LINC01224靶向miR-125b对口腔鳞癌细胞增殖及凋亡的影响

Effects of LINC01224 targeting miR-125b on the proliferation and apoptosis of oral squamous cell carcinoma cells

目的探讨长链非编码RNA(LncRNA)LINC01224是否通过调控微小RNA-125b(miR-125b)的表达,从而影响口腔鳞癌(OSCC)细胞增殖及凋亡。方法采用实时荧光定量聚合酶链反应(qRT-PCR)检测OSCC患者癌组织及癌旁组织中LINC01224的表达水平;体外培养OSCC细胞系CAL-27,将si-NC、si-LINC01224、si-LINC01224与anti-miR-NC、si-LINC01224与anti-miR-125b转染至CAL-27细胞;甲基噻唑基四唑(MTT)试验检测细胞增殖能力;流式细胞术检测细胞周期与细胞凋亡率;双荧光素酶报告试验验证LINC01224与miR-125b的靶向结合关系;western blot检测半胱氨酰天冬氨酸特异性蛋白酶3前体蛋白(pro-caspase-3)、增殖标记蛋白细胞增殖核抗原67(Ki67)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(clv-caspase-3)、P21蛋白的表达水平。结果与癌旁组织相比,OSCC患者癌组织中LINC01224的表达水平(1.00±0.05 vs 2.43±0.17)显著升高(t=94.345,P<0.05),miR-125b的表达水平(0.98±0.06 vs 0.22±0.02)显著降低(t=14.838,P<0.05);与si-NC组比较,si-LINC01224组细胞存活率[(100.02±6.73)%vs(47.94±4.69)%]显著降低(t=19.047,P<0.05),G 1期细胞比例[(31.03±3.01)%vs(42.29±4.12)%]显著增加(t=6.615,P<0.05),S期细胞比例[(34.18±3.38)%vs(23.49±2.57)%]显著减少(t=7.553,P<0.05),细胞凋亡率[(8.10±0.92)%vs(24.17±1.74)%]显著升高(t=24.494,P<0.05),Ki67、pro-caspase-3蛋白水平显著降低(P<0.05),P21、clv-caspase-3蛋白水平显著升高(P<0.05);双荧光素酶报告试验证实LINC01224与miR-125b靶向结合;与si-LINC01224+anti-miR-NC组比较,si-LINC01224+anti-miR-125b组细胞存活率[(48.03±4.57)%vs(90.01±5.59)%]显著升高(t=17.442,P<0.05),细胞凋亡率[(24.11±1.58)%vs(12.81±1.12)%]显著降低(t=17.504,P<0.05),G 1期细胞比例[(42.27±4.10)%vs(35.09±3.18)%]显著减少(t=4.151,P<0.05),S期细胞比例[(23.53±2.54)%vs(30.03±2.96)%]显著增加(t=4.999,P<0.05),pro-caspase-3、Ki67蛋白水平显著升高(P<0.05),clv-caspase-3、P21蛋白水平显著降低(P<0.05)。结论LINC01224能够靶向调控miR-125b的表达,从而促进OSCC细胞增殖及抑制细胞凋亡。

Objective To investigate whether LINC01224,a long non-coding RNA(LncRNA),affects the proliferation and apoptosis of oral squamous cell carcinoma(OSCC)cells by regulating the expression of microRNA-125b(miR-125b).Methods The expression levels of LINC01224 in the OSCC tissues and adjacent tissues of OSCC patients were detected by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR).The OSCC cell line CAL-27 cells were cultured in vitro,and si-NC,si-LINC01224,si-LINC01224+anti-miR-NC,and si-LINC01224+anti-miR-125b were transfected into CAL-27 cells,respectively.The proliferation of CAL-27 cells was detected by the methyl thiazolyl tetrazolium(MTT)method.Flow cytometry was used to detect cell cycle and apoptosis rate.The targeted binding relationship between LINC01224 and miR-125b was verified by the double luciferase report assay.The expression levels of pro-caspase-3,Ki67,clv-caspase-3 and P21 proteins were detected by Western blot.Results Compared with adjacent tissues,the expression levels of LINC01224 in OSCC tissues were significantly increased([1.00±0.05]vs[2.43±0.17],t=94.345,P<0.05),while the expression levels of miR-125b were significantly reduced([0.98±0.06]vs[0.22±0.02],t=14.838,P<0.05).Compared with the si-NC group,the cell survival rates of the si-LINC01224 group were significantly reduced([100.02±6.73]%vs[47.94±4.69]%,t=19.047,P<0.05),the proportions of G 1 phase cells were significantly increased([31.03±3.01]%vs[42.29±4.12]%,t=6.615,P<0.05),the proportions of S-phase cells were significantly decreased([34.18±3.38]%vs[23.49±2.57]%,t=7.553,P<0.05),the apoptosis rates were significantly increased([8.10±0.92]%vs[24.17±1.74]%,t=24.494,P<0.05),the levels of Ki67 and pro-caspase-3 proteins were significantly reduced(P<0.05),and the levels of P21 and clv-caspase-3 proteins were significantly increased(P<0.05).The dual luciferase report assay confirmed that LINC01224 could bind with miR-125b.Compared with the si-LINC01224+anti-miR-NC group,the cell survival rates of the si-LINC01224+anti-miR-125b group were significantly increased([48.03±4.57]%vs[90.01±5.59]%,t=17.442,P<0.05),the cell apoptosis rates were significantly reduced([24.11±1.58]%vs[12.81±1.12]%,t=17.504,P<0.05),the proportions of G 1 phase cells were significantly decreased([42.27±4.10]%vs[35.09±3.18]%,t=4.151,P<0.05),the proportions of S-phase cells were significantly increased([23.53±2.54]%vs[30.03±2.96]%,t=4.999,P<0.05),the levels of pro-caspase-3 and Ki67 proteins were significantly increased(P<0.05),and the levels of clv-caspase-3 and P21 proteins were significantly decreased(P<0.05).Conclusion LINC01224 can promote the proliferation and inhibit apoptosis of OSCC cells by regulating the expression of miR-125b.