 5.93,P ＜0.05).In the wound-healing assay,the cell migration distance was significantly shorter in the experimental group than in the blank group and negative control group at 24 and 48 hours (all P ＜ 0.05).Conclusion Beclin 1 overexpression can markedly inhibit the proliferation,invasion and migration of SK-MEL-2 cells.

Wound Age Estimation by Neutrophil Migration Distance / 法医学杂志

Objective To explore the application of neutrophil migration distance for wound age estimation of skeletal muscles in rats, and to provide methodological basis for follow-up study in future. Methods The skeletal muscle contusion model was established in rats, and the control group and the 2, 4, 6 h post-traumatic groups were set. The law of response of neutrophils that participated in the inflammation after injury was detected by immunohistochemical staining, and the relationship between neutrophil migration distance and injury time was detected by TissueFAXS PLUS software. Results The skeletal muscle was obviously infiltrated with neutrophils 2-6 h after injury. The positive rate of neutrophil was （28.75±0.94）% at 2 h post-traumatic, and reached the peak （45.50±3.63）% at 4 h post-traumatic, then decreased to （31.92±1.56）% at 6 h post-traumatic. The neutrophil migration distances increased with the progress of inflammation, and reached （124.80±12.32） μm, （229.03±21.45） μm and （335.04±16.75） μm at 2 h, 4 h and 6 h, respectively. Conclusion There is a relationship of neutrophil infiltrated number and migration distance and wound age within the 2-6 h after skeletal muscle injury, which could be used for the inference of skeletal muscle wound age.

Mechanisms of matrine inhibiting proliferation and migration of gastric cancer MGC-803 cells / 肿瘤

Objective: To investigate the molecular mechanisms of matrine (MAT) inhibiting the proliferation and migration of gastric cancer MGC-803 cells. Methods: MGC-803 cells were treated with different concentrations of MAT. MTT assay was used to test the proliferation activity of MGC-803 cells. The colony-forming assay was used to detect the colony formation ability of MGC-803 cells. Wound healing assay and Transwell chamber assay were used to detect the lateral and vertical migration abilities of MGC-803 cells, respectively. The cell cycle distribution was detected by FCM. The protein expression levels of epithelial-mesenchymal transition (EMT) markers and matrix metalloproteinase (MMP) family members (MMP2 and MMP9) in MGC-803 cells were detected by Western blotting. Results: MAT significantly inhibited the proliferation (P < 0.01), colony formation (P < 0.05) and lateral and vertical migration (both P < 0.01) of MGC-803 cells, and blocked the cell cycle at G0/G1 phase (P < 0.01). MAT significantly decreased the expression levels of Vimentin (P < 0.05), Snail (P < 0.01), MMP2 (P < 0.01) and MMP9 (P < 0.01) proteins, while the expression level of E-cadherin protein was up-regulated (P < 0.05). Conclusion: MAT may effectively suppress the proliferation and migration of gastric cancer MGC-803 cells, and block cell cycle at G0/G1 phase. The mechanism may be related to the regulation of EMT-related protein expression.

YAP1 knockdown in cancer-associated fibroblasts inhibits migration and invasion of breast cancer MDA-MB-231 cells / 肿瘤

Objective: To investigate the effects of Yes-associated protein 1 (YAP 1) gene knockdown in cancer-associated fibroblast (CAF) on the migration and invasion of breast cancer MDAMB- 231 cells. Methods: The expression of YAP1 in breast primary CAF and paired normal fibroblast (NF) in mRNA microarray was analyzed by bioinformatics methods. To verify the authenticity and accuracy of the microarray results, the expressions of YAP1 mRNA and protein in primary and immortalized NF and CAF were detected by real-time fluorescent quantitative PCR and Western blotting. A co-cultured system including breast cancer MDA-MB-231 cells and the conditioned medium of NF or CAF was used, and the effect of fibroblasts on the invasion ability of breast cancer cells was detected by Transwell chamber assay. CAF cells were transfected with the plasmids carrying the specific shRNA targeting YAP 1 gene, then the expressions of YAP1 mRNA and protein in CAF were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The effects of YAP 1 gene interference in CAF on the migration and invasion of MDA-MB-231 cells cultured with CAF conditioned medium were detected by scratch wound healing and Transwell chamber assay, respectively. CAF cells were treated with Hippo pathway inhibitor XMU-MP-1, then the expressions of total YAP1 protein and phosphorylated YAP1 protein in CAF were detected by Western blotting, as well as the invasion ability of MDA-MB-231 cells co-cultured with the CAF conditioned medium was measured by Transwell chamber assay. The expressions of transforming growth factor-beta 1 (TGF -β 1) and interleukin 6 (IL 6), two target genes of YAP1 and the cytokines associated with cell migration and invasion, in CAF with YAP 1 gene interference were detected by real-time fluorescent quantitative PCR. The effect of YAP1-knocked CAF on the extracellular matrix remodeling was tested by Collagen gel contraction assay. Results: Compared with primary NF, the expression of YAP 1 gene was significantly upregulated in primary CAF of breast cancer according mRNA microarray (P < 0.05). The higher expressions of YAP1 mRNA and protein in primary and immortalized CAF were confirmed by real-time fluorescent quantitative PCR (P < 0.05) and Western blotting (P < 0.01). The invasion ability of breast cancer MDA-MB-231 cells was significantly promoted in the coculture system with the conditioned medium of CAF as compared with NF (P < 0.01). The CAF cell line transfected with YAP1 shRNA was successfully constructed, and the expressions of YAP1 mRNA and protein were successfully stably knocked down in CAF (both P < 0.01). Compared with the YAP1-unknocked CAF control group, the migration (P < 0.05) and invasion (P < 0.01) abilities of breast cancer MDA-MB-231 cells were significantly weakened after culture with the conditioned medium of YAP1-knocked CAF. The phosphorylation level of YAP1 protein was decreased in CAF treated with Hippo pathway inhibitor XMU-MP-1 (P < 0.01), while the invasion ability of MDA-MB-231 cells was significantly enhanced after coculture with XMU-MP-1-treated CAF (P < 0.05). The expression levels of TGF-β1 and IL6 mRNAs were down-regulated in YAP1-knocked CAF (both P < 0.05), and the collagen gel shrinking ability of extracellular matrix was significantly decreased in YAP1-knocked CAF group (P < 0.05). Conclusion: The stable knockdown of YAP1 expression in CAF can inhibit the migration and invasion of breast cancer MDA-MB-231 cells through down-regulating the expressions of TGF-β1 and IL6 and remodeling the extracellular matrix.

Expression of linc01296 in hepatocellular carcinoma tissues and its role in proliferation, migration and invasion of hep3b cells / 肿瘤

Objective: To investigate the expression of long noncoding RNA (lncRNA) LINC01296 in hepatocellular carcinoma (HCC) tissues and cells, and to explore its effects on the proliferation, migration and invasion of Hep3B cells. Methods: The expression of LINC01296 in liver cancer cells, HCC tissues and para-cancerous tissues was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) database and real-time quantitative PCR. Moreover, the expression of LINC01296 in HCC cells and the normal hepatocyte cells was detected by real-time fluorescent quantitative PCR. After the lentivirus carrying LINC01296-overexpression or-silencing vector was infected into Hep3B cells, the cell proliferation, migration and invasion were detected by CCK-8 assay and Transwell chamber assay, respectively. The effect of LINC01296 on the expressions of epithelial-mesenchymal transition (EMT)-related proteins in Hep3B cells was detected by Western blotting. Results: The expression level of LINC01296 was significantly up-regulated in HCC tissues and cells (both P < 0.05). The overexpression of LINC01296 significantly promoted the proliferation, migration, invasion and EMT activities of Hep3B cells (all P < 0.01), whereas the results were opposite when LINC01296 was silenced (all P < 0.01). Conclusion: The expression level of LINC01296 is up-regulated in HCC tissues and cells, and it can promote the proliferation, migration and invasion of Hep3B cells by enhancing the activities of EMT.

Inhibitory effects of wnt inhibitor IWR-1 on proliferation and migration of osteosarcoma MG-63 cells / 肿瘤

Objective: To investigate the effects of Wnt inhibitor IWR-1-endo (IWR-1) on the proliferation and migration of human osteosarcoma MG-63 cells, and to explore the possible mechanism. Methods: MG-63 cells were treated with different concentrations of IWR-1 (2, 4, 8 and 16 μmol/L). Then the growth inhibitory rates of MG-63 cells was determined by CCK-8 assay. The cell cycle and apoptosis rate of MG-63 cells were detected by FCM method. The migration ability of MG-63 cells was abserved by scratch wound healing assay. The expression levels of β-catenin and cyclin D1 mRNAs were detected by real-time fluorescent quantitative PCR. The expression levels of Axis inhibition protein 1 (AXIN1), β-catenin, phospho-β-catenin (p-β-catenin) and cyclin D1 proteins were detected by Western blotting. Results: The proliferation inhibitory rate of MG-63 cells after IWR-1 treatment increased in a time-and concentration-dependent manner (all P < 0.01). The proportion of MG-63 cells treated with IWR-1 in G0/G1 phase were increased (P < 0.05), and the apoptosis rate of MG-63 cells increased significantly with the increase of IWR-1 concentration (P < 0.01). IWR-1 reduced significantly the scratch healing rate of MG-63 cells (P < 0.01), decreased the mRNA (both P < 0.01) and protein (both P < 0.05) expression levels of β-catenin and cyclin D1, and increased the expression levels of AXIN 1 and p-β-catenin proteins (both P < 0.05). Conclusion: IWR-1 can inhibit the proliferation and migration of MG-63 cells by blocking Wnt/β-catenin signaling pathway.

Inhibitory effect of lycorine on osteosarcoma and its underlying mechanism / 肿瘤

Objective: To investigate the inhibitory effect of lycorine on human osteosarcoma 143B cells and its underlying molecular mechanism. Methods: Osteosarcoma 143B cells were treated with 0-8 μmol/L lycorine, respectively. Then the effect of lycorine on the proliferation of 143B cells was detected by crystal violet staining, MTT and colony-forming assay. The migration and invasion abilities of 143B cells were detected by scratch wound healing assay and Transwell assay, respectively. The apoptosis of 143B cells was measured by hoechst 33258 staining. The expression levels of migration-and invasion-related proteins matrix metalloproteinase-7 (MMP-7) and MMP-9, as well as apoptosis-related proteins Bcl-2, Caspase 3 and cleaved Caspase 3 (c-Caspase 3) were detected by Western blotting. Furthermore, the transcription activity of T cell factor/ lymphoid enhancer factor (TCF/LEF) was detected by luciferase reporter gene system. The expression levels of β-catenin and its downstream target molecules Cyclin D1 and c-Myc in Wnt/β-catenin signaling pathway were detected by Western blotting. Results: Lycorine significantly inhibited the proliferation, migration and invasion abilities of osteosarcoma 143B cells, and promoted apoptosis (all P < 0.01). The expression levels of migration-and invasion-related proteins MMP-7 and MMP-9 in 143B cells treated with lycorine were down-regulated remarkably (all P < 0.05), and the expression of apoptosis-related protein Bcl-2 was down-regulated, while the expressions of Caspase 3 and c-Caspase 3 were up-regulated (all P < 0.05). The transcription activity of TCF/LEF was significantly decreased (P < 0.01), and the expression levels of β-catenin, c-Myc and Cyclin D1 were all decreased (all P < 0.05) after lycorine treatment. Conclusion: Lycorine may inhibit the proliferation, migration and invasion of human osteosarcoma 143B cells, and promote apoptosis by blocking the activity of Wnt/β-catenin signaling pathway.

SNHG16 facilitates proliferation and migration of hepatocellular carcinoma cells by Wnt/β-catenin signaling pathway / 肿瘤

Objective: To investigate the expression of long non-coding RNA (lncRNA) small nucleolar RNA host gene 16 (SNHG16) in hepatocellular carcinoma (HCC) tissues and cells, and to explore the effects of SNHG16 expression regulation on the proliferation and migration of HCC cells as well as the underlying molecular mechanisms. Methods: The cancer tissues and adjacent tissues were collected from 38 patients with HCC. The real-time fluorescent quantitative PCR was used to detected the expression of SNHG16 in 38 cases of clinical HCC tissue samples and their adjacent tissues, 4 kinds of HCC cell lines and normal hepatocellular cell line. The relationship between SNHG16 expression and the clinicopathological features of HCC patients was analyzed. The SNHG16 overexpression or SNHG16-shRNA recombinant lentivirus was infected into Hep-3B or SK-Hep-1 cells, respectively. The up- or down-regulation of SNHG16 expression in Hep-3B or SK-Hep-1 cells was verified by real-time fluorescent quantitative PCR. The effects of SNHG16 expression regulation on the proliferation and migration of HCC cells were determined by CCK-8 and Transwell chamber experiments, respectively. The expressions of key proteins in Wnt/β-catenin signaling pathway were detected by Western blotting. Xenograft tumor experiment was used to determine the effect of SNHG16 on the tumorigenic ability of HCC cells in nude mice. Results: The expression level of SNHG16 in HCC tissues and HCC cells was significantly higher than that in the adjacent tissues (P < 0.001) and normal hepatocellular cells (P < 0.05), respectively. The expression of SNHG16 in HCC tissues was associated with tumor size (P < 0.01), TNM stage (P < 0.01) and alanine aminotransaminase (ALT) expression level (P < 0.05). After the infection with SNHG16 overexpression recombinant lentivirus, the expression of SNHG16 was significantly up-regulated in Hep-3B cells (P < 0.001), the proliferation and migration of Hep-3B cells were significantly promoted (both P < 0.01), and the expressions of β-catenin and c-myc proteins were up-regulated (both P < 0.01). After the infection with SNHG16-shRNA recombinant lentivirus, the expression of SNHG16 was dramatically downregulated in SK-Hep-1 cells (P < 0.001), the proliferation and migration of SK-Hep-1 cells were significantly inhibited (both P < 0.001), and the expressions of β-catenin (P < 0.05) and c-myc (P < 0.01) proteins were down-regulated. In addition, the tumorigenic ability of Hep- 3B cells with SNHG16 over-expression was significantly enhanced in nude mice (P < 0.01), while the tumorigenic ability of SK-Hep-1 cells with SNHG16 knock-down was significantly weakened in nude mice (P < 0.05). Conclusion: SNHG16 is highly expressed in HCC tissues and cell lines, and may promote the proliferation and migration of HCC cells through Wnt/β-catenin signaling pathway.

Effect of water-soluble components of atmospheric fine particulate matter PM2.5 on proliferation, migration and melanogenesis of a human melanocyte line PIG1 / 中华皮肤科杂志

Objective@#To evaluate the effect of water-soluble components of atmospheric fine particulate matter PM2.5 on proliferation, migration, tyrosinase activity and melanin content of a human melanocyte line PIG1.@*Methods@#PM2.5 was collected during haze weather in heating seasons, and processed into suspensions. PIG1 melanocytes were cultured and divided into 5 experimental groups and 1 control group. PIG1 melanocytes in the 5 experimental groups were treated with 10, 20, 50, 100 and 200 mg/L PM2.5 suspensions respectively for 48 hours, while cells in the control group were not treated with PM2.5 suspensions. In cell migration assay, there was only 1 experimental group treated with 10 mg/L PM2.5 suspensions. After treatment, methyl thiazol tetrazolium (MTT) assay, micropore filtration assay, DOPA oxidase assay and NaOH lysis method were performed to determine the cell proliferation rate, migration rate, tyrosinase activity and melanin content respectively. Statistical analysis was carried out by using t test for comparison of means of two samples, one-way analysis of variance for means of multiple samples, Student-Newman-Keuls (SNK) -q test for multiple comparisons, and linear correlation analysis for analysis of correlations.@*Results@#Compared with the control group ([100 ± 1.41]%) , the proliferation rate of PIG1 cells significantly decreased in the 20-, 50-, 100- and 200-mg/L PM2.5 groups ([93.41 ± 2.13]%, [88.31 ± 1.3557]%, [79.75 ± 1.89]%, [69.83 ± 2.50]% respectively, all P < 0.05) . Linear correlation analysis showed that the proliferation rate and tyrosinase activity of PIG1 cells decreased with the increase in PM2.5 concentrations (r = -0.98, -0.93, respectively, both P < 0.01) . After the treatment with 10 mg/L PM2.5, the migration rate of PIG1 cells significantly decreased (66.23% ± 1.11%) compared with the control group ([76.86 ± 1.81]%, t = 7.55, P < 0.01) . With the increase in PM2.5 concentrations (50-200 mg/L) , the melanin content of PIG1 cells gradually decreased (r = -0.97, P < 0.01) .@*Conclusion@#Atmospheric fine particulate matter PM2.5 can affect the normal functions of melanocytes by inhibiting their proliferation and migration, and reducing their tyrosinase activity and melanin content.

Effect of human stromal vascular fraction gel on the treatment of patients with skin depressed scar and its mechanism / 中华烧伤杂志

Objective@#To observe content of cytokine in human stromal vascular fraction gel (SVF-GEL) and effect of SVF-GEL on biological behaviors of epidermal and dermal cells in vitro and clinical efficacy of SVF-GEL.@*Methods@#(1) SVF-GEL was prepared using liposuction aspirates harvested from females who received abdomen liposuction in author′s unit. SVF-GEL (1 mL) and high-glucose Dulbecco′s modified eagle medium (DMEM, 1 mL) were respectively cultured for 24 h with high-glucose DMEM containing 10% fetal calf serum, 10 g/L penicillin, and 10 g/L streptomycin, denoted as SVF-GEL group and negative control group, with 6 samples in each group. Content of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in the supernatant was determined by enzyme-linked immunosorbent assay. (2) A number of 5×105 human skin fibroblasts (HSF) and HaCaT cells in logarithmic phase were inoculated and cultured in Transwell chambers for 12 h. All Transwell chambers containing cells were divided into SVF-GEL group (0.5 mL SVF-GEL was added for co-culture) and control group (0.5 mL high-glucose DMEM was added for co-culture), with 9 samples in each group for HSF and HaCaT cells. Scratch assay was performed after culture for 24 h, and residual scratch width was observed at post scratch hour (PSH) 0 (immediately), 24, and 48. Cell migration distance was measured at PSH 24 and 48. After culture for 24, 48, and 72 h, the number of living cell was counted using cell counter. (3) From June 2018 to June 2019, SVF-GEL was applied clinically to treat 15 patients with depressed scars on face, including 2 males and 13 females, aged 19 to 42 years. Survival condition of SVF-GEL and whether complications or not were observed 6 months after surgery. Before surgery and 6 months after surgery, depressed degree, color, and pliability of scar were compared. Vancouver Scar Scale (VSS) was employed to access color, vascularity, and pliability before surgery and 6 months after surgery, and total score was calculated. The number of patients with complete satisfaction or satisfaction was counted six months after surgery. Data were processed with analysis of variance of factorial design, paired samples t test, and Wilcoxon rank sum test.@*Results@#(1) The content of EGF in SVF-GEL group and negative control group was (316.6±12.8) and (3.4±0.6) pg/mL, and the content of VEGF in SVF-GEL group and negative control group was (568.67±12.19) and (4.93±0.16) pg/mL, with statistically significant differences between the two groups (t=48.777, 92.485, P<0.01). (2) Residual scratch widths of HSF and HaCaT in SVF-GEL group and control group were decreased gradually along with time elapse, in which those in SVF-GEL group at PSH 24 and 48 were less than those in control group. At PSH 24 and 48, cell migration distances of HSF and HaCaT in SVF-GEL group were more than those in control group (tHSF=-20.304, -43.516, tHaCaT=-15.060, -8.684, P<0.01). After culture for 24, 48, and 72 h, the number of living cell of HSF and HaCaT in SVF-GEL group was significantly more than that in control group (tHSF=-3.374, -6.809, -18.036, tHaCaT=-4.793, -6.028, -8.141, P<0.05 or P<0.01). (3) Six months after surgery, SVF-GEL grafted into patients survived well without complications, and depressed degree of scar ameliorated obviously with lightened pigmentation and softer texture as compared with before surgery. Compared with those before surgery, VSS scores of color, vascularity, and pliability, and total score of 15 patients with depressed scars on face were obviously decreased 6 months after surgery (Z=-2.06, -2.07, -2.07, t=-15.811, P<0.05 or P<0.01). One patient was satisfied with the clinical outcome, and the rest 14 patients were completely satisfied with the clinical outcomes.@*Conclusions@#SVF-GEL contains cytokines EGF and VEGF, which can enhance cell migration ability and proliferation ability of HSF and HaCaT cells and have obvious effects on depressed scars for clinical application.