Effect Of RNA Interference Of Nanog On Human Colorectal Cancer Stem Cells

Colorectal cancer is one of the most common malignant tumors of digestive tract in the world,with mortality rate at the third place in tumor-related death.The incidence and mortality rate of colorectal cancer have increased in our country year by year trend.So far,surgical resection combined with radiotherapy and chemotherapy remains the main treatment for colorectal cancer,but patients still appear postoperative tumor recurrence and metastasis,seriously endangering the health and life of patients.Therefore,it is urgently required to develop more suitable and effective method for treatment of colorectal cancer.In recent years,with the deep research of malignant tumors and the putting forward of cancer stem cells(CSC)theory,it has been found that there are very few cancer stem cells with "stem cell properties" like self-renewal and multiple differentiation potential,which are closely related to tumor development,metastasis and recurrence.The CSC theory proposes that CSCs are the initiating cells of tumors,which may be the origin of malignant tumors and only targeting treatment of CSCs may eventually cure the tumor.Stemness factors play an important role in self-renewal and differentiation of stem cells,in which the stem cell transcription factor Nanog not only plays a vital role in maintaining the pluripotency and self-renewal of embryonic stem cells,but also expresses abnormally in multiple tumors.Existing studies show that the high expression of Nanog is closely related to the poor prognosis of tumors,suggesting that Nanog may promote the development of tumor by maintaining the stemness properties of cancer stem cells.Therefore,in order to explore the biological effect of Nanog on colorectal cancer stem cells,in this study,Ep CAM and CD44 were used as markers to screen human colorectal cancer stem cells(CCSC)of Ep CAM+CD44+ HCT-116 from human colorectal cancer cell line HCT-116 by immunomagnetic beads.RNA interference(RNAi)was used to silence the expression of Nanog in colorectal cancer stem cells and we explored its effect on the biological behavior of colorectal cancer stem cells.Results show that Ep CAM+CD44+ HCT116 were suspended and cultured in serum-freeDMEM/F12 medium,and formed tumor microspheres from single cells gradually,which then re-adhered and differentiate after serum was added to the medium.The Real-time PCR analysis shows that the expression of Nanog in Ep CAM+CD44+ HCT-116 cells was significantly higher than that of unsorted HCT-116 cells(P<0.01);after transfection of Nanog si RNA for 48 h,the expression of Nanog was significantly decreased at both m RNA level and protein level(P<0.01,P<0.05).The MTS proliferation assays show that Nanog silencing could significantly inhibit the proliferation of Ep CAM+CD44+ HCT-116(P<0.01);the Annexin V-FITC/PI double staining method distinguishes the early and late apoptotic cells from the perspective of the integrity of the cell membrane,results show that the percentage of early and late apoptotic cells increased after Nanog gene silencing in Ep CAM+CD44+ HCT-116;the JC-1 method reflects the apoptosis from the point of view of cell membrane potential,results show that the cell membrane potential was significantly decreased after Nanog gene silencing and the proportion of apoptotic cells increased;the Real-time PCR analysis shows that the m RNA expression level of anti-apoptotic gene Bax in Ep CAM+CD44+ HCT-116 was significantly decreased(P<0.05)after Nanog gene silencing,while the m RNA expression level of pro-apoptotic genes Bax and Caspase-3 was significantly increased(P<0.01);and the western blot results show that the expression of cleaved-caspase3 protein in Ep CAM+CD44+ HCT-116 was significantly increased after Nanog gene silencing(P<0.05).The transwell chamber results show that silencing Nanog gene significantly inhibited the invasion of Ep CAM+CD44+ HCT-116(P<0.01);meanwhile,the Real-time PCR analysis shows that the m RNA expression level of pro-invasion genes MMP-2 and MMP-9 were significantly decreased after Nanog gene silencing(P<0.05,P<0.01),while the m RNA expression level of anti-invasive gene Timp-1 was significantly increased(P<0.01).The tumor xenograft assays show that,the tumor size and tumor weight of Nanog si RNA group were significantly lower than those of NC si RNA and mock groups(P<0.05),and the mice survival time of Nanog si RNA group was significantly longer than those of NC si RNA and mock groups(P <0.05).Taken together,our studies reveal that Nanog silencing can suppress the proliferation and invasion of colorectal cancer stem cells,and induce apoptosis,as well as reduce the tumorigenic capacity of colorectal cancer stem cells,which provide experimental basis fortargeted therapy of colorectal cancer stem cells.