The Effects of miR-96-5p Targeting and Regulating FBXW7 Expression on the Proliferation and Apoptosis of Tongue Squamous Cell Carcinoma Cells
WEI Qing1*, SUN Yang1, FU Shuang2, XING Shuo3, ZHU Qingxiao2
This study investigated the effects of miR-96-5p targeting and regulating the expression of FBXW7 (F-box and WD repeat domain containing 7) on the proliferation and apoptosis of TSCC (tongue squamous cell carcinoma) cells. The abnormal expression of miR-96-5p and FBXW7 in human TSCC was detected by RT-qPCR. In vitro culture of human TSCC cell lines SCC15 and CAL-27, and explore their correlation with the clini- copathological characteristics of patients. Cells were randomly divided into the Blank control group, the NC-miR-96-5p group, the anti-miR-96-5p group, the miR-96-5p group, the anti-miR-96-5p+si-NC group, and the anti-miR-96-5p+si-FBXW7 group. After grouping and transfection, the expression of miR-96-5p and FBXW7 in the cells was detected by RT-qPCR and Western blot. Cell proliferation was detected by CCK-8 and colony formation assays. Cell apoptosis was detected by flow cytometry and Hoechst 33342/PI double staining. The expression of proteins related to cell proliferation and apoptosis was detected by immunofluorescence staining. The effect of miR-96-5p targeting FBXW7 expression on the growth of TSCC solid tumors was detected through nude mouse experiments. The targeted regulatory effect of miR-96-5p on FBXW7 was verified by the dual-luciferase reporter gene and RIP (RNA immunoprecipitation) assay. The results showed that compared with the paired adjacent tissues, miR-96-5p was clearly higher in human TSCC tissues, while the expression of FBXW7 was clearly lower (P<0.05), and these changes were correlated with tumor size, lymph node metastasis, and clinical stage in TSCC patients. Compared with the Blank control group, the expression of miR-96-5p, cell viability, clone formation rate, positive expression of Ki67, and the value of Bcl-2/Bax in the anti-miR-96-5p group were lower, while the expression of FBXW7 protein and mRNA, apoptosis index, and apoptosis rate were higher (P<0.05); the changing trends of each index in the miR-96-5p group were opposite to those in the anti-miR-96-5p group; the results of nude mouse experiments showed that downregulation of miR-96-5p could inhibit the growth of TSCC solid tumors by upregulating FBXW7 expression. Knockdown of FBXW7 could reverse the anti-proliferative and pro-apoptotic effects of downregulation on TSCC cells. The results indicated that down-regulation of miR-96-5p can inhibit the proliferation of TSCC cells and promote their apoptosis by up-regulating the expression of FBXW7.
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