LncRNA SNHG15通过miR-483-3p/DDIT4轴调节非小细胞肺癌细胞的顺铂敏感性和上皮间质转化

LncRNA SNHG15 regulates cisplatin sensitivity and epithelial⁃mesenchymal transition in non⁃small cell lung cancer cells through miR⁃483⁃3p/DDIT4 axis

目的 探究LncRNA SNHG15对非小细胞肺癌(NSCLC)细胞的顺铂(DDP)敏感性和上皮间质转化的影响以及miR-483-3p/DNA损伤诱导转录物4(DDIT4)轴在其中发挥的作用。方法 体外培养NSCLC细胞(A549)、NSCLC耐药细胞(A549/DDP),检测两种细胞对DDP的IC50及LncRNA SNHG15、miR-483-3p、DDIT4 mRNA与蛋白表达,将A549/DDP细胞随机分为A549/DDP组、si NC组、si SNHG15组、si SNHG15+inhibitor NC组、si SNHG15+miR-483-3p inhibitor组、si SNHG15+miR-483-3p inhibitor+si NC组、si SNHG15+miR-483-3p inhibitor+si DDIT4组;蛋白印迹分析法检测各组细胞E-cad、N-cad、DDIT4蛋白表达变化;双荧光素酶报告实验验证miR-483-3p与LncRNA SNHG15、DDIT4的靶向关系。结果 与A549细胞相比,A549/DDP细胞对DDP的IC50、LncRNA SNHG15、DDIT4 mRNA与蛋白升高,miR-483-3p水平降低(P <0.05);A549/DDP组细胞间黏附明显被破坏;si SNHG15组、si SNHG15+miR-483-3p inhibitor+si DDIT4组细胞排列相对紧密,呈现上皮细胞特性;si SNHG15+miR-483-3p inhibitor组较si SNHG15组细胞排列松散、扩散细胞多;与A549/DDP组相比,si SNHG15组A549/DDP细胞增殖抑制率、E-cad表达升高,侵袭细胞数、迁移细胞数、N-cad、DDIT4表达降低(P <0.05);与si SNHG15组相比,si SNHG15+miR-483-3p inhibitor组A549/DDP细胞增殖抑制率、E-cad表达降低,侵袭细胞数、迁移细胞数、N-cad、DDIT4表达升高(P <0.05)。结论沉默A549/DDP细胞中SNHG15表达可通过调控miR-483-3p/DDIT4,抑制A549/DDP细胞增殖、侵袭、迁移、EMT,并降低A549/DDP细胞对DDP的耐药性。

Objective To investigate the impacts of LncRNA SNHG15 on cisplatin(DDP)sensitivity and epithelial⁃mesenchymal transition of non⁃small cell lung cancer(NSCLC)cells and the role of miR⁃483⁃3p/DNA damage⁃induced transcript 4(DDIT4)axis.Methods NSCLC cells(A549)and NSCLC drug⁃resistant cells(A549/DDP)were cultured in vitro,the IC50 of the two cells to DDP and the mRNA and protein expression of LncRNA SN⁃HG15,miR⁃483⁃3p and DDIT4 in the two cells were detected.A549/DDP cells were randomly divided into A549/DDP group,si NC group,si SNHG15 group,si SNHG15+inhibitor NC group,si SNHG15+miR⁃483⁃3p inhibitor group,si SNHG15+miR⁃483⁃3p inhibitor+si NC group,and si SNHG15+miR⁃483⁃3p inhibitor+si DDIT4 group.Western blot was used to detect the changes of E⁃cad,N⁃cad and DDIT4 protein expression in each group anddual⁃luciferase reporter assay was used to verify the targeting relationship of miR⁃483⁃3p with LncRNA SNHG15 and DDIT4.Results Compared with A549 cells,the IC50,LncRNA SNHG15,DDIT4 mRNA and protein of DDP in A549/DDP cells increased,and the miR⁃483⁃3p level decreased(P<0.05).In A549/DDP group,intercellular adhesion was significantly destroyed.The cells of si SNHG15 group and si SNHG15+miR⁃483⁃3p inhibitor+si DDIT4 group were relatively closely arranged,showing epithelial characteristics.The cells in the si SNHG15+miR⁃483⁃3p inhibitor group were loosely arranged and diffused as compared with the si SNHG15 group.Compared with A549/DDP group,the inhibition rate of A549/DDP cell proliferation and the expression of E⁃cad in si SNHG15 group increased,while the number of invasive cells,the number of migrating cells,the expression of N⁃cad and DDIT4 decreased(P<0.05).Compared with the si SNHG15 group,the proliferation inhibition rate and E⁃cad expression of A549/DDP cells in the si SNHG15+miR⁃483⁃3p inhibitor group decreased,and the number of invasive cells,the number of migrating cells,N⁃cad and DDIT4 expression increased(P<0.05).Conclusion Silencing the expression of SNHG15 in A549/DDP cells can inhibit the proliferation,invasion,migration and EMT of A549/DDP cells,and reduce the drug resistance of A549/DDP cells to DDP by regulating miR⁃483⁃3p/DDIT4.