circKDM4C对肺癌细胞增殖和迁移侵袭影响

Effects of circKDM4C on proliferation,migration and invasion of lung cancer cells

目的本研究探讨circKDM4C在肺癌中的表达及生物学功能。方法通过qRT-PCR检测circKDM4C在肺癌细胞系A549、H1299、H1650、H1975及肺正常上皮细胞BEAS-2B中的表达。siRNA转染A549和H1975,沉默circKDM4C,获得4组细胞系,分别为A549si-circKDM4C组和其对应的对照组A549si-NC,H1975si-circKDM4C组和其对应的对照组H1975si-NC。CCK-8和Transwell assay检测4组细胞的增殖、迁移和侵袭情况。流式细胞术检测细胞周期。蛋白质印迹法检测细胞周期抑制蛋白(p53、Rb、p21)和上皮间质转化(EMT)相关蛋白(N-cadherin、E-cadherin、Vimentin)的表达。circKDM4C在肺正常上皮细胞与在各肺癌细胞中的表达进行差异分析,A549si-circKDM4C组与A549si-NC组,H1975si-circKDM4C组与H1975si-NC组在细胞增殖、迁移、侵袭、细胞周期及相关蛋白表达水平进行差异分析,差异分析采用t检验。结果 circKDM4C在肺癌细胞A549、H1299、H1650和H1975的相对表达量分别为0.52±0.04、0.26±0.03、0.31±0.03和0.48±0.05,均低于正常肺上皮细胞BEAS-2B的表达(1.00±0.09),t值分别为19.21、16.29、31.14和25.41,均P<0.01。转染siRNA后,与对照组A549si-NC组和H1975si-NC组相比相比,A549si-circKDM4C组(t=7.45,P=0.023)和H1975si-circKDM4C组(t=16.33,P=0.027)中的circKDM4C相对表达量均降低,差异有统计学意义。转染si-circKDM4C后,A549si-circKDM4C组和H1975si-circKDM4C组细胞细胞增殖能力提高。Transwell迁移结果显示circKDM4C沉默后,细胞相对迁移数目A549si-circKDM4C组(2.71±0.20)比A549si-NC组(1.02±0.08)高,t=17.67,P=0.009;H1975si-circKDM4C组(3.02±0.13)同样比H1975si-NC组(1.01±0.05)高,t=26.27,P=0.004。Transwell侵袭结果显示侵袭相对细胞数量A549si-circKDM4C组(2.54±0.15)比A549si-NC组(1.04±0.10)高,t=21.62,P=0.007;H1975si-circKDM4C组(3.73±0.32)比H1975si-NC组(1.02±0.10)高,t=25.74,P=0.002。circKDM4C沉默后,促进了细胞周期,降低了周期抑制蛋白p53、Rb和p21的表达;此外N-cadherin和Vimentin表达增加,E-cadherin表达降低,促进了肺癌细胞上皮间质转化。结论 circKDM4C抑制了肺癌细胞的增殖、迁移和侵袭为肺癌患者提供了潜在的治疗靶点。

Objective The expression of circKDM4 Cand its biological function in lung cancer were investigated in this study.Methods Lung cancer cell lines A549,H1299,H1650,H1975,and lung normal epithelial cells BEAS-2 Bwere employed in this research,and the expression of circKDM4 Cwas detected in these cells by qRT-PCR.Lung cancer cells A549 and H1975 were transfected with siRNAs to silencing circKDM4 C,four groups,A549 si-circKDM4 Cgroup,A549 si-NC group,H1975 si-circKDM4 Cgroup and H1975 si-NC group,were acquired.CCK-8 and Transwell assay were used to determine the ability of proliferation,migration and invasion.Cell cycle was detected by flow cytometry.Western blot was employed to measure the protein expression of cyclin-dependent kinase inhibitors(p53,Rb and p21)and protein related to epithelial-mesenchymal transition(N-cadherin,E-cadherin and Vimentin).The differences in the expression of KIFC1 between normal lung epithelial cell line and each lung cancer cell line,the difference of proliferation,migration,invasion,cell cycle and protein expression between the A549 si-circKDM4 Cgroup and A549 si-NC group,and the difference between the H1975 si-circKDM4 Cgroup and H1975 si-NC group were analyzed.Student’s t-test was used for the difference analysis.Results The relative expressions of circKDM4 Cin lung cancer cells A549,H1299,H1650 and H1975 were(0.52±0.04),(0.26±0.03),(0.31±0.03)and(0.48±0.05),respectively,which were lower than that in normal lung epithelial cells BEAS-2 B(1.00±0.09),and the t values were 19.21,16.29,31.14 and 25.41,respectively,all P<0.01,and the differences were statistically significant.Compared with si-NC group,the expression of circKDM4 Cwas decreased in A549 and H1975 after transfecting with si-circKDM4 C,t=7.45,16.33,P=0.023,0.027,respectively.Moreover,the ability of proliferation increased after transfecting with si-circKDM4 C.Transwell migration assay results showed that after circKDM4 Csilencing,the relative number of cells in A549 si-circKDM4 Cgroup(2.71±0.20)was higher than that in the A549 si-NC group(1.02±0.08),t=17.67,P=0.009.The relative number of cells in H1975 si-circKDM4 Cgroup(3.02±0.13)was higher than that in the H1975 si-NC group(1.01±0.05),t=26.27,P=0.004.Transwell invasion results showed that the number of invasion cells in A549 si-circKDM4 Cgroup(2.54±0.15)was higher than that in the A549 siNC group(1.04±0.10),t=21.62,P=0.007.The relative number of invasion cells(3.73±0.32)in the H1975 si-circKDM4 Cgroup was higher than that in the H1975 si-NC group(1.02±0.10),t=25.74,P=0.002.After circKDM4 Cwas silenced,the cell progression was enhanced,and the cyclin-dependent kinase inhibitors protein expression of p53,Rb and p21 were decreased.The expression of N-cadherin and Vimentin was increased after silencing circKDM4 C,while the expression of E-cadherin was decreased,which promotes EMT of lung cancer cells.Conclusions The proliferation,migration and invasion of lung cancer were inhibited by circKDM4 C.CircKDM4 Cplayed a tumor-suppressor in lung cancer,which may provide a potential therapeutic target for lung cancer patients.