miR-105 inhibits cell proliferation,migration and invasion abilities in non-small-cell lung cancer cells by targeting and negative regulation of KIFC1

AIM:To study the effects of microRNA-105(miR-105) on the cell proliferation, migration and invasion abilities of non-small-cell lung cancer (NSCLC) H460 cells, and further to explore its mechanism. METHODS:The expression of miR-105 and kinesin family member C1 (KIFC1) mRNA in the NSCLC tissues and adjacent tissues and cells was detected by RT-qPCR. The protein expression of KIFC1 in the NSCLC tissues, adjacent normal tissues and cells was determined by Western blot. The H460 cells were divided into miR-105 group (transfection with miR-105 mimics), miR-negative control (NC) group (transfection with miR-NC), inhibitor-NC group (transfection with NC of inhibitor), inhibitor-miR-105 group (transfection with miR-105 inhibitor), si-NC group (transfection with NC siRNA), si-KIFC1 group (transfection with KIFC1 siRNA), miR-105+vector group (miR-105 mimics and pcDNA 3.1 co-transfection) and miR-105+KIFC1 group (miR-105 mimics and pcDNA 3.1-KIFC1 co-transfection). The cell proliferation was measured by MTT assay and colony formation assay. The migration and invasion abilities were detected by Transwell methods. The relative luciferase acitivity was evaluated by double luciferase reporter assay. RESULTS:Compared with the adjacent tissues, the expression of miR-105 was significantly decreased and the expression of KIFC1 was significantly increased in NSCLC tissues (P<0.05). Compared with human normal embryonic lung fibroblasts MRC-5, the expression of miR-105 in the H460 cells was significantly decreased, and the expression of KIFC1 was significantly increased (P<0.05). miR-105 inhibited the relative luciferase activity of H460 cells with wild-type KIFC1 and negatively regulated the protein expression of KIFC1. Over-expression of miR-105 and knockdown of KIFC1 expression significantly inhibited the proliferation, migration and invasion abilities of H460 cells. Over-expression of KIFC1 reversed the inhibitory effect of miR-105 on the cell proliferation, migration and invasion abilities of H460 cells. CONCLUSION:miR-105 inhibits the proliferation, migration and invasion abilities of NSCLC cells. The mechanism may be related to targeting and negatively regulating expression of KIFC1.     

Toll-like receptor 4 promotes migration and invasion abilities of human non-small-cell lung cancer cells

AIM:To evaluate the expression and biological role of Toll-like receptor 4 (TLR4) in human non-small-cell lung cancer (NSCLC) cells. METHODS:The mRNA and protein levels of TLR4 in NSCLC tissue were exa-mined by RT-qPCR, Western blot, and immunohistochemistry. After treating the A549 cells and SPC-A-1 cells with TLR4 stimulator lipopolysaccharide (LPS) and inhibitor TAK-242, RT-qPCR, Western blot and flow cytometry were performed to detect the expression of TLR4. The migration and invasion abilities were detected by Transwell assay, and the mRNA expression of matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor (VEGF) was also detected. RESULTS:The mRNA and protein levels of TLR4 were higher in the NSCLC tissue than those in the noncancerous tissue (P<0.01). LPS stimulation significantly increased the mRNA and protein expression levels of TLR4 in the NSCLC cell lines A549 and SPC-A-1 (P<0.01). The LPS-induced TLR4 activation enhanced the migration and invasion abilities of A549 cells and SPC-A-1 cells (P<0.01). LPS increased the expression levels of MMP-2, MMP-9 and VEGF in the A549 cells and SPC-A-1 cells (P<0.01). Moreover, the expression levels of TLR4, MMP-2, MMP-9 and VEGF, as well as the migration and invasion abilities of the cells were blocked by TAK-242 (P<0.01). CONCLUSION:TLR4 might be involved in the migration and invasion of NSCLC cells, and TLR4 inhibition might be considered as a therapeutic target for treatment of NSCLC.     

miR-485-5p inhibits viability and migration and invasion abilities of hepatocellular carcinoma Hep3B cells by targeting SOX5

AIM: To investigate the effects of microRNA-485-5p (miR-485-5p) on the viability, migration and invasion abilities of hepatocellular carcinoma cells and the underlying mechanism. METHODS: The expression levels of sex determining region Y-box 5 (SOX5) mRNA and miR-485-5p in the hepatocellular carcinoma Hep3B cells were detected by RT-qPCR with normal hepatocyte THLE-3 as control. Western blot was used to measure the expression levels of SOX5, proliferating cell nuclear antigen (PCNA), Ki67, cyclin D1 and matrix metalloproteinase-2 (MMP-2). The viability of Hep3B cells was measured by MTT assay. The migration and invasion abilities of the Hep3B cells were detected by Transwell assay. Dual-luciferase reporter assay system was applied to verify the relationship between miR-485-5p and SOX5. RESULTS: Compared with the control cells, the expression level of miR-485-5p was decreased in hepatocellular carcinoma cells Hep3B, Huh7 and HCCLM3 (P<0.05), while the expression of SOX5 at mRNA and protein levels were significantly increased (P<0.05). Over-expression of miR-485-5p inhibited the viability, migration and invasion of Hep3B cells. miR-485-5p targeted the 3′-UTR of SOX5 and negatively regulated the expression of SOX5. Knocking-down of SOX5 expression inhibited the viability, migration and invasion of Hep3B cells. Over-expression of SOX5 partially reversed the inhibitory effect of miR-485-5p over-expression on the viability, migration and invasion of Hep3B cells. CONCLUSION: miR-485-5p inhibits the viability, migration and invasion of Hep3B cells by targeting SOX5 gene. miR-485-5p is a potential molecular target for hepatocellular carcinoma.     

Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

miR-556-3p regulates viability,migration and invasion of endometrial cancer cells by targeting SASH1

AIM To investigate whether microRNA-556-3p （miR-556-3p） regulates the viability, migration and invasion of endometrial cancer cells by targeting SASH1 gene. METHODS The expression of miR-556-3p, and the mRNA and protein levels of SASH1 in endometrial cancer tissues were detected by RT-qPCR and Western blot. Anti-miR-556-3p or pcDNA-SASH1 was transfected into endometrial cancer Ishikawa cells. The cell viability was detected by MTT assay, the migration and invasion abilities of the cells were detected by Transwell chamber method, and the protein expression levels of cyclin D1, p21, matrix metalloproteinase-2 （MMP-2） and MMP-9 were detected by Western blot. StarBase prediction and dual-luciferase reporter experiments were used to analyze the targeting relationship between miR-556-3p and SASH1. Anti-miR-556-3p and si-SASH1 were co-transfected into the Ishikawa cells, and their effects on cell viability, migration and invasion were examined by the methods described above. RESULTS Compared with adjacent tissues, the expression of miR-556-3p in endometrial cancer tissues was increased significantly, and the expression of SASH1 at mRNA and protein levels was decreased significantly （P<0.05）. Inhibition of miR-556-3p expression or induction of SASH1 over-expression obviously reduced the viability of Ishikawa cells, the number of migratory cells, the number of invasive cells and the protein levels of cyclin D1, MMP-2 and MMP-9, and dramatically increased the protein level of p21 （P<0.05）. miR-556-3p targeted SASH1 and negatively regulated its expression. Knock-down of SASH1 expression reversed the inhibitory effect of miR-556-3p expression inhibition on the viability, migration and invasion of Ishikawa cells. CONCLUSION Inhibition of miR-556-3p expression suppresses the viability, migration and invasion of endometrial cancer cells. The mechanism is related to the regulation of its target gene SASH1.     

miR-137 regulates invasion,migration and apoptosis of breast cancer MDA-MB-231 cells through TWIST1

AIM: To investigate the effect and its molecular mechanism of microRNA-137(miR-137) on the invasion, migration abilities and apoptosis of breast cancer cells. METHODS: miR-137 mimimics were transfected into the breast cancer MDA-MB-231 cells. The expression of miR-137 was detected by RT-qPCR. Apoptosis was analyzed by flow cytometry. The invasion and migration abilities were detected by Transwell assays. The protein levels of matrix metalloproteinase 9 (MMP-9), cleaved caspase-3 (C-caspase-3) and Bax were determined by Western blot. Bioinformatics software was used to predict that TWIST1 might be the target gene of miR-137 and then it was conformed by luciferase reporter gene identification. The effect of miR-137 mimics on TWIST1 protein expression was evaluated by Western blot. TWIST1 over-expression vector and miR-137 mimics were co-transfected into the MDA-MB-231 cells, and then the apoptosis, invasion, migration abilities and the protein levels of MMP-9, C-caspase-3 and Bax were determined. RESULTS: In the miR-137 mimics transfected MDA-MB-231 cells, the expression level of miR-137 and the apoptosis rate were increased, the cell invasion and migration abilities were decreased, the protein levels of C-caspase-3 and Bax were increased, the protein expression of MMP-9 was decreased (P<0.05). In addition, the target regulation of TWIST1 by miR-137 was identified by luciferase reporter assay. Moreover, the expression of TWIST1 in the MDA-MB-231 cells was inhibited by miR-137 mimics. Compared with the MDA-MB-231 cells co-transfected with negative control vector and miR-137 mimics, the protein expression levels of TWIST1 and MMP-9 in the MDA-MB-231 cells co-transfected with TWIST1 over-expression vector and miR-137 mimics were increased, the protein levels of C-caspase-3 and Bax and the apoptosis rate were decreased, the cell invasion and migration abilities were increased. CONCLUSION: miR-137 inhibits the invasion, migration abilities and induces apoptosis of breast cancer cells through targeting TWIST1.     

miR-140-3p inhibits viability,invasion and migration of non-small-cell lung cancer A549 cells by targeting PD-L1

AIM: To explore the target relationship between microRNA-140-3p (miR-140-3p) and programmed cell death ligand 1 (PD-L1) and their effect on the viability, migration and invasion of non-small-cell lung cancer A549 cells.METHODS: RT-qPCR was used to detect the miR-140-3p expression in HLF-1, A549 and H1299 cells, and then the A549 cells with the most significant difference were selected as the subsequent research object. TargetScan software and dual-luciferase reporter assay were performed to predict and confirm the target relationship between miR-140-3p and PD-L1. RT-qPCR and Western blot were used to determine the effects of miR-140-3p mimic and inhibitor on PD-L1 expression level. MTT assay was used to detect the viability of A549 cells. Transwell assay was performed to detect the migration and invasion abilities of the A549 cells.RESULTS: miR-140-3p was significantly down-regulated in the A549 cells and H1299 cells (P<0.05). Transfection with miR-140-3p mimic decreased the expression of PD-L1 and inhibited the viability, migration and invasion of the A549 cells. Transfection with pcDNA3.0-PD-L1 reversed the inhibitory effect of miR-140-3p on the viability, migration and invasion of the A549 cells.CONCLUSION: miR-140-3p inhibits the viability, migration and invasion of A549 cells by targeting PD-L1.     

MicroRNA-497 inhibits viability,invasion and migration of thyroid papillary cancer cells

AIM:To investigate the underlying mechanisms of mircoRNA-497 (miR-497) inhibiting the viability, migration and invasion of papillary thyroid cancer (PTC) cells. METHODS:TargetScan 6.0 was used to predict the potential targets of miR-497. The target gene was confirmed by luciferase reporter assay, RT-qPCR and Western blot. The expression levels of miR-497 and its target gene in the PTC tissues were detected by RT-qPCR. Gene transfection, MTT assay, and cell migration and invasion assays were used to investigate the effects of miR-497 and its target gene on PTC cell viability, migration and invasion. RESULTS:AKT3 was demonstrated to be the direct target gene of miR-497. In addition, AKT3 expression was higher in the PTC tissues than that in normal tissues (P<0.05) and negatively correlated with miR-497 expression (r=-0.573 7, P<0.01). Furthermore, down-regulation of AKT3 also suppressed cell viability, migration and invasion of PTC, which played similar roles of miR-497 over-expression in PTC cells. CONCLUSION:miR-497 inhibits the viability, migration and invasion of PTC cells by directly targeting AKT3.     

Mechanism of microRNA-138-5p inhibiting proliferation,migration and invasion of lung cancer cells

AIM: To investigate the mechanism of microRNA-138-5p (miR-138-5p) inhibiting the proliferation, migration and invasion abilities of lung cancer cells.METHODS: The lung cancer A549 and H460 cells were transfected with miR-NC (control group) or miR-138-5p (experimental group). The bioinformatic analysis was performed to predict the target genes of miR-138-5p.The expression levels of miR-138-5p, forkhead box protein C1 (FOXC1) mRNA and vimentin mRNA were detected by RT-qPCR. The protein expression of FOXC1, vimentin, E-cadherin, N-cadherin and β-catenin was determined by Western blot. MTS method and colony formation assay were used to detect cell viability and proliferation ability. Wound healing assay and Transwell assay were used to detect cell migration and invasion ability.RESULTS: Over-expression of miR-138-5p significantly reduced the expression of FOXC1 and vimentin at mRNA and protein levels (P<0.05). The expression of E-cadherin and β-catenin were up-regulated and the expression of N-cadherin was down-regulated. The proliferation, migration and invasion abilities of the lung cancer cells were inhibited by the over-expression of miR-138-5p.CONCLUSION: miR-138-5p inhibits the proliferation, migration and invasion abilities of lung cancer cells by targeting FOXC1 and vimentin. It may be a potential target for lung cancer gene therapy.     

Down-regulation of lncRNA PCAT1 suppresses oral squamous cell carcinoma cell proliferation,growth, invasion,migration and epithelial-mesenchymal transition

AIM: To investigate the effects of the long non-coding RNA (lncRNA) PCAT1 on the oral squamous cell carcinoma (OSCC) cell proliferation, growth, invasion and migration, and to explore the underlying mechanisms. METHODS: The PCAT1 siRNA was transfected by Lipofectmine 2000, and RT-qPCR and Western blot were performed to determine the mRNA and protein expression of relevant genes, respectively. CCK-8 assay and colony formation assay were used to measure OSCC cell proliferation and growth, respectively. The cell invasion and migration assays were used to measure the invasive and migratory abilities of the OSCC cells, respectively. RESULTS: PCAT1 was significantly up-regulated in OSCC tissues and cells compared with normal adjacent tissues and normal human oral keratinocyte cells, respectively (P<0.05). PCAT1 siRNA transfection suppressed the expression of PCAT1 in Tca8113 and TSCCa cells (P<0.05). Knockdown of PCAT1 in Tca8133 cells and TSCCa cells significantly suppressed the cell proliferation, invasion and migration abilities (P<0.05). In addition, knockdown of PCAT1 in Tca8133 cells and TSCCa cells also suppressed the mRNA and protein levels of ZEB-1, N-cadherin and vimentin, and increased the mRNA and protein expression of E-cadherin (P<0.05). CONCLUSION: Knockdown of PCAT1 suppresses cell proliferation and migration abilities, and the effect of PCAT1 on OSCC cells may be associated with epithelial-mesenchymal transition.     

lncRNA TTN-AS1 regulates the viability and invasion of lung adenocarcinoma A549 cell via miR-519d-3p/MMP2 pathway

AIM To investigate the expression level of long noncoding RNA （lncRNA） TTN antisense RNA 1 （TTN-AS1） in lung adenocarcinoma tissues and the effects of TTN-AS1 silencing on the viability and invasion of lung adenocarcinoma A549 cells. METHODS RT-qPCR was used to detect the expression of TTN-AS1, microRNA-519d-3p （miR-519d-3p） and matrix metalloproteinase 2 （MMP2） mRNA in 32 cases of lung adenocarcinoma and adjacent normal tissues. The untransfected A549 cells were divided into blank group, si-NC group （with si-NC transfection） and si-lncRNA group （with silencing of lncRNA TTN-AS1 expression）, with n=5 in each group. The effects of TTN-AS1 silencing on the viability and invasion of A549 cells were detected by CCK8 and Transwell methods. The targeting regulatory effects of TTN-AS1 on miR-519d-3p and miR-519d-3p on MMP2 were determined by dual-luciferase reporter assay, RNA immunoprecipitation test, RT-qPCR and Western blot. RESULTS The expression level of TTN-AS1 in 32 cases of lung adenocarcinoma tissues is notably higher than that in the adjacent normal tissues （P<0.05）. Silencing of TTN-AS1 in A549 cells significantly suppressed the cell viability and invasion. TTN-AS1 negatively regulated the expression of miR-519d-3p via sponging and absorbing miR-519d-3p. MMP2 is the target gene of miR-519d-3p and can be negatively regulated by miR-519d-3p. Overexpression of MMP2 partially reversed the inhibitory effect of TTN-AS1 silencing and miR-519d-3p overexpression on the invasion of A549 cells. CONCLUSION The lncRNA TTN-AS1 is overexpressed in lung adenocarcinoma tissues, and it regulates lung adenocarcinoma A549 cell viability and invasion via miR-519d-3p/MMP2 pathway.     

Expression of miRNA-22 in ovarian cancer and effect of miRNA-22 over-expression on SKOV-3 ovarian cancer cell proliferation,migration and invasion

AIM: To examine the expression of miRNA-22 in the ovarian tissues and the effect of miRNA-22 over-expression on the proliferation, migration and invasion in SKOV-3 cells. METHODS: The expression levels of miRNA-22 in different ovarian tissues and SKOV-3 cells were determined by qPCR. miRNA-22 was over-expressed by transfection of miRNA-22 mimic. The cell viability was examined by CCK-8 assay. The cell migration was measured by wound healing test. The cell invasion was analyzed by Transwell assay. The protein expression levels of VEGF and P53 were determined by Western blot. RESULTS: Compared with the normal ovarian tissue, the expression level of miRNA-22 was remarkably decreased in the ovarian tumor tissues. After transfection with miRNA-22 mimic, the expression level of miRNA-22 in the SKOV-3 cells was significantly increased, while the cell viability, migration and invasion were obviously decreased. Moreover, the protein expression of VEGF and P53 was dramatically inhibited after over-expression of miRNA-22. CONCLUSION: The decreased miRNA-22 expression may be correlated with the development of ovarian can-cer. Over-expression of miRNA-22 decreases the cell viability, migration and invasion by reducing the protein expression of VEGF and P53.     

Effect of inhibiting lncRNA LINC01503 on viability,migration and invasion of lung cancer cells by targeted regulation of miR-335-5p

AIM To investigate the effects of long non-coding RNA (lncRNA) LINC01503 on the viability, migration and invasion of lung cancer cells and its mechanism. METHODS Human lung carcinoma H1299 cells were divided into si-NC group (transfected with si-NC), si-LINC01503 group (transfected with si-LINC01503), pcDNA group (transfected with pcDNA), pcDNA-LINC01503 group (transfected with pcDNA-LINC01503), miR-NC group (transfected with miR-NC), miR-335-5p group (transfected with miR-335-5p mimics), si-LINC01503+anti-miR-NC group (co-transfected with si-LINC01503 and anti-miR-NC), si-LINC01503+anti-miR-335-5p group (co-transfected with si-LINC01503 and anti-miR-335-5p), miR-NC+WT-LINC01503 group (co-transfected with miR-NC and WT-LINC01503), miR-NC+MUT-LINC01503 group (co-transfected with miR-NC and MUT-LINC01503), miR-335-5p+WT-LINC01503 group (co-transfected with miR-335-5p and WT-LINC01503) and miR-335-5p+MUT-LINC01503 group (co-transfected with miR-335-5p and MUT-LINC01503). The expression of miR-335-5p and LINC01503 was detected by RT-qPCR. Western blot was used to detect protein expression. MTT assay was used to detect cell viability. Transwell assay was used to detect the migration and invasion abilities. Dual-luciferase reporter assay was used to confirm the targeted relationship between LINC01503 and miR-335-5p. RESULTS Compared with normal tissues, the expression of LINC01503 was significantly increased in the lung cancer tissues, and the expression of miR-335-5p was significantly decreased (P<0.05). Compared with stage I/II , the expression level of LINC01503 in the lung cancer tissues of stage III/IV was significantly increased, and the expression of miR-335-5p was significantly decreased (P<0.05). The patients with high expression of LINC01503 had lower short-term survival rates than those with low expression of LINC01503 (P<0.05). Compared with normal human bronchial epithelial cell line BEAS-2B, the expression of miR-335-5p in lung cancer cell lines H1299, A549 and SPC-A-1 were significantly decreased, and the expression of LINC01503 was significantly increased (P<0.05). Over-expression of miR-335-5p and inhibition of LINC01503 expression inhibited the viability, migration and invasion of H1299 cells, and inhibited the protein expression of cyclin D1, matrix metalloproteinase-2 （MMP-2） and MMP-9 (P<0.05). LINC01503 targeted and regulated miR-335-5p expression, and interfering with miR-335-5p expression reversed the inhibitory effect of inhibiting LINC01503 expression on the viability, migration and invasion of H1299 cells. CONCLUSION Inhibition of lncRNA LINC01503 inhibits the viability, migration and invasion of lung cancer cells. The mechanism may be related to the targeted regulation of miR-335-5p.     

Long non-coding RNA PVT1 regulates effect of miR-551 on migration and invasion abilities of ovarian cancer through Wnt signaling pathway

AIM: To investigate the expression of long non-coding RNA PVT1 in ovarian cancer and the role of PVT1 in migration and invasion abilities of ovarian cancer cells.METHODS: The expression of PVT1 in ovarian cancer tissue, normal ovarian tissue and different ovarian cancer cell lines was detected by qPCR. Transwell assay was used to detect the invasion ability of ovarian cancer cells after PVT1 silencing. The migration ability of the ovarian cancer cells after PVT1 silencing was detected by scratch test. The interaction between PVT1 and microRNA (miR)-551 was analyzed by dual-luciferase reporter assay. The effect of miR-551-inhibitor on the invasion and migration abilities of ovarian cancer cells after PVT1 silencing was detected by Transwell assay and scratch test. The expression of Wnt signaling pathway-related proteins was determined by Western blot after PVT1 silencing. The effects of PVT1 silencing on tumor weight and volume of ovarian cancer were examined by subcutaneous tumor transplantation in nude mice.RESULTS: The expression of PVT1 in ovarian cancer tissue was significantly higher than that in normal ovarian tissue (P<0.05). The expression level of PVT1 in ovarian cancer cell line ES-2 was the highest. PVT1 silencing inhibited the invasion and migration abilities of the ovarian cancer cells. After PVT1 silencing, miR-551-inhibitor promoted the invasion and migration abilities of the ovarian cancer cells. The expression of Wnt signaling pathway-related proteins was decreased after PVT1 silencing (P<0.05). Compared with negative control group, the tumor volume and weight in PVT1-siRNA group were significantly decreased (P<0.05).CONCLUSION: PVT1 plays an important role in the development of ovarian cancer. PVT1 regulates the invasion and migration abilities of ovarian cancer cells through Wnt signaling pathway.     

miR-129-3p targets Smad3 to inhibit viability and migration of NIH3T3 cells during TGF-β-induced transformation into myofibroblasts

AIM: To explore the effects of microRNA-129-3p (miR-129-3p) on the viability and migration of NIH3T3 cells during transforming growth factor-β (TGF-β)-induced transformation into myofibroblasts and the underlying molecular mechanisms. METHODS: RT-qPCR was used to examine the relative expression of miR-129-3p in renal cell carcinoma (RCC)-adjacent tissues and fibrotic renal tissue. NIH3T3 cells were stimulated with TGF-β to transform into myofibroblasts, and miR-129-3p expression level was detected. After transfection with miR-129-3p mimics for 48 h in vitro, the cell viability was measured by MTT assay, the protein expression level of Ki-67 was determined by Western blot, and the cell migration was observed by wound healing assay. The direct target of miR-129-3p was predicted by online database TargetScan and confirmed by dual-luciferase reporter assay. The expression level of target protein was further confirmed by Western blot. RESULTS: Compared with the RCC-adjacent tissues, the expression of miR-129-3p was down-regulated in fibrotic renal tissue (P<0.01). In TGF-β-induced NIH3T3 cell transformation into myofibroblasts, the expression of miR-129-3p was also decreased (P<0.01). Transfection with miR-129-3p mimics followed by TGF-β stimulation in the NIH3T3 cells inhibited the viability, Ki-67 expression and migration. TargetScan analysis showed miR-129-3p had binding sites in the 3'-UTR of Smad3, which was confirmed by dual-luciferase reporter assay. The results of Western blot further confirmed that miR-129-3p affected the expression of Smad3. CONCLUSION: miR-129-3p inhibits the viability and migration ability of NIH3T3 cells during TGF-β-induced transformation into myofibroblasts by directly targeting Smad3.     

Effects of miR-337 targeting p53 expression on autophagy and migration ability of colon cancer cells

AIM: To investigate the effect of microRNA-337 (miR-337) on the autophagy and migration ability of colon cancer cells, and to explore its possible mechanism involving targeting p53 expression. METHODS: The me-thod of immunohistochemistry was used to detect the protein expression of beclin-1, LC3B and p53 in colon cancer tissues. The correlations between the protein expression of beclin-1/LC3B and clinicopathological features, and the correlations between the protein expression of p53 and beclin-1/LC3B were analyzed. After knock-down of p53 expression by small interfering RNA, the formation of autophagiosomes was observed under electron microscope in colon cancer cell line HCT116, and the protein expression of beclin-1 and LC3B was determined by Western blot. The miRNAs targeting p53 were predicted and screened by bioinformatics, and their expression in HCT116 cells was verified by RT-qPCR. Luciferase reporter assay was used to detect the regulatory effect of miR-337 on p53 gene. The protein expression of p53, beclin-1 and LC3B was determined by Western blot, and the migration ability of HCT116 cells after miR-337 over-expression was detected by Transwell assay. RESULTS: The protein expression of beclin-1 and LC3B in the colon cancer tissues was decreased, which was significantly related to the occurrence, development, invasion and metastasis of colon cancer. The expression of p53 was increased in the colon cancer tissues, which was negatively correlated with the protein expression of beclin-1 and LC3B. Knock-down of p53 gene expression increased the protein expression of beclin-1 and LC3B (P<0.05). Over-expression of miR-337 down-regulated the expression of p53, up-regulated the protein expression of beclin-1 and LC3B, and decreased the migration ability of HCT116 cells (P<0.05). CONCLUSION: miR-337 promotes autophagy and inhibits migration ability of colon cancer cells, and the mechanism may be related to targeted inhibition of p53 expression.     

Sphingosine kinase 1 enhances invasion and migration of non-small-cell lung cancer cells by ERK1/2 signaling pathway

AIM To explore the effects of sphingosine kinase 1 (SphK1) on the migration and invasion of non-small-cell lung cancer (NSCLC) cells and its mechanism. METHODS Thirty-one tumor specimens, which were surgically resected and routinely histologically confirmed as NSCLC, and matched adjacent lung tissues were selected. Immunohistochemical staining and RT-qPCR were used to detect the expression of SphK1. The pcDNA3.1-SphK1 vector (SphK1 group), empty pcDNA3.1 vector control (NC group), SphK1 siRNA (siSphK1 group) or control siRNA (siNC group) was transfected into human lung adenocarcinoma A549 cells, and the protein levels of SphK1, E-cadherin, fibronectin and p-ERK1/2 were determined by Western blot. The effects of over-expression of SphK1 and inhibition of ERK1/2 on migration and invasion of A549 cells were evaluated by Transwell assays. RESULTS SphK1 was highly expressed in the NSCLC tissues and was associated with tumor stage. SphK1 over-expression significantly promoted the migration and invasion of A549 cells, increased the protein levels of p-ERK1/2 and fibronectin, and decreased the protein expression of E-cadherin (P<0.05), but the opposite result was observed after SphK1 interference. The ERK1/2 inhibitor U0126 significantly inhibited the up-regulation of p-ERK1/2 and fibronectin levels and the down-regulation of E-cadherin expression induced by SphK1 over-expression, and also inhibited the invasion and migration of A549 cells promoted by SphK1 over-expression (P<0.05). CONCLUSION SphK1 may reduce E-cadherin protein levels, increase fibronectin protein levels, and promote the invasion and migration of NSCLC cells through ERK1/2 signaling pathway.