| BackgroundColon cancer is the second leading cause of cancer-related death in the world.Most colon cancers are slow-growing and originate from precancerous lesions,and clinical intervention is always challenging for advanced colon cancers.At present,the main treatments include surgery,chemotherapy,radiotherapy,biotherapy,immunotherapy and so on.If important regulatory factors for tumor development can be identified in the genome network,it may have significant potential applications for the detection,monitoring,diagnosis and treatment of colon cancer.Long non-coding RNA is the general term for a class of non-coding RNA whose length is greater than 200 nt.By interacting with DNA,RNA and protein,it can exert an important influence on DNA replication,RNA transcription,protein localization,translation,stability,activity and other stages,thus regulating the state and survival of tumor cells.It can be concluded that long non-coding RNAs may be the key tandem molecules in the development of tumors,so it is necessary to further study them.Long non-coding RNA HEIH(Lnc RNA high expression in hepatocellular carcinoma)is a carcinogenic Lnc RNA with high expression that has been characterized and studied for the first time in the tissues of patients with primary hepatocellular carcinoma.Its full length is1681 bp.It is located in chromosome 5q35.3 and contains an exon,which is mainly distributed in the nucleus and cytoplasm.In the previous stage,our research group found that the expression of HEIH is uneven in pan-cancer tissues,and the overall expression level of HEIH is significantly down-regulated in colon cancer tissues.However,there have been no significant reports on the low expression of HEIH in tumors.To further clarify the role of HEIH in colon cancer,we detected the expression of HEIH in colon cancer tissues and cell lines,and the results showed that the expression of HEIH was down-regulated in colon cancer tissues and cell lines,indicating that HEIH may be a functional Lnc RNA molecule in colon cancer.Next,our research work will focus on HEIH for the regulation and related mechanisms of colon cancer.Specifically,the effect of HEIH on the function of colon cancer cells,the specific mechanism of HEIH downregulation in colon cancer,the interaction between HEIH and YBX3,the verification and research of downstream target genes of YBX3,animal experiments and so on.Objective1.To clarify the expression and clinical significance of Lnc RNA HEIH in colon cancer tissues and cells;to preliminarily identify the correlation between HEIH and YBX3 through co-analysis.2.To evaluate the effects of Lnc RNA HEIH overexpression on the proliferation,migration,and invasion of colon cancer cells DLD1 and HCT116.3.To explore the cause of down-regulated expression of Lnc RNA HEIH in colon cancer cells.4.To clarify the combination and regulation relationship between Lnc RNA HEIH and YBX3;to identify the downstream target gene PPARα and evaluate its role and influence on the development of colon cancer.Methods1.93 postoperative colon cancer and adjacent tissues were collected from Xinqiao Hospital,with 3 cases randomly selected from each of the TNM stages 1-4,for a total of 12 cases.The expression of HEIH was quantified by q PCR in colon cancer tissues from different sources,including the Mec DNA-HCol A095Su01 containing 100 colon cancer tissues,15 of which had paired adjacent tissues.HEIH expression was quantitatively detected by q PCR in cell lines LOVO,HCT116,SW48,SW620,LS-174 T,DLD1,HT-29,HCT-15 and SW480,including colon cancer stage 1-4,and FHC cell lines of normal colon epithelial cells as controls.In terms of bioinformatics,multiple cancer data sets from TCGA were analyzed,and the value of HEIH in tumor diagnosis was evaluated using receiver operating characteristic(ROC)curves.The expression of HEIH was analyzed using Kaplan-Meier plots in relation to overall survival(OS),disease-specific survival(DSS),and progression-free interval(PFI).Violin plots were generated with R ggplot2 software to show the HEIH expression levels of colon cancer patients with different clinical features.GO and KEGG enrichment analyses of DEGs were performed using the cluster Profiler package and displayed in chord diagrams.2.Lentiviral vector overexpressing HEIH gene was constructed,and transfected DLD1 and HCT116 after accurate sequencing,and stable overexpressing HEIH cell lines were screened.The effect of HEIH on cell proliferation was evaluated by clonal formation test and CCK8 method,the effect of HEIH on cell invasion was evaluated by Transwell test,and the effect of HEIH on cell migration was evaluated by cell scratch test.3.HEIH’ upstream gene ZFP62,downstream gene MGAT1 and adjacent gene sequences were defined,and primers of each segment of genes were designed.q PCR was used to detect the expression of each gene,and western blot was used to detect the expression of ZFP62 and MGAT1 proteins.The locations of adjacent Cp G islands upstream of HEIH were predicted,and primers for methylation and non-methylation of Cp G islands were designed.The methylation modification status of Cp G islands was detected by specific methylation PCR.The binding sequences of CTCF and HEIH upstream genes were predicted and primers were designed.The binding states of CTCF and HEIH sequences were detected by Chip-PCR.To predict HEIH transcription factors,construct si RNA of each predictive transcription factor,and detect HEIH expression after interfering with each transcription factor in FHC;The binding sequence of YY1 on the HEIH promoter was predicted and verified by Chip-PCR.4.HEIH sequence pull-down probe was designed,and HEIH binding proteins were obtained by pull-down RNA.Then,the expression of YBX3 was detected by western blot.Intracellular localization of HEIH and YBX3 was determined by in situ hybridization.The expression of YBX3 was detected by western blot using HEIH wild type,antisense chain and mutant chain.Interference or overexpression plasmids of YBX3 and HEIH were designed to transfect FHC or tumor cells,respectively.The expressions of YBX3 and HEIH were detected by western blot or q PCR.RIP sequencing was performed on YBX3,and the possible protein binding RNA PPARα,PPARδ,PKN1,TGFBR2 were screened out and identified by RIPq PCR.After overexpression of YBX3 in FHC or interference of YBX3 in tumor cells,the expression of PPARα was detected by q PCR and the effect of PPARα on the proliferation,migration and invasion of tumor cells was evaluated.Results1.Clinical significance and bioinformatics analysis of HEIH for long non-coding RNA.⑴ HEIH,as an independent diagnostic factor,had a large area under the ROC curve(AUC)in pan-cancer,some of which could reach 1,and the area under the ROC curve of HEIH in colon cancer was 0.875.The results showed that HEIH had good diagnostic accuracy in various types of tumors and colon cancer.⑵ The HEIH expression in colon cancer cell lines,including LOVO,HCT116,SW48,SW620,LS-174 T,DLD1,HT-29,HCT-15 and SW480,was significantly down-regulated compared with FHC,and HCT116 and DLD1 were most significantly down-regulated.⑶ The expression of HEIH was generally down-regulated in colon cancer tissues,and the expression decreased with the increase of colon cancer stage.The overall expression was lower in lymph node metastasis or distant metastasis.The lower the HEIH expression,the shorter the overall survival time of patients.The results indicated that HEIH expression was closely related to malignant clinical features and prognosis of patients.⑷ The single gene DEGs screening of HEIH and YBX3 in colon cancer showed that HEIH expression in colon cancer tissue was negatively correlated with YBX3 expression,and the Pearson correlation coefficient of both was 0.44.After Hub network construction by DEGs,among the three hub gene networks with the highest scores of related differentially expressed genes in HEIH and YBX3,there are two almost identical co-regulatory networks.This indicated that HEIH was closely related to YBX3 in regulation and function.2.Long non-coding RNA HEIH inhibited the proliferation,invasion and migration of colon cancer cells.⑴ The overexpressing HEIH lentivirus vector was successfully constructed and transfected into HCT116 and DLD1 cell lines.After screening,HEIH expression was detected.The results showed that the HEIH expression level was significantly increased in HCT116 and DLD1 cell strains with stable overexpression of HEIH compared with the blank group and wild group.These results indicated that the lentiviral vector that overexpressed HEIH had a good ability to overexpress HEIH.⑵ Compared with the blank group of HCT116 and DLD1,the clone formation ability,migration ability and invasion ability of HCT116 and DLD1 cell strains with stable overexpression of HEIH were significantly reduced.The results indicated that HEIH could inhibit the malignant characteristics of colon cancer cells.3.Reasons for down-regulated expression of long-chain non-coding RNA HEIH in colon cancer cells.⑴ HEIH is an intergenic Lnc RNA.Compared with FHC cells,m RNA and protein expression of the upstream encoding gene ZFP62 of HEIH were up-regulated in colon cancer cell lines LOVO and SW620.Starting from the downstream sequence of ZFP62,the expression of HEIH upstream gene,HEIH,HEIH downstream sequence and MGAT1 was down-regulated at the same time.These results suggested that the regulatory site of HEIH down-regulated in colon cancer cells might be located between the ZFP62 and HEIH.⑵ Methylation modification of Cp G1 and Cp G2 in HEIH upstream of FHC,LOVO and SW620 cells was detected,the results showed that the Cp G1 rich region was in a hypomethylated state in normal FHC cells while in a hypermethylated state in LOVO and SW620 cells.The Cp G2 region is hypermethylated in FHC cells and hypomethylated in LOVO and SW620.These results suggested that HEIH expression may be regulated by upstream epigenetics.⑶ It was predicted that CTCF bound to the upstream DNA sequence of HEIH,which is specifically subdivided into P1 to P5,and the verification in SW620 cells showed that CTCF bound to P2 and P3 regions significantly.The repeated results of FHC,LOVO and SW620 cell lines suggested that CTCF protein had stronger binding ability to P3 in tumor cell lines.The expression of HEIH might be regulated by CTCF.⑷ To predict and screen HEIH transcription factors,including YY1,TEAD2,FOXA2,SP1 and MAZ,si RNA of each transcription factor was constructed and transfected into FHC cells.The results showed that interfering with YY1 expression could significantly downregulate HEIH expression level.YY1 was the transcription factor of HEIH.4.The imbalance of HEIH-YBX3-PPARα axis of long non-coding RNA promoted the development of colon cancer.(1)YBX3 was confirmed as a binding protein of HEIH by RNA pull-down and mass spectrometric detection in SW620 and LS-174 t cell lines.HEIH antisense chains could not bind to YBX3,and the binding efficiency of HEIH with YBX3 decreased significantly after local mutation.After interfering HEIH expression in FHC cells,YBX3 expression was significantly up-regulated.After overexpression of HEIH in LOVO and SW620,the expression of YBX3 was significantly down-regulated.After overexpression of YBX3 in FHC cells,the expression of HEIH was significantly down-regulated.After interfering with YBX3 in LOVO and SW620,HEIH expression was significantly up-regulated.The results showed that HEIH and YBX3 directly combined and negatively regulated the expression of each other.(2)HEIH and YBX3 were mainly co-located in the cytoplasm.HEIH and YBX3 might be involved in the development of colon cancer at the post-transcriptional level.(3)YBX3 binding m RNAs,including PPARα,PKN1,TGFBR2,PPARδ and so on,were found by YBX3 protein RIP sequencing.Quantitative detection results showed that the binding amount of YBX3 and PPARα was relatively large.After overexpression of YBX3 in FHC cells and interference of YBX3 expression in LOVO and SW620 cells,the expression of PPARα was reversed.PPARα was a binding m RNA of YBX3,and YBX3 could negatively regulate PPARα expression.(4)Compared with the control group,PPARα could inhibit the invasion ability of FHC,LOVO and SW620 cells.PPARα could inhibit the migration ability of LOVO and SW620 cells.PPARα could inhibit the proliferation of LOVO and SW620 cells.Conclusions1.Lnc RNA HEIH is down-regulated in colon cancer tissues and cell lines,and its expression is closely related to tumor staging,metastasis,survival and so on.HEIH has a good diagnostic value for colon cancer.2.The up-regulated expression of Lnc RNA HEIH in colon cancer cells can inhibit the proliferation,migration and invasion of tumor cells.3.Compared with FHC,the difference in CTCF binding ability caused by the hypermethylation of Cp G1 and the hypomethylation of Cp G2 in the upstream coding region of Lnc RNA HEIH in colon cancer cells is the key factor for the low expression of Lnc RNA HEIH in colon cancer cells.4.HEIH and YBX3 bind and have a bidirectional negative regulatory effect,and the two factors may have a common downstream regulatory network.YBX3 directly binds to downstream PPARα m RNA and negatively regulates its expression,and down-regulation of PPARα promotes the development of colon cancer. |
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