siRNA靶向干扰PTPRO对结直肠癌HCT116细胞增殖与侵袭影响
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摘要
目的结直肠癌是常见的消化道恶性肿瘤之一,近年来结直肠癌发病率上升趋势,已仅次于肺癌。本研究通过应用小分子干扰RNA(small interfering RNA,siRNA)技术,靶向干扰膜结合受体型蛋白酪氨酸磷酸酶O(protein tyrosine phosphatase-receptor-type O,PTPRO),观察其对结直肠癌HCT116细胞增殖、侵袭的影响。方法培养结直肠癌HCT116细胞,随机分为实验组、阴性对照组、空白组,利用脂质体2000将PTPRO-siRNA体外转染至实验组细胞内、将无关序列NC-siRNA体外转染至阴性对照组细胞内,空白组不予转染。转染48h后,利用荧光定量PCR和蛋白质印迹法分别检测各组PTPRO的mRNA和蛋白表达情况;另取细胞,同法分组、转染,采用CCK-8法、Transwell小室细胞侵袭实验检测细胞的增殖、侵袭能力的变化。结果实验组、阴性对照组和空白组PTPRO mRNA的相对表达量分别为(0.112±0.031)、(0.776±0.077)和(0.972±0.074),差异有统计学意义,F=247.344,P<0.001。实验组、阴性对照组和空白组PTPRO的相对表达量分别为(0.150±0.045)、(0.366±0.050)和(0.434±0.049),差异有统计学意义,F=47.391,P<0.001。各组细胞增殖能力随着转染时间的延长逐渐提高,且实验组转染细胞增殖能力均高于同期阴性对照组及空白组,均P<0.05。实验组穿膜细胞数(104.400±9.978)显著高于阴性对照组的(63.300±6.513)及空白组的(62.100±7.317),差异有统计学意义,F=266.970,P<0.001。结论 siRNA靶向干扰PTPRO对结直肠癌HCT116细胞增殖及侵袭有促进作用,PTPRO可能是结直肠癌的抑癌因子之一,具有抑制肿瘤生长、侵袭的作用。
OBJECTIVE Colorectal cancer is one of the most common malignant tumors of digestive tract.In recent years,the incidence of colorectal cancer has increased,which is second only to lung cancer.The study is to investigate the effects of siRNA-mediated inhibition of the membrane-bound receptor protein tyrosine phosphatase O(PTPRO)expression on cell proliferation and invasion in human colorectal cancer cell line HCT116.METHODS The colorectal cancer cell line HCT116 were cultured and randomly divided into three groups:the experimental group,the negative control group and the blank group.PTPRO-siRNA was transfected into the experimental group cells by Lipofectamine○R2000 in vitro,the irrelevant sequence NC-siRNA was transfected into negative control group in vitro,and the blank group was not transfected.After transfection for 48 h,the expression of PTPRO-mRNA and PTPRO were detected by real-time PCR and Western blot.Other cells were separated and transfected in the same way.The changes of cell proliferation and invasive ability were detected by CCK-8 and Transwell cell invasion assay.RESULTS The relative expression levels of PTPRO mRNA in the experimental group,the negative control group and the blank group were respectively(0.112±0.031),(0.776±0.077)and(0.972±0.074),and the difference was statistically significant,F=247.344,P<0.001.The relative expression levels of PTPRO in the experimental group,the negative control group and the blank group were respectively(0.150±0.045),(0.366±0.050)and(0.434±0.049),and the difference was statistically significant,F=47.391,P<0.001.The cells proliferation ability of each group increased gradually with the prolongation of transfection time,and the proliferative ability of the transfected cells in the experimental group was higher than that in the negative control group and the blank group(P<0.05).The cells invasive ability of the experimental group was significantly higher than that of the negative control group and the blank group(P<0.05).CONCLUSIONS SiRNA-mediated inhibition of PTPRO can promote the proliferation and invasion of colorectal cancer HCT116 cells.PTPRO may be one of the tumor suppressor factors of colorectal cancer,which has the function of inhibiting tumor growth and invasion.
OBJECTIVE Colorectal cancer is one of the most common malignant tumors of digestive tract.In recent years,the incidence of colorectal cancer has increased,which is second only to lung cancer.The study is to investigate the effects of siRNA-mediated inhibition of the membrane-bound receptor protein tyrosine phosphatase O(PTPRO)expression on cell proliferation and invasion in human colorectal cancer cell line HCT116.METHODS The colorectal cancer cell line HCT116 were cultured and randomly divided into three groups:the experimental group,the negative control group and the blank group.PTPRO-siRNA was transfected into the experimental group cells by Lipofectamine○R2000 in vitro,the irrelevant sequence NC-siRNA was transfected into negative control group in vitro,and the blank group was not transfected.After transfection for 48 h,the expression of PTPRO-mRNA and PTPRO were detected by real-time PCR and Western blot.Other cells were separated and transfected in the same way.The changes of cell proliferation and invasive ability were detected by CCK-8 and Transwell cell invasion assay.RESULTS The relative expression levels of PTPRO mRNA in the experimental group,the negative control group and the blank group were respectively(0.112±0.031),(0.776±0.077)and(0.972±0.074),and the difference was statistically significant,F=247.344,P<0.001.The relative expression levels of PTPRO in the experimental group,the negative control group and the blank group were respectively(0.150±0.045),(0.366±0.050)and(0.434±0.049),and the difference was statistically significant,F=47.391,P<0.001.The cells proliferation ability of each group increased gradually with the prolongation of transfection time,and the proliferative ability of the transfected cells in the experimental group was higher than that in the negative control group and the blank group(P<0.05).The cells invasive ability of the experimental group was significantly higher than that of the negative control group and the blank group(P<0.05).CONCLUSIONS SiRNA-mediated inhibition of PTPRO can promote the proliferation and invasion of colorectal cancer HCT116 cells.PTPRO may be one of the tumor suppressor factors of colorectal cancer,which has the function of inhibiting tumor growth and invasion.
引文
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[16]Espejo R,Jeng YJ,Paulucci-Holthauzen A,et al.PTP-PEST targets a novel tyrosine site in p120catenin to control epithelial cell motility and Rho GTPase activity[J].J Cell Sci,2014,127(3):497-508.
[2]Murata Y,Mori M,Kotani T,et al.Tyrosine phosphorylation of R3subtype receptor-type protein tyrosine phosphatases and their complex formations with Grb2or Fyn[J].Genes Cells,2010,15(5):513-524.
[3]Liao WH,Cheng CH,Hung KS,et al.Protein tyrosine phosphatase receptor type O(Ptpro)regulates cerebellar formation during zebrafish development through modulating Fgf signaling[J].Cell Mol Life Sci,2013,70(13):2367-2381.
[4]Asbagh LA,Vazquez I,Vecchione L,et al.The tyrosine phosphatase PTPRO sensitizes colon cancer cells to anti-EGFR therapy through activation of SRC-mediated EGFR signaling[J].Oncotarget,2014,5(20):10070-10083.
[5]Lau KH,Stiffel V,Amoui M.An osteoclastic protein-tyrosine phosphatase regulates the beta3-integrin,syk,and shp1signaling through respective src-dependent phosphorylation in osteoclasts[J].Am J Physiol Cell Physiol,2012,302(11):C1676-1686.
[6]Zhang W,Hou J,Wang X,et al.PTPRO-mediated autophagy prevents hepatosteatosis and tumorigenesis[J].Oncotarget,2015,6(11):9420-9433.
[7]Li SY,Li R,Chen YL,et al.Aberrant PTPRO methylation in tumor tissues as a potential biomarker that predicts clinical outcomes in breast cancer patients[J].BMC Genet,2014,15:67.
[8]Mori Y,Yin J,Sato F,et al.Identification of genes uniquely involved in frequent microsatellite instability colon carcinogenesis by expression profiling combined with epigenetic scanning[J].Cancer Res,2004,64(7):2434-2438.
[9]Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2)6:115-132.
[10]Lee CHA,Kong JC,Ismail H,et al.Systematic review and metaanalysis of objective assessment of physical fitness in patients undergoing colorectal cancer surgery[J].Dis Colon Rectum,2018,61(3):400-409.
[11]Fang W.Chemotherapy in patient with colon cancer after renal transplantation:a case report with literature review[J].Medicine(Baltimore),2018,97(5):e9678.
[12]Chau R,Jenkins MA,Buchanan DD,et al.Determining the familial risk distribution of colorectal cancer:A data mining approach[J].Fam Cancer,2016,15(2):241-251.
[13]Massague J,Obenauf AC.Metastatic colonization by circulating tumour cells[J].Nature,2016,529(7586):298-306.
[14]Hsu SH,Motiwala T,Roy S,et al.Methylation of the PTPRO Gene in Human Hepatocellular Carcinoma and Identification of VCP as Its Substrate[J].J Cell Biochem,2013,114(8):1810-1818.
[15]Ming F,Sun Q.Epigenetically silenced PTPRO functions as a prognostic marker and suppressor in human lung squamous cell carcinoma[J].Mol Med Rep,2017,16(1):746-754.
[16]Espejo R,Jeng YJ,Paulucci-Holthauzen A,et al.PTP-PEST targets a novel tyrosine site in p120catenin to control epithelial cell motility and Rho GTPase activity[J].J Cell Sci,2014,127(3):497-508.