Down-regulation of microRNA-181b has protective effect on cerebral ischemic injury of mice

AIM: To explore the role of microRNA-181b(miR-181b) in ischemic injury and autophagy protein 5(Atg5) levels of mice. METHODS: Oxygen-glucose depletion(OGD) model in N2A cells to mimic ischemic injury in vitro was established. A middle cerebral artery occlusion(MCAO) model to mimic ischemic injury in vivowas also induced in mice. The N2A cell apoptosis after OGD was assessed by in situ cell death detection kit. The Atg5 and caspase-9 expressions were determined by Western blotting. Luciferase reporter assay was performed to identify the direct binding of miR-181b with 3'-UTR of Atg5 mRNA. RESULTS: The alteration of miR-181b expression level by transfection with pre-miR-181b or anti-miR-181b significantly affected N2A cell apoptosis(P<0.05). Accordingly, the changes of miR-181b levels significantly altered the protein level of Atg5(P<0.05). Co-transfection of the luciferase reporters with pre-miR-181b or anti-miR-181b resulted in the inhibition or enhancement of the luciferase activities of luciferase expressing plasmid containing 3'-UTR of Atg5 mRNA (P<0.05). In addition, the miR-181b antagonist significantly reduced the cleaved caspase-9 levels in cerebral ischemic cortex of the mice after MCAO (P<0.05). CONCLUSION: Down-regulation of miR-181b plays an important role in ischemic injury of mice through regulating Atg5 protein level.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

Effects of miR-130a on viability and apoptosis of rat basilar arterial smooth muscle cells

AIM: To investigate the regulatory effects of microRNA (miR)-130a on the biological characteristics of rat basilar arterial smooth muscle cells (BASMCs) and its underlying mechanisms. METHODS: The expression of miR-130a in rat BASMCs was measured by real-time PCR. After knockdown of miR-130a with inhibitor in the BASMCs, the cell viability, cell cycle distribution and apoptosis were detected by CCK-8 assay and flow cytometry. The expression of cell cycle-and apoptosis-related molecules, such as cyclin D1, cyclin-dependent kinase 2 (CDK2), p21, Bcl-2 and cleaved caspase-3/caspase-3 at protein levels was determined by Western blot. The growth arrest-specific homeobox protein (Gax) expression at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: AngiotensionⅡ (AngⅡ) up-regulated the expression of miR-130a and down-regulated the expression of Gax (P<0.05). Transfection with miR-130a inhibitor partly reversed the increase in AngⅡ-induced cell viability and promoted the Gax expression. Furthermore, the early cell apoptotic rate was significantly increased after down-regulation of miR-130a (P<0.05), and the protein levels of cyclin D1, CDK2, Bcl-2 and p-Rb were significantly decreased, accompanied with the up-regulation of p21 and cleaved caspase-3 (P<0.05). CONCLUSION: Down-regulation of miR-130a restrains the viability and promotes the apoptosis of BASMCs by promoting Gax expression and regulating cell cycle-and apoptosis-related molecules, suggesting that miR-130a may be a potential therapeutic target of brain vascular remodeling during hypertension.     

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.     

Regulatory effects of miR-195 on biological behaviors of lung cancer A549 cells

AIM: To investigate the regulatory effects of microRNA (miR)-195 on the biological behaviors, such as viability, apoptosis and migration, of lung cancer A549 cells, and to explore the related mechanisms. METHODS: After miR-195 mimics were transfected into the A549 cells, the cell viability, cell cycle distribution and apoptosis were measured by CCK-8 assay and flow cytometry. Transwell assay was used to detect cell migration ability. Furthermore, the protein levels of cyclin D1, CDK2, Bcl-2 and p-Rb/Rb were determined by Western blot. Dual-luciferase reporter assay was used to screen and identify the possible target genes of miR-195. RESULTS: Over-expression of miR-195 in the A549 cells inhibited the cell viability and induced cell cycle arrest, accompanied with the decrease in the cell migration ability and the increase in the apoptotic rate (P<0.05). Furthermore, the protein levels of cyclin D1, CDK2, Bcl-2 and p-Rb were significantly decreased (P<0.05). Dual-luciferase reporter assay demonstrated that MYB was a potential target gene of miR-195. Over-expression of MYB in the A549 cells partially reversed the effects of miR-195 on the cell viability, apoptosis and migration. CONCLUSION: miR-195 inhibits lung cancer A549 cell growth and migration, and promotes cell apoptosis by targeting MYB gene.     

Expression and significance of miR-193b in cervical cancer

AIM: To investigate the expression of microRNA-193b(miR-193b) in the cervical tissues, and further to explore the effect of silencing miR-193b on diamminedichloroplatinum(DDP)-treated HeLa cell viability. METHODS: The expression levels of miR-193b in different cervical tissues were examined by qPCR. After transfection of miR-193b-inhibitor, the cell migration was determined by Transwell assay, the sensitivity of HeLa cells to DDP was measured by MTT assay, the protein levels of phosphate and tension homology deleted on chromsome ten(PTEN), protein kinase B(Akt), p-Akt and p-glycoprotein(P-gp) were determined by Western blot. RESULTS: The mRNA level of miR-193b was significantly increased in the cervical cancer tissues compared with normal cervical tissues(P<0.05). Knockdown of miR-193b obviously inhibited migration and enhanced sensitivity to DDP of HeLa cells(P<0.05). Additionally, after transfection of miR-193b-inhibitor, the expression of PTEN was increased, whereas the protein levels of p-Akt and P-gp were decreased(P<0.05). CONCLUSION: miR-193b is highly expressed in the cervical cancer tissues. Inhibition of miR-193b augments the sensitivity to DDP of HeLa cells, at least in part, through PTEN-PI3K/Akt signaling pathway.     

Effect of miR-23b-3p on viability and apoptosis of rheumatoid arthritis synovial fibroblasts by targeting XIAP

AIM:To investigate the effect of microRNA-23b-3p (miR-23b-3p) on the viability and apoptosis of rheumatoid arthritis synovial fibroblasts by targeting X-linked inhibitor of apoptosis protein (XIAP). METHODS:The expression of miR-23b-3p and XIAP was detected by RT-qPCR. The TargetScan was used to predict the targeting regulatory relation between miR-23b-3p and XIAP, and then the regulatory relation was confirmed by dual-luciferase reporter assay. After the miR-23b-3p mimic and inhibitor were transfected into the cells, the expression of miR-23b-3p and XIAP was detect by RT-qPCR. The effect of miR-23b-3p on the viability and apoptosis was measured by CCK-8 assay and flow cytometry. The protein expression levels of Ki67 and Bcl-2 were determined by Western blot. RESULTS:The expression level of miR-23b-3p was down-regulated significantly (P<0.05), and XIAP was up-regulated significantly in rheumatoid arthritis synovial fibroblasts (P<0.05). The miR-23b-3p mimic significantly inhibited XIAP expression and the cell viability, promoted the apoptosis, and down-regulated the expression of Ki67 and Bcl-2 (P<0.05). The effects of miR-23b-3p inhibitor were the opposite. CONCLUSION:miR-23b-3p inhibits the viability and promotes apoptosis of rheumatoid arthritis synovial fibroblasts by targeting XIAP.     

Expression of miR-625-3p in colorectal carcinoma tissues and cells

AIM: To investigate the expression of microRNA-625-3p (miR-625-3p) in colorectal carcinoma (CRC) and its underlying mechanism. METHODS: Quantitative real-time PCR was employed to detect the levels of miR-625-3p expression in different CRC cell lines, CRC tissues and pair-matched adjacent normal tissues. The relationships between the expression levels of miR-625-3p and the patients' clinicopathological parameters were estimated. The effects of miR-625-3p on the apoptosis and the cell mitotic cycle of CRC cells were analyzed with propidium iodide staining and flow cytometry. The effect of miR-625-3p on the apoptosis-related proteins was analyzed by Western blot. RESULTS: The expression level of miR-625-3p in the CRC tissues was higher than that in the pair-matched adjacent normal tissues (P<0.05). The expression of miR-625-3p in the CRC tumor tissues was significantly correlated with the tumor infiltrative depth, TNM stage and distant metastasis (P<0.05). The expression levels of miR-625-3p in CRC SW620 cells were higher than that in SW480 cells. The CRC cell mitotic cycle was significantly inhibited and cell apoptosis was significantly promoted when the expression of miR-625-3p was inhibited (P<0.05). The expression of Bax protein didn't change and the expression of Bcl-2 protein increased after miR-625-3p mimics were transfected into CRC SW620 cells(P<0.05). CONCLUSION: miR-625-3p may be a promising approach for the treatment of CRC by promoting cell proliferation and inhibiting apoptosis.     

miR-153 promotes LPS-induced myocardial H9C2 cell injury by targeting SORBS2

AIM: To study the effects of microRNA-153 (miR-153) on inflammatory factors, cell viability and apoptosis of embryonic rat H9C2 cardiomyocytes induced by lipopolysaccharide (LPS), and to explore its mechanism. METHODS: The injury model of H9C2 cells was established by LPS stimulation. The H9C2 cells were divided into anti-miR-Con group (transfected with anti-miR-Con), anti-miR-153 group (transfected with anti-miR-153), pcDNA group (transfected with pcDNA), pcDNA-SORBS2 group (transfected with pcDNA-SORBS2), anti-miR-153+si-Con group (co-transfected with anti-miR-153 and si-Con) and anti-miR-153+si-SORBS2 group (co-transfected with anti-miR-153 and si-SORBS2), and treated with LPS after transfection. The expression of miR-153 and SORBS2 mRNA in the cells was detected by RT-qPCR. The viability of H9C2 cells was measured by MTT assay. The protein expression of SORBS2 in the H9C2 cells was determined by Western blot. The contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were detected by ELISA. The apoptosis of the H9C2 cells was analyzed by flow cytometry. The targeting relationship between miR-153 and SORBS2 was verified by dual-luciferase reporter assay. RESULTS: The LPS-induced H9C2 cell injury model was successfully constructed. Compared with PBS group, the expression of miR-153 was significantly increased and the expression of SORBS2 was significantly decreased in the H9C2 cells treated with LPS. The inhibition of miR-153 and over-expression of SORBS2 decreased the contents of TNF-α and IL-6 and the level of apoptosis, but increased the cell viability. miR-153 inhibited the luciferase activity of the H9C2 cells containing wild-type SORBS2. Inhibition of SORBS2 reversibly inhi-bited the anti-inflammatory effects of miR-153 on LPS-induced H9C2 cells and increased the viability of the cells. CONCLUSION: miR-153 promotes the secretion of inflammatory factors, induces apoptosis, and inhibits the viability of H9C2 cells induced by LPS, thus enhancing the damage. Its mechanism may be related to targeting SORBS2, which will provide new targets for the treatment of myocardial injury.     

Effects of miRNA-25 on proliferation of human esophageal squamous-cell carcinoma cell line TE1

AIM: To investigate the effects and mechanisms of microRNA-25(miRNA-25) on the proliferation of human esophageal squamous-cell carcinoma cell line TE1. METHODS: The abundance of miRNA-25 in different tissues was measured by RT-PCR. After silencing or over-expression of miRNA-25 with mimics or inhibitor in TE1 cells, the cell proliferation, cell cycle distribution and the expression of cyclin E1 and cyclin-dependent kinase 2(CDK2) at mRNA and protein levels were measured by CCK-8 assay, BrdU detection, flow cytometry, RT-PCR and Western blot, respectively. RESULTS: miRNA-25 was prominent in esophageal mucosal tissue and highly expressed in TE1 cells (P<0.05). Over-expression of miRNA-25 increased TE1 cell proliferation, promoted the cell cycle progression and enhanced the entrance of the cells into S phase (P<0.05). Inverse results were obtained after down-regulation of miRNA-25(P<0.05). Furthermore, the expression of cyclin E1 and CDK2 at mRNA and protein levels was significantly increased after over-expression of miRNA-25, but decreased after down-regulation of miRNA-25(P<0.05). CONCLUSION: miRNA-25 enhances cell cycle transition by increasing the expression of cyclin E1 and CDK2, thus accelerating TE1 cell proliferation. This study provides a novel mechanism by which miRNA-25 increases the proliferation of human esophageal squamous-cell carcinoma cell line TE1, suggesting that down-regulation of miRNA-25 may be a potential new therapeutic strategy for treating esophageal squamous-cell carcinoma.     

miR-221 promotes proliferation of lung cancer A549 cells by down-regulating PTEN

AIM: To explore the effect of microRNA-221 (miR-221) on the proliferation of lung cancer A549 cells, and to investigate its mechanism. METHODS: The A549 cells were transfected with miR-221 mimics by Lipofectamine 2000. The expression of miR-221 was detected by RT-qPCR. The expression of PTEN at mRNA and protein le-vels was detected by RT-qPCR and Western blot, respectively. The cell proliferation was examined by CCK-8 assay and colony formation assay. The 3'-UTR of PTEN was cloned into luciferase reporter vector and its enzymatic activity was detected to verify whether miR-221 targeted to PTEN. RESULTS: The expression level of miR-221 in the A549 cells was significantly increased after transfection with miR-221 mimics as compared with negative control group and blank group (P<0.01). The mRNA and protein levels of PTEN were significantly down-regulated compared with control group and blank group (P<0.05). In addition, miR-221 over-expression significantly promoted the proliferation of A549 cells (P<0.05). Moreover, miR-221 inhibited the enzymatic activity of luciferase reporter vector of PTEN. CONCLUSION: Over-expression of miR-221 significantly promotes the proliferation ability of human lung cancer A549 cells by down-regulation of PTEN.     

Effect of low expression of miR-21 on apoptosis of HepG2 cells induced by matrine

AIM: To explore whether miR-21 low expression enhances the effect of matrine (MAT) on the apoptosis of hepatocellular carcinoma cells.METHODS: Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of miR-21 in the HepG2 cells treated with different concentrations of MAT. The effect of miR-21 on MAT-induced HepG2 cell apoptosis was analyzed by flow cytometry. The mRNA and protein expression of Bcl-2 and Bax in the HepG2 cells treated with MAT was determined by RT-qPCR and Western blot.RESULTS: The expression of miR-21 increased with the increasing concentration of MAT. Low expression of miR-21 promoted MAT-induced apoptosis, and enhanced the expression of Bax at mRNA and protein levels (P<0.05), while inhibited the expression of Bcl-2 at mRNA and protein levels (P<0.05).CONCLUSION: Low expression of miR-21 enhances MAT-induced HepG2 cell apoptosis by inhibiting the expression of Bcl-2 and promoting Bax expression.     

miR-222 enhances HBx-HepG2 cell growth via regulation of BCL2L13 gene

AIM: To investigate the regulation of miR-222 on BCL2L13 gene and its effect on the growth and apoptosis of HBx-HepG2 cells, and to explore the underlying molecular mechanisms. METHODS: The expression level of miR-222 was detected by RT-qPCR. The HBx-HepG2 cell growth was examined by MTT and colony formation assays. The cell cycle and apoptosis were analyzed by flow cytometry. The recombination vector pmirGLO-BCL2L13 was constructed, and dual-luciferase reporter experiment was performed to validate the target of miR-222. RESULTS: The expression level of miR-222 in the HBx-HepG2 cells was significantly higher than that in the L02 cells (P<0.05). Over-expression of miR-222 enhanced HBx-HepG2 cell growth, changed cell cycle, and inhibited apoptosis (P<0.05). Knockdown of miR-222 reduced HBx-HepG2 cell growth, changed cell cycle, and increased cell apoptotic rate (P<0.05). BCL2L13 was down-regulated in the HBx-HepG2 cells as compared with L02 cells (P<0.05), and knockdown of miR-222 in the HBx-HepG2 cells increased the expression level of BCL2L13 (P<0.05). The results of dual-luciferase reporter assay and restore experiment showed that miR-222 negatively regulated the expression of BCL2L13 via targeting 3'UTR of BCL2L13, resulting in the promotion of HBx-HepG2 cell growth. CONCLUSION: miR-222 enhances HBx-HepG2 cell growth via down-regulation of BCL2L13.     

Mechanism of miR-30c over-expression inhibiting malignant phenotypes of cervical cancer cells

AIM:To investigate the molecule mechanism of microRNA (miR)-30c over-expression inhibiting malignant phenotypes of cervical cancer cells. METHODS:Cervical cancer cell lines C33A, HeLa, SiHa and CaSki were transfected with pGenesil-1-miR-30c plasmid using Lipofectamine 2000 kit, and the expression of miR-30c was determined by TaqMan real-time PCR. The cell viability inhibition rate, colony formation ability, migration rate and apoptotic rate were measured by MTT assay, colony formation assay, Transwell experiment, and flow cytometry with Annexin V-FITC staining. The protein expression of Bax, Bcl-2, matrix metalloproteinase (MMP)-9, MMP-13 and tissue inhibitor of metalloprotei-nase-1 (TIMP-1) was detected by Western blot. RESULTS:The expression levels of miRNA-30c in the cervical cancer cell lines transfected with pGenesil-1-miR-30c plasmid were significantly higher than those in negative control groups (cell lines transfected with pGenesil-1 plasmid) (P<0.01). Significantly increased cell viability inhibition rate, and decreased colony formation ability and migration rate were found in the cervical cancer cell lines over-expressing miR-30c as compared with negative control groups (P<0.05). The apoptotic rate in the cervical cancer cell lines over-expressing miR-30c was dramatically higher than that in control groups (P<0.05). Over-expression of miR-30c in cervical cancer cells promoted the protein expression of Bax and TIMP-1, and decreased the protein expression of Bcl-2 and MMP-13 (P<0.05 or P<0.01). CONCLUSION:Over-expression of miR-30c significantly inhibits the viability and migration, and induces apoptosis of cervical cancer cells. The mechanism may be related to activating apoptosis pathway and inhibiting MMP-13 protein expression.     

Berberine enhances mitomycin C-induced cell cycle arrest and apoptosis of T24 bladder cancer cells

AIM: To observe the effects of the combination of berberin (Ber) and mitomycin C (MMC) on the cell cycle arrest and apoptosis of T24 bladder cancer cells and the underlying mechanisms. METHODS: The T24 cells were exposed to MMC in the presence or absence of difference concentrations of Ber. The viability of the T24 cells was determined by CCK-8 assay. The cell cycle distribution was detected by flow cytometry. The apoptosis was analyzed by flow cytometry with Annexin V-FITC/PI staining, and the protein expression levels of cyclin D1, survivin, CDK2, CDK4, p21 and p27 were determined by Western blot. RESULTS: CCK-8 experiments showed that Ber enhanced the inhibitory effect of MMC on the viability of T24 cells. The results of flow cytometry showed that Ber also enhanced the blockade effect of MMC on T24 cells in G₀/G₁ phase (P<0.05). Compared with the MMC group, Ber increased the expression of p21 and p27 up-regulated by MMC, and decreased the expression of cynlin D1, CDK2 and CDK4 (P<0.05). Meanwhile, Ber promoted MMC to inhibit the expression of survivin (P<0.05). Ber increased the apoptosis of T24 cells induced by MMC (P<0.05). CONCLUSION: Ber significantly enhances the inhibitory effect of MMC on the viability of T24 cells. The mechanism may be related to up-regulation of p21 and p27, thereby inhibiting the expression of cyclin D1, CDK-2 and CDK-4. At the same time, Ber inhibits the protein expression of survivin, which eventually leads to cell arrest in G₀/G₁ phase and promotes apoptosis.     

Inhibitory effect of anti-miR-196a on invasion and migration of human pancreatic cancer PANC-1 cells

AIM:To investigate the expression of miR-196a in different pancreatic cancer cell lines and to observe the effect of anti-miR-196a on the biological behaviors of human pancreatic cancer PANC-1 cells. METHODS:The expression of miR-196a in the pancreatic cancer cells was examined by real-time quantitative PCR. Anti-miR-196a was chemically synthesized and transfected into PANC-1 cells by Lipofectamine 2000. The cell proliferation was measured by CCK-8 assay. The apoptosis was determined by flow cytometry. Matrigel invasion assay was performed to examine the migration and invasion of the tumor cells. The wild-type and mutant-type NF-κB inhibitor α(NFKBIA) 3'UTR luciferase reporter vectors were constructed. The relative activity of Renilla luciferase was detected to confirm the binding site of miR-196a on NFKBIA mRNA. RESULTS:The expression of miR-196a in human pancreatic cancer cell lines was significantly higher than that in human pancreatic ductal epithelial H6c7 cells. The expression of anti-miR-196a in miR-196a group was down-regulated. After transfected with miR-196a, no change of cell proliferation and apoptosis was observed, but the abilities of invasion and migration were reduced(P<0.01). Compared with negative control, wild-type NFKBIA3'UTR or mutant-type NFKBIA 3'UTR, cotransfection of anti-miR-196a and wild-type NFKBIA 3'UTR significantly increased the relative activity of Renilla luciferase. CONCLUSION:miR-196a is one of the oncomiRs and may be a target microRNA of human pancreatic cancer for gene therapy.     

Estradiol promotes growth of decidua derived mesenchymal stem cells by inhibiting miR-16 via estrogen receptor α

AIM: To study the effect of estradiol (E₂) on the viability of mesenchymal stem cells (MSCs) derived from the decidua of the placenta by regulating the expression of microRNA-16 (miR-16). METHODS: The concentration of E₂ in the peripheral blood of normal pregnant women and the patients with severe preeclampsia (PE) was measured. The effects of E₂ at different concentrations on the viability of MSCs were analyzed. The effect of E₂ at different concentrations on the expression of miR-16 in the MSCs was detected, and which estrogen receptor (ER) mediated the regulatory effect of E₂ on miR-16 expression was determined. RESULTS: The concentration of E₂ in peripheral blood of the patients with severe PE was significantly decreased (P<0.01). After treatment with E₂ at 5, 10 and 100 nmol/L for 48 h, the viability of MSCs was increased (P<0.05). The expression level of miR-16 was down-regulated in the MSCs treated with E₂ at 5, 10 and 100 nmol/L for 12 h. After treatment with E₂ at 10 nmol/L for different time (0 h, 3 h, 6 h, 12 h and 24 h), the expression level of miR-16 in the MSCs showed a clear time-dependent downward trend. E₂ significantly promoted the viability of MSCs, and the cell viability was significantly reversed after miR-16 pretreatment. Pretreatment with estrogen receptor antagonists ICI 182780 and tamoxifen for 6 h attenuated the inhibitory effect of E₂ on miR-16 expression. Only ERα agonist propyl pyrazole triol significantly inhibited the expression of miR-16 in MSCs but ERβ agonist diarylpropionitrile did not. CONCLUSION: E₂ promotes the growth of decidua-derived MSCs by inhibiting miR-16 via ERα.     

Effects of rat mesenchymal stem cells modified by CGRP on proliferation and phenotype transformation of vascular smooth muscle cells in vitro

AIM：To explore the effects of rat mesenchymal stem cells (MSCs) modified by calcitonin gene-related peptide (CGRP) on the proliferation and phenotype transformation of rat vascular smooth muscle cells (VSMCs) in vitro. METHODS：Rat MSCs and VSMCs were isolated, cultured and identified. The MSCs were infected by lentivirus which carried genes encoding enhanced green fluorescent protein (EGFP) and CGRP. The expression levels of CGRP in CGRP-modified MSCs were detected using real-time PCR and enzyme-linked immunosorbent assay (ELISA). The prolife-ration and migratory abilities of VSMCs were evaluated by MTT assay, Trypan blue staining and scratch test. The expression levels of α-smooth muscle actin (α-SMA) and osteopontin (OPN) were assessed by Western blotting. RESULTS：Compared with MSCs and MSCs-EGFP groups, the expression levels of CGRP in MSCs-CGRP group were markedly increased (P<0.01). The results of MTT assay and scratch test demonstrated that the proliferation and migratory abilities of VSMCs in MSCs-CGRP group were significantly inhibited compared with MSCs and MSCs-EGFP groups (P<0.05). Furthermore, cell viability was >90% shown by Typan blue staining. Western blotting showed that the expression of α-SMA was increased and that of OPN was decreased in MSCs-CGRP group compared with MSCs and MSCs-EGFP groups (P<005). CONCLUSION：CGRP-modified MSCs could secrete CGRP protein and inhibit the proliferation and migration of VSMCs, which may be associated with deterring the phenotype transformation from contractile type to synthetic type. These results lay a foundation for gene therapy in vivo.