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.     

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.     

IL-17 promotes viability and migration ability of endometrial carcinoma cells by inhibiting miR-195-5p expression

AIM:To investigate the potential mechanism of interleukin-17 (IL-17) promoting the viability, migration and invasion of human endometrial carcinoma cells. METHODS:The expression of IL-17 and microRNA-195-5p (miR-195-5p) in the human endometrial carcinoma and benign uterine lesion samples were detected by RT-qPCR. The expression of miR-195-5p in human endometrial carcinoma HEC-1-B cells after treatment with IL-17 at different concentrations for 48 h was detected by RT-qPCR. The viability, migration and invasion of HEC-1-B cells after treatment with IL-17 at 100 μg/L or transfection of miR-195-5p mimics were detected by MTT assay and Transwell assays. The viability, migration and invasion of HEC-1-B cells after over-expression of miR-195-5p combined with 100 μg/L IL-17 intervention were also observed. RESULTS:The expression of IL-17 was increased while the expression of miR-195-5p was decreased in the human endometrial carcinoma samples (P<0.05). The expression of miR-195-5p in the HEC-1-B cells after treatment with IL-17 at 10,100 and 300 μg/L for 48 h was significantly decreased (P<0.05). The results of MTT assay and Transwell experiments indicated that IL-17 at 100 μg/L enhanced the viability, migration and invasion of HEC-1-B cells, while over-expression of miR-195-5p resulted in the opposite effect. CONCLUSION:Over-expression of miR-195-5p inhibits the enhancing effects of IL-17 on the viability, invasion and migration of HEC-1-B cells.     

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.     

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.     

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.     

Paeonol affects viability and migration ability of hepatocellular carcinoma cells by up-regulating miR-424-3p and inhibiting PI3K/AKT signaling pathway

AIM To investigate the effect of paeonol on the viability and migration ability of hepatocellular carcinoma cells and its molecular mechanism. METHODS Human hepatocellular carcinoma Hep3B cells was treated with paeonol at different concentrations (50, 100, 200 and 400 mg/L). The cell viability was measured by CCK-8 assay to determine the optimal drug concentration. The Hep3B cells were divided into normal control (NC) group, paeonol group, miR-NC group, miR-424-3p group, paeonol+anti-miR-NC and paeonol+anti-miR-424-3p group. The expression level of miR-424-3p was detected by RT-qPCR. The migration ability was detected by Transwell assay. The protein levels of cyclin D1, matrix metalloproteinase 2 (MMP2), MMP9 and PI3K/AKT signaling pathway-related molecules were determined by Western blot. RESULTS Paeonol intervention inhibited the viability of Hep3B cells in a concentration-dependent manner (P<0.05). The concentration of paeonol at 200 mg/L was selected for the following study. Paeonol intervention inhibited the protein expression of MMP2 and MMP9 in the Hep3B cells, and inhibited the migration ability of the Hep3B cells. Paeonol intervention promoted the expression of miR-424-3p in the Hep3B cells (P<0.05). Over-expression of miR-424-3p inhibited the expression of cyclin D1, MMP2 and MMP9 in the Hep3B cells and inhibited cell viability and migration ability (P<0.05). Inhibition of miR-424-3p reversed the effect of paeonol on the viability and migration ability of the Hep3B cells (P<0.05). Paeonol inhibited phosphorylation levels of PI3K and AKT in the Hep3B cells and inhibited the activation of PI3K/AKT signaling pathway (P<0.05). Inhibition of miR-424-3p reversed the effect of paeonol on PI3K/AKT signaling pathway in the Hep3B cells (P<0.05). CONCLUSION Paeonol inhibits the viability and migration ability of hepatocellular carcinoma cells by up-regulating miR-424-3p and inhibiting PI3K/AKT signaling pathway.     

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.     

miR-509 regulates growth,invasion and migration of human hepatocellular carcinoma LM3 cells and survival of nude mice

AIM: To investigate the effect of microRNA-509 (miR-509) on the growth, invasion and migration of human hepatocellular carcinoma (HCC) LM3 cells and survival of tumor-bearing nude mice. METHODS: LM3 cells were transferred with miR-509 mimic and pcDNA Ras-related C3 botulinum toxin substrate 1 (pcRac1), and the expression of Rac1 was measured by Western blot. The relationship between miR-509 and Rac1 was determined by luciferase reporter assay. The invasion ability was determined by Transwell assay, and the migration ability was measured by wound healing assay. Xenograft model of HCC was established by subcutaneous injection with LM3 cells into nude mice. The survival rate of the mice were recorded and the protein level of Rac1 was determined by Western blot. RESULTS: miR-509 mimic inhibited the expression of Rac1 in the LM3 cells (P<0.05). pcRac1 attenuated the effect of miR-509 on Rac1. miR-509 also alleviated luciferase activity of wild Rac1 (P<0.05). Meanwhile, miR-509 mimic decreased the number of invasive LM3 cells and inhibited the migration of LM3 cells (P<0.05). In addition, over-expression of miR-509 up-regulated survival rate of model mice and decreased the protein level of Rac1 in the tumor tissue (P<0.01). CONCLUSION: miR-509 inhibits the invasion and migration of HCC cells and promotes the survival of tumor-bearing nude mice through inhibiting the expression of Rac1.     

Sinomenine inhibits viability,migration and invasion of human ovarian cancer SKOV3 cells

AIM:To investigate the effect of sinomenine on the viability, migration and invasion of human ovarian cancer SKOV3 cells and its possible mechanism. METHODS:The SKOV3 cells were treated with sinomenine at different concentrations for 12 h, 24 h and 48 h. CCK-8 assay was employed to detect the effects of sinomenine on the viability of the SKOV3 cells. Flow cytometry was used to analyze the cell cycle distribution. The cell migration and invasion abilities were measured by Transwell assay. Western blot was used to determine the protein levels of cyclin A, cyclin D1, E-cadherin and matrix metalloproteinase-9 (MMP-9). RESULTS:Sinomenine remarkably inhibited the viability of SKOV3 cells and IOSE80 cells in a time-dependent and dose-dependent manner (P<0.05), and the IC₅₀ values of 48 h were 2.12 mmol/L and 17.35 mmol/L, respectively. In a dose-dependent manner, sinomenine induced G₀/G₁ and S phase arrest in SKOV3 cells (P<0.05), suppressed the migration and invasion abilities of SKOV3 cells (P<0.05), down-regulated the protein levels of cyclin A, cyclin D1 and MMP-9 (P<0.05), and up-regulated the protein level of E-cadherin (P<0.05). CONCLUSION:Sinomenine inhibits the viability, migration and invasion of human ovarian cancer SKOV3 cells most likely via down-regulation of the protein levels of cyclin A, cyclin D1 and MMP-9, and up-regulation of the protein level of E-cadherin.     

miR-24-3p targeting KLF6 gene affects viability and apoptosis of esophageal cancer cells via IL-6/STAT3 signaling pathway

AIM: To investigate the effect of microRNA-24-3p (miR-24-3p) on the viability and apoptosis of esophageal cancer cells. METHODS: The expression of miR-24-3p and KLF6 mRNA in the esophageal cancer cells TE11, Eca109 and EC9706 were detected by RT-qPCR. The protein expression of KLF6 was determined by Western blot. EC9706 cells were transfected with anti-miR-24-3p and KLF6 siRNA. The cell viability was measured by MTT assay, the apoptotic rate was analyzed by flow cytometry, and the proliferation, apoptosis and IL-6/STAT3 signaling pathways related proteins were determined by Western blot. The level of IL-6 was measured by ELISA. The dual luciferase reporter gene assay was used to verify the relationship between miR-24-3p and KLF6. RESULTS: The levels of miR-24-3p were up-regulated in the esophageal cancer cells TE11, Eca109 and EC9706 (P < 0.05), and the expression of KLF6 at mRNA and protein levels was down-regulated (P < 0.05). Knock-down of miR-24-3p expression inhibited the cell viability, induced apoptosis, and inhibited the protein levels of CDK4, cyclin D1, CDC25A, p-STAT3, Bcl-2 and IL-6, and promoted the protein expression of caspase-3 and Bax in EC9706 cells. CONCLUSION: miR-24-3p targets KLF6 gene to affect the viability and apoptosis of esophageal cancer cells by regulating IL-6/STAT3 signaling pathway.     

Effect of Linc00152 targeting miR-376c-3p on viability,apoptosis and radiosensitivity of cervical cancer cells

AIM: To investigate the effect of Linc00152 on the viability, apoptosis and radiosensitivity of cervical cancer cells. METHODS: RT-qPCR was used to detect the expression levels of Linc00152 and microRNA-376c-3p(miR-376c-3p) in human cervical cancer HeLa cells and SiHa cells, and normal cervical Ect1/E6E7 cells. The cervical cancer HeLa cells with low Linc00152 expression or miR-376c-3p over-expression were established. MTT assay, flow cytometry, colony formation assay and Western blot were used to determine the cell viability, apoptosis, radiosensitivity and related protein expression. The dual-luciferase reporter assay was used to verify the regulatory relationship between Linc00152 and miR-376c-3p in the HeLa cells. RESULTS: Compared with the Ect1/E6E7 cells, Linc00152 was up-regulated in the HeLa cells and SiHa cells, and miR-376c-3p was down-regulated (P < 0.05). Low expression of Linc00152 or over-expression of miR-376c-3p inhibited the viability of HeLa cells, induced apoptosis, enhanced the radiosensitivity, inhibited the protein expression of cyclin D and Bcl-2, and promoted the protein expression of P21 and Bax (P < 0.05). Linc00152 negatively regulated miR-376c-3p expression in the HeLa cells, and inhibition of miR-376c-3p expression reversed the effect of low expression of Linc00152 on HeLa cell viability, apoptosis and radiosensitivity. CONCLUSION: Linc00152 is highly expressed in the cervical cancer cells. Linc00152 affects the viability, apoptosis and radiosensitivity of HeLa cells by targeting miR-376c-3p, which is a potential diagnosis and treatment target for cervical cancer.     

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.     

Celastrol blocks cell cycle of A549 cells by regulating miR-17-5p/miR-155-5p and targeting cyclin D1

AIM: To investigate the effect of celastrol on the cell cycle of human lung adenocarcinoma A549 cells and to probe into its mechanisms.METHODS: A549 cells were exposed to celastrol at gradient concentrations. The cell viability and apoptosis were detected by MTT assay and flow cytometry, respectively, and the median lethal concentration (LC₅₀) of celastrol was screened. The A549 cells were treated with celastrol at LC₅₀, and the cell cycle was detected by flow cytometry. The expression of cyclin D1 was determined by Western blot, and the expression of microRNA (miR)-17-5p and miR-155-5p was detected by real-time PCR. The correlation between cyclin D1 and miR-17-5p or miR-155-5p was predicted by bioinformatics software. After miR-17-5p mimics/miR-155-5p mimics/mutant-miR-17-5p/mutant-miR-155-5p and pcDNA-GFP-cyclin D1-3'UTR were cotransfected into the A549 cells, the changes of GFP expression were evaluated by fluorescence microscopy and flow cytometry. Finally, after miR-17-5p mimics or miR-155-5p mimics were transfeced into the A549 cells, the expression of miR-17-5p and miR-155-5p was detected by real-time PCR, and the protein level of cyclin D1 was determined by Western blot. RESULTS: With the increasing concentration of celastrol, the viability inhibition rate and apoptotic rate of the A549 cells were increased, indicating that celastrol effectively inhibited the growth of A549 cells and induced apoptosis. The LC₅₀ of celastrol was almost 3 μmol/L. After treatment with celastrol at LC₅₀, the A549 cell cycle was arrested at G₁ phase, the protein expression of cyclin D1 was down-regulated (P<0.01), and the expression levels of miR-17-5p and miR-155-5p were significantly increased (P<0.01). The results of bioinformatics software prediction indicated that there were binding sites for miR-17-5p and miR-155-5p in the 3'-UTR of cyclin D1. After cotransfected with miR-17-5p or miR-155-5p and pcDNA-GFP-cyclin D1-3'UTR into the A549 cells, the expression of GFP declined (P<0.05). After miR-17-5p or miR-155-5p mimics were transfected into A549 cells, the results of real-time PCR showed this treatment significantly increased the miRNA expression (P<0.01), and the results of Western blot showed the transfection inhibited cyclin D1 expression (P<0.01).CONCLUSION: Celastrol blocks the A549 cells at G₁ phase, inhibits the viability and induces apoptosis, which may be caused by up-regulating the expression of miR-17-5p and miR-155-5p, and then down-regulating cyclin D1 expression. This study provides a new theoretical basis for the treatment of non-small-cell lung cancer with celastrol.     

Effect of formononetin on viability,migration and invasion of ovarian cancer cells in vitro

AIM To observe the effect of formononetin on the viability, migration and invasion of ovarian cancer cells, and to explore its mechanism. METHODS Human ovarian serous cystadenocarcinoma SKOV-3 cells were cultured in vitro. The cells were treated with formononetin at 0, 25, 50 and 100 μmol/L for 48 h. The cell viability was measured by MTS assay. The migration and invasion abilities of the SKOV-3 cells were detected by scratch wound assay and Transwell assay. RT-qPCR and Western blot were used to detect the mRNA and protein levels of E-cadherin and matrix metalloproteinase-9 (MMP-9). RESULTS The viability of SKOV-3 cells was decreased with the increase in the formononetin concentration compared with control group (P<0.01). The wound migration distance of the cells in 50 μmol/L formononetin group was less than that in control group (P<0.01). The number of invasive SKOV-3 cells across the Transwell sub-compartment was significantly decreased in 50 μmol/L formononetin group compared with control group (P<0.01). The mRNA and protein levels of E-cadherin in 50 μmol/L formononetin group were significantly higher than those in control group (P<0.01), while the mRNA and protein levels of MMP-9 in 50 μmol/L formononetin group were significantly lower than those in control group (P<0.01). CONCLUSION Formononetin inhibits the migration and invasion abilities of ovarian cancer SKOV-3 cells by increasing expression of E-cadherin and decreasing expression of MMP-9.     

Pulsatilla saponin A affects proliferation and radiosensitivity of breast cancer cells by regulating miR-24-3p/RNF2 expression

AIMTo investigate the effect of Pulsatilla saponin A on proliferation and radiosensitivity of breast cancer cells and its mechanism. METHODSHuman breast cancer MCF-7 cells were treated with Pulsatilla saponin A at concentrations of 0, 5, 10, 15 and 20 mg/L and transfected with microRNA-24-3p (miR-24-3p) over-expression vector or inhibitory expression vector. The proliferation and radiosensitivity of the MCF-7 cells were measured by MTT assay and colony formation assay. The miR-24-3p expression and ring finger protein 2 （RNF2） mRNA level were detected by RT-qPCR. The protein expression of RNF2 was determined by Western blot. The luciferase reporter assay was used to detect the targeting relationship between miR-24-3p and RNF2. RESULTSCompared with control group (0 mg/L), the proliferation inhibitory rate of the MCF-7 cells was significantly increased in 5, 10, 15 and 20 mg/L Pulsatilla saponin A groups (P<0.05). The survival score of the MCF-7 cells treated with Pulsatilla saponin A was significantly decreased after irradiation, and the expression of RNF2 was significantly decreased (P<0.05). miR-24-3p targeted RNF2 and negatively regulated its expression. When the MCF-7 cells were simultaneously treated with Pulsatilla saponin A and miR-24-3p, the cell survival curve significantly shifted down. Inhibition of miR-24-3p expression reversed the proliferation-inhibiting and radiation-sensitizing effects of Pulsatilla saponin A on the MCF-7 cells. CONCLUSION Pulsatilla saponin A may affect the proliferation and radiosensitivity of breast cancer cells through miR-24-3p/RNF2 signaling pathway.     

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC, pcDNA-FEZF1-AS1, anti-miR-NC, anti-miR-363-3p, miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1, Ki67 and cleaved caspase-3. RESULTS Compared with control group, the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）, and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group, the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group, the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group, the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）, the apoptotic rate was significantly increased （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）, and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

lncRNA-MALAT1 targets and downregulates miR-570-3p expression to promote proliferation of gastric cancer cells

AIM:To investigate whether long non-coding RNA MALAT1 (lncRNA-MALAT1) targets and down-regulates microRNA-570-3p (miR-570-3p) expression to further promote the proliferation of gastric cancer cells. METHODS:Gastric cancer cell line SGC7901 was cultured in vitro and divided into 3 groups:blank control, si-MALAT1 and si-MALAT1 NC. The si-MALAT1 and si-MALAT1 NC groups were transfected with MALAT1 siRNA and its negative control, respectively. The cell proliferation was evaluated by MTS assay. The expression of miR-570-3p was detected at different time points in the pure SGC7901 gastric cancer cell line, and the expression of lncRNA-MALAT1 and miR-570-3p in different groups was detected by RT-qPCR. The potential complementary binding sites of lncRNA-MALAT1 and miR-570-3p were predicted by RegRNA. The MALAT1 gene and its mutant fragment were cloned into luciferase reporter vector psiCHECK-2. Restriction enzyme analysis and sequencing were used to identify whether the recombinant plasmids carrying MALAT1 or MALAT1-Mut were successfully constructed. miR-570-3p mimic, miR-570-3p inhibitor, miR-570-3p mimic negative control and miR-570-3p inhibitor negative control were co-transfected into the 293T cells with the luciferase repor-ters containing MALAT1 or MALAT1-Mut. Dual-luciferase reporter assay was performed to detect luciferase activity in different groups in order to verify the relationship between lncRNA-MALAT1 and miR-570-3p. RESULTS:Compared with blank control group and si-MALAT1 NC group, the A₄₉₀ value in si-MALAT1 group was significantly decreased (P<0.01). The expression of miR-570-3p presented an obvious declining trend over time. The expression of lncRNA-MALAT1 in si-MALAT1 group was remarkably decreased, whereas the expression of miR-570-3p was obviously increased. The dual-luciferase reporter assay indicated that the MALAT1 reporter luciferase activity decreased significantly in miR-570-3p mimic group compared with mimic negative control (P<0.01), and the luciferase activity of MALAT1 reporter was obviously up-regulated in miR-570-3p inhibitor group compared with miR-570-3p mimic group (P<0.01). However, miR-570-3p mi-mic, miR-570-3p inhibitor, miR-570-3p mimic negative control and miR-570-3p inhibitor negative control showed no effect on the luciferase activity of MALAT1-Mut reporter. CONCLUSION:lncRNA-MALAT1 targets and down-regulates miR-570-3p expression to further promote the proliferation of gastric cancer cells.     

CASC2/miR-18a/BTG3 signaling inhibits cell migration and invasion of non-small cell lung cancer

AIM: To explore the function and molecular mechanism of long non-coding RNA CASC2 in non-small-cell lung cancer (NSCLC) cell migration and invasion. METHODS: RT-qPCR and Western blot were used to detect the expression of CASC2, microRNA-18a (miR-18a) and BTG3 in human bronchial epithelial cell line 16-HBE, and NSCLC cell lines A549 and H1299. The interaction between CASC2 and miR-18a or miR-18a and BTG3 was predicted by bioinformatics software and verified by double-luciferase reporter assays. Transwell assays were performed to detect the migration and invasion abilities of the NSCLC cells. RT-qPCR and Western blot were used to determine the regulatory effects of CASC2 on miR-18a and BTG3 expression. RESULTS: Compared with 16-HBE cells, the expression of CASC2 and BTG3 was significantly down-regulated in the NASCL cells, while miR-18a was significantly over-expressed (P<0.05). CASC2 acted as a molecular sponge for miR-18a, and BTG3 was verified to be a target gene of miR-18a. Up-regulation of CASC2 inhibited the migration and invasion abilities of NSCLC cells, while exogenous restoration of miR-18a stimulated cell migration and invasion abilities. In addition, exogenously over-expressed miR-181a reversed the promoting effect of CASC2 on BTG3 protein expression. CONCLUSION: CASC2 promotes BTG3 expression by negatively regulating miR-18a, and then inhibits the migration and invasion abilities of NSCLC cells.