miR-181d-5p对人结肠癌细胞凋亡的影响及机制
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摘要
目的:探讨微小RNA-181d-5p(microRNAmiR-181d-5p)对人结肠癌细胞凋亡的影响及机制。方法:采用miR-181d-5p抑制物转染人结肠癌细胞系HCT116细胞,流式细胞术检测细胞凋亡情况,Real-time PCR和Western blot技术检测细胞miR-181d-5p、第10染色体同源缺失性磷酸酶-张力蛋白基因(PTEN)、黏附斑激酶(FAK)、B细胞淋巴瘤-2(Bcl-2)、半胱天冬蛋白酶3 (Caspase-3) mRNA和PTEN、FAK、p-FAK、Bcl-2、激活型Caspase-3(cleaved Caspase-3)蛋白表达,Targetscan软件在线预测及双荧光素酶实验分析miR-181d-5p和PTEN的调控关系。结果:与阴性对照组比较,miR-181d-5p抑制物组miR-181d-5p mRNA表达明显下调(P<0.01),凋亡细胞明显增加(P<0.01),PTEN mRNA表达水平明显上调(P<0.01),Bcl-2 mRNA表达水平明显下调(P<0.01),PTEN、cleaved Caspase-3蛋白表达水平明显升高(P<0.01),p-FAK、Bcl-2蛋白表达水平显著下调(P<0.01),其余各组之间差异均无统计学意义(P>0.05);Targetscan软件预测显示PTEN基因3'UTR含有miR-181d-5p的结合位点,双荧光素酶结果显示与突变型PTEN 3'UTR联合miR-181d-5p模拟物组相比,野生型PTEN3'UTR联合miR-181d-5p模拟物组荧光素酶活性明显降低,差异有高度统计学意义(P<0.01)。结论:miR-181d-5p可通过靶向调控PTEN抑制人结肠癌细胞凋亡,可成为结肠癌临床分子靶向治疗的潜在靶点。
Objective:To investigate the effect and mechanism of miR-181 d-5 p on apoptosis of human colon cancer cells.Methods:Human colon cancer cell line HCT116 was transfected with miR-181 d-5 p inhibitor.Apoptosis was detected by flow cytometry.Real-time PCR and Western blot were adopted to detect the protein expression of miR-181 d-5 p,PTEN,FAK,Bcl-2,Caspase-3 mRNA and PTEN,FAK,p-FAK,Bcl-2,cleaved Caspase-3 proteins.Targetscan analysis software online prediction and dual luciferase reporter gene system was used to analyze the regulatory relationship of miR-181 d-5 p and PTEN.Results:Compared with the negative control group,the expression of miR-181 d-5 p mRNA was down-regulated in the miR-181 d-5 p inhibitor group(P<0.01),the apoptotic cells were significantly increased(P<0.01),and the PTEN mRNA expression level was significantly upregulated(P<0.01);Bcl-2 mRNA expression level was significantly down-regulated(P<0.01),PTEN and cleaved Caspase-3 protein expression levels were significantly increased(P<0.01),pFAK,Bcl-2 protein expression levels were significantly down-regulated(P<0.01);the differences between the groups were not statistically significant(P>0.05).Targetscan software predicted that the3'UTR of PTEN gene contained the binding site of miR-181 d-5 p,and the dual luciferase results showed that compared with the mutant PTEN 3'UTR + miR-181 d-5 p mimic group,the luciferase activity of the wild PTEN 3'UTR + miR-181 d-5 p mimic group was significantly lower,difference was statistically significant(P<0.01).Conclusion:miR-181 d-5 p may inhibit the apoptosis of human colon cancer cells by targeting PTEN,which could be the potential target of clinical molecular targeted therapy for treating colon cancer.
Objective:To investigate the effect and mechanism of miR-181 d-5 p on apoptosis of human colon cancer cells.Methods:Human colon cancer cell line HCT116 was transfected with miR-181 d-5 p inhibitor.Apoptosis was detected by flow cytometry.Real-time PCR and Western blot were adopted to detect the protein expression of miR-181 d-5 p,PTEN,FAK,Bcl-2,Caspase-3 mRNA and PTEN,FAK,p-FAK,Bcl-2,cleaved Caspase-3 proteins.Targetscan analysis software online prediction and dual luciferase reporter gene system was used to analyze the regulatory relationship of miR-181 d-5 p and PTEN.Results:Compared with the negative control group,the expression of miR-181 d-5 p mRNA was down-regulated in the miR-181 d-5 p inhibitor group(P<0.01),the apoptotic cells were significantly increased(P<0.01),and the PTEN mRNA expression level was significantly upregulated(P<0.01);Bcl-2 mRNA expression level was significantly down-regulated(P<0.01),PTEN and cleaved Caspase-3 protein expression levels were significantly increased(P<0.01),pFAK,Bcl-2 protein expression levels were significantly down-regulated(P<0.01);the differences between the groups were not statistically significant(P>0.05).Targetscan software predicted that the3'UTR of PTEN gene contained the binding site of miR-181 d-5 p,and the dual luciferase results showed that compared with the mutant PTEN 3'UTR + miR-181 d-5 p mimic group,the luciferase activity of the wild PTEN 3'UTR + miR-181 d-5 p mimic group was significantly lower,difference was statistically significant(P<0.01).Conclusion:miR-181 d-5 p may inhibit the apoptosis of human colon cancer cells by targeting PTEN,which could be the potential target of clinical molecular targeted therapy for treating colon cancer.
引文
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[12]赵娟霞,龚勇,杨淑梅,等. MiR-181d靶向LCRG1调控喉癌细胞Hep2增殖与迁移的机制[J].临床与实验病理学杂志,2018,34(9):8-12.
[13]SAUNDERS I T,MIR H,NEERAJ KAPUR N. et al.Emodin inhibits colon cancer by altering BCL-2 family proteins and cell survival pathways[J]. Cancer Cell Int,2019,19(1):98.
[14]SVERCHINSKY D V,NIKOTINA A D,KOMAROVA E Y. et al. Etoposide-induced apoptosis in cancer cells can bereinforced by an uncoupled link between Hsp70and caspase-3[J]. Int J Mol Sci,2018,9(9):2519.
[15]WU B,LIANG J. Pectolinarigenin promotes functional recovery and inhibits apoptosis in rats following spinal cord injuries[J]. Exp Ther Med,2019,17(5):3877-3882.
[16]HUANG X,TANG F,WENG Z,et al. MiR-591 functions as tumor suppressor in breast cancer by targeting TCF4 and inhibits Hippo-YAP/TAZ signaling pathway[J]. Cancer Cell International,2019,19(1):108.
[17]詹晓娟,戴益琛.抑癌基因PTEN在大肠癌中的功能及机制[J].临床和实验医学杂志,2018,17(5):449-452.
[18]LV P C,JIANG A Q,ZHANG W M,et al. FAK inhibitors in Cancer,a patent review[J]. Expert Opinion on Therapeutic Patents,2017,28(1):1.
[19]WANG S,CHENG Z,YAGN X,et al. Effect of wild type PTEN gene on proliferation and invasion of multiple myeloma[J]. International Journal of Hematology,2010,92(1):83-94.
[20]CAVAZZONI A,MONICAS S L,ALFIERI R,et al. Enhanced efficacy of AKT and FAK kinase combined inhibition in squamous cell lung carcinomas with stable reduction in PTEN[J]. Oncotarget,2017,8(32):53068-53083.
[2]WANG B,LU F Y,SHI R H,et al. MiR-26b regulates5-FU-resistance in human colorectal cancer via down-regulation of Pgp[J]. Am J Cancer Res,2018,8(12):2518-2527.
[3]XU T,ZHANG K,SHI J,et al. MicroRNA-940 inhibits glioma progression by blocking mitochondrial folate metabolism through targeting of MTHFD2[J]. Am J Cancer Res,2019,9(2):250-269.
[4]SHEN L M,SONG Z W,HUA Y,et al. MiR-181d-5p promotes neurite outgrowth in PC12 Cells via PI3K/Akt pathway[J]. Cns Neuroscience&Therapeutics,2017,23(11):894.
[5]ZHANG G,LIU D,LONG G,et al. Downregulation of microRNA-181d had suppressive effect on pancreatic cancer development through inverse regulation of KNAIN2[J]. Tumour Biology the Journal of the International Society for Oncodevelopmental Biology&Medicine,2017,39(4):1-10.
[6]GUO X,ZHU Y,HONG X,et al. MiR-181d and c-mycmediated inhibition of CRY2 and FBXL3 reprograms metabolism in colorectal cancer[J]. Cell Death&Disease,2017,8(7):e2958.
[7]BAROUDIi O,BENAMMER-ELGAAIED A. Involvement of genetic factors and lifestyle on the occurrence of colorectal and gastric cancer[J]. Critical Reviews in Oncology,2016,(107):72-81.
[8]宋涛,王铎,肖嘎.卡培他滨联合奥沙利铂术前新辅助化疗对结肠癌的临床疗效[J].贵州医科大学学报,2017,42(9):1107-1110.
[9]耿培亮.纤维粘连蛋白EDA片段调控结肠癌SW480细胞增殖、凋亡和糖酵解及其机制研究[D].重庆:第三军医大学,2011.
[10]ZHOU W,LI X,LIU F,et al. MiR-135a promotes growth and invasion of colorectal cancer via metastasis suppressor 1 in vitro[J]. Acta Biochim Biophys Sin(Shanghai),2012,44(10):838-846.
[11]臧晓方,李骎,王卫国,等. MiR-181a靶向RASSF1A促进骨肉瘤细胞的生长和转移[J].中南大学学报(医学版),2016,41(8):789-795.
[12]赵娟霞,龚勇,杨淑梅,等. MiR-181d靶向LCRG1调控喉癌细胞Hep2增殖与迁移的机制[J].临床与实验病理学杂志,2018,34(9):8-12.
[13]SAUNDERS I T,MIR H,NEERAJ KAPUR N. et al.Emodin inhibits colon cancer by altering BCL-2 family proteins and cell survival pathways[J]. Cancer Cell Int,2019,19(1):98.
[14]SVERCHINSKY D V,NIKOTINA A D,KOMAROVA E Y. et al. Etoposide-induced apoptosis in cancer cells can bereinforced by an uncoupled link between Hsp70and caspase-3[J]. Int J Mol Sci,2018,9(9):2519.
[15]WU B,LIANG J. Pectolinarigenin promotes functional recovery and inhibits apoptosis in rats following spinal cord injuries[J]. Exp Ther Med,2019,17(5):3877-3882.
[16]HUANG X,TANG F,WENG Z,et al. MiR-591 functions as tumor suppressor in breast cancer by targeting TCF4 and inhibits Hippo-YAP/TAZ signaling pathway[J]. Cancer Cell International,2019,19(1):108.
[17]詹晓娟,戴益琛.抑癌基因PTEN在大肠癌中的功能及机制[J].临床和实验医学杂志,2018,17(5):449-452.
[18]LV P C,JIANG A Q,ZHANG W M,et al. FAK inhibitors in Cancer,a patent review[J]. Expert Opinion on Therapeutic Patents,2017,28(1):1.
[19]WANG S,CHENG Z,YAGN X,et al. Effect of wild type PTEN gene on proliferation and invasion of multiple myeloma[J]. International Journal of Hematology,2010,92(1):83-94.
[20]CAVAZZONI A,MONICAS S L,ALFIERI R,et al. Enhanced efficacy of AKT and FAK kinase combined inhibition in squamous cell lung carcinomas with stable reduction in PTEN[J]. Oncotarget,2017,8(32):53068-53083.