miR-216a-3p miR-128-3p通过调控RGS17抑制口腔鳞状细胞癌细胞表型恶化的机制研究

Study on the Mechanism of miR-216a-3p and miR-128-3p Inhibiting the Phenotypic Deterioration of Oral Squamous Cell Carcinoma Cells by Regulating RGS17

目的:观察miR-216a-3p、miR-128-3p对口腔鳞状细胞癌(OSCC)细胞表型表达的影响,并探究其潜在的作用机制。方法:运用实时荧光定量逆转录聚合酶链式反应(RT-qPCR)检测癌组织、癌旁组织、人正常口腔角质形成细胞(hNOK)、OSCC癌细胞(SCC-9、SCC-25、HN4)中miR-216a-3p、miR-128-3p、G蛋白信号调节器17(RGS17)的表达;脂质体法将mimic-NC组(转染mimic)、mimic-miR-216a-3p组(转染miR-216a-3p mimic)、mimic-miR-128-3p组(转染miR-128-3p mimic)、si-NC组(转染si-NC)、si-RGS17组(转染si-RGS17)、mimic-miR-216a-3p+pcDNA组(共转染miR-216a-3p mimic和pcDNA)、mimic-miR-216a-3p+pcDNA-RGS17组(共转染miR-216a-3p mimic和pcDNA-RGS17)、mimic-miR-128-3p+pcDNA组(共转染miR-128-3p mimic和pcDNA)、mimic-miR-128-3p+pcDNA-RGS17组(共转染miR-128-3p mimic和pcDNA-RGS17)转染SCC-9细胞。5-溴-2-脱氧尿嘧啶(EdU)染色、克隆形成实验检测细胞增殖能力;Transwell实验检测细胞迁移侵袭能力;膜联蛋白V(Annexin V)/碘化丙啶(PI)染色检测细胞凋亡能力;双荧光素酶报告实验、RNA沉降实验验证靶向关系;蛋白免疫印迹(WB)实验检测RGS17蛋白。结果:与癌旁组织或hNOK细胞相比,OSCC组织(Ⅰ、Ⅱ、Ⅲ期)、癌细胞(SCC-9、SCC-25、HN4)中miR-216a-3p、miR-128-3p表达显著降低,RGS17表达显著升高(P<0.05)。与mimic-NC组相比,mimic-miR-216a-3p、mimic-miR-128-3p组细胞EdU阳性率、克隆形成率、迁移侵袭细胞数均显著降低,细胞凋亡率显著升高(P<0.05)。si-RGS17组细胞具有与mimic-miR-216a-3p、mimic-miR-128-3p细胞相似的变化。miR-216a-3p、miR-128-3p共同靶向负调控RGS17。过表达RGS17显著减弱miR-216a-3p、miR-128-3p对SCC-9细胞增殖、迁移侵袭抑制和凋亡促进作用。结论:miR-216a-3p、miR-128-3p抑制OSCC细胞增殖、迁移侵袭,促进凋亡,这与靶向RGS17有关。

Objective:To observe the effects of miR-216a-3p and miR-128-3p on the phenotypic expression of OSCC cells,and explore their potential mechanisms.Methods:The expression of miR-216a-3p,miR-128-3p and G protein signal regulator 17(RGS17)in cancer tissues,adjacent tissues,human normal oral keratinocytes(hNOK)and OSCC cancer cells(SCC-9,SCC-25,HN4)were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR);mimic NC group(transfected mimic),mimic-miR-216a-3p group(transfected miR-216a-3p mimic),mimic-miR-128-3p group(transfected miR-128-3p mimic),si-NC group(transfected si-NC),si-RGS17 group(transfected si-RGS17),mimic-miR-216a-3p+pcDNA group(co-transfected miR-216a-3p mimic and pcDNA),and mimic-miR-216a-3p+pcDNA-RGS17 group(co-transfected miR-216a-3p mimic and pcDNA-RGS17)were transfected into SCC-9 cells.5-bromo-2-deoxyuracil(EdU)staining and clonogenic assay were used to detect cell proliferation;Transwell test was used to detect the ability of cell migration and invasion;Annexin V/propidium iodide(PI)staining was used to detect the ability of apoptosis;Double luciferase report assay and RNA sedimentation assay were used to verify the targeting relationship;The RGS17 protein was detected by Western blot(WB).Results:Compared with adjacent tissues or hNOK cells,the expression of miR-216a-3p and miR-128-3p in OSCC tissues(stageⅠ,Ⅱ,Ⅲ)and cancer cells(SCC-9,SCC-25,HN4)was significantly decreased,and the expression of RGS17 was significantly increased(P<0.05).Compared with the mimic-NC group,the positive rate of EdU,the rate of clone formation,the number of migrating and invasive cells in the mimic-miR-216a-3p and mimic-miR-128-3p groups were significantly lower,and the apoptosis rate was significantly higher(P<0.05).The cells in si-RGS17 group had similar changes to those in mimic-miR-216a-3p and mimic-miR-128-3p cells.miR-216a-3p and miR-128-3p jointly targeted and negatively regulated RGS17.Overexpression of RGS17 significantly attenuated the proliferation,migration-invasion inhibition and apoptosis-promoting effects of miR-216a-3p and miR-128-3p on SCC-9 cells.Conclusion:MiR-216a-3p and miR-128-3p could inhibit the proliferation,migration and invasion of OSCC cells and promote apoptosis,which may be related to targeting RGS17.