Mechanism of long non-coding RNA GATA3-antisense RNA 1 in regulating the proliferation, migration and invasion of colorectal cancer SW620 cells
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摘要:目的
探讨长链非编码RNA (lncRNA) GATA3-反义RNA 1(lncRNA GATA3-AS1)是否通过靶向微小RNA-574-3p (miR-574-3p)调控结直肠癌细胞SW620的增殖、迁移和侵袭。
方法采用逆转录-定量聚合酶链反应(RT-qPCR)检测43例结直肠癌组织及其对应癌旁组织中lncRNA GATA3-AS1和miR-574-3p表达情况。根据转染物的不同, 将SW620细胞分为si-NC组、si-GATA3-AS1组、miR-NC组、miR-574-3p组、si-GATA3-AS1+anti-miR-NC组、si-GATA3-AS1+anti-miR-574-3p组。采用细胞计数试剂盒8(CCK-8)法检测细胞增殖活性，采用克隆形成实验检测细胞克隆形成数，采用细胞划痕实验检测划痕愈合率，采用Transwell实验检测细胞侵袭能力，采用蛋白质印迹法(Western blot)检测迁移侵袭相关蛋白E-cadherin和N-cadherin表达。采用双荧光素酶报告基因实验检测lncRNA GATA3-AS1与miR-574-3p的靶向关系。
结果结直肠癌组织中lncRNA GATA3-AS1表达量高于癌旁组织, miR-574-3p表达量低于癌旁组织，差异有统计学意义(P < 0.05)。si-GATA3-AS1组SW620细胞的增殖活性、克隆形成数、划痕愈合率、侵袭细胞数和N-cadherin蛋白表达量均低于si-NC组, E-cadherin蛋白表达量高于si-NC组，差异有统计学意义(P < 0.05)。miR-574-3p组SW620细胞的增殖活性、克隆形成数、划痕愈合率、侵袭细胞数和N-cadherin蛋白表达量均低于miR-NC组, E-cadherin蛋白表达量高于miR-NC组，差异有统计学意义(P < 0.05)。lncRNA GATA3-AS1靶向调控miR-574-3p的表达。si-GATA3-AS1+anti-miR-574-3p组SW620细胞的增殖活性、克隆形成数、划痕愈合率、侵袭细胞数和N-cadherin蛋白表达量均高于si-GATA3-AS1+anti-miR-NC组, E-cadherin蛋白表达量低于si-GATA3-AS1+anti-miR-NC组，差异有统计学意义(P < 0.05)。
结论lncRNA GATA3-AS1在结直肠癌中表达上调，可通过靶向调控miR-574-3p促进结直肠癌细胞SW620的增殖、迁移和侵袭。
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关键词:
- 结直肠癌细胞 /
- 长链非编码RNA GATA3-反义RNA 1 /
- 微小RNA-574-3p /
- 增殖 /
- 迁移 /
- 侵袭
Abstract:ObjectiveTo explore whether long noncoding RNA (lncRNA) GATA3-antisense RNA 1 (lncRNA GATA3-AS1)regulates the proliferation, migration, and invasion of colorectal cancer SW620 cells by targeting microRNA-574-3p(miR-574-3p).
MethodsReverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine the expressions of lncRNA GATA3-AS1 and miR-574-3p in colorectal cancer tissues of 43 cases and para-cancerous tissue. The SW620 cells were divided into si-NC group, si-GATA3-AS1 group, miR-NC group, miR-574-3p group, si-GATA3-AS1+anti-miR-NC group, si-GATA3-AS1+anti-miR-574-3p group according to different transfection. The cell counting kit-8 (CCK-8) method was used to detect cell proliferation activity, the clone formation experiment to measure the number of cell clones, the scratch test to examine the scratch healing rate, the Transwell experiment to analyze cell invasion, and the Western blot to determine the expression of migration and invasion related proteins E-cadherin and N-cadherin. The dual luciferase reporter experiment was used to detect the targeting relationship between lncRNA GATA3-AS1 and miR-574-3p.
ResultsThe expression of lncRNA GATA3-AS1 in colorectal cancer tissues was higher, and the expression of miR-574-3p decreased compared with that in the adjacent tissues (P < 0.05). SW620 cells in the si-GATA3-AS1 group had lower proliferation activity, the number of clone formation, scratch healing rate, the number of invasive cell, and N-cadherin protein expression than the si-NC group, but the expression of E-cadherin protein was higher than that of si-NC group (P < 0.05). SW620 cell proliferation activity, the number of clone formation, scratch healing rate, the number of invasive cells, and N-cadherin protein expression in the miR-574-3p group were lower than those in the miR-NC group, and the E-cadherin protein expression level was higher than that of the miR-NC group (P < 0.05). LncRNA GATA3-AS1 targeted and regulated the expression of miR-574-3p. The proliferation activity, the number of clone cells, scratch healing rate, number of invasive cells and N-cadherin protein expression of SW620 cells in the si-GATA3-AS1+anti-miR-574-3p group were all higher than those in si-GATA3-AS1+anti-miR-NC group, while the expression level of E-cadherin protein was lower than that of the si-GATA3-AS1+anti-miR-NC group (P < 0.05).
ConclusionLncRNA GATA3-AS1 is up-regulated in colorectal cancer, and can promote the proliferation, migration and invasion of colorectal cancer cells SW620 by targeting miR-574-3p regulation.
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图 2 干扰lncRNA GATA3-AS1表达对结直肠癌细胞SW620迁移和侵袭能力的影响
A: 细胞划痕实验检测划痕愈合率; B: Transwell实验检测细胞侵袭能力; C: Western blot检测迁移侵袭相关蛋白E-cadherin和N-cadherin表达。
表 1 lncRNA GATA3-AS1、miR-574-3p在结直肠癌组织和癌旁组织中的表达(x±s)
组织 n lncRNA GATA3-AS1 miR-574-3p 癌旁组织 43 1.00±0.08 1.00±0.05 结直肠癌组织 43 4.24±0.30* 0.38±0.04* lncRNA GATA3-AS1: 长链非编码RNA GATA3-反义RNA 1; miR-574-3p: 微小RNA-574-3p。与癌旁组织比较, *P < 0.05。 
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表 2 干扰lncRNA GATA3-AS1表达对结直肠癌细胞SW620增殖能力的影响(x±s)
组别 n lncRNA GATA3-AS1 OD450 nm 细胞克隆形成数/个 si-NC组 3 1.00±0 1.03±0.08 86.66±6.53 si-GATA3-AS1组 3 0.24±0.02* 0.51±0.04* 37.33±3.65* OD450 nm: 450 nm波长处光密度。与si-NC组比较, *P < 0.05。
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表 3 干扰lncRNA GATA3-AS1表达对SW620细胞迁移和侵袭能力的影响(x±s)
组别 n 划痕愈合率/% 侵袭细胞数/个 E-cadherin蛋白 N-cadherin蛋白 si-NC组 3 70.78±6.13 107.33±10.62 0.19±0.02 0.73±0.06 si-GATA3-AS1组 3 29.31±2.11* 51.66±4.81* 0.59±0.04* 0.28±0.03* 与si-NC组比较, *P < 0.05。
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表 4 双荧光素酶报告基因实验检测结果(x±s)
转染物 n WT-GATA3-AS1 MUT-GATA3-AS1 miR-NC 3 0.97±0.06 0.99±0.07 miR-574-3p模拟物 3 0.35±0.03* 0.98±0.05 与miR-NC比较, *P < 0.05。
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表 5 miR-574-3p过表达对结直肠癌细胞SW620增殖、迁移和侵袭能力的影响(x±s)
组别 n miR-574-3p OD450 nm 细胞克隆形成数/个 划痕愈合率/% 侵袭细胞数/个 E-cadherin蛋白 N-cadherin蛋白 miR-NC组 3 1.00±0 1.02±0.07 89.66±7.03 71.84±6.12 108.66±11.61 0.16±0.02 0.75±0.05 miR-574-3p组 3 2.78±0.25* 0.60±0.06* 43.66±4.33* 32.58±3.17* 59.33±4.94* 0.51±0.04* 0.32±0.04* 与miR-NC组比较, *P < 0.05。
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表 6 抑制miR-574-3p表达对干扰lncRNA GATA3-AS1表达的SW620细胞增殖、迁移和侵袭的影响(x±s)
组别 miR-574-3p OD450 nm 细胞克隆形成数/个 划痕愈合率/% 侵袭细胞数/个 E-cadherin蛋白 N-cadherin蛋白 si-GATA3-AS1+anti-miR-NC组 1.00±0 0.50±0.04 35.99±3.39 27.92±2.49 49.33±4.11 0.58±0.05 0.27±0.03 si-GATA3-AS1+anti-miR-574-3p组 0.42±0.04* 0.88±0.07* 74.44±6.61* 61.34±5.11* 87.44±6.62* 0.28±0.03* 0.61±0.05* 与si-GATA3-AS1+anti-miR-NC组比较, *P < 0.05。
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