出 处:《山东医药》2020年第16期38-42,共5页Shandong Medical Journal
摘 要:目的探讨癌症易感候选基因19(CASC19)低表达对口腔鳞状细胞癌(OSCC)细胞增殖、凋亡的影响及机制。方法常规培养人口腔角质细胞NOK及OSCC细胞SCC9、HSC3、CAL-27,并用实时荧光定量PCR法确定SCC9细胞作为实验细胞。取对数生长期SCC9细胞并随机分组,用脂质体法将CASC19过表达对照质粒(pcDNA)、CASC19过表达质粒(pcDNA-CASC19)、CASC19抑制表达对照质粒(si-NC)、CASC19抑制表达质粒(si-CASC19)、miR-802阴性对照质粒(miR-NC)、miR-802模拟物(miR-802)分别转染至pcDNA组、pcDNA-CASC19组、si-NC组、si-CASC19组、miR-NC组、miR-802组;将si-CASC19与miR-802抑制剂阴性对照质粒(anti-miR-NC)、miR-802抑制剂(anti-miR-802)共转染至si-CASC19+anti-miR-NC组、si-CASC19+anti-miR-802组,用实时荧光定量PCR法检测细胞内miR-802、CASC19表达确认转染效率。用双荧光素酶报告基因实验检测lncRNA CASC19与miR-802的靶向关系,Western blotting法检测细胞内细胞周蛋白D1(Cyclin D1)、p21、B细胞淋巴瘤/白血病-2蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)表达,MTT法检测细胞增殖活性,流式细胞术检测细胞凋亡率。结果CASC19与miR-802存在结合位点。与pcDNA组比较,pcDNA-CASC19组miR-802相对表达量低(P<0.05);与si-NC组比较,si-CASC19组miR-802相对表达量高。细胞培养48、72 h,与si-NC组比较,si-CASC19组OD值低,细胞凋亡率高,Cyclin D1、Bcl-2蛋白表达低,p21、Bax蛋白表达高(P均<0.05)。与si-NC组比较,si-CASC19组培养48、72 h细胞增殖活力低,细胞凋亡率高,Cyclin D1、Bcl-2蛋白相对表达量低,p21、Bax蛋白相对表达量高(P均<0.05);与si-CASC19+anti-miR-NC组比较,si-CASC19+anti-miR-802组培养48、72 h细胞增殖活力高,细胞凋亡率低,Cyclin D1、Bcl-2蛋白相对表达量高,p21、Bax蛋白相对表达量低(P均<0.05)。结论CASC19低表达可抑制OSCC细胞增殖并促进其凋亡,其机制可能与靶向调控miR-802表达有关。Objective To investigate the effects of low expression of cancer susceptibility candidate gene 19(CASC19)on the proliferation and apoptosis of oral squamous cell carcinoma(OSCC)cells and its mechanism.Methods Human oral keratinocyte NOK and OSCC cells SCC9,HSC3,CAL-27 were routinely cultured,and the expression of CASC19 and miR-802 in the cells was detected by real-time fluorescent quantitative PCR.SCC9 cells in the logarithmic growth phase were taken and randomly grouped.CASC19 over-expression control plasmid(pcDNA),CASC19 over-expression plasmid(pcDNA-CASC19),CASC19 inhibition expression plasmid(si-NC),CASC19 inhibition expression plasmid(si-CASC19),miR-802 negative control plasmid(miR-NC),and miR-802 mimic(miR-802)were transfected into SCC9 cells by liposome method,and they were recorded as the pcDNA group,pcDNA-CASC19 group,si-NC group,si-CASC19 group,miR-NCgroup,and miR-802 group,respectively.The si-CASC19 combined with miR-802 inhibitor negative control plasmid(anti-miR-NC)and miR-802 inhibitor(anti-miR-802)were co-transfected into SCC9 cells,which are recorded as the si-CASC19+anti-miR-NC group and si-CASC19+anti-miR-802 group.Real-time fluorescence quantitative PCR was used to detect the expression of miR-802 and CASC19;dual luciferase reporter assay was used to detect the targeting relationship between lncRNA CASC19 and miR-802.Western blotting was used to detect cyclin D1(Cyclin D1),p21,and B cell lymphocytes tumor/leukemia-2(Bcl-2),Bcl-2 related X protein(Bax)expression;MTT assay was used to detect cell proliferation activity,and flow cytometry was used to detect apoptosis rate.Results CASC19 and miR-802 had binding sites.Compared with the pcDNA group,the relative expression of miR-802 in the pcDNA-CASC19 group was lower(P<0.05);compared with the si-NC group,the relative expression of miR-802 in the si-CASC19 group was higher.At 48 and 72 h after culture,compared with the si-NC group,the si-CASC19 group had lower OD value,higher apoptosis rate,lower Cyclin D1,Bcl-2 protein expression,and higher p21 and
关 键 词:口腔鳞状细胞癌 癌症易感候选基因19 微小RNA-802 细胞增殖 细胞凋亡
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