LINC01139靶向miR-1262影响卵巢癌细胞增殖和侵袭及迁移的机制

LINC01139 targets miR-1262 to affect the proliferation.invasion and migration of ovarian cancer cells

目的探讨长链基因间非编码RNA01139(LINC01139)在卵巢癌细胞增殖、侵袭和迁移中的可能机制。方法采用基因表达谱交互分析(GEPIA2)数据库分析LINC01139在乳腺癌组织的表达。qRT-PCR检测正常卵巢IOSE-80以及卵巢癌SKOV3、A2780、OVCAR3和A547细胞的LINC01139、微小RNA1262(miR-1262) mRNA表达。RNA结合蛋白免疫沉淀实验(RIP)检测LINC01139在细胞中的定位。Starbase数据库预测LINC01139与miR-1262的靶向关系,荧光素酶报告基因验证二者的结合情况。4种卵巢癌细胞中LINC01139 mRNA表达最高的为SKOV3。使用LipofectamineTM2000将靶向LINC01139长链非编码RNA(lncRNA)的siRNA#1、siRNA#2、siRNA#3及加扰的阴性对照siRNA转染SKOV3细胞,得到敲低组1、敲低组2、敲低组3和敲低对照组,未进行转染的细胞作为空白对照组。qRT-PCR检测各组miR-1262 mRNA表达,筛选miR-1262 mRNA表达最高的敲低组2。向敲低组2加入miR-1262抑制剂,CCK8法、细胞克隆实验、Transwell和划痕实验检测敲低对照组、敲低组2、敲低组2+miR-1262抑制剂组(敲低组2+抑制组)及敲低组2+抑制对照组的细胞增殖能力、克隆形成数目、结晶紫染色、侵袭和迁移能力。结果 GEPIA2数据库预测LINC01139在卵巢癌表达上调,在正常卵巢细胞IOSE-80(1.000±0.059)以及卵巢癌细胞系SKOV3(3.031±0.139)、A2780(2.470±0.218)、OVCAR3(2.803±0.075)和A547(2.010±0.074)中表达上调,LINC01139 mRNA在SKOV3细胞中表达最高,F=119.4,P<0.001;故选择SKOV3细胞进行下一步实验。核质分离实验显示,LINC01139主要位于细胞质中,t=16.70,P<0.001。免疫沉淀实验表明,LINC01139可以作为内源性RNA与miRNA相互作用,t=42.52,P<0.001。Starbase数据库分析显示,LINC01139和miR-1262存在7个互补碱基。双荧光素酶报告基因实验结果显示,miR-1262能与pmirGLO-LINC01139-WT结合,抑制荧光信号强度,t=15.42,P<0.001;LINC01139进行定点突变后,荧光信号强度不变,t=1.08,P=0.526。miR-1262 mRNA在卵巢癌细胞SKOV3(0.302±0.015)、A2780(0.509±0.037)、OVCAR3(0.406±0.024)和A547(0.437±0.028)中表达均低于正常卵巢细胞(0.980±0.050),均P<0.001;在SKOV3细胞中表达最低,F=191.5,P<0.001;敲低LINC01139后,PCR测定敲低组2表达量最低,F=139.2,P<0.001;选择敲低组2进行下一步实验。PCR检测显示,敲低LINC01139后miR-1262表达量增强,F=203.8,P<0.001。CCK-8实验表明,在转染24、48和72 h后,敲低组细胞增殖活性低于对照组,P24 h=0.027,P48 h<0.001,P72 h<0.001;在转染48、72 h后,与敲低组+抑制对照组相比,敲低组2+抑制组细胞增殖活性升高,P48 h=0.004,P72 h=0.001。克隆实验表明,敲低LINC01139后结晶及克隆数减少,t=10.46,P<0.001;而在加入miR-1262抑制剂后,逆转了这种变化,F=36.6,P<0.001。Transwell实验检测显示,敲低LINC01139后紫色结晶明显减少,P<0.001;而在加入miR-1262后紫色结晶增多,P<0.001。对比正常组,敲低组2细胞迁移能力下降,P<0.001;而在加入miR-1262抑制剂后,逆转LINC01139抑制细胞迁移的能力,F=52.5,P<0.001。结论 miR-1262可抑制卵巢癌细胞的增殖、侵袭和迁移能力,LINC01139通过下调miR-1262表达影响卵巢癌细胞的增殖、侵袭和迁移能力。

Objective To investigate the possible mechanism of the long-chain non-coding RNALINC01139 in the proliferation,invasion and migration of ovarian cancer cells.Methods The expression of LINC01139 in breast cancer tissues was analyzed by gene expression profile interaction analysis(GEPIA2)database.qRT-PCR was used to detect the mRNA expression of LINC01139 and microRNA1262(miR-1262)in IOSE-80 normal ovary and SKOV3,A2780,OVCAR3 and A547 ovarian cancer cells.RNA binding protein immunoprecipitation assay(RIP)was used to detect the localization of LINC001139 in cells.Starbase database predicted the targeting relationship between LINC001139 and miR-1262,and luciferase reporter gene verified the binding of LINC001139 and miR-1262.SKOV3 had the highest expression of LINC01139 mRNA among the four types of ovarian cancer cells.siRNA#1,SIRNA#2,siRNA#3 targeting LINC01139 were transfected into SKOV3 cells using LipofectamineTM2000 as well as interfered negative control siRNA,and knockdown group 1,knockdown group 2,knockdown group 3,and knockdown control group were obtained.Untransfected cells served as blank control group.qRT-PCR was used to detect the expression of miR-1262 mRNA in each group,and the knockdown group 2 with the highest expression of miR-1262 mRNA was screened.MiR-1262 inhibitor was added to knockdown group 2,CCK8 assay,cell cloning assay,Transwell assay and scratch assay were used to detect cell proliferation,number of clone formation,crystal violet staining,invasion and migration in knockdown control group,knockdown group 2,knockdown group 2+miR-1262 inhibitor group and knockdown group 2+inhibition group.Results GEPIA2 database predicted the upregulation of LINC01139 in ovarian cancer.The expression was up-regulated in normal ovarian cells IOSE-80(1.000±0.059)and ovarian cancer cell lines SKOV3(3.031±0.139),A2780(2.470±0.218),OVCAR3(2.803±0.075)and A547(2.010±0.074).mRNA expression of LINC01139 was the highest in SKOV3 cells(F=119.4,P<0.001).Therefore,SKOV3 cells were selected for the next experiment.Nuclear/cytoplasmic separation assay showed that LINC01139 was mainly located in the cytoplasm(t=16.70,P<0.001).Immunoprecipitation assay showed that LINC01139 could interact with miRNA as endogenous RNA(t=42.52,P<0.001).Starbase database analysis showed that LINC01139 and miR-1262 had 7 complementary bases.Dual luciferase reporter gene assay results showed that miR-1262 could bind to pmirGlo-LINC01139-WT and inhibit the fluorescence signal intensity(t=15.42,P<0.001);After site-directed mutation of LINC01139,the fluorescence signal intensity remained unchanged(t=1.080,P=0.526).miR-1262 expression in ovarian cancer cells SKOV3(0.302±0.015),A2780(0.509±0.037),OVCAR3(0.406±0.024)and A547(0.437±0.028)was lower than that in normal ovarian cells(0.980±0.050,P<0.001);The expression was the lowest in SKOV3 cells(F=191.5,P<0.001).After knockdown of LINC01139,the expression of knockdown group 2 was the lowest by PCR(F=139.2,P<0.001);Knockout group 2 was selected for the next experiment.PCR detection showed that the expression of miR-1262 was enhanced after LINC01139 knockdown(F=203.8,P<0.001).CCK-8 assay showed that the proliferation activity of knockdown group was lower than that of control group 24,48 and 72 hafter transfection(P24 h=0.027,P48 h <0.001,P72 h <0.001);After transfection for 48 and 72 h,cell proliferation activity in knockdown group 2+inhibition group increased compared with knockdown +inhibition control group(P48 h=0.004,P72 h=0.001).Cloning experiments showed that crystallization and number of clones decreased after knocking down LINC01139(t=10.46,P<0.001);However,the addition of miR-1262 inhibitor reversed this change(F=36.6,P<0.001).Transwell test showed that purple crystals were significantly reduced after knocking down LINC01139(P<0.001);The number of purple crystals increased after the addition of miR-1262(P<0.001).Compared with normal group,cell migration ability of knockdown group 2 decreased(P<0.001);After addition of miR-1262 inhibitor,the inhibition ability of LINC01139 on cell migration was reversed(F=52.5,P<0.001).Conclusions miR-1262 can inhibit the proliferation,invasion and migration of ovarian cancer cells.LINC01139 can affect the proliferation,invasion and migration of ovarian cancer cells by down-regulating the expression of miR-1262.