miR-338-3p通过靶向PPM1B促进口腔癌细胞凋亡

miR-338-3p promotes apoptosis in oral cancer cells by targeting PPM1B

目的探讨miR-338-3p在口腔鳞状细胞癌(OSCC)中的作用及其调节细胞增殖、迁移和凋亡的机制。方法以正常口腔组织或成人牙龈成纤维细胞(HGF)作为对照组,RT-qPCR法检测OSCC组织或OSCC细胞(TSCCA,CAL-27,Tb3.1)中miR-338-3p和PPM1B mRNA的表达情况;免疫组化法检测OSCC组织与正常组织PPM1B表达量;过表达miR-338-3p后,MTT法检测OSCC细胞增殖、Annexin V-FITC-PI双染试剂盒检测OSCC的凋亡;Western blot法检测PPM1B与凋亡相关蛋白Bax和Claved-caspase 3、Bcl-2的表达;采用生物信息学软件Target scan网站和荧光素酶检测法探索miR-338-3p和PPM1B潜在的靶向关系。通过敲除和过表达PPM1B探索其对OSCC细胞增殖、迁移和凋亡的影响。结果OSCC组织和细胞中miR-338-3p表达下调,而PPM1B mRNA与蛋白水平均升高(P<0.05)。过表达miR-338-3p可抑制OSCC细胞的增殖并诱导细胞凋亡(P<0.05)。Western blot检测显示,miR-338-3p可以促进Bax和Claved-caspase 3并抑制PPM1B、Bcl-2的表达(P<0.05)。敲除PPM1B也抑制了细胞的生长并诱导细胞凋亡(P<0.05)。荧光素酶报告基因检测显示PPM1B和miR-338-3p之间有直接结合(P<0.05)。过表达PPM1B部分逆转了miR-338-3p对OSCC细胞增殖、凋亡的作用(P<0.05)。结论miR-338-3p在OSCC患者和OSCC细胞系中表达下调。过表达miR-338-3p通过靶向PPM1B,抑制OSCC细胞增殖并诱导细胞凋亡。

Objective To investigate the role of miR-338-3p in OSCC and its mechanism of regulating cell proliferation,migration and apoptosis.Methods Normal oral tissues or adult gingival fibroblasts(HGF)were used as control groups and RT-qPCR was used to detect the mRNA expressions of miR-338-3p and PPM1Bin OSCC tissues or OSCC cells(TSCCA,CAL-27,Tb3.1).Immunohistochemical method was used to detect the expression of PPM1Bin OSCC tissue and normal tissue.After overexpression of miR-338-3p,MTT assay was used to detect OSCC cell proliferation,Annexin V-FITC-PI double staining kit was added to detect OSCC apoptosis.Western blot was used to detect the expression of PPM1Band apoptosis-related proteins Bax,Claved-caspase 3and Bcl-2.The potential targeting relationship between miR-338-3p and PPM1Bwas explored by bioinformatics software Target scan website and luciferase assay.The effects of PPM1Bon the proliferation,migration and apoptosis of OSCC cells were explored by knocking out and overexpressing PPM1B.Results The expression of miR-338-3p was down-regulated in OSCC tissues and cells,while the levels of PPM1BmRNA and protein were both increased(P<0.05).Overexpression of miR-338-3p could inhibit the proliferation of OSCC cells and induce apoptosis(P<0.05).Western blot analysis showed that miR-338-3p could promote Bax and Claved-caspase 3and inhibit the expression of PPM1Band Bcl-2(P<0.05).Knockout of PPM1Balso inhibited cell growth and induced apoptosis(P<0.05).In addition,luciferase reporter gene assay showed direct binding between PPM1Band miR-338-3p(P<0.05).Overexpression of PPM1Bpartially reversed the effects of miR-338-3p on OSCC cell proliferation and apoptosis(P<0.05).Conclusion The expression of miR-338-3p is down-regulated in OSCC patients and OSCC cell lines.Overexpression of miR-338-3p inhibited OSCC cell proliferation and induced apoptosis by targeting PPM1B.