摘要
目的:探讨Ras-结合结构域家族蛋白1A(Ras-association domain family1A,RASSF1A)对结直肠癌细胞DLD-1凋亡、增殖、线粒体自噬及细胞黏附的影响以及可能的作用机制。方法:采用亚硫酸氢盐测序(bisulfite sequencing PCR,BSP)法检测5株结直肠癌细胞(DLD-1、HCT116、HT-29、Lo Vo和SW480)中RASSF 1A基因的甲基化情况,实时荧光定量PCR检测DLD-1、HCT116和HT-29细胞中RASSF1A mRNA的表述水平。采用脂质体法将携带有RASSF 1A基因的重组载体p EAK13-RASSF1A转入DLD-1细胞;随后分别采用FCM法、DCFH-DA探针法、JC-1法及CCK-8法检测RASSF1A过表达对DLD-1细胞凋亡、细胞中活性氧(reactive oxygen species,ROS)水平、线粒体膜电位及细胞增殖的影响,蛋白质印迹法检测凋亡、线粒体自噬、细胞增殖及黏附相关蛋白的表达水平。结果:DLD-1细胞中RASSF1A基因发生甲基化,DLD-1细胞中未能检测到RASSF1A mRNA的表达。在DLD-1细胞中瞬时转入重组p EAK13-RASSF1A过表达质粒恢复RASSF1A表达后,在24、48和72h时均检测发现DLD-1细胞的凋亡率明显提高(P均<0.05)。Bcl-2和Bax蛋白的表达水平均明显下调(P均<0.001),剪切型PARP蛋白的表达水平明显提高(P<0.05)。72 h时,LATS/YAP通路中LATS1(P<0.05)和YAP1(P<0.001)蛋白的表达水平均明显下调。48 h时DLD-1细胞的线粒体膜电位水平降低(P<0.05),同时PRKN蛋白的表达水平增加(P<0.05),并且观察到24 h时LC3-Ⅱ蛋白的表达水平增加(P<0.05)和p62蛋白的表达水平降低(P<0.01)。RASSF1A过表达抑制了DLD-1细胞的增殖(P均<0.01),同时下调RAS和RAF蛋白的表达水平(P均<0.001)。同时,48 h时PI3K、p-PI3K以及其下游的m TOR、p-m TOR蛋白的表达水平下调(P均<0.01)。48 h时,RASSF1A过表达能够下调β-actin蛋白的表达水平(P<0.01),同时下调β-连环蛋白(β-catenin)和黏着斑蛋白(vinculin)蛋白的表达水平(P均<0.01)。结论:RASSF1A通过促进结直肠癌DLD-1细胞的凋亡和线粒体自噬以及抑制其增殖和细胞黏附而发挥抑癌作用。
Objective:To investigate the effects of Ras-association domain family 1A(RASSF1A)on apoptosis,proliferation,mitochondrial autophagy and cell adhesion of colorectal cancer DLD-1 cells and the possible mechanisms.Methods:Bisulfite sequencing PCR(BSP)was used to detect the methylation of RASSF 1A gene in five colorectal cancer cells(DLD-1,HCT116,HT-29,LoVo and SW480),and the RASSF1A mRNA expression levels in DLD-1,HCT116 and HT-29 cells were detected by realtime fluorescent quantitative-PCR.The recombinant plasmid pEAK13-RASSF1A carrying RASSF 1A gene was transferred into DLD-1 cells by liposome method,the effects of RASSF1A overexpression on apoptosis,reactive oxygen species(ROS)level in cells,mitochondrial membrane potential and cell proliferation of DLD-1 cells were detected by FCM method,DCFH-DA probe method,JC-1 method and CCK-8 method,and the apoptosis,mitochondrial autophagy,cell proliferation and the expressions of adhesion-related proteins were detected by Western blotting.Results:Methylation of RASSF 1A gene in DLD-1 cells was observed and the expression of RASSF1A mRNA was undetectable in DLD-1 cells.After transient transfection of the pEAK13-RASSF1A overexpression plasmid to restore RASSF1A expression in DLD-1 cells,a significant increase in apoptotic rate was observed in DLD-1 cells at 24,48 and 72 h(all P<0.05).The expression level of Bcl-2 and Bay proteins were significantly decreased(both P<0.001),and the expression level of cleaved-PARP protein was significantly increased(P<0.05).The LATS/YAP pathway showed significantly lower protein levels of both LATS1(P<0.05)and YAP1(P<0.001)at 72 h.At 48 h,RASSF1A reduced mitochondrial membrane potential level in DLD-1 cells(P<0.05),and an increase in PRKN protein level(P<0.05)was observed.LC3-Ⅱprotein level was increased(P<0.05)and p62 protein level was decreased(P<0.01)at 24 h.Overexpression of RASSF1A could inhibit the proliferation of DLD-1 cells(P<0.01),meanwhile the protein levels of RAS and RAF were decreased(both P<0.001).The expression levels of PI3K and p-PI3K and their downstream mTOR and p-mTOR proteins were downregulated(all P<0.01).Overexpression of RASSF1A was able to reduce the protein expression ofβ-actin at 48 h(P<0.01),meanwhile the protein expression levels ofβ-catenin and vinculin was significantly reduced(both P<0.01).Conclusion:RASSF1A plays an anti-cancer role by promoting apoptosis and mitochondrial autophagy of rectal cancer DLD-1 cells,and inhibiting their proliferation and cell adhesion.
作者
胡斐
陈丽
马萍
唐文如
盛苗苗
HU Fei;CHEN Li;MA Ping;TANG Wenru;SHENG Miaomiao(Laboratory of Molecular Genetics of Aging&Tumor,Medical School,Kunming University of Science and Technology,Kunming 650500,Yunnan Province,China)
出处
《肿瘤》
CAS
CSCD
北大核心
2021年第10期670-683,共14页
Tumor
