摘要
目的探讨RNA甲基化酶甲基转移酶样蛋白3(METTL3)对宫颈癌干细胞(CCSCs)干性及生物学行为的影响。方法取对数生长期的宫颈癌细胞系Siha细胞,采用分选流式细胞仪分选CD44和CD133阳性细胞作为CCSCs组,CD44和CD133阴性细胞作为Non-CCSCs组;采用实时定量聚合酶链式反应(qPCR)和蛋白质印迹法分别检测2组细胞中NANOG同源框蛋白(Nanog)、乙醛脱氢酶家族成员A1(ALDHA1)和SRY相关HMG盒蛋白2(SOX2)的mRNA和蛋白表达,成球实验检测CCSCs成球能力。取对数生长期的CCSCs组细胞,随机分为NC-CCSCs组(不转染任何质粒)和SH-M-CCSCs组(转染SH-METTL3质粒);采用qPCR和蛋白质印迹法分别检测2组细胞中METTL3、Nanog、SOX2、ALDHA1的mRNA和蛋白表达,采用Transwell实验检测细胞侵袭能力,采用细胞计数试剂盒-8实验、平板克隆形成实验检测细胞增殖能力,采用蛋白质印迹法检测细胞中凋亡相关蛋白B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、caspase-3的表达。结果CCSCs组细胞中Nanog、SOX2、ALDHA1 mRNA及蛋白相对表达量均显著高于Non-CCSCs组(P<0.05)。CCSCs组细胞观察到干细胞球形成。NC-CCSCs组细胞中METTL3、Nanog、SOX2、ALDHA1 mRNA及蛋白相对表达量均显著高于SH-M-CCSCs组(P<0.05)。SH-M-CCSCs组细胞侵袭数显著低于NC-CCSCs组(P<0.05)。SH-M-CCSCs组细胞增殖能力显著低于NC-CCSCs(P<0.05)。SH-M-CCSCs组细胞中凋亡相关蛋白Bcl-2/Bax比值显著低于NC-CCSCs组,caspase-3蛋白相对表达量显著高于NC-CCSCs组(P<0.05)。结论RNA甲基化酶METTL3可以增加CCSCs干性,提高CCSCs增殖、成球和侵袭能力,并抑制CCSCs凋亡。
Objective To explore the effects of RNA methlylase methyltransferase-like protein 3(METTL3)on the stemness and biological behavior of cervical cancer stem cells(CCSCs).Method Logarithmic-phase cervical cancer cell line Siha cells were sorted using a flow cytometer sorter.CD44 and CD133 double-positive cells were collected as the CCSCs group,while CD44 and CD133 double-negative cells were designated as the Non-CCSCs group.Real-time quantitative polymerase chain reaction(qPCR)and Western blot were performed to detect mRNA and protein expression levels of NANOG homeobox protein(Nanog),aldehyde dehydrogenase family member A1(ALDHA1),and SRY related HMG box protein 2(SOX2)in cells in the two groups.Spheroidization experiments were conducted to assess the spheroidization ability of CCSCs.Logarithmic-phase CCSCs were selected and randomly divided into the NC-CCSCs group(without transfection of any plasmid)and the SH-M-CCSCs group(transfected with SH-METTL3 plasmid).qPCR and Western blot were performed to detect mRNA and protein expression levels of METTL3,Nanog,SOX2 and ALDHA1.Cell invasion ability was examined using the Transwell assay,cell proliferation ability was assessed through cell counting kit-8 assay and plate cloning assay,and apoptosis-related protein expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax)and caspase-3 were detected using the Western blot technique.Results The relative expression levels of Nanog,SOX2,and ALDHA1 mRNA and protein in the CCSCs group were significantly higher than those in the Non-CCSCs group(P<0.05).The formation of stem cell balls was observed in CCSCs group.The relative expression levels of METTL3,Nanog,SOX2,and ALDHA1 mRNA and protein in the NC-CCSCs group were significantly higher than those in the SH-M-CCSCs group(P<0.05).The number of cell invasions in the SH-M-CCSCs group was significantly lower than that in the NC-CCSCs group(P<0.05).The cell proliferation ability in the SH-M-CCSCs group was significantly lower than that in the NC-CCSCs group(P<0.05).The relative expression level of apoptosis-related proteins Bcl-2/Bax in SH-M-CCSCs group cells was significantly lower than that in the NC-CCSCs group,while the relative expression level of caspase-3 protein was significantly higher than that in the NC-CCSCs group(P<0.05).Conclusion RNA methylase METTL3 can increase the stemness of CCSCs,enhance their proliferation,spheroidization,and invasion abilities,and inhibit CCSCs apoptosis.
作者
李鹏辉
谢佩
郭祥翠
韩若冰
刘娜娜
钟加滕
王倩青
LI Penghui;XIE Pei;GUO Xiangcui;HAN Ruobing;LIU Nana;ZHONG Jiateng;WANG Qianqing(Xinxiang Central Hospital/Fourth Clinical College of Xinxiang Medical University,Xinxiang 453000,Henan Province,China;Henan Provincial Key Medical Laboratory of Cervical Disease,Xinxiang 453000,Henan Province,China;Xinxiang Cervical Disease Engineering Technology Research Center,Xinxiang 453000,Henan Province,China;Department of Pathology,School of Basic Medicine,Xinxiang Medical University,Xinxiang 453003,Henan Province,China)
出处
《新乡医学院学报》
2025年第8期625-630,636,共7页
Journal of Xinxiang Medical University
基金
河南省科技攻关计划项目(编号:222102520012)。
关键词
甲基转移酶样蛋白3
宫颈癌干细胞
干性
生物学行为
methyltransferase-like protein 3
cervical cancer stem cells
stemness
biological behavior
