干扰LncRNA LRRC75A-AS1抑制肺癌细胞发生发展研究

Effect of Inhibition of LncRNA LRRC75A-AS1 on the development of lung cancer cells

目的:探讨LncRNA LRRC75A-AS1/miR-22-3p对肺癌细胞增殖、凋亡、迁移的影响及其可能作用机制。方法:采用qRT-PCR法检测肺癌组织与癌旁组织中LRRC75A-AS1、miR-22-3p的表达水平;体外培养人肺癌细胞A549,si-NC、si-LRRC75A-AS1、si-LRRC75A-AS1与anti-miR-NC、si-LRRC75A-AS1与miR-22-3p inhibitor转染入A549细胞;采用qRT-PCR法检测A549细胞中LRRC75A-AS1、miR-22-3p的表达水平;采用MTT实验与平板克隆形成实验分别检测细胞增殖及克隆形成能力;采用流式细胞术检测细胞凋亡率;采用划痕实验检测细胞迁移能力;双荧光素酶报告实验检测LRRC75A-AS1与miR-22-3p的靶向关系;Western blotting法检测Cleaved-caspase3蛋白表达量。结果:与癌旁组织比较,肺癌组织中LRRC75A-AS1的表达水平[(1.01±0.13)vs(4.59±0.34)]升高(P<0.01),miR-22-3p的表达水平[(1.00±0.12)vs(0.28±0.04)]降低(P<0.01);与si-NC组比较,si-LRRC75A-AS1组细胞存活率[100%vs(54.45±2.21)%]降低(P<0.01),克隆形成数[(121.00±5.10)个vs(58.67±2.49)个]减少(P<0.01),凋亡率[(8.42±0.34)%vs(25.59±0.80)%]升高(P<0.01),Cleaved-caspase3蛋白水平[(0.20±0.01)vs(0.68±0.05)]升高(P<0.01),划痕愈合率[(66.71±1.43)%vs(32.56±0.95)%]降低(P<0.01);双荧光素酶报告实验证实LRRC75A-AS1可充当miR-22-3p的竞争性内源RNA;与si-LRRC75A-AS1+anti-miR-NC组比较,si-LRRC75A-AS1+miR-22-3p inhibitor组细胞存活率[(54.61±2.28)%vs(88.75±3.22)%]升高(P<0.01),克隆形成数[(57.67±2.49)个vs(106.67±3.68)个]增多(P<0.01),凋亡率[(25.63±0.99)%vs(13.11±0.55)%]降低(P<0.01),划痕愈合率[(32.52±0.98)%vs(55.30±1.18)%]升高(P<0.01),Cleaved-caspase3蛋白水平[(0.67±0.06)vs(0.30±0.03)]降低(P<0.01)。结论:干扰LRRC75A-AS1表达可能通过上调miR-22-3p从而减弱肺癌细胞增殖、迁移及克隆形成能力,并能够诱导细胞凋亡。

Objective:To explore the effect of LncRNA LRRC75A-AS1/miR-22-3p on the proliferation,apoptosis and migration in lung cancer cells.Methods:The qRT-PCR method was used to detect the expressions of LRRC75A-AS1 and miR-22-3p in lung cancer tissues and adjacent tissues.Human lung cancer cells A549 were cultured in vitrol,si-NC,si-LRRC75A-AS1,si-LRRC75A-AS1 and anti-miR-NC,si-LRRC75A-AS1 and miR-22-3p inhibitor were transfected into A549 cells.The qRT-PCR method was used to detect the expressions of LRRC75A-AS1 and miR-22-3p in A549 cells.MTT assay and plate clone formation experiment were used to detect cell proliferation and clone formation ability,respectively.Flow cytometry was used to detect the apoptosis rate.Scratch test was used to detect cell migration ability.The dual luciferase reporter experiment was used to detect the targeting relationship between LRRC75A-AS1 and miR-22-3p.Western blotting was used to detect the expression of cleaved-caspase3 protein.Results:Compared with adjacent tissues,the expression of LRRC75A-AS1 in lung cancer tissues was increased[(1.01±0.13)vs(4.59±0.34)](P<0.01),and the expression level of miR-22-3p was decreased[(1.00±0.12)vs(0.28±0.04)](P<0.01).Compared with the si-NC group,the cell survival rate of the si-LRRC75A-AS1 group was reduced[100%vs(54.45±2.21)%](P<0.01),the number of clone formation was reduced[(121.00±5.10)vs(58.67±2.49)](P<0.01),the apoptosis rate was increased[(8.42±0.34)%vs(25.59±0.80)%](P<0.01),the protein level of cleaved-caspase3 was increased[(0.20±0.01)vs(0.68±0.05)](P<0.01),and the rate of scratch healing was decreased[(66.71±1.43)%vs(32.56±0.95)%](P<0.01).The dual luciferase report experiment confirmed that LRRC75A-AS1 could act as a competitive endogenous RNA for miR-22-3p.Compared with the si-LRRC75A-AS1+anti-miR-NC group,the cell survival rate of the si-LRRC75A-AS1+miR-22-3p inhibitor group was increased[(54.61±2.28)%vs(88.75±3.22)%](P<0.01),the number of clones was increased[(57.67±2.49)vs(106.67±3.68)](P<0.01),the apoptosis rate was decreased[(25.63±0.99)%vs(13.11±0.55)%](P<0.01),the scratch healing rate was increased[(32.52±0.98)%vs(55.30±1.18)%](P<0.01),and the protein level of cleaved-caspase3 was decreased[(0.67±0.06)vs(0.30±0.03)](P<0.01).Conclusions:Inhibiting the expression of LRRC75A-AS1 may reduce the proliferation,migration and clone formation ability of lung cancer cells by up-regulating miR-22-3p,which can induce cell apoptosis.