摘要
目的:探讨长链非编码RNA GAS6反义RNA1(long non-coding RNA GAS6 antisense RNA1, lncRNA GAS6-AS1)调节miR-370-3p/精子发生相关蛋白2 (spermatogenesis-associated protein 2, SPATA2)轴对卵巢癌细胞增殖、迁移、侵袭、凋亡和上皮间质转化(epithelial mesenchymal transformation, EMT)的影响。方法:qRT-PCR、Western blot分别检测癌旁组织、卵巢癌组织、人正常卵巢上皮细胞IOSE80及卵巢癌细胞系HO-8910、SKOV3、A2780中GAS6-AS1、miR-370-3p及SPATA2蛋白表达。将SKOV3细胞分为:对照组(NC组)、 si-NC组、si-GAS6-AS1组、mimic NC组、miR-370-3p mimic组、si-GAS6-AS1+inhibitor NC组、si-GAS6-AS1+miR-370-3p inhibitor组,qRT-PCR检测细胞中GAS6-AS1、miR-370-3p表达;CCK-8法检测细胞增殖;流式细胞术检测细胞凋亡;划痕愈合实验检测细胞迁移;Transwell实验检测细胞侵袭;Western blot检测SPATA2、细胞周期素D1(CyclinD1)、Bcl-2相关X蛋白(Bcl-2-associated X,Bax)、E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)、神经钙黏蛋白(N-cadherin)表达;双荧光素酶报告基因实验检测GAS6-AS1与miR-370-3p、 miR-370-3p与SPATA2的关系。结果:在卵巢癌组织和细胞中GAS6-AS1、SPATA2蛋白高表达,miR-370-3p低表达,且在SKOV3细胞中GAS6-AS1、SPATA2蛋白表达量最高,miR-370-3p表达水平最低,因此,选择SKOV3细胞为后续研究对象。与NC组、si-NC组比较,si-GAS6-AS1组GAS6-AS1、OD450值(24 h、48 h、72 h)、划痕愈合率、侵袭细胞数、SPATA2、CyclinD1、Vimentin、N-cadherin蛋白表达降低,miR-370-3p表达、细胞凋亡率、Bax、E-cadherin蛋白表达升高(P<0.05);与NC组、mimic NC组比较,miR-370-3p mimic组OD450值(24 h、48 h、72 h)、划痕愈合率、侵袭细胞数、SPATA2、CyclinD1、Vimentin、N-cadherin蛋白表达降低,miR-370-3p表达、细胞凋亡率、Bax、E-cadherin蛋白表达升高(P<0.05);miR-370-3p inhibitor减弱了沉默GAS6-AS1对SKOV3细胞增殖、迁移、侵袭及EMT的抑制及对细胞凋亡的促进作用。GAS6-AS1与mi
Objective:To investigate the impacts of long non-coding RNAGAS6 antisense RNA1(lncRNA GAS6-AS1)on proliferation,migration,invasion,apoptosis and epithelial mesenchymal transformation(EMT)of ovarian cancer cells by regulating miR-370-3p/spermatogenesis-associated protein 2(SPATA2)axis.Methods:qRT-PCR and Western blot were used to detect the expression of GAS6-AS1,miR-370-3p,and SPATA2 proteins in adjacent cancer tissues,ovarian cancer tissues,human normal ovarian epithelial cells IOSE80,and ovarian cancer cell lines HO-8910,SKOV3,and A2780,respectively.SKOV3 cells were grouped into:Control group(NC group),si-NC group,si-GAS6-AS1 group,mimic NC group,miR-370-3p mimic group,si-GAS6-AS1+inhibitor NC group,and si-GAS6-AS1+miR-370-3p inhibitor group,qRT-PCR was applied to detect the expression of GAS6-AS1 and miR-370-3p in cells.CCK-8 method was applied to detect cell proliferation.Flow cytometry was applied to detect cell apoptosis.Scratch healing test was applied to detect cell migration.Transwell experiment was applied to detect cell invasion.Western blot was applied to detect the protein expression of SPATA2,CyclinD1,Bcl-2-associated X(Bax),E-cadherin,Vimentin,and N-cadherin.Double Luciferase reporter gene experiment was applied to detect the relationship between GAS6-AS1 and miR-370-3p,and between miR-370-3p and SPATA2.Results:In ovarian cancer tissues and cells,GAS6-AS1 and SPATA2 proteins were highly expressed,while miR-370-3p was lowly expressed,and GAS6-AS1 and SPATA2 protein expression levels were the highest and miR-370-3p expression level was the lowest in SKOV3 cells,therefore,SKOV3 cells were selected as the follow-up research object.Compared with the NC group and si-NC group,the GAS6-AS1,OD450 value(24 h,48 h,72 h),scratch healing rate,number of invasive cells,expression of SPATA2,CyclinD1,Vimentin,N-cadherin proteins in the si-GAS6-AS1 group decreased,the expression of miR-370-3 p,cell apoptosis rate,and expression of Bax and E-cadherin proteins increased(P<0.05).Compared with the NC group and mimic NC
作者
贾奕娟
王中显
王冬花
龚世雄
JIA Yijuan;WANG Zhongxian;WANG Donghua;GONG Shixiong(Department of Gynecology,Wuhan First Hospital,Hubei Wuhan 430022,China.)
出处
《现代肿瘤医学》
CAS
2024年第3期424-431,共8页
Journal of Modern Oncology
基金
湖北省科学技术厅2021年省科技计划项目(编号:2021CFB576)。
