| Background and objectiveLiver cancer is one of the most common tumors in the world.It is characterized by high malignancy,strong invasiveness,rapid progression and poor prognosis.However,the reason of hepatocarcinogenesis remains unclear.There are many theories have been put forward for the origin of liver cancer,the most important one is tumor initiating cells(TICs)theory.Tumor initiating cells,also named cancer stem cells(CSCs),are a small subset of cells in the tumor bulk,with the characteristics of tumor cells and stem cells.TICs can activate several signaling pathways,possess the potential of self-renewal and differentiation,promote tumor development,metastasis and recurrence,also liver TICs are related to drug resistance.However,the mechanism of liver TICs self renewal remains to be further explored.The regulators of TICs are obscure.Treatment regemin for liver cancer includes transplantation,surgical resection,interventional therapy,targeted drugs,chemotherapy,radiotherapy,im-munotherapy and so on,but the overall survival is still unsatisfied.Current treatment rarely target TICs,which may be the main reason of tumor recurrence,metastasis and drug resistance.Adenomatous polyposis coli(APC)is a tumor suppressor gene as an antagonist of Wnt/p-catenin signaling and plays a pivotal role in tumorigenesis and Wnt/β-catenin activation.However,how to regulate the expression of APC is not clear.LncRNAs are non-coding RNAs with length of more than 200 nucleotides,can regulate gene expression through multiple layers and play a key role in multiple diseases including tumor.Many lncRNAs are involved in the activation of Wnt/β-catenin signaling pathway.However,whether lncRNAs participate in APC expression and regulate Wnt/β-catenin remain unclear.Here,we found a long non-coding RNA,LncAPC,located near the APC locus.For the first time,our study comprehensively analyzed LncAPC expression and function in liver cancer and CSCs,explored the effect of LncAPC on TICs self-renewal and Wnt/β-catenin signaling pathway.Furthermore,the molecular mechanism of LncAPC was studied in tumorigenesis.Our work may provide a new target for the treatment of TICs.This study is divided into five parts.Part One:Study of lncRNA selection and expression;Part Two:Functions of LncAPC;Part Three:The effect of LncAPC on Wnt/p-catenin signaling pathway.Part Four:Molecular mechanism of LncAPC functions.Part Five:LncAPC-EZH2-APC can be targeted for liver TICs elimination.Part One Study of IncRNA selection and expressionMethods:1.APC protein level in peri-tumor tissue(P),early hepatocellular carcinoma(eHCC)and advanced hepatocellular carcinoma(aHCC)samples were examined through Western blotting.Liver TICs were enriched using(Fluorescence Activated Cell Sorting)FACS with tumor marker CD 133,and expression of APC was detected through Realtime qPCR.Oncospheres and non-spheres were crushed and subjected into Western blotting for APC examination.2.Five lncRNAs were found near APC locus through UCSC website.3.LncRNAs were silenced with antisense oligos(ASOs),followed by APC examination.ASO knockdown efficiency and the expression levels of APC were examined by.Real-time qPCR.The target gene lncRNA was determined.4.Peri-tumor,early HCC(eHCC)and advanced HCC(aHCC)samples were used for Realtime qPCR and in situ hybridization to detect LncAPC expression.5.Liver TICs were enriched by FACS with TIC surface marker CD133 or sphere formation assay,the expression levels of LncAPC were detected through Realtime qPCR.6.Fluorescence in situ hybridization(FISH)was used to show LncAPC in oncospheres and non-spheres.Results:1.Results of Western blotting showed that APC expression in aHCC was lower than eHCC and peri-tumor tissues.These results were confirmed by Realtime qPCR test of liver TICs through FACS with tumor marker CD133(P<0.05).In addition,Western blotting results of APC examination in oncospheres and non-spheres support the conclusion.2.Five lncRNAs were found near the APC locus through UCSC website.3.Results of Realtime qPCR showed expression of lncRNAs were silenced after ASO knocking down in liver cancer cells and samples(P<0.05).Furthermore,the expression of APC in Lncl silenced cells increased(P<0.05),and no significant change was found in Lnc2-Lnc5 silenced cells.Therefore,we fosuced on Lncl and named it LncAPC.4.The ratio of LncAPChigh cells and lncAPC photon intensity were higher in aHCC than eHCC and peri-tumor tissues by Realtime qPCR(P<0.05).The results were aslo confirmed by in situ hybridization,and LncAPC was mainly located in nucleus.The correlation between LncAPC and APC expression level was negative.5.Liver TICs were enriched using FACS with surface marker CD 133,the expression levels of LncAPC was higher through Realtime qPCR in CD133+ cells than CD133’cells(P<0.05).Also,the expression levels of LncAPC were higher in oncosphere cells than non-sphere cells.6.Expression level of LncAPC in oncospheres was higher than non-spheres by Fluorescence in situ hybridization(FISH).Part Two Functions of LncAPCMethods:1.LncAPC silenced cells were established using ASOs and the expression of LncAPC was tested through Northern blotting,followed by sphere formation assays.2.LncAPC silenced cells were used for serial sphere formation assays.Four passages of sphere formation assays were performed.3.LncAPC silenced cells were injected subcutaneously into BALB/c nude mice,followed by measurements of tumor weight for xenograft growth assays.4.Different numbers of LncAPC silenced cells were injected subcutaneously into BALB/c nude mice,and the ratios of tumor-free mice were calculated for tumor initiating assays.5.LncAPC overexpressed cells were established using lentiviral vectors and the expression of LncAPC was tested through Realtime qPCR,followed by sphere formation assays.6.LncAPC overexpressed cells were injected subcutaneously into BALB/c nude mice,followed by measurements of tumor weight for xenograft growth assays.7.Different numbers of LncAPC overexpressed cells were injected subcutaneously into BALB/c nude mice,and the ratios of tumor-free mice were calculated for tumor initiating assays.Results:1.We established LncAPC silenced cells using ASOs,followed by sphere formation assays.The ratio of tumor initiating cells in LncAPC silenced cell group is lower than the control group(P<0.05).LncAPC depleted cells showed impaired sphere formation capacity.2.LncAPC silenced cells were used for serial sphere formation assays.Four passages of sphere formation assays were performed.The ratio of sphere initiating cells is lower in LncAPC silenced cell group than the control group(P<0.05).3.LncAPC silenced cells were injected subcutaneously into BALB/c nude mice and xenograft growth assay was performed.The tumor weight in LncAPC silenced cell group was lighter than the control group(P<0.05).4.Different numbers of LncAPC silenced cells were injected subcutaneously into BALB/c nude mice and tumor initiating assays were performed.The ratio of tumor-free mice in LncAPC silenced cell group was higher than the control group(P<0.05).5.LncAPC overexpressed cells were established using lentiviral vectors,followed by sphere formation assays.The ratio of tumor initiating cells in LncAPC silenced cell group is higher than the control group(P<0.05).6.LncAPC overexpressed cells were injected subcutaneously into BALB/c nude mice and xenograft growth assay was performed.The tumor weight in LncAPC overexpressed cell group was heavier than the control group(P<0.05).7.Different numbers of LncAPC overexpressed cells were injected subcutaneously into BALB/c nude mice and tumor initiating assays were performed.The ratio of tumor initiating cells in LncAPC overexpressed cell group is lower than the control group(P<0.05).Part Three The influences of LncAPC on Wnt/p-catenin Signaling PathwayMethods:1.Expression levels of indicated genes in LncAPC silenced and overexpressed TICs were shown as heat map.The target genes of NFκB,Wnt/β-catenin,Notch and Hedgehog pathways were detected.2.TOPFlash assay was used to examine the activity of Wnt/β-catenin signaling pathway in LncAPC silenced cells and control cells.3.Western blotting was used to detect the expression of endonuclear β-catenin in LncAPC silenced and overexpressed cells.4.APC was deleted using CRISPR/Cas9 approach.The knockout efficiency was detected by Western blotting.β-actin was considered as control and Axin2 was considered as target gene of Wnt/β-catenin signaling pathway.5.lncAPC expression levels in APC knockout cells were examined by Realtime qPCR.6.Silencing or overexpressing LncAPC in APC knockout cells,expression levels of target gene in four major signaling pathways were detected and shown as heatmap.Results:1.The target genes of NFκB,Wnt/β-catenin,Notch and Hedgehog pathways were detected and shown as heatmap.Wnt/β-catenin was inhibited in LncAPC silenced cells,and activated in LncAPC overexpressed cells.2.TOPFlash assay results showed impaired activation of Wnt/β-catenin signaling pathway in LncAPC silenced cells by ASOs than the control group(P<0.05).3.Western blotting results displayed that β-catenin expression level was lower in nuclear compared with cytoplasm in LncAPC silenced cells,higher in nuclear compared with cytoplasm in LncAPC overexpressed cells.4.APC was deleted using CRISPR/Cas9 approach and knockout efficiency was detected by Western blotting.The expression of Axin2 was increased after APC knocked out.5.LncAPC expression levels had no significant change in APC knockout cells compared with the control group by Realtime qPCR(P>0.05).6.LncAPC knocking down and overexpressing were performed in APC knockout cells,and the expression levels of indicated target genes were detected and shown as heat map.Neither knockdown nor overexpression of LncAPC had effect on the target gene activity of Wnt/p-catenin signaling pathway after APC knocked out.Part Four Molecular mechanisms of LncAPCMethods:1.RNA pulldown assay was used to confirm LncAPC binding proteins and mass spectrometry analysis was for protein identification.2.EZH2 expression levels in the indicated cells were examined by realtime qPCR.EZH2 protein levels in peri-tumor,eHCC and aHCC were examined through western blotting.Oncospheres and non-spheres were crushed and subjected to western blotting for EZH2 examination.3.The interaction between EZH2 and LncAPC was expolred by RNA pulldown and western blotting.LncAPC truncates were constructed and RNA pulldown assays were performed,followed by western blotting with EZH2 antibody.4.RNA electrophoretic migration assay(REMSA)was performed to further confirm the interaction between EZH2 and LncAPC by using lncAPC#2 as probes.5.RNA immunoprecipitation(RIP)was performed with EZH2 antibody or control IgG,followed by Realtime qPCR for the enrichment of LncAPC.6.The interaction between EZH2 and LncAPC was also examined through double FISH staining.7.Oncospheres were treated with LncAPC ASOs and EZH2 inhibitors(GSK126 and GSK343).APC,Axin2,c-Myc antibodies were used for Western blotting.APC expression levels in the indicated treated cells were examined by Realtime qPCR.8.ChIP and ChIRP assays were performed with EZH2 antibody and LncAPC probes.The enrichment of APC promoter was examined by Realtime qPCR.9.The interaction between EZH2 and APC promoter was detected by Chromatin Immunoprecipitation(ChIP)assasy with EZH2 antibody.LncAPC silenced and control spheres were used for ChIP assays.HCC samples were used for EZH2 ChIP assays,and the enrichment of APC promoter was detected by Realtime qPCR.Then the correlation between EZH2-APC promoter binding intensity and LncAPC expression was explored.10.H3K9Me3,H4K20Me3 and H3K27Me3 antibodies were used for detecting the APC promoter in the indicated cells.LncAPC silenced cells were used for ChIP assay and APC promoter enrichment was examined by realtime qPCR,The global levels of H3K9Me3,H4K20Me3 and H3K27Me3 were detected by Western blotting.11.LncAPC overexpressing(oeLncAPC)cells were used for ChIP assays with H3K27me3,and APC promoter enrichment was detected by Realtime qPCR.LncAPC silencing and overexpressing cells were used for ChIP assays with H3K27Ac and H3K4me3 antibodies,and APC promoter enrichment was detected by Realtime qPCR.12.EZH2 inhibited cells were used for ChIP assay and APC promoter enrichment was examined by Realtime qPCR.The global levels of H3K9Me3,H4K20Me3 and H3K27Me3 were detected by Western blotting.13.HCC samples were used for H3K9Me3,H4K20Me3 and H3K27Me3 ChIP assays,and the enrichment of APC promoter was detected by Realtime qPCR.Then the correlation between LncAPC expression and histone modifications was explored.Results:1.LncAPC,LncAPC-AS were obtained through vitro transcription and used for RNA pulldown assays.The specific band in LncAPC eluate was identified EZH2 by mass spectrum.2.EZH2 was highly expressed in liver tumor tissues and cell lines examined by Realtime qPCR.EZH2 protein levels were highly expressed in aHCC than eHCC and peri-tumor tissues examined through Western blotting.Also,EZH2 was highly expressed in oncospheres than non-spheres by Western blotting examination.3.The interaction between EZH2 and LncAPC was confirmed by results of RNA pulldown and Western blotting.LncAPC truncates were constructed and RNA pulldown assays were performed,followed by Western blotting with EZH2 antibody,the physiology interaction was confirmed between EZH2 and LncAPC#2.4.RNA electrophoretic migration assay(REMSA)was performed by using LncAPC#2 as probe,the interaction between EZH2 and LncAPC was definitely determined.5.RNA immunoprecipitation(RIP)was performed with EZH2 antibody or control IgG antibody,the enrichment of LncAPC was found by Realtime qPCR in EZH2 group(P<0.05).6.The results Double FISH staining showed the interaction and co-localization between LncAPC and EZH2.7.Oncospheres were treated with LncAPC ASOs and EZH2 inhibitors(GSK126 and GSK343)for Western blotting.APC expression levels were higher in indicated treated cells than control group by Realtime qPCR examination(P<0.05),and the Wnt/β-catenin signaling pathway was inhibited.8.ChIP and ChIRP assays were performed with EZH2 antibody and LncAPC probes.The enrichment of APC promoter was found in both of the two assays by Realtime qPCR examination.EZH2 and LncAPC could bind to the same region of APC promoter.9.The interaction between EZH2 and APC promoter was detected by ChIP assasy with EZH2 antibody.The enrichment of APC promoter was observed in LncAPC silenced group(P<0.05).HCC samples were used for EZH2 ChIP assays,the enrichment of APC promoter was detected by Realtime qPCR.There was a positive correlation between EZH2-APC promoter binding intensity and LncAPC expression.10.H3K9Me3,H4K20Me3 and H3K27Me3 antibodies were used for detecting APC promoter in the indicated cells.LncAPC silenced cells were used for ChIP assay and APC promoter enrichment was examined by Realtime qPCR.The results indicated that transcription was more active in LncAPC silenced cell group(P<0.05).The global levels of H3K9Me3,H4K20Me3 and H3K27Me3 had no significant change by Western blotting.11.LncAPC overexpressing cells were used for ChIP assay with H3K27me3,and enrichment of APC promoter was detected by Realtime qPCR.LncAPC silencing and overexpressing cells were used for ChIP assay with H3K27Ac and H3K4me3 antibodies,increasing APC promoter enrichment was detected in LncAPC silencing cells and decreasing APC promoter enrichment was found in LncAPC overexpressing cells by realtime qPCR(P<0.05).12.EZH2 inhibited cells were used for ChIP assay,increasing APC promoter enrichment was detected compared to control cells by realtime qPCR(P<0.05).The global levels of H3K9Me3 and H4K20Me3 had no significant change by Western blotting,but H3K27Me3 decreased because of EZH2 mediated.13.HCC samples were used for H3K9Me3,H4K20Me3 and H3K27Me3 ChIP assays,and the enrichment of APC promoter was detected by Realtime qPCR,a positive correlation was explored between LncAPC expression and enrichment of APC promoter.Part Five LncAPC-EZH2-APC could be a target for liver TICs elimination Methods:1.APC knockout cells were established using CRISPR/Cas9 approach,followed by sphere formation assays.2.Liver TICs were treated with EZH2 inhibitors(GSK126 and GSK343)or Wnt/β-catenin inhibitors(Wiki4 and XAV-939),followed by sphere formation assays.3.EZH2 and Wnt/β-catenin inhibited cells were subcutaneously injected into 6-week-old BALB/c nude mice,and tumor weight was detected 1 month later for xenograft growth assay.Six mice were used in each group.4.Immunohistochemistry was performed to detect the expression of c-Myc,APC and H3K27me3.C-Myc is a well-accepted functional marker of liver TICs.5.Liver TIC numbers was detected through FACS in EZH2 and Wnt/p-catenin inhibited cells.Results:1.APC knockout cells were established using CRISPR/Cas9 approach,increased oncospheres after APC knockout was found by sphere formation assays(P<0.05).2.Liver TICs were treated with EZH2 or Wnt/β-catenin signaling pathway inhibitors,impaired sphere formation was detected after both treatments through sphere formation assays(P<0.05).3.EZH2 and Wnt/β-catenin inhibited cells were subcutaneously injected into 6-week-old BALB/c nude mice,and smaller tumor weight was detected 1 month later by xenograft growth assay(P<0.05).4.Immunohistochemistry was performed,the expression levels of c-Myc and the ratios of c-Mychgh cells were reduced after inhibition of EZH2 and Wnt/β-catenin.EZH2 inhibition led to APC overexpression and H3K27me3 impairment.5.Decreased liver TIC numbers was detected through FACS in EZH2 and Wnt/β-catenin inhibited cells.Conclusions:1.We found a non-coding RNA,LncAPC,could promote liver tumor initiating cells self-renewal.2.LncAPC inhibits APC expression and activates Wnt/β-catenin signaling pathway.3.LncAPC inhibits APC transcription through recruiting EZH2 to APC promoter region.4.LncAPC-EZH2-APC could be targeted for liver cancer stem cells elimination. |
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