Effects of miR-9-5p on the migration, invasion and epithelial-mesenchymal transition process in cervical squamous cell carcinoma / 中南大学学报(医学版)

OBJECTIVES@#Cervical squamous cell carcinoma is the most common cancer in female reproductive system. This study aims to explore the effect of microRNA-9-5p (miR-9-5p) on the migration, invasion, and epithelial-mesenchymal transition (EMT) process of cervical squamous cells.@*METHODS@#Bioinformatics were used to predict the miRNAs that could bind to E-cadherin (E-cad). The Cancer Genome Atlas (TCGA) database was used to analyze and extract significantly differentially expressed miRNAs from part of cervical squamous cell carcinoma tissues and normal cervical tissues, and miR-9-5p was selected as the main research target. The translated regions (UTR) of wild-type E-cad (E-cad-WT 3'-UTR) or the 3'-UTR of mutant E-cad (E-Cad-MUT 3'-UTR) was transfected with miR-9-5p mimic normal control (NC), and miR-9-5p mimic was co-transfected human embryonic kidney cells (293T). The relationship between miR-9-5p and E-cad was detected by double luciferase assay. The expression of miR-9-5p in normal cervical epithelial cell lines (H8) and cervical squamous cell lines (C33A, siha, caski and Me180) were detected by quantitative real-time PCR. Then, the experiments were divided into groups as follows: a block control group, an overexpression control group (mimic-NC group), a miR-95p overexpression group (mimic group), an inhibitory expression control group (inhibitor-NC group), and a miR-9-5p inhibitory expression group (inhibitor group). The changes of migration ability were detected by scratch assay. Transwell invasion assay was used to analyze the changes of invasion ability, and the mRNA and protein changes of E-cad and vimentin were detected by quantitative real-time PCR and Western blotting.@*RESULTS@#MiR-9-5p had a targeting binding relationship with E-cad. Compared with the normal cervical tissue H8 cell line, the miR-9-5p was highly expressed in cervical cancer cell lines (C33A, siha, caski and Me180) (all P<0.05). The luciferase activity of E-cad-MUT was increased compared with that of E-cad-WT in miR-9-5p mimic cells (P<0.05). Compared with the blank control group, the protein and mRNA expressions of E-cad were decreased in the miR-9-5p mimic group (both P<0.05), which were increased in the miR-9-5p inhibitor group (both P<0.05). Compared with H8 cell line, the miR-9-5p was highly expressed in the cervical squamous cell lines (all P<0.05). Compared with the mimic-NC group, the distance of wound healing, the number of caski and Me180 cells invaded below the membrane, and the mRNA and protein expressions of vimentin were all increased in the miR-9-5p mimic group (all P<0.05), while the mRNA and protein of E-cad were decreased (both P<0.05). Compared with the inhibitor-NC group, the distance of wound healing, the number of caski and Me180 cells invading the membrane, and the mRNA and protein expressions of vimentin were decreased in the miR-9-5p inhibitor group (all P<0.05), but the mRNA and protein expressions of E-cad were increased (both P<0.05).@*CONCLUSIONS@#The miR-9-5p is highly expressed in cervical squamous cell carcinoma, which can increase the migration and invasion ability, and promote the EMT process of cancer cells.

Protective effect of miR-9-5p regulating transient receptor potential melastatin 7 on myocardial ischemia-reperfusion in rats / 解剖学报

Objective To investigate the effect of microRNA-9-5p (miR-9-5p) regulating transient receptor potential melastatin 7 (TRPM7) on myocardial ischemia-reperfusion (MIR) in rats. Methods Thirty-two SD rats were divided into sham operation group, model group, miR-9-5p overexpression group and empty vector control group. The MIR model was established by ligation of left coronary artery. The sham operation group was not ligated. miR-9-5p agomir and agomir NC were injected into tail vein 24 hours before model establishment in miR-9-5p overexpression group and empty vector control group. The myocardial injury was observed by HE staining. The expression of miR-9-5p was detected by Real-time PCR. The serum levels of interleukin(IL)-6, tumor necrosis factor alpha(TNF-α), IL-1β, creatine kinase isoenzyme MB (CK-MB), cardiac troponin Ⅰ (cTnI), lactate dehydrogenase (LDH) and the contents of malondialdehyde (MDA) and superoxide dismutase (SOD) in myocardium were measured were measured by ELISA. Cardiomyocyte apoptosis was detected by TUNEL. Double luciferase assay verified the relationship between miR-9-5p and TRPM7. The protein expressions of TRPM7, Bcl-2, Bcl-2 associated X (Bax), phosphorylated nuclear factor kappa-B 65 (p-NF-κB p65) and toll like receptor 4 (TLR4) were detected by Western blotting. Results The expression of miR-9-5p was low in myocardial tissue of rats (P<0.05). Overexpression of miR-9-5p could reduce the expression levels of CK-MB, cTnI and LDH, and improve the degree of myocardial injury. Compared with the model group, the apoptosis rate, Bax protein expression, MDA, IL-6, TNF-α and IL-1β contents in myocardial cells of miR-9-5p overexpression group decreased, while Bcl-2 protein expression and SOD content increased (P<0.05). The result of dual luciferase assay showed that TRPM7 was the target gene of miR-9-5p, and the protein expressions of TRPM7, p-NF-κB p65 and TLR4 in miR-9-5p overexpression group were lower than those in model group (P<0.05). Conclusion MiR-9-5p can inhibit myocardial cell apoptosis, oxidative stress and inflammation induced by myocardial ischemia-reperfusion, and inhibit TLR4/NF-κB pathway by regulating TRPM7.