lncRNA CASC2C靶向Wnt/β-catenin信号通路影响口腔鳞癌顺铂敏感性的实验研究

Effect of lncRNA CASC2C targeted Wnt/β-catenin signaling pathway on cisplatin sensitivity in oral squamous cell carcinoma

目的探讨长链非编码RNA癌易感性候选基因2C(lncRNA CASC2C)对Wnt/β-catenin信号通路的负性调控作用,以及对口腔鳞癌细胞株顺铂(DDP)耐药的影响。方法体外培养FaDu细胞和FaDu顺铂耐药细胞(FaDu/DDP),将lncRNA CASC2C序列和无序序列分别转染至FaDu/DDP细胞中作为过表达组和阴性对照(NC)组,另设空白对照组(CTL),并通过实时荧光定量PCR(QPCR)法进行验证。采用MTT法检测不同浓度DDP(3.125、6.25、12.5、25、50、100μmol/L)对各组细胞增殖的影响。流式细胞术法检测CTL组、NC组、过表达组细胞凋亡情况。采用Western blotting法检测各组细胞中Wnt/β-catenin信号通路相关蛋白Axin1、c-myc、cyclin D1、β-catenin表达情况。结果FaDu/DDP细胞lncRNA CASC2C的表达量为0.67±0.045,明显低于FaDu细胞(P<0.05)。MTT法结果显示,DDP对FaDu细胞和FaDu/DDP细胞的IC50分别为14.471μmol/L和39.886μmol/L。FaDu/DDP的耐药指数为2.756。QPCR结果显示,过表达组FaDu/DDP细胞lncRNA CASC2C的相对表达量为1.47±0.139,明显高于CTL组和NC组(P<0.05)。不同浓度DDP(3.125、6.25、12.5、25、50、100μmol/L)对过表达组FaDu/DDP细胞增殖抑制率分别为(5.46±1.29)%、(10.31±1.34)%、(22.69±2.15)%、(40.83±3.40)%、(69.71±3.67)%、(73.54±4.10)%,明显高于CTL组和NC组(P<0.05)。过表达组FaDu/DDP细胞FZD8蛋白、c-myc蛋白、cyclin D1蛋白、β-catenin蛋白表达量分别为0.66±0.17、0.84±0.12、0.78±0.15、0.52±0.16,低于CTL组和NC组;而Anxin蛋白表达量为1.12±0.16,高于CTL组和NC组,差异有统计学意义(P<0.05)。结论上调lncRNA CASC2C表达可逆转口腔鳞癌顺铂耐药细胞株FaDu/DDP的耐药性,其作用机制可能是通过负性调控FZD8的表达,从而抑制Wnt/β-catenin信号通路的激活。

Objective To investigate the negative regulation of Wnt/β-catenin signaling pathway by lncRNA CASC2C,a candidate gene for long non-coding RNA cancer susceptibility,and the effect on drug resistance of FaDu/DDP cells in oral squamous cell carcinoma.Methods FaDu cells and FaDu DDP drug-resistant cells were cultured in vitro,and lncRNA CASC2C and disordered sequence were transfected into FaDu/DDP cells respectively as overexpression group and negative control group(NC),which were verified by real-time fluorescence quantification(QPCR)method.And blank control group(CTL group)was set up.MTT assay was used to determine the effects of different DDP concentrations(3.125,6.25,12.5,25,50,100μmol/L)on cell proliferation.Flow cytometry was used to detect the apoptosis of FaDu/DDP cells in CTL group,NC group and overexpression group.Western blotting was used to detect the differences in the expression of Wnt/β-catenin signaling pathway related proteins Axin1,c-myc,cyclin D1,β-catenin molecules in each group.Results The expression of lncRNA CASC2C in FaDu/DDP cells was 0.67±0.045,which was significantly lower than that in FaDu cells(P<0.05).MTT assay showed that the IC50 of DDP on FaDu and FaDu/DDP cells was 14.471μmol/L and 39.886μmol/L,respectively.The resistance index of FaDu/DDP was 2.756.QPCR results showed that the relative expression of lncRNA CASC2C of FaDu/DDP cells in the overexpression group was 1.47±0.139,which was significantly higher than that of CTL group and NC group(P<0.05).The inhibitory rates of FaDu/DDP cells in the overexpression group with different DDP concentrations(3.125,6.25,12.5,25,50 and 100μmol/L)were(5.46±1.29)%,(10.31±1.34)%,(22.69±2.15)%,(40.83±3.40)%,(69.71±3.67)%and(73.54±4.10)%,respectively,which were significantly higher than those in the CTL group and the NC group(P<0.05).The expression levels of FZD8,c-myc,cyclin D1 andβ-catenin in FaDu/DDP cells in the overexpression group were 0.66±0.17,0.84±0.12,0.78±0.15 and 0.52±0.16,respectively,which were lower than those in the CTL group and the NC group.The expression level of Anxin was 1.12±0.16,higher than that of CTL group and NC group,and the difference was statistically significant(P<0.05).Conclusion Upregulation of lncRNA CASC2C can reverse the drug resistance of FaDu/DDP,and its mechanism of action may be to negatively regulate the expression of FZD8,thereby inhibiting the activation of Wnt/β-catenin signaling pathway.