外泌体miR-512-3p通过下调Gas2促进TSCC细胞增殖的研究

Study on the effect of exosome miR-512-3p on the proliferation of tongue squamous cell carcinoma cell lines by down-regulating Gas2

目的探讨舌鳞状细胞癌(TSCC)患者唾液外泌体对TSCC细胞增殖的影响及其相关分子机制。方法采用差速超速离心法收集舌癌患者和健康对照者唾液外泌体。用透射电镜和Western blot鉴定收集得到的沉淀。将唾液外泌体与TSCC细胞共培养,检测细胞增殖、GAS2和外泌体中微RNA-512-3p(miR-512-3p)的表达变化,并验证GAS2为miR-512-3p的靶基因。结果透射电镜和Western blot检测结果显示,提取的外泌体符合其结构特征和生物学特征。噻唑蓝(MTT)法检测结果显示,相比于对照组,TSCC组患者唾液外泌体明显促进TSCC细胞增殖(P<0.05)。Western blot结果显示,TSCC组患者唾液外泌体中cylinD1和增殖细胞核抗原(PCNA)表达水平明显升高(P<0.05);qPCR结果显示,TSCC患者唾液外泌体中miR-512-3p水平较健康对照者明显升高(P<0.001),并且miR-512-3p过表达组的外泌体可明显促进细胞增殖(P<0.05),其细胞p53表达水平降低(P<0.001)。荧光素酶报告基因检测结果显示,GAS2为miR-512-3p靶基因。结论TSCC患者唾液外泌体可通过miR-512-3p促进癌细胞增殖,其可能机制是通过靶向GAS2基因,并促进p53的降解。

Objective To explore the effect of salivary exosomes from patients with tongue squamous cell carcinoma(TSCC)on TSCC cell proliferation and its related molecular mechanisms.Methods The salivary exosomes of tongue cancer patients and healthy controls were collected by using differential ultracentrifugation.The collected precipitates were identified by transmission electron microscopy and Western blot.The salivary exosomes were co-cultured with TSCC cell lines to detect cell proliferation,the expression of GAS2 and miR-512-3p expression changes in exosomes,and verify whether GAS2 was the target gene of miR-512-3p.Results The results of transmission electron microscopy and Western blot showed that the extracted exosomes conformed to their structural and biological characteristics.The results of MTT assay showed that,compared with the control group,the salivary exosomes in the TSCC group significantly promoted the proliferation of TSCC cell(P<0.05).Western blot results showed that the expression levels of cylinD1 and proliferating cell nuclear antigen(PCNA)in salivary exosomes of the TSCC group were significantly increased(P<0.05).The qPCR results showed that the levels of miR-512-3p in salivary exosomes of TSCC patients were significantly higher than those in healthy controls(P<0.001),and exosomes in the miR-512-3p overexpression group significantly promoted cell proliferation(P<0.05),and the p53 expression level in the miR-512-3p overexpression group was decreased(P<0.001).The results of luciferase reporter gene assays showed that GAS2 was the miR-512-3p target gene.Conclusion The salivary exosomes of TSCC patients can promote the proliferation of cancer cells through miR-512-3p,and the possible mechanism might be to target the GAS2 gene and promote the degradation of p53.