| Backgroud and ObjectiveColorectal cancer(CRC)is a common malignant tumor in the digestive system.In recent years,with the change of people's life style and diet structure,the incidence of colorectal cancer is increasing year by year.Approximately 1.2 million patients worldwide are diagnosed with colorectal cancer each year,and more than 600 thousand patients died directly or indirectly of CRC.Early signs of CRC is not obvious,symptoms often appear late and prone to metastasis,then the prognosis is poor.This is the main reason for the high mortality rate.The molecular mechanism of the occurrence of CRC is not completely clear.Now,it is believed that CRC is a process with multi-gene involved in.Like ras oncogene activation,anti oncogene p53 inactivation,APC mutations,these are coding genes.With the development of genomics and gene chip,some genes encoding the RNA were found through genome sequencing,but these RNA is not translated into protein,said for non coding RNA(Non-coding RNA),.Non encoding RNA including rRNA,tRNA,snRNA,snoRNA,microRBNA and so on.Most of them the functions were unknown.One class of transcript RNA molecules longer than 200nt which does not code for proteins,regulate gene expression in the form of RNA in multiple levels(table epigenetic regulation,regulation of transcription and post transcriptional regulation)called long-chain non coding RNA(lncRNA).Respect to the protein coding gene and small molecule RNA,lncrna research is just in the infancy stage,lncrna initially was considered as "noise" of the transcribed genome,was a by-product of RNA polymerase II and was not have a biological function.Recent studies have indicated that IncRNA plays an important role in regulating cell proliferation,cell cycle,cell differentiation,apoptosis and so on.IncRNA abnormal expression is closely related to human disease,especially in cancer and has been found in a variety of tumors,including breast cancer,stomach cancer,lung cancer,prostate cancer,etc.These lncRNA mainly involved in tumorigenesis,growth,invasion,metastasis and recurrence process.This is suggesting that lncRNA may play a role as oncogenes or tumor suppressor genes in the development of tumors.Compared with previous studies of microRNA(miRNA),lncRNA still belongs to a relatively unknown field,most of the lncRNA function and molecular regulation mechanism is not clear,Even if several lncRNAs associated with CRC were found,it is just the tip of the iceberg.Therefore,there is still a need to continue looking for functional lncRNA in CRC,and explore its features to learn more of CRC.Cancer susceptibility candidate 11(CASC11),located in the chromosome 8q24,33kb length,is a novel lncRNA.The function of CASC11 has not been reported yet.Several lncRNAs were found to be located in the 8q24 gene desert and all of them played an important role in tumor progression.CCAT1 has been reported to located in the super-enhancer region to regulate c-Myc expression.In addition,CCAT1 can also be regulated by c-Myc.CCAT2 can enhance WNT signaling activity and regulat c-Myc expression.PC AT1 has also been reported to promote prostate cancer cell proliferation through regulating c-Myc.Previous studies have showed that some lncRNAs may affect the expression of the gene located to their chromosomal neighborhood.CASC11 is the closest gene to c-Myc,and c-Myc was located in the promoter of CASC11.In addition,sequence variant on 8q24 rs16902359 confers susceptibility to lymphoma.And we found CASC11 transcript encompasses the rs16902359 SNP.Therefore,we hypothesized that the lncRNA CASC11 may also have a role in cancer.Therefor,in this study we will detect CASC11 expression level in CRC,determine the relationship between CASC11 expression and clinical pathological parameter,analyse the function and molecular mechanism of CASC11 in growth and metastasis of CRC,and explore the upstream regulating mechanisims of CASC11.Methods1.The expression of CASC11 in CRC tissues and cells(1)Real-time quantitative PCR(qRT-PCR)was employed to determine the expression of CASC11 in 36 pair samples from fresh CRC biopsies and CRC cell lines SW480,SW620,HT29,LOVO,Ls174t,RKO,HCT116 and colorectal epithelum cell line FHC.(2)The relationship between CASC11 expression and clinical parameters,including tumor size,serosal invasion,lymph metastasis,and tumor-node-metastasis(TNM)stage.2.The role of CASC11 in growth and metastasis of CRC in vitro and in vivo(1)CASC11 knockdown Lentivital vector was purchased from company and CASC11 overexpression plasmid was constructed.The results from sequencing showed insertion sequence was correct and there was no mutation or loss.Then Lentiviral vectors were transfected to SW480 and SW620 for establishing stable CASC11 knockdown cell lines.The plasmids were transfected to SW620 and RKO for establing transient CASC11 overexpression cell lines.The results were confirmed by using qRT-PCR.(2)The proliferation and invasion ability of celll lines with CASC11 overexpression and knockdown were measured by CCK8,colony formation,flow cytometry,transwell and scratch wound healing assays.(3)SW480-pLV-shCASC11,SW480-PLV-shNC were injected subcutaneously into the left or right flank of nude mice.After 4 weeks,we analyzed primary tumor growth.For metastasis model,cells were injected into the tail vein of nude mice,eight weeks later,the mice were sacrificed,and their liver and lung were removed and formalin fixed for histological analysis.3.The relationship between the CASC11 and the target gene(1)RNA-pull-down assay was adopted to identify the CASC11 interacting proteins.We found several bands were pulled down by vitro-transcribed biotinylated CASC11 sense transcript using silver staining.Heterogeneous ribonucleoprotein K(hnRNP-K),one of the proteins identified by mass spectrometry,was confirmed by western blotting.(2)Inhibition or overexpression CASC11,then measure the expression level of hnRNP-K.Determine the change of CASC11 to explore whether CASC11 could regulate the expression of hnRNP-K.(3)Subcellular fractionation location assay was used to examin the location of CASC11 in cell.Then,we speculate the function of CASC11 rely on its location in cell.To further determine whether CASC11 regulate the synthesis or degradation of hnRNP-K mRNA,we enhanced or inhibited CASC11 expression,treated the cells with actinomycin D or dimethylsulfoxide to block new RNA synthesis,then measured the stability of hnRNP-K.(4)In addition,knockdown or upregulate the expression level of hnRNP-K,then determine the expression level of CASC11.qRT-PCR and IHC were used to measure the expression of hnRNP-K in CRC tissue;correlation analysis was used to explore the relationship between CASC11 and hnRNP-K.4.The role of compound CASC11 and hnRNP-K in activating WNT signaling(1)GEO database and GSEA were used to analyse the enrichment of WNT pathway in CRC patients with high CASC11 expression.(2)Luciferase activity assay was used to detect the activity of WNT signaling,qRT-PCR and Western blot were used to measured doenstream WNT pathway targets.(3)Nuclear protein extraction assay was used to determine the location of ?-catenin and hnRNP-K after changing the expression level of CASC11 in cell.Then,we examine whether an important role of hnRNP-K in this process.(4)Protein co-immunoprecipitation(COIP)was used to explore the interaction between hnRNP-K and the key genes in WNT pathway.Then,we suppose the possible mechanisms of hnRNP-K in process that CASC11 regulate WNT pathway.5.The upstream regulating mechanism of CASC11 in CRC(1)Transfac database was used to predict the interaction between promoter region of CASC11 and c-Myc;CHIP and luciferase activity assays were used to confirm it.(2)QRT-PCR was used to measure the expression of CASC11 after increasing or decreasing c-Myc expression.correlation analysis was used to explore the relationship between CASC11 and c-Myc.(3)The UCSC database was used to predict acetylation in promoter region of c-Myc,Chip assay was used to confirm the acetylation region of CASC11 promoter,then,the correlation between CASC11 expression and the degree of acetylation was analysed.6.Statistical analysesStatistical analyses were performed using SPSS20.0 software.The differences between groups were tested by using a two-tailed Student's t-test.Data was presented as the mean ± SD of at least 3 independent experiments.One-Way ANOVA was used for multiple comparisons with SNK OR LSD.Relationships between CASC11 expression and clinicopathologic characteristics were determined by ?e test.The Pearson's correlation coefficient was used to measure the linear relationship between the expression levels of CASC11 and hnRNP-K or c-Myc in CRC tissues.Differences were considered significant if p<0.05:*,p<0.05;**,p<0.01;***,p<0.001.Results1.CASC11 overexpression in CRC associated with tumor size,serosal invasion,lymph metastasis,and TNM stage(1)CASC11 is up-regulated in colorectal cancer tissues and cellsThe results of qRT-PCR showed CASC11 expression was indeed higher in 32 of 36 cases of CRC specimens compared with those of the adjacent normal mucosa tissues(p<0.001).The expression levels of CASC11 were also up-regulated in investigated CRC cell lines(LOVO,M5,LS174t,RKO,SW620,SW480,HT29,HCT116)compared with normal colorectal epithelium cell FHC.(2)The relationship between CASC11 expression and clinicopathological parameters correlation analysis showed that CASC11 expression was positively associated with tumor size,serosal invasion,lymph metastasis,and tumor-node-metastasis C(TNM)stage in CRC.Furthermore,there is no relationship between CASC11 expression and age,gender,differentiation.2.The role of CASC11 in the development of CRC(1)The construction of the CASC11 knockdown Lentuviral vectors and overexpression plasmid The molecular cloning technologies were used to construct the CASC11 knockdown Lentuviral vectors and overexpression plasmid.The results from sequencing showed insertion sequence was correct and there was no mutation or loss.Then,lentiviral vectors were transfected to SW480 and SW620 for establishing stable CASC11 knockdown cell lines.QRT-PCR showed CASC11 were significantly reduced both in SW480 and SW620 cells(P<0.001).The plasmids were transfected to SW620 and RKO for establing transient CASC11 overexpression cell lines.The results were confirmed by qRT-PCR(P<0.001).(2)The effects of CASC11 knockdown and overexpression on proliferation in CRC cellsCCK8 proliferation assays revealed that knockdown of CASC11 significantly decreased cell growth(P<0.001)and overexpression of CASC11 increased cell growth(P<0.05).Similarly,colony formation capacity was suppressed after the downregulation of CASC11(P<0.05).Furthermore,the number of cells in the G1 or G2/M phase was assessed by flow cytometry,we found pLV-shCASC11 cells increased at the G1 phase and induced G1 phase cell cycle arrest significantly compared with the pLV-shNC cells.Xenograft model showed that mice in SW480/shCASC11 group developed smaller tumors than those in SW480/shNC group(P<0.01),IHC assay confirmed that Ki-67 proliferation index in the SW480/shCASC11-xenografted tumors was lower than that in SW480/shNC-xenografted tumors(P<0.001).(3)The effects of CASC11 knockdown and overexpression on metastasis in CRC cellsWound healing and transwell assay results revealed repression of CASC11 could attenuate the migration of SW480 and SW620 cells(P<0.001),overexpression of CASC11 could enhance the migration of SW480 and SW620 cells(P<0.05).Wound healing assay showed knockdown of CASC11 could inhibit the migration of SW480 and SW620 cells(P<0.001).To determine the effect of CASC11 on the metastasis of CRC in vivo,SW480/shNC and SW480/shCASC11 cells were injected into tail vein.Eight weeks later,mice were killed and lung and liver metastases were examined.None of the mice in SW480/shCASCI11 group had lung and hepatic metastatic nodule.However,40%(2 of 5)of mice in the SW480/shNC group had lung metastatic nodules,and 80%(4 of 5)of these mice had hepatic metastatic foci.The number of hepatic metastatic nodules in mice of the SW480/shCASC11 group was obviously reduced compared with the SW480/shNC group(P<0.05)3.CASC11 is associated with protein hnRNP-K and increases its expression(1)hnRNP-K is a target gene of CASC11RNA-pull-down assay was adopted to identify the CASC11 interacting proteins.We found several bands were pulled down by vitro-transcribed biotinylated CASC11 sense transcript using silver staining.Heterogeneous ribonucleoprotein K(hnRNP-K),one of the proteins identified by mass spectrometry,was confirmed by western blotting.Next,we investigated CASC11 and hnRNP-K binding in vivo by RNA-binding protein immunoprecipitation experiments.We observed CASC11 enrichment using the hnRNP-K antibody versus a nonspecific antibody(IgG control).(2)CASC11 can regulate the expression of hnRNP-KFurthermore,we detected a downregulation of hnRNP-K RNA and protein levels upon CASC11 repression in SW480 and SW620 cells.While we found higher hnRNP-K expression at both RNA and protein levels in cells overexpressing CASC11.(3)CASC11 can enhance the stability of hnRNP-KWe examined the subcellular fractionation location of CASC11 in CRC cells and found a higher expression in the cytosol versus the nucleus.The resluts suggest CASC11 may play an important role in regulating mRNA stability.To further determine whether CASC11 regulate the synthesis or degradation of hnRNP-K mRNA,we enhanced or inhibited CASC11 expression in SW620 cells,treated the cells with actinomycin D in different period.We found that inhibition of CASC11 decreased stability of hnRNP-K mRNA.Conversely,CASC11 overexpression increased stability of hnRNP-K.This suggested that CASC11 can enhance the stability of hnRNP-K.(4)There is a positive correlation between CASC11 expression and hnRNP-K expressionWe found knockdown of hnRNP-K inhibited CASC11 mRNA levels whereas overexpression of hnRNP-K boosted up CASC11 transcription.qRT-PCR and IHC results showed hnRNP-K mRNA levels were significantly increased in colorectal cancer tissues in comprarison with the adjacent normal tissues.Correlation analysis indicated a positive correlation between CASC11 expression and hnRNP-K expression(r = 0.570)(p<0.001).4.CASC11 directly target hnRNP-K to activate WNT signaling in CRC cells(1)The analysis of upregulation pathway in CRC with higher CASC11 expressionThrough gene set enrichment analysis(GSEA),we observed WNT signaling pathway was correlated with CASC11 expression in GSE8671(P<0.05).(2)CASC11 can regulate WNT signaling activity and regulate downstream genes of WNT pathwayWe used a TOP-Flash luciferase assay and observed that downregulation of CASC11 inhibited WNT/?-catenin pathway activity(P<0.05).On the other hand,overexpression of CASC11 upregulated WNT/?-catenin signalling activity(P<0.05).We next detected the expression of downstream WNT pathway targets such as ?-catenin,c-Myc,CycinDl and MMP7.We found that inhibition of CASC11 decreased the expression of ?-catenin,c-Myc,CyclinD1 and MMP7.On the contrary,overexpression of CASC11 increased the expression of these relative proteins.(3)CASC11 can active WNT pathway through hnRNP-KIn order to identify whether CASC11 increases nuclear localization of ?-catenin through hnRNP-K,we changed the expression levels of CASCII in SW480 and SW620 cells and examined the protein level and nuclear translocation of ?-catenin and hnRNP-K.Overexpression of CASC11 increased the nuclear translocation of ?-catenin and hnRNP-K proteins.Conversely,knockdown of CASC11 decreases the nuclear translocation of ?-catenin and hnRNP-K proteins.There were no obvious changes in cytoplasm protein levels of ?-catenin and hnRNP-K.We also examined whether hnRNP-K was necessary for CASC11-induced nuclear localization of?-catenin.Reintroduction of hnRNP-K in CASC11 depleting cells increase nuclear translocation of ?-catenin and hnRNP-K.Moreover,redepletion of hnRNP-K abolished the promoting effect of CASC11.These data suggested that CASC 11 could increase ?-catenin nuclear accumulation relying on hnRNP-K.(4)The possible mechanisim of CASC 11 activation on WNT through hnRNP-KTo futher ascertain the roles of hnRNP-K in ?-catenin nuclear accumulation,we detected whether hnRNP-K could interact with ?-catenin,AXIN,GSK3? and TCF4 to form a complex by COIP.Because the components of AXIN and GSK3? induced the degradation of ?-catenin,the interaction between hnRNP-K and ?-catenin,AXIN,GSK3p suggested hnRNP-K may protect P-catenin from being degradated.Previous study has shown hnRNP-K can shuttle between nucleus and cytoplasm.Moreover,we found there was an interaction between hnRNP-K and TCF4.These results suggested ?-catenin nuclear accumulation activates WNT signaling through complex forms with hnRNP-K.Whether CASC 11 also keeps complex form with hnRNP-K and?-catenin in this process and the hypothesis about the role of hnRNP-K in ?-catenin nuclear accumulation need more experiments to confirm.5.The upstream regulating mechanism of CASC11 in CRC(1)C-Myc can regulate CASC11 expression as transcript factorTo examine how transcription of CASC 11 was regulated,we analyzed the potential transcript factor binding sites in the promoter regions of CASC11(http://www.gene-regulation.com/index2)and found that one E-box element which could be possible binding sites recognized by c-Myc.It was observed that c-Myc effectively stimulated the luciferase activity of CASC11 promoter in HEK293 and SW480 cells.Additionally,ChIP assay showed that c-Myc could directly bind the region(-1274?-1279bp)in the promoter of CASC11.Moreover,Cell lines with ectopic c-Myc expression showed higher CASC11 expression levels.On the contrary,knockdown of c-Myc decreased the expression of C ASC11 in SW480 and SW620 cells.Finally,we also found CASC11 expression was positively correlated with c-Myc in CRC tissues.(2)c-Myc increases promoter histone acetylation to enhance CASC11 expressionWe performed a computational screen of the promoter regions of CASC11(https://genome.ucsc.edu/cgi-bin/hgGatewav)and found an obvious Histone H3 Lysine 27 acetylation(H3K27AC)region in the promoter of CASC11.CHIP assays confirmed that acetylation occurred in the CASC11 promoter.Previously,histone acetyltmnsferse(HATs)was found to be recruited by c-Myc.In order to verify c-Myc can enhance acetylation of CASC11 promoter,we knocked down c-Myc in SW620 cells,and found inhibition of c-Myc decreased the level of H3K27AC in the E-box region of CASC11 promoter.To further delineated the role of histone acetylation in c-Myc mediating transcriptional induction of CASC11,we knocked down c-Myc in SW620 cells and treated with histone deacetylase(HDAC)inhibitor TSA.We found TSA increased expression of CASC11 in sh-c-Myc cells.These results suggest that histone acetylation to enhance CASC11 expression.Conclusion1.CASC11 overexpression in CRC associated with tumor size,serosal invasion,lymph metastasis,and TNM stage.We reasoned that CASC11 may play a role as oncogene.2.The overexpression of CASC11 enhances growth and migration ability of CRC cells in vitro and in vivo;while the knockdown of CASC11 inhibits growth and migration ability.3.CASC11 directly interacts with hnRNP-K and increases its expression by enhancing the stability of hnRNP-K.4.WNT/?-catenin signaling is activated by CASC11 through elevating the complex of hnRNP-K and ?-catenin nuclear accumulation.5.c-Myc increases promoter histone acetylation to enhance C ASC11 expression. |
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