lncRNA CASC19通过miR-152-3p上调HMGA2影响骨肉瘤发生发展的机制研究

Effects of lncRNA CASC19 on the pathogenesis and development of osteosarcoma by up-regulating HMGA2 through miR-152-3p

目的探讨长链非编码RNA癌症易感基因19(lncRNA CASC19)通过miR-152-3p/高迁移率族蛋白A2(HMGA2)对骨肉瘤发生发展的影响。方法实验设置si-NC组、si-CASC19组、pcDNA-NC组、pcDNA-CASC19组、miR-NC组、miR-152-3p组、anti-miR-NC组、anti-miR-152-3p组、si-CASC19+anti-miR-NC组、si-CASC19+anti-miR-152-3p组、si-CASC19+anti-miR-152-3p+si-NC组、si-CASC19+anti-miR-152-3p+si-HMGA2组。实时荧光定量PCR(qRT-PCR)检测lncRNA CASC19、miR-152-3p、HMGA2 mRNA表达水平;蛋白质印迹(Western blot)法检测HMGA2、细胞周期蛋白D1(CyclinD1)、裂解的半胱氨酸天冬氨酸蛋白酶-3(Cleaved-caspase-3)蛋白表达;细胞计数试剂盒8(CCK-8)检测细胞活性;流式细胞术检测细胞凋亡;双荧光素酶报告实验验证CASC19和miR-152-3p以及miR-152-3p和HMGA2的靶向关系。结果骨肉瘤细胞Saos2、U2OS、HOS中lncRNA CASC19高表达,miR-152-3p低表达,HMGA2高表达(P<0.05)。相较于si-NC组,si-CASC19组骨肉瘤细胞Saos2中lncRNA CASC19表达水平降低,HMGA2表达水平降低,CyclinD1表达水平降低,Cleaved-caspase-3表达水平升高,细胞存活率降低,细胞凋亡率升高差异均有统计学意义(P<0.05)。相较于miR-NC组,miR-152-3p组骨肉瘤细胞Saos2中miR-152-3p表达水平升高,HMGA2表达水平降低,CyclinD1表达水平降低,Cleaved-caspase-3表达水平升高,细胞存活率降低,细胞凋亡率升高差异均有统计学意义(P<0.05)。通过Starbase软件预测显示CASC19可直接靶向结合miR-152-3p,miR-152-3p可直接靶向结合HMGA2。双荧光素酶报告基因验证结果显示,共转染miR-152-3p和野生型CASC19和HMGA2的载体时,荧光素酶活性显著下降(P<0.05),而共转染miR-152-3p和靶向突变的CASC19和HMGA2的载体时,miR-152-3p对荧光素酶活性的抑制作用丧失。抑制CASC19表达后miR-152-3p表达水平升高(P<0.05),过表达CASC19后miR-152-3p表达水平下降(P<0.05);过表达miR-152-3p,HMGA2表达水平下降(P<0.05),抑制miR-152-3p表达,HMGA2表达水平升高(P<0.05)。相较于si-CASC19+anti-miR-NC组,si-CASC19+anti-miR-152-3p组Saos2细胞中miR-152-3p表达水平降低,CyclinD1表达水平升高,Cleaved-caspase-3表达水平降低,细胞存活率升高,细胞凋亡率降低差异均有统计学意义(P<0.05)。与si-CASC19+anti-miR-152-3p+si-NC组相比,si-CASC19+anti-miR-152-3p+si-HMGA2组HMGA2表达水平降低,CyclinD1表达水平降低,Cleaved-caspase-3表达水平升高,细胞存活率降低,细胞凋亡率升高,差异均有统计学意义(P<0.05)。结论抑制lncRNA CASC19通过上调miR-152-3p进而下调HMGA2的表达抑制骨肉瘤细胞增殖,促进细胞凋亡。

Objective To investigate the effects of long chain non-coding RNA cancer susceptibility 19(lncRNA CASC19)on the occurrence and development of osteosarcoma through miR-152-3p/high mobility group protein AT hook 2(HMGA2).Methods The experiment was divided in to si NC group,si CASC19 group,pcDNA NC group,pcDNA-CASC19 group,miR-NC group,miR-152-3p group,anti miR-NC group,anti miR-152-3p group,si-CASC19+anti miR-NC group,si-CASC19 anti miR-152-3p group,si-CASC19+anti miR-152-3p+si NC group si-CASC19+anti miR-152-3p+si HMGA2 group.The expression levels of lncRNA CASC19,miR-152-3p and HMGA2 mRNA were detected by Real time fluorescent quantitative PCR(qRT PCR).The protein expressions of HMGA2,cyclin D1(cyclinD1),and cleaved cysteine aspartic protease 3(Cleaved-caspase-3)were examined by Western Blot.The cell viability was detected by cell counting kit-8(CCK-8).The apoptosis was detected by flow cytometry.The targeted relationship between CASC19 and miR-152-3p,between miR-152-3p and HMGA2 was verified by the dual luciferase report assay.Results The expression levels of lncRNA CASC19 and HMGA2 in osteosarcoma cells Saos2,U2OS and HOS were significantly increased,however,the expression levels of miR-152-3p were significantly decreased(P<0.05).As compared with those in si NC group and si-CASC19 group,the expression levels of IncRNA CASC19,HMGA2,CyclinD1 as well as the cell viability were significantly decreased,however the expression levels of Cleaved Caspase 3 as well as apoptosis rate were significantly increased in osteosarcoma cell Saos2(P<0.05).As compared with those in miR-NC goup and miR1523p group,the expression levels of miR-152-3p,Cleaved Caspase 3 as well as apoptosis rate were significantly increased,however,the expression level of HMGA2 and cyclinD1 as well as the cell viability were significantly decreased(P<0.05).The fluoresein enzymatic activity was significantly decreased after cotransfected miR-152-3p,wild type CASC19 and HMGA2 into the carrier(P<0.05).After the inhibition of CASC19 expression,the expression levels of miR-152-3p were significantly increased(P<0.05),In overexpression CASC19,the expression levels of miR-152-3p were significantly decreased(P<0.05).After overexpression of miR-152-3p,the expression levels of HMGA2 were significantly decreased(P<0.05).As compared with those in si-CASC19+anti miR-NC group,the expression levels of miR-152-3p,Cleaved Caspase 3 of Saos2 cells as wel as apoptosis rate in si-CASC19+anti miR-152-3p group were significantly decreased,however the expression levels of CyclinD1 and the cell viability were significantly increased(P<0.05).As compared with those in si-CASC19+anti miR-152-3p+si NC group,the expression levels of HMGA2,cyclinD1 and the cell viability in si-CASC19+anti miR-152-3p+si HMGA2 group were significantly decreased,however the expression levels of Cleaved Caspase 3 and the apoptosis rate were significantly increased(P<0.05).Conclusion The inhibition of lncRNA CASC19 can inhibit osteosarcoma cell proliferation and promote apoptosis by up-regulating miR-152-3p and down-regulating HMGA2 expression.