MicroRNA-200a affects malignant biological behaviors of breast cancer cells by inhibiting epithelial-mesenchymal transition

AIM: To investigate the effect of microRNA-200a (miR-200a) on the malignant biological beha-viors of breast cancer cells and its regulatory mechanism. METHODS: The expression of miR-200a in human breast can-cer cell lines MDA-MB-231, MDA-MB-468 and MCF-7, and normal human mammary epithelial cell line MCF-10A was detected by RT-qPCR. CCK-8 assay was used to detect the viability of MDA-MB-231 cells after transfection with miR-200a mimic or miR-200a inhibitor. Flow cytometry method and Transwell assay were used to detect the apoptosis and invasive ability of MDA-MB-231 cells after transfection with miR-200a mimic or miR-200a inhibitor. The expression of SIP1, E-cadherin, N-cadherin, Snail, Twist, ZEB1 and ZEB2 at mRNA and protein levels was determined by RT-qPCR and Western blot. RESULTS: Compared with MCF-10A cells, the lowest expression of miR-200a was observed in the MDA-MB-231 cells (P<0.05). Over-expression of miR-200a attenuated the viability of MDA-MB-231 cells (P<0.05), increased apoptosis (P<0.05) and decreased the invasion ability (P<0.05). The expression of SIP1, N-cadherin, Snail, Twist, ZEB1 and ZEB2 at mRNA and protein levels was also significantly down-regulated, while the mRNA and protein expression of E-cadherin was significantly increased (P<0.05). Transfection with miR-200a inhibitor reversed the above results. CONCLUSION: Up-regulation of miR-200a inhibits the viability and invasion ability of MDA-MB-231 cells and promotes the apoptosis of MDA-MB-231 cells. miR-200a may regulate the biological behaviors of breast cancer by inhibiting epithelial-mesenchymal transition.     

Effects of luteolin on invasion,migration and adhesion of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the effects of luteolin on invasion, migration and adhesion of human hepatocelluar carcinoma HepG2 cells.METHODS: HepG2 cells were cultured and treated with luteolin at 10, 20 and 40 μmol/L respectively. The invasion capability was examined by cell invasion assay. The migration ability was examined by wound healing assay. The adhesion capability was measured by adhesion assay. The protein levels of E-cadherin, N-cadherin, vimentin and Snai1 were determined by Western blot analysis.RESULTS: Luteolin inhibited the invasion, migration and adhesion ability of HepG2 cells in vitro in a dose-dependent manner. After treatment with luteolin, the expression of E-cadherin was increased significantly and the expression of N-cadherin, vimentin and Snai1 were decreased significantly.CONCLUSION: Luteolin inhibits the invasion, migration and adhesion ability of human hepatocelluar carcinoma HepG2 cells. The mechanism may be related to the regulatory effects of luteolin on epithelial-mesenchymal transition.     

Effect of NEU3 on biological behaviors of human prostate cancer DU145 cells

AIM:To explore the effects of neuraminidase 3 (NEU3) on the viability, invasion and apoptosis of human prostate cancer DU145 cells and the molecular mechanism. METHODS:The human prostate cancer DU145 cells were divided into blank control group and treatment group. The cells in treatment group were treated with either neuraminidase inhibitor DANA, or NEU3 small interfering RNA (siRNA) to knock down the expression of NEU3. The cell viability was measured by CCK-8 assay. The cell invasion ability was detected by Transwell assay. The effects of the treatments on the mRNA level of Bcl-2 were detected by qPCR. The effects of the treatments on the protein levels of matrix metalloproteinase 2 (MMP2) and apoptotic inhibitory protein Bcl-2 were determined by Western blot. Apoptosis of the cells was analyzed by flow cytometry. RESULTS:The protein level of NEU3 and the apoptotic rate in DANA group were not significantly different from those in blank control group. The viability of DANA-treated DU145 cells was increased, and the invasion ability, MMP2 protein level, and Bcl-2 mRNA and protein levels were all decreased in these cells, compared with blank control group. On the other hand, the levels of NEU3 protein and Bcl-2 mRNA and protein in NEU3 siRNA group were significantly decreased compared with blank control group, while the viability and apoptotic rate of the cells with NEU3 siRNA transfection were increased (P<0.05). However, the protein expression of MMP2 and the invasion ability of the cells were not significantly changed after NEU3 siRNA treatment. CONCLUSION:The inhibition of NEU3 in enzyme activity and expression decreases the viability, and enhances the apoptosis of human prostate cancer DU145 cells. However, it has no obvious effect on the invasion ability of DU145 cells.     

Expression of miR-203 in human tongue carcinoma tissues and its in-fluence on viability and invasion ability of Tca8113 cells

AIM: To study the expression of miR-203 in tongue carcinoma tissues and the effect of miR-203 over-expression on the viability and invasion ability of Tca8113 cells.METHODS: Twenty-eight pairs of tongue carcinoma tissues and adjacent nontumor tissues were collected, and the clinicopathological characters were analyzed. miR-203 was detected in the tongue tissues of 28 patients with tongue carcinoma by real-time PCR. miR-203 mimics and scramble were transfected into Tca8113 cells by Lipofectamine 2000. The expression of miR-203 was detected in Tca8113, Tca8113-miR-203 mimics and Tca8113-scramble cells by RT-qPCR. The cell viability was measured by CCK-8 assay. The cell invasion ability was determined by Transwell chamber invasion experiment.RESULTS: miR-203 expression was significantly down-regulated in the tongue carcinoma tissues compared with those in the adjacent nontumor tissues. The expression of miR-203 was associated with TNM stage and lymph node metastasis. Up-regulation of miR-203 inhibited the viability and invasion ability of Tca8113 cells.CONCLUSION: miR-203 suppresses the growth and invasion of tongue carcinoma cells. miR-203 may be a potential therapeutic target for treating human tongue cancer.     

Clinical significance of miRNA-326 expression in gastric cancer and effect of up-regulation of its expression on viability and apoptosis of gastric cancer cells

AIM: To investigate the clinical significance of microRNA-326 (miRNA-326) expression in gastric carcinoma and the effect of up-regulation of its expression on the viability and apoptosis of gastric cancer cells. METHODS: The expression of miRNA-326 in 55 tissue samples of gastric cancer was detected by RT-qPCR, and the relationship between the expression and the clinicopathological features was analyzed. The expression of miRNA-326 in gastric cancer BGC-823 cells was detected by RT-qPCR. The BGC-823 cells were transfected by liposome method, and randomly divided into normal control group (untransfected), mimic-NC group (transfected with negative control mimic) and miRNA-326 mimic group (transfected with miRNA-326 mimic). After up-regulation of miRNA-326 expression, the cell viability was measured by CCK-8 assay, and the apoptosis of the cells was analyzed by flow cytometry. The protein levels of matrix metalloprotein 9 (MMP-9), p21, cyclin D1, Bcl-2 and cleaved caspase-3 were determined by Western blot, and the mRNA expression of cyclin D1 was detected by RT-qPCR. Whether CCND1 (the gene of cyclin D1) was the target gene of miRNA-326 was evaluated by dual-luciferase reporter assay. RESULTS: The expression of miRNA-326 in the gastric cancer tissues was significantly lower than that in the adjacent tissues (P<0.05). The miRNA-326 expression had a significant correlation with the tumor size, lymph node metastasis, differentiation, and clinical stages (P<0.05), but it had no correlation with the age and sex of the patients. Moreover, the expression of miRNA-326 was also closely related to the survival rate of the patients (P<0.05). The expression of miRNA-326 in the BGC-823 cells was significantly lower than that in the normal gastric mucosa GES-1 cells (P<0.05). Compared with normal control group, the expression of miRNA-326 in mimic-NC group did not change significantly, while that in miRNA-326 mimic group was increased significantly (P<0.05). Compared with normal control group, the cell viability in miRNA-326 mimic group was significantly decreased, and the apoptosis was increased (P<0.05). In addition, compared with normal control group, the protein levels of MMP-9, cyclin D1 and Bcl-2, and the mRNA expression of cyclin D1 in miRNA-326 mimic group were decreased, while the protein levels of p21 and cleaved caspase-3 were increased (P<0.05). However, no significant difference of above protein and mRNA levels between mimic-NC group and normal control group was observed. Compared with mimic-NC+miR-326 mimic group, the activity of luciferase in the cells transfected with pmiR-CCND1-WT plasmid was significantly decreased (P<0.05), but that in the cells transfected with pmiR-CCND1-Mut plasmid did not change significantly. CONCLUSION: The expression level of miRNA-326 in gastric cancer tissues is low, and it may promote cell viability and inhibit cell apoptosis by targeting CCND1.     

Effect of GPNMB on proliferation,apoptosis and invasion of human hepatoma cells

AIM：To investigate the effect of glycoprotein nonmetastatic melanoma protein B (GPNMB) on the proliferation, apoptosis and invasion of human hepatoma HepG2 cells and its molecular mechanisms. METHODS：The GPNMB siRNA and GPNMB-overexpressing vector were constructed, and then transfected into HepG2 cells. MTT assay, flow cytometry and Transwell chamber were used to determine the effects of GPNMB down-regulation and up-regulation on the proliferation, apoptosis and invasive ability of HepG2 cells. RESULTS：The proliferation of HepG2 cells was obviously promoted by the up-regulation of GPNMB. No effect of GPNMB on the apoptosis of HepG2 cells was observed. The invasion of HepG2 cells was also significantly promoted by the up-regulation of GPNMB. When integrin β1 was silenced by siRNA, the promoting effect of GPNMB on the proliferation and invasive ability of HepG2 cells was significantly suppressed. CONCLUSION: GPNMB may promote the proliferation and invasion of HepG2 cells by the interaction with integrin β1, and may be used as a potential therapeutic target in liver cancer.     

Effect of miR-375 on viability,cell cycle and apoptosis of colon cancer HCT116 cells

AIM: To investigate the effect of microRNA-375 (miR-375) on the viability, cell cycle and apoptosis of HCT116 cells.METHODS: The expression of miR-375 in different colorectal cancer cell lines was detected by real-time PCR. The miR-375 mimics was transfected into HCT116 cells by Lipofectamine^TM 2000. The mRNA expression of miR-375 and AEG-1 was detected by real-time PCR. The HCT116 cell viability was detected by MTT assay. The changes of apoptosis and cell cycle distribution were analyzed by flow cytometry.RESULTS: Real-time PCR showed that miR-375 expression was the lowest in HCT116 among 4 colorectal cancer cell lines. The expression level of miR-375 significantly increased in miR-375 mimics group compared with that in the negative control group. The high expression level of miR-375 significantly inhibited the mRNA expression of AEG-1. After transfection with miR-375 mimics, the cell viability was inhibited, the apoptotic rate was increased, the proportion of G₁-stage cells was increased, and the proportion of S-stage cells was decreased.CONCLUSION: miR-375 inhibits the viability, mediates the cell cycle arrest and promotes the apoptosis of colon cancer HCT116 cells. miR-375 may act as a tumor suppressor in colorectal cancer by inhibiting AEG-1.     

Association of miR-497 and prognosis in patients with renal cell carcinoma and its effects on proliferation,apoptosis and invasion of human renal cell carcinoma cell line 786-0

ATM: To investigate the association of microRNA-497 (miR-497) and prognosis in the patients with renal cell carcinoma (RCC) and its effects on the proliferation, apoptosis and invasion of human RCC cell line 786-0. METHODS: Paired RCC and adjacent non-tumor tissue specimens were surgically collected from 80 patients who were diagnosed with primary RCC between 2011 and 2015. The expression of miR-497 in the paired RCC and adjacent non-tumor tissue specimens was detected by real-time PCR. Recurrence-free survival was estimated using the Kaplan-Meier method and compared using the log-rank test. The 786-0 cells were transfected with miR-497 mimics or scramble control miRNA. The proliferation, apoptosis and invasion abilities of the transfected cells were assessed by MTT assay, Trypan blue exclusion, flow cytometry and Transwell chamber experiment. The protein expression of miR-497-targeted gene cyclin D1 in the transfected cells was quantified by Western blotting. RESULTS: miR-497 was down-regulated in the RCC specimens compared with the adjacent tissues. miR-497 was down-regulated in the RCC 786-0 cells compared with the HK-2 cells. By the end of the study, 74 cases were followed up. The follow-up rate was 92.5%. Median follow-up was 29 months (ranging from 2 months to 48 months). The 3-year recurrence-free survival rates of the patients with high and low miR-497 expression were 71.2% and 40.1%, respectively. Over-expression of miR-497 resulted in significant suppression effect on RCC cell proliferation, invasion and the expression of cyclin D1. CONCLUSION: Low expression of miR-497 was correlated with poor prognosis in the RCC patients. miR-497 inhibits proliferation and invasion of RCC 786-0 cells and its mechanism is associated with the down-regulation of cyclin D1.     

Ursolic acid inhibits migration and invasion of human lung cancer A549 cells by targeting miRNA-133a

AIM: To investigate the effects of ursolic acid (UA) on the migration and invasion of human lung cancer cell line A549, and to explore its mechanism. METHODS: The cell viability was detected by MTT assay. The expression of miRNA-133a was detected in the A549 cells treated with UA by real-time PCR. The miRNA-133a mimics and inhibitor were transfected into the A549 cells, and the transfection efficiency was analyzed by real-time PCR. The cell migratory and invasive abilities were determined by wound healing and Transwell methods, respectively. RESULTS: The viability of the human lung cancer A549 cells was significantly inhibited by UA in a dose-dependent manner (P<0.05). IC₅₀ of UA (24 h) for lung cancer A549 cells was 31.04 μmol/L. UA treatment significantly inhibited the migratory and invasive abilities of A549 cells in a concentration-dependent manner, accompanied by significantly elevation of miRNA-133a expression. The mimics and inhibitor of miRNA-133a significantly upregulated and downregulated the expression of miRNA-133a in the transfected A549 cells, respectively. In addition, the viability of the A549 cells was decreased extremely after tansfected with the miRNA-133a mimics (P<0.01), so did the results of the cell migration and invasion test. The A549 cells tansfected with the miRNA-133a inhibitor showed an opposite changes of the cell viability, migration and invasion. CONCLUSION: UA inhibited the viability, migration and invasion of lung cancer A549 cells by elevating the expression of miRNA-133a.     

PTBP1 promotes migration and invasion of liver cancer cells through EMT pathway

AIM: To investigate the molecular mechanism of polypyrimidine tract-binding protein 1 (PTBP1) promoting the migration and invasion of hepatoma cells. METHODS: The differentially expressed splicing proteins in different cell lines were screened by qPCR and Western blot. The difference of the expression of PTBP1 between liver cancer and normal liver tissues was analyzed by bioinformatics. Wound-healing and Transwell assays were used to study the effect of PTBP1 on the migration and invasion of hepatoma cells, and Western blot was used to detect the effect of PTBP1 on epithelial-mesenchymal transition (EMT) signaling pathway. RESULTS: Compared with the HepG2 cells, the expression of splicing factor PTBP1 was significantly increased in the HCCLM3 cells with high metastatic ability (P<0.05), and the expression level of PTBP1 in hepatocellular carcinoma tissues was significantly higher than that in normal tissues (P<0.05). Over-expression of PTBP1 significantly increased the migration and invasion of HCCLM3 cells (P<0.05), increased the expression of mesenchymal marker proteins N-cadherin and vimentin (P<0.05), and promoted the EMT process of liver cancer cells. CONCLUSION: PTBP1 promotes the migration and invasion of liver cancer cells by promoting the EMT pathway of liver cancer cells.     

Effects of shRNA targeting SIAH2 gene on proliferation and apoptosis of human hepatoma cell line HepG2

AIM：To investigate the effects of shRNA-mediated seven in absentia homolog 2 (SIAH2; one of ubiquitin ligases) gene silencing on cell cycle and apoptosis of human hepatoma cell line HepG2. METHODS：The specific recombinant vector pGenesil-SIAH2 was transiently transfected into HepG2 cells with Lipofectamine 2000. RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of SIAH2. MTS assay was employed to evaluate the cell proliferation. Flow cytometry was used to determine the cell cycle and apoptosis of the transfected cells. RESULTS：Compared with control groups, the mRNA and protein levels of SIAH2 were reduced by pGenesil-SIAH2 transfection in HepG2-SIAH2 group. The proliferation of HepG2-SIAH2 cells was significantly inhibited. The percentage of G1-phase cells and the early apoptotic rate were significantly higher in HepG2-SIAH2 cells. CONCLUSION: Tansfection of pGenesil-SIAH2 effectively inhibits the proliferation of HepG2 cells, arrests the cells in G1 phase and induces apoptosis, indicating an experimental basis of SIAH2-targeting gene therapy for hepatocellular carcinoma.     

Effects of XPD on expression of Rb and MAD2 in human hepatoma HepG2 cells

AIM：To investigate the effects of xeroderma pigmentosum group D (XPD) protein on the growth of human hepatoma HepG2 cells and the expression of retinoblastoma (Rb) and mitotic arrest deficient 2 (MAD2) proteins. METHODS：The recombinant plasmid pEGFP-N2-XPD and empty plasmid pEGFP-N2 were transfected into HepG2 cells by LipofectamineTM 2000. The cells were divided into 4 groups including blank control group, liposome group, pEGFP-N2 group (N2 group) and pEGFP-N2-XPD group (XPD group). The expression of XPD, Rb and MAD2 at mRNA and protein levels was detected by RT-PCR and Western blotting. The cell growth was measured by MTT assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. RESULTS：Overexpression of XPD up-regulated the expression of Rb, and down-regulated the expression of MAD2 at mRNA and protein levels. XPD inhibited the proliferation of HepG2 cells and exacerbated the apoptosis. XPD prevented the hepatoma cells from G1 stage to S stage. CONCLUSION：XPD suppresses the growth of hepatoma cells, up-regulates the expression of Rb, and down-regulates the expression of MAD2.     

Expression of miRNA-181a in human lung adenocarcinoma cells and its effect on cell function

AIM: To study the expression of microRNA (miRNA)-181a in different human lung adenocarcinoma cell lines, and to investigate the effect of miRNA-181a on cell function and its mechanism in human lung adenocarcinoma drug resistant cell A549/DDP. METHODS: Real-time PCR was used to detect the expression of miRNA-181a in BEAS-2B cells, A549 cells and A549/DDP cells. The A549/DDP cells were transfected with pGenesil-miRNA-181a eukaryotic expression plasmid. At the same time, the untransfection group and negative transfection group were also set up. The expression of miRNA-181a, cell viability, cell growth inhibition and apoptosis rate during cis-diamminedichloroplatinum (DDP) treatment, cell cycle, cell invasion, the protein expression of miRNA-181a target genes bcl-2 and p53 in the A549/DDP cells were determined by real-time fluorescence quantitative PCR, MTT assay, flow cytometry, Transwell method and Western blot, respectivly. RESULTS: The expression of miRNA-181a in A549 cells and A549/DDP cells was significantly lower than that in BEAS-2B cells, and the lowest expression level was observed in A549/DDP cells (P<0.05). The expression of miRNA-181a in A549/DDP cells was significantly increased after transfection with pGenesil-miRNA-181a (P<0.05). The cell viability, cell cycle and invasion ability of the A549/DDP cells were inhibited after miRNA-181a transfection (P<0.05). The cell growth inhibition rate and apoptotic rate of the A549/DDP cells were increased (P<0.05). The expression of Bcl-2 was reduced, but the expression of P53 was increased after transfection with miRNA-181a in A549/DDP cells (P<0.05). CONCLUSION: miRNA-181a may be correlated with the development of human lung adenocarcinoma. miRNA-181a can serve as a new target for treatment of lung cancer.     

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.     

MicroRNA-330 inhibits growth and migration of gastric cancer cells by directly targeting EGR-2

AIM:To explore the function and significance of microRNA-330 (miR-330) in the development of gastric cancer. METHODS:Forty-eight cases of gastric cancer tissues and paired adjacent tissues were collected in Department of Oncology, Affiliated Hospital of Gansu University of Chinese Medicine, and the expression levels of miR-330 were detected by RT-qPCR. The expression levels of miR-330 in the gastric cancer cells and human gastric epithelial GES-1 cells were evaluated by RT-qPCR. The viability, colony formation and migration of gastric cancer cells after transfected with miR-330 inhibitor or miR-330 mimic were analyzed by CCK-8 assay, colony formation assay and Transwell assay, respectively. Furthermore, miR-330 target gene was predicted by miRanda target gene prediction database. RESULTS:miR-330 expression was down-regulated both in gastric cancer tissues and gastric cancer cells (P<0.05). The expression levels of miR-330 were negatively associated with the tumor size, lymph metastasis, pathological grade stage and T stage (P<0.05). The viability, colony formation and migration of gastric cancer cells were significantly increased after transfected with miR-330 inhibitor (P<0.05). However, the viability, colony formation and migration of gastric cancer cells were significantly decreased after transfected with miR-330 mimic (P<0.05). Furthermore, EGR-2 was the direct target gene of miR-330. CONCLUSION:miR-330 suppresses gastric cancer cell growth and migration, and the mechanism may be related to its direct target gene EGR-2, suggesting that miR-330 may be used as a potential new target for diagnosis and targeted therapy for gastric cancer.     

Long non-coding RNA TTTY15 expression in osteosarcoma and its effect on viability and invasion ability of osteosarcoma cells

AIM:To observe the expression of long noncoding RNA TTTY15 in osteosarcoma tissues and cell lines and to explore its effect on the viability and invasion ability of osteosarcoma cell lines. METHODS:qPCR was used to detect the expression of TTTY15 in 11 cases of osteosarcoma and its adjacent tissues. The mRNA levels of TTTY15 in osteosarcoma cell lines (143B, Saos2, MG-63, U2OS and HOS) and human osteoblast cell line hFOB1.19 were also tested. TTTY15 was down-regulated after transfected with small interfering RNA in MG-63 cells, the cell line with the highest level of TTTY15. The effect of TTTY15 knockdown on the viability of MG-63 cells was measured by CCK-8 assay. The cell cycle distribution was analyzed by flow cytometry. The effect of TTTY15 knockdown on the cell invasion ability was detected by Transwell assay. The levels of miR-216b-5p and FOXM1 mRNA were detected by qPCR, and the changes of the related proteins were determined by Western blot. RESULTS:Compared with the adjacent tissues, the expression of TTTY15 increased in the osteosarcoma tissues (P<0.01). Compared with the human osteoblast cell line, the expression of TTTY15 increased in the osteosarcoma cell lines (P<0.05), and the level of TTTY15 in the MG-63 cells was the highest (P<0.01). After knockdown of TTTY15 expression in the MG-63 cells, the cell viability was decreased (P<0.05), cell cycle progression was inhibited (P<0.01), and the cell invasion ability was decreased (P<0.01). The expression of miR-216b-5p was increased (P<0.01) and the expression of FOXM1 mRNA was decreased (P<0.01). The protein expression of FOXM1, CDK4, cyclin D1, MMP-2 and N-cadherin was decreased, while the protein expression of E-cadherin was increased (P<0.05). CONCLUSION:The expression of TTTY15 is increased in the osteosarcoma tissues and cell lines. The low expression of TTTY15 inhibits the cell viability and invasion ability of osteosarcoma cells. The possible mechanism is that the knockdown of TTTY15 expression results in the increase in miR-216b-5p expression and the down-regulation of FOXM1 expression.     

Effects of USP9X down-regulation on apoptosis and invasion ability of gastric carcinoma AGS cells

AIM: To investigate the effects of ubiquitin-specific peptidase 9, X-linked (USP9X) down-regulation on apoptosis and invasion ability in gastric carcinoma cells, and to explore its possible molecular mechanisms. METHODS: USP9X small interfering RNA (siRNA) and control siRNA were used to be transfected into gastric carcinoma AGS cells. The cells were divided into 3 groups, including untreated AGS group, control siRNA group and USP9X siRNA group. The expression of USP9X at mRNA and protein levels in the AGS cells with different treatments was determined by real-time PCR and Western blot. The cell viability was analyzed by CCK-8 assay. Flow cytometry and Boyden chamber were employed to examine the apoptosis and invasion ability of the AGS cells. RESULTS: USP9X siRNA significantly down-regulated the expression of USP9X at mRNA and protein levels in the AGS cells. Down-regulation of USP9X markedly induced apoptosis and reduced invasion ability of the gastric carcinoma AGS cells. Notably, down-regulation of USP9X significantly reduced the protein expression of Mcl-1 and MMP-2, but markedly increased the protein level of Bax. CONCLUSION: USP9X may be a key regulator for apoptosis and invasion in gastric carcinoma.     

miRNA-126 inhibits proliferation,migration and invasion of human lung cancer A549 cells via EGFR/AKT/mTOR pathway

AIM: To investigate the effects of microRNA(miRNA)-126 on the proliferation, migration and invasion of human lung cancer cell lines, and to explore its mechanism. METHODS: The A549 cells were transfected with miRNA-126 agomir by Lipofectamine 2000. The expression of miRNA-126 was detected by real-time PCR. The cell activity was detected by MTT assay. The number of viable A549 cells was counted by the method of Trypan blue exclusion. The cell colony-forming capability was determined by cell colony formation test. The cell migration and invasion abilities were assayed by wound healing and Transwell methods, respectively. The protein levels of p-EGFR, EGFR, p-AKT, AKT, p-mTOR and mTOR were determined by Western blot. RESULTS: The expression level of miRNA-126 was significantly increased in the A549 cells compared with negative control(NC) group and control group(P<0.01). The proliferation of A549 cells was decreased extremely after transfected with the miRNA-126 agomir(P<0.01), so did the result of the cell colony-formation test. The migration and invasion abilities of the lung cancer cells were also significantly inhibited. The protein levels of p-EGFR, p-AKT and p-mTOR were significantly down-regulated compared with NC group and control group(P<0.01). CONCLUSION: Over-expression of miRNA-126 significantly inhibits the proliferation, migration and invasion ability of human lung cancer A549 cells by down-regulation of EGFR/AKT/mTOR pathway.