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我国肺癌发病率与死亡率逐年上升,目前肺癌发病机制尚未完全阐明,长链非编码RNA(LncRNA)是一种长度超过220 nt的非编码RNA,LncRNA与微小RNA(miRNA)之间存在相互作用,其基因序列上含有多个miRNA的结合位点,并可通过调控miRNA转录过程从而参与肿瘤发生及发展过程,已知敲低LncRNA XIST通过激活miR-335/SOD2/ROS信号途径而抑制非小细胞肺癌的发展[1]。LncRNA Gm15290通过与肿瘤抑制因子miR-615-5p直接相互作用从而促进肺癌细胞的增殖和侵袭[2]。LncRNA MALAT1通过调节miR-124/STAT3轴促进非小细胞肺癌的发展[3]。LncRNA TUC338通过激活MAPK途径促进肺癌的侵袭[4]。LncRNA LRRC75A-AS1通过调控BAALC从而促进三阴性乳腺癌细胞的增殖和侵袭[5]。但LRRC75A-AS1在肺癌中的表达及其可能作用机制尚未阐明。靶基因预测显示miR-22-3p与LRRC75A-AS1存在结合位点,miR-22-3p在非小细胞肺癌中表达水平降低,LncRNA NNTAS1通过调节miR-22-3p/YAP1轴促进非小细胞肺癌的进展[6]。但LRRC75A-AS1/miR-22-3p分子轴在肺癌发生过程中的作用机制尚未阐明。因此,本研究主要探讨LRRC75A-AS1对肺癌细胞增殖、凋亡、迁移的影响及其对miR-22-3p的调控作用。
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与癌旁组织比较,肺癌组织中LRRC75A-AS1的表达水平升高(P < 0.01),miR-22-3p的表达水平降低(P < 0.01)(见表 1)。
分组 n LRRC75A-AS1 miR-22-3p 癌旁组织 43 1.01±0.13 1.00±0.12 肺癌组织 43 4.59±0.34 0.28±0.04 t — 64.49 37.33 P — < 0.01 < 0.01 表 1 LRRC75A-AS1和miR-22-3p在肺癌中的表达比较(x±s)
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与si-NC组比较,si-LRRC75A-AS1组细胞存活率降低(P < 0.01),克隆形成数减少(P < 0.01),凋亡率升高(P < 0.01),Cleaved-caspase3蛋白水平升高(P < 0.01)(见表 2)。
分组 存活率/% 克隆形成数/个 凋亡率/% Cleaved-caspase3 si-NC 100.00 121.00±5.10 8.42±0.34 0.20±0.01 si-LRRC75A-AS1 54.45±2.21 58.67±2.49 25.59±0.80 0.68±0.05 t 35.70 19.02 34.21 16.31 P < 0.01 < 0.01 < 0.01 < 0.01 表 2 干扰LRRC75A-AS1对A549增殖凋亡的影响(x±s; ni=3)
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与si-NC组比较,si-LRRC75A-AS1组划痕愈合率降低(P < 0.01),miR-22-3p的表达水平升高(P < 0.01)(见表 3)。
分组 LRRC75A-AS1 miR-22-3p 划痕愈合率/% si-NC 0.99±0.05 0.99±0.04 66.71±1.43 si-LRRC75A-AS1 0.23±0.01 3.47±0.07 32.56±0.95 t 25.82 53.28 34.45 P < 0.01 < 0.01 < 0.01 表 3 干扰LRRC75A-AS1对A549迁移的影响(x±s; ni=3)
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LncBase v.2预测显示LRRC75A-AS1与miR-22-3p存在结合位点(见图 1)。miR-22-3p过表达可降低野生型载体WT-LRRC75A-AS1的荧光素酶活性(P < 0.01),而对突变型载体MUT-LRRC75A-AS1的荧光素酶活性无明显影响(P>0.05)(见表 4)。
分组 WT-LRRC75A-AS1 MUT-LRRC75A-AS1 miR-NC 0.98±0.05 1.00±0.05 miR-22-3p 0.24±0.01 0.99±0.03 t 25.14 0.30 P < 0.01 >0.05 表 4 双荧光素酶报告实验(x±s; ni=3)
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与si-LRRC75A-AS1+anti-miR-NC组比较,si-LRRC75A-AS1+miR-22-3p inhibitor组细胞存活率升高(P < 0.01),克隆形成数增多(P < 0.01),凋亡率降低(P < 0.05),划痕愈合率升高(P < 0.01),Cleaved-caspase3蛋白水平降低(P < 0.01)(见表 5)。
分组 存活率/% 克隆形成数/个 凋亡率/% 划痕愈合率/% Cleaved-caspase3 si-LRRC75A-AS1+anti-miR-NC 54.61±2.28 57.67±2.49 25.63±0.99 32.52±0.98 0.67±0.06 si-LRRC75A-AS1+miR-22-3p inhibitor 88.75±3.22 106.67±3.68 13.11±0.55 55.30±1.18 0.30±0.03 t 14.99 19.10 19.15 25.72 9.55 P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 表 5 抑制miR-22-3p可逆转干扰LRRC75A-AS1对A549增殖、凋亡、迁移的影响(x±s; ni=3)
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
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摘要:
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。 Abstract:ObjectiveTo explore the effect of LncRNA LRRC75A-AS1/miR-22-3p on the proliferation, apoptosis and migration in lung cancer cells. MethodsThe 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. ResultsCompared 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). ConclusionsInhibiting 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. -
Key words:
- lung neoplasms /
- LncRNA LRRC75A-AS1 /
- miR-22-3p /
- proliferation /
- apoptosis /
- migration
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表 1 LRRC75A-AS1和miR-22-3p在肺癌中的表达比较(x±s)
分组 n LRRC75A-AS1 miR-22-3p 癌旁组织 43 1.01±0.13 1.00±0.12 肺癌组织 43 4.59±0.34 0.28±0.04 t — 64.49 37.33 P — < 0.01 < 0.01 表 2 干扰LRRC75A-AS1对A549增殖凋亡的影响(x±s; ni=3)
分组 存活率/% 克隆形成数/个 凋亡率/% Cleaved-caspase3 si-NC 100.00 121.00±5.10 8.42±0.34 0.20±0.01 si-LRRC75A-AS1 54.45±2.21 58.67±2.49 25.59±0.80 0.68±0.05 t 35.70 19.02 34.21 16.31 P < 0.01 < 0.01 < 0.01 < 0.01 表 3 干扰LRRC75A-AS1对A549迁移的影响(x±s; ni=3)
分组 LRRC75A-AS1 miR-22-3p 划痕愈合率/% si-NC 0.99±0.05 0.99±0.04 66.71±1.43 si-LRRC75A-AS1 0.23±0.01 3.47±0.07 32.56±0.95 t 25.82 53.28 34.45 P < 0.01 < 0.01 < 0.01 表 4 双荧光素酶报告实验(x±s; ni=3)
分组 WT-LRRC75A-AS1 MUT-LRRC75A-AS1 miR-NC 0.98±0.05 1.00±0.05 miR-22-3p 0.24±0.01 0.99±0.03 t 25.14 0.30 P < 0.01 >0.05 表 5 抑制miR-22-3p可逆转干扰LRRC75A-AS1对A549增殖、凋亡、迁移的影响(x±s; ni=3)
分组 存活率/% 克隆形成数/个 凋亡率/% 划痕愈合率/% Cleaved-caspase3 si-LRRC75A-AS1+anti-miR-NC 54.61±2.28 57.67±2.49 25.63±0.99 32.52±0.98 0.67±0.06 si-LRRC75A-AS1+miR-22-3p inhibitor 88.75±3.22 106.67±3.68 13.11±0.55 55.30±1.18 0.30±0.03 t 14.99 19.10 19.15 25.72 9.55 P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 -
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