LINC01224靶向miR-125b对口腔鳞癌细胞增殖及凋亡的影响

摘要：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)，G1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)，G1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。

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

Abstract:?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 G1?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 G1?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.