lncRNA OIP5-AS1通过海绵吸附miR-143-3p调控COL1A1促进胃癌发展

LncRNA OIP5-AS1 promotes gastric cancer development by targeting COL1A1 through miR-143-3p

目的探究长链非编码RNA(lncRNA)Opa相互作用蛋白5反义RNA1(OIP5-AS1)对胃癌发展的调控机制。方法qRT-PCR检测lncRNA OIP5-AS1在正常胃细胞系(GES-1)和胃癌细胞系(AGS和SGC7901)中的表达水平;构建OIP5-AS1敲低载体、miR-143-3p抑制剂(miR-143-3p inhibitor)以及Ⅰ型胶原α1(COL1A1)敲低载体并转染胃癌细胞系,运用CCK-8实验、伤口愈合实验、Transwell实验检测lncRNA OIP5-AS1、miR-143-3p、COL1A1在胃癌细胞增殖、迁移、侵袭中的作用;采用双荧光素酶报告基因、qRT-PCR、Western blot实验检测lncRNA OIP5-AS1以及miR-143-3p、COL1A1之间的靶向调控作用。结果OIP5-AS1在胃癌细胞AGS、SGC7901中表达水平分别是GES-1细胞的6.10和5.53倍,差异有统计学意义(P<0.01)。同时,敲低OIP5-AS1会抑制胃癌细胞增殖[AGS:(0.71±0.07)比(1.20±0.11);SGC7901:(1.02±0.10)比(1.80±0.10),P均<0.05]、迁移[AGS:(29.01±3.68)%比(72.04±6.82)%;SGC7901:(23.97±2.24)%比(62.02±3.73)%,P均<0.01]和侵袭[AGS:(35.68±6.96)个比(278.33±11.85)个;SGC7901:(62.74±12.77)个比(248.65±12.03)个,P均<0.01]。双荧光素酶报告基因证实OIP5-AS1靶向作用miR-143-3p并下调其表达水平,miR-143-3p可负调控COL1A1的表达(P<0.01)。进一步研究发现,敲降OIP5-AS1通过靶向上调miR-143-3p对COL1A1的抑制作用,抑制胃癌细胞增殖[AGS:(0.73±0.05)比(1.14±0.10);SGC7901:(1.21±0.09)比(1.59±0.09),P均<0.01]、迁移[AGS:(28.00±4.32)%比(73.67±4.50)%;SGC7901:(24.33±2.05)%比(67.67±4.11)%,P均<0.01]和侵袭[AGS:(57.00±6.16)个比(261.33±16.42)个;SGC7901:(65.00±7.26)个比(249.33±10.66)个,P均<0.01]。结论lncRNA OIP5-AS1通过海绵吸附miR-143-3p进而上调COL1A1促进胃癌细胞增殖、迁移和侵袭,为胃癌临床诊断与治疗提供了潜在的标靶。

Objective To investigate the regulatory mechanism of long non-coding RNA(lncRNA)Opa-interacting protein 5 antisense RNA 1(OIP5-AS1)on gastric cancer development.Methods qRT-PCR was performed to analyze level of lncRNA OIP5-AS1 mRNA in normal gastric(GES-1)and gastric cancer(AGS and SGC7901)cell lines.The OIP5-AS1 knockdown vector,miR-143-3p inhibitor and COL1A1 knockdown vector were constructed and transfected into gastric cancer cell lines.The CCK-8,wound healing and Transwell assays were used to assess the effects of lncRNA OIP5-AS1,miR-143-3p,and COL1A1 on gastric cancer cell viability,migration,and invasion,respectively.Dual-luciferase reporter gene,qRT-PCR,and Western blot assays were used to assess the targeting regulation among lncRNA OIP5-AS1,miR-143-3p,and COL1A1,respectively.Results Level of OIP5-AS1 was 6.10 and 5.53 folds higher in gastric cancer cells AGS and SGC7901 than in GES-1 cells,respectively(P<0.01).However,knockdown of OIP5-AS1 expression inhibited gastric cancer cell viability(AGS,0.71±0.07 vs.1.20±0.11;SGC7901,1.02±0.10 vs.1.80±0.10;All P<0.05),migration(AGS,29.01±3.68 vs.72.04±6.82%;SGC7901,23.97±2.24 vs.62.02±3.73%;All P<0.01),and invasion(AGS,35.68±6.96 vs.278.33±11.85;SGC7901,62.74±12.77 vs.248.65±12.03;All P<0.01).The dual-luciferase gene reporter assay confirmed that OIP5-AS1 was able to directly target miR-143-3p and downregulated miR-143-3p expression;however,miR-143-3p was able to downregulate COL1A1 expression(P<0.01).Knockdown of OIP5-AS1 expression suppressed COL1A1 expression by targeted miR-143-3p upregulation and inhibited gastric cancer cell viability(AGS,0.73±0.05 vs.1.14±0.10;SGC7901,1.21±0.09 vs.1.59±0.09;All P<0.01),migration(AGS,28.00±4.32 vs.73.67±4.50%;SGC7901,24.33±2.05 vs.67.67±4.11%;All P<0.01),and invasion(AGS,57.00±6.16 vs.261.33±16.42;SGC7901,65.00±7.26 vs.249.33±10.66;All P<0.01).Conclusion The lncRNA OIP5-AS1 was able to upregulate COL1A1 expression through the sponge adsorption of miR-143-3p and subsequently,induce gastric cancer cell viability,migration,and invasion.This study provides a potentially novel target for future control of gastric cancer.