| Research background and purpose:Colorectal cancer(CRC)is the third most common malignancy worldwide,and metastasis is responsible for the majority of CRC-related mortality.The plasticity of epithelial and mesenchymal features involves the epithelial-mesenchymal transition(EMT)and the reverse process,mesenchymal-epithelial transition(MET).Although there still is debate concerning the role of EMT in the metastasis of certain types of cancers,EMT is generally considered to be a crucial step in the metastatic cascades of many types of malignancies,including CRC.The cancer stem cell(CSC)hypothesis,in which a subpopulation of cancer cells that exhibit stem-like features sustain tumor formation,metastasis,and resistance to therapy,has been proposed to explain the functional heterogeneity and carcinogenesis of cancer.Several studies have investigated the proteincoding genes and their products that participate in the “stemness” maintenance and tumorigenicity of CRC progenitor cells.Thus,it is important to identify the regulatory mechanisms and signaling pathways involved in CRC progenitor cells to develop novel reagents to target the refractory CRC progenitor cell population.Nanog is a transcription factor(TF)that is involved in the self-renewal of embryonic stem cells(ESCs)and is a critical factor for the maintenance of the undifferentiated state of pluripotent cells.Extensive data in the literature show that the Nanog gene is aberrantly expressed during the development of malignancy in cancer cells.Overexpression of Nanog in colorectal cancer(CRC)cells results in a more mesenchymal phenotype with the qualities required for migration to secondary sites,and clinically,it is associated with lower recurrence-free survival.Our preliminary study showed that(1)mi R-200 s was down-regulated in human colorectal cancer samples and colon cancer stem cells;(2)Nanog m RNA was abnormally elevated in human colorectal cancer samples and the increased Nanog expression was accompanied by EMT occurrence in colorectal cancer cells.There are a lot of reports in literature have shown that the mi R-200 family is involved in cancer stem cell function in collaboration with the ZEB family and plays a key role in regulating the development of various tumor metastases by controlling the EMT process of tumor cells.The balance between epithelial mesenchymal transition and its reversal process,mesenchymal transition(MET),is an accurate adjustment of the fate of tumor cells,and plays an important role in regulating the development of tumor cells as modulators in cancer invasion and metastasis.Recent data show that cancer stem cells(CSCs)with a high capacity for metastasis usually exhibit epithelial-mesenchymal transition.This phenomenon is defined by the loss of epithelial morphology and the acquisition of a mesenchymal phenotype.In addition,several micro RNAs(mi RNAs),predominantly mi R-200,regulate EMT by forming double-negative feedback loops.Nanog needs the co-operation of other factors or molecules to potentiate tumorigenic activity and modulate malignant transformation and metastasis.It is known that EMT plays an essential role in cancer metastasis,and restoration of the MET program should efficiently slow dissemination of tumor cells.The mi R-200 family is important in specifying an epithelial or a mesenchymal state not only during embryonic development but also in tumorigenesis,and these mi RNAs play important regulatory roles in the plasticity between epithelial and mesenchymal features.At present,the molecular transcriptional mechanism of the mi R-200 family is largely unknown.In this report,we demonstrated that enforced expression of Nanog in colon cancer cells was related to mi RNA-200 family-related repression of colon cancer cells and identified that Nanog can transcriptionally repress mi R-200 family member expression.These results indicated that the Nanog/mi R-200 axis can modulate plasticity between epithelial and mesenchymal features in colon cancer cells.They also suggest that Nanogmi R-200 axis may be good therapeutic targets for the control of CRC,especially for metastasis.Research methods:1.Sixty-two patients with colorectal cancer who were scheduled for either colonoscopy or surgical resection were enrolled in this study.Cancerous samples were collected by biopsy or from the resected tissue.The fresh samples were immediately stored in liquid nitrogen for further extraction of total m RNA,protein or nuclear protein.The Caco2,LS174 T,Lovo,HT-29,HCT116,SW480 and SW620 human colonic adenocarcinoma cell lines were obtained from the Chinese ATCC.Colon cancer cells were cultured for extraction of total m RNA and protein.2.Sixty-two samples of colorectal cancer tissues and their adjacent normal tissues,and seven colon cancer cell lines were reverse-transcripted for future quantitation of Nanog m RNA,and mi R-200 c and mi R-200 b expression levels using quantitative real-time PCR(SYBR)analysis.And the correlation analysis between Nanog and mi R-200 c and mi R-200 b was performed using Pearson coefficient.Western blot was used to detect Nanog protein in seven colon cancer cell lines.3.Lentivirus particles expressing Nanog were produced by GenePharma Co.,Ltd.SW480 and LS174 T cells were transfected with the lentivirus particles using the LV5 vector with a Nanog CDS region insert.Cells that were stably transfected with GFP were sorted using flow cytometry or isolated using puromycin selection.The HT-29 and HCT116 cells seeded in 6-well plates were transfected with Nanog si RNA,as well as the scrambled controls,using the Fu GENE HD transfection reagent.The effeciency of overexpression or interference of Nanog was verified at the nucleic acid and protein levels.4.The expression of mi R-200 c and mi R-200 b in Nanog over-expressed cell lines and si RNA-Nanog cells were detected by real time q PCR(SYBR).5.The influence of Nanog on cell proliferation was analyzed by MTT assay.The effect of Nanog on cell migration and invasion was analyzed by Transwell chamber in Nanog enforced over-expressed cells,Nanog interfered cells and their corresponding control cells.6.The effect of Naong on the vivo tumorigenesis of nude mice was analyzed by transplanted nude mice subcutaneously.7.The changes of EMT biomarkers(E-cadherin,Zeb1,Zeb2 and N-cadherin)in Nanog enforced expressed colon cancer cells and Nanog interfered cells and the corresponding control cells were detected by real-time q PCR and Western-blot.8.Nanog transcription binding sites of mi R-200 c and mi R-200 b promoter regions were predicted by bioinformatics and software of transcription factor binding sites,and the eight truncated mutants of mi R-200 c and mi R-200 b promoters containing dual luciferase reporter gene were constructed.9.The effect of Nanog on the transcriptional activity of mi R-200 c and mi R-200 b promoters was analyzed by dual luciferase reporter assay compared with their controls.10.Ch IP assays were performed using a Ch IP Assay Kit according to the manufacturer’s instructions.Soluble chromatin was prepared from the cultured cells.The chromatin was immunoprecipitated with an anti-Nanog antibody.11.The p GL3-basic vector containing the mi R-200 c promoter(-767 to +44bp)with one Nanog potential binding site(agg AATGggc)and the pGL3-basic vector containing the mi R-200 b promoter(-640 to +120bp)and one Nanog potential binding site(gccCATTggg)served as a wild-type construct for the generation of p GL3-767/+44MUT construct,which harbors mutations in the Nanog potential binding site(agg CCTGggc)and p GL3-640/+120MUT construct,which harbors mutations in the Nanog potential binding site(gcc CAGGggg)via PCR-based site-directed mutagenesis.12.The effect of mi R-200 s mimics on the proliferation,migration and invasion of colon cancer cell lines with Nanog over-expression were confirmed by MTT assay and Transwell chamber assay.13.Real-time q PCR and Western-blot were used to detect the effect of mi R-200 s mimics on EMT of colon cancer cells with Nanog over-expression.Result:1.Correlation between Nanog,mi R-200 c and mi R-200 b expression levels in colon cancer(CRC)cell lines and CRC samples.Nanog m RNA expression was negatively correlated with mi R-200 c and mi R-200 b expression in colon cancer cell lines.Nanog m RNA expression was negatively correlated with mi R-200 c and mi R-200 b expression in human colorectal cancer samples.2.Over-expression or interference of Nanog in CRC cell lines was established successfully.Nanog expression in colon cancer cell lines ranging from high to low levels were SW620,HCT116,HT-29,Caco2,LS174 T,SW480 and Lo Vo,respectively.Then,we established Nanog-enforced LS174 T and SW480 stable expression strain successfully.Nanog relative m RNA levels in Lv-Nanog/SW480 or Lv-Nanog/LS174 T cells increased about 56-fold or 17-fold compared their controls.There was a significant increase of Nanog protein levels in Lv-Nanog/SW480 or Lv-Nanog/LS174 T cells compared their controls.The mi R-200 c relative levels in Lv-Nanog/SW480 or Lv-Nanog/LS174 T cells decreased 14-fold or 4-fold compared their controls and mi R-200 b relative levels in LvNanog/SW480 or Lv-Nanog/LS174 T cells decreased 3-fold or 3.7-fold compared their controls.Knockdown of Nanog using si RNA targeting method indicated that Nanog relative m RNA levels in Nanog interfered HT-29(si RNA-Nanog/HT-29)or HCT116(si RNA-Nanog/HCT116)cells decreased about 8-fold or 11-fold compared their controls using the most efficient one of six si RNA sequences.There was a significant decrease of Nanog protein levels in si RNA-Nanog/HT-29 or si RNA-Nanog/HCT116 cells compared their controls.3.Changed Nanog expression in colon cancer cells altered mi R-200 c and mi R-200 b expression.The mi R-200 c relative levels in si RNA-Nanog/HT-29 or si RNANanog/HCT116 cells increased 8-fold or 5-fold compared their controls and mi R-200 b relative levels in si RNA-Nanog/HT-29 or si RNA-Nanog/HCT116 cells increased 4-fold or 3.8-fold compared their controls.The results provided the first evidence that Nanog could affect mi R-200 c and mi R-200 b expression in colon cancer cells.4.Nanog overexpression in colon cancer cells affects their cellular behavior and in vivo tumorigenicity.The proliferation rates of Nanog/SW480 or Nanog/LS174 T from days 1 to 4 after seeding were significantly higher than their control cells.Nanog/SW480 or Nanog/LS174 T cells migrated and invaded significantly more than the mock transfectants.The volume and mass of in vivo developed tumors in athymic nude mice using Lv-Nanog/SW480 or Lv-Nanog/LS174 T cells were significantly higher than control.5.Nanog expression in colon cancer cells induced EMT.Compared with their control cells,Lv-Nanog/SW480 or Lv-Nanog/LS174 T cells exhibited decreased m RNA level of E-cadherin,which is considered to be an epithelial marker,and increased m RNA levels of ZEB1,ZEB2 and N-cadherin,which are considered to be mesenchymal markers.The protein level of the epithelial marker E-cadherin was decreased.However,the mesenchymal markers N-cadherin,ZEB1 and ZEB2 were increased in both LvNanog/SW480 and Lv-Nanog/LS174 T cells compared with their control cells.The m RNA level of E-cadherin was increased,but the ZEB1,ZEB2 and N-cadherin m RNA levels were reduced in the si RNA-Nanog/HT-29 and si RNA-Nanog/HCT116 cells compared with their control cells.There was an increase in E-cadherin protein level and a decrease in ZEB1,ZEB2 and N-cadherin protein levels in the si RNA-Nanog/HT-29 and si RNANanog/HCT116 cells compared with their control cells.These data provide the first evidence that Nanog enforced expression in colon cancer cells results in EMT characterized by the gain of mesenchymal markers and the loss of an epithelial marker.However,the exact mechanism(s)of how Nanog induced EMT of colon cancer cells remains unclear.6.Nanog repressed mi R-200 family member transcription.Compared with control cells,significantly lower luciferase activity was observed in both Lv-Nanog/SW480 and Lv-Nanog/LS174 T cells when using the pGL3-mi R-200 c promoter encompassing-1482 to +44,-1440 to +44 and-767 to +44 relative to the putative TSS.These results indicated that multiple elements located between-767 and-671 contributed to the activity of the mi R-200 c promoter in Lv-Nanog/SW480 and Lv-Nanog/LS174 T cells relative to their control cells.The findings suggest that Nanog functions as a transcriptional repressor for mi R-200 c expression in colon cancer cells.Compared with control cells,significantly lower luciferase activity was observed in both Lv-Nanog/SW480 and Lv-Nanog/LS174 T cells when using the p GL3-mi R-200 c promoter encompassing-1574 to +120,-1088 to +120 and-640 to +44 relative to the putative TSS.These results indicated that multiple elements located between-640 and-585 contributed to the activity of the mi R-200 b promoter in Lv-Nanog/SW480 and Lv-Nanog/LS174 T cells relative to their control cells.The findings suggest that Nanog functions as a transcriptional repressor for mi R-200 b expression in colon cancer cells.7.Nanog bound to the promixal promoters of mi R-200 family members.The binding of Nanog to the proximal promoters of the mi R-200 c and mi R-200 b genes was determined compared with negative controls with clear evidence of Nanog binding to these proximal promoters based on Ch IPs 1–3 experiments and Ch IPs1–6 experiments.Experiment Ch IP3 indicated that the binding of Nanog to its potential binding site in the mi R-200 c promoter was increased in both Lv-Nanog/SW480 and Lv-Nanog/LS174 T cells compared with control cells.Similarly,experiments Ch IP 4 indicated that the binding of Nanog to its potential binding site in the mi R-200 b promoter was increased in both LvNanog/SW480 and Lv-Nanog/LS174 T cells compared with control cells.8.Identification of the Nanog binding sites in the promixal promoters of mi R-200 gene.We chose to use the mi R-200 c promoter-Luc construct(-767 to +44)in which there are one potential Nanog binding site(agg AATGggc)to produce its mutants(agg CCTGggc)(-767/+44MUT).Cells expressing the luciferase reporter driven by the wild-type mi R-200 c promoter-Luc construct(p GL3--767/+44)had a significantly lower levels of luciferase activity in both Lv-Nanog/SW480 and Lv-Nanog/LS174 T cells compared with their controls.The construct with a mutation of potential Nanog binding site(p GL3--767/+44MUT)showed a similar luciferase activity in both Lv-Nanog/SW480 and Lv-Nanog/LS174 T cells compared with their controls.Similarly,Cells expressing the luciferase reporter driven by the wild-type mi R-200 b promoter-Luc construct(p GL3--640/+120)had a significantly lower levels of luciferase activity in both Lv-Nanog/SW480 and Lv-Nanog/LS174 T cells compared with their controls.The construct with a mutation of potential Nanog binding site(p GL3--640/+120MUT)showed a similar luciferase activity in both Lv-Nanog/SW480 and Lv-Nanog/LS174 T cells compared with their controls.These experiments indicates that the potential Nanog binding site in the proximal promoter of mi R-200 c and mi R-200 b functions as a binding sites for Nanog and Nanog binding transcriptionally represses mi R-200 c and mi R-200 b c expression.9.mi R-200 c or mi R-200 b mimics led to the partial reversal of EMT in Nanog enforced expressed colon cancer cells.To confirm Nanog induced EMT by its repression of mi R-200 c or mi R-200 b,we used a mi R-200 c or mi R-200 b mimics to the transfect LvNanog/SW480 and Lv-Nanog/LS174 T cells.An increase in the levels of the epithelial marker(E-cadherin)and a decrease in the mesenchymal markers(N-cadherin,ZEB1 and ZEB2)were observed in both the mi R-200 c mimics transienly transfected LvNanog/SW480 and Lv-Nanog/LS174 T cells and mi R-200 b mimics transienly transfected Lv-Nanog/SW480 and Lv-Nanog/LS174 T cells.The results of the migration and invasion assay indicated that transfection of Lv-Nanog/SW480 and Lv-Nanog/LS174 T cells with either a mi R-200 c or mi R-200 b mimics led to a decrease in their proliferation,migration and invasiveness.These data provide further evidence of how Nanog promotes the mi R-200c/b-dependent EMT in colorectal cancer cells. |
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