| Backgroud and ObjectivesColorectal cancer?CRC?is the third most common cancer worldwide.The incidence and mortality of colorectal cancer is gradually increasing,and ranking the fifth in all of malignant tumors in China.It is great need to explore the molecular mechanisms of colorectal cancer,and to find the biomarkers for early detection and diagnosis.A growing number of studies have shown that long non-coding RNA?lncRNA?is closely related to the development of colorectal cancer.However,most of the traditional biological research focuses on the function of a single lncRNA,and it is difficult to elaborate the whole of the biological system.Therefore,the specific mechanism of lncRNA in the development and progression of colorectal cancer still needs further investigation.In this study,we explore the gene modules closely related to colorectal cancer development using weighted gene co-expression network analysis?WGCNA?MIR17HG was then selected using transcription factor regulatory network analysis.We then explore the effect of MIR17HG on the biological phenotype of colorectal cancer cells and regulation mechanism of MIR17HG.At the same time,the role of lncRNA MIR17HG in immunotherapy of colorectal cancer and the potential mechanism were explored.In conclusion,we aim to clarify the pathogenesis of colorectal cancer,discover the biomarkers for early prognosis in colorectal cancer patients,and provides clues for the treatment of colorectal cancer Methods1.The tissue samples used for lncRNA and mRNA microarray analysis were obtained from 6 CRC patients and 6 adenoma patients.We constructed WGCNA co-expression regulation network analysis,GO and KEGG Pathway analysis,for further screening CRC related lncRNA2.qRT-PCR was used to validate key molecules in the expanded colorectal cancer and adenoma patient samples.The regulatory network was validated by siRNA or silencer transfection experiments.Immunohistochemistry?IHC?and in situ hybridization?ISH?assay were used to explore the relationship between candidate genes and the prognosis of colorectal cancer patients3.The MIR17HG stably knockdown colorectal cancer cell line was constructed and used to evaluate the effects of MIR17HG on the cell proliferation,invasion,and migration Subcutaneous tumorigenesis was performed to evaluate the effect of MIR17HG knockdown on tumor formation and metastasis capacity of colorectal cancer cells in nude mice.Colitics-associated cancer model,ISH,chromatin immunoprecipitation?ChIP?,qRT-PCR were used to explore the transcriptional regulation of RELA/p65 on MIR17HG using Rela/p65 intestinal conditional knockout mice.The bioinformatics prediction databases?miRBase and TargetScan?,dual luciferase reporter gene?DLR?,RNA-binding protein immunoprecipitation?RIP?assay were used to explore the molecular regulation mechanism of MIR17HG/miR-375/RELA/p65 signal axis4.Expression levels of miR-17-92 clust was analysis using TCGA dababase and the expanded colorectal cancer and adenoma patient samples.The Chromosome location and sequence analysis,overexpression or knockdown of MIR17HG/miR-17-5p,qRT-PCR,DLR,colony formation,transwell,tumorigenesis of nude mice and so on were performed to explore the role of MIR17HG/miR-17-5p/BLNK signal axis in colorectal cancer development5.The binding of MIR17HG to PD-L1 protein was predicted by RPIseq bioinformatics database and evaluated by RNA-pull down assay.The regulation mechanism of MIR17HG on PD-L1 was explored by qRT-PCR,Western Blot and IHC assay.IHC assay was used to explore the relationship between PD-L1 and prognosis of colorectal cancer patientsResults1.Bioinformatics analysis of lncRNA and mRNA expression profile microarray in colorectal cancer1.1 Construction of colorectal cancer lncRNA and mRNA expression profile microarrayAccording to the fold change?FC?>2,and P<0.05,a total of 1009 lncRNAs and 957 mRNAs were up-regulated,956 lncRNAs and1177 mRNAs were down-regulated in colorectal adenoma tissues when compared with normal tissues.Atotal of 367 lncRNAs and 1631 mRNAs were up-regulated,748 lncRNAs and 571 mRNAs were down-regulated in colorectal tumor tissues when compared with normal tissues.1.2 Construction of WGCNA regulation networkA total of 27 modules were identified based on One-step network construction of WGCNA analysis.Wilcoxon test results revealed that turquoise,yellow,and light yellow module gene were gradually increased or decreased from adjacent,colorectal adenoma to CRC tissue samples.DEGs in both turquoise and yellow showed significantly enriched KEGG pathways?P<0.05?,including immunity-associated pathways;however,there was no markedly enriched KEGG pathways in light yellow.gene-TF regulatory network analysis showed that RELA/p65 transcriptionally regulated 9 lncRNAs and 19 mRNAs involved in the immune pathways in turquoise and yellow module.2.Identification of MIR17HG in colorectal cancer development2.1 RELA/p65 and differential IncRNA expression and regulatory relationship in colorectal tissueqRT-PCR results showed increasing expression patterns for MIR17HG and LINC00460,with gradually decreasing expression levels of XLOC006495,from adjacent to adenoma,and CRC tissues?P<0.05?.Moreover,the expression levels of RELA/p65 were gradually accumulated from adjacent to adenoma,and CRC tissues?P<0.001?.MIR17HG levels were significantly blunted after RELA/p65 knockdown in all tested CRC cell lines?P<0.01?.Correspondingly,RELA/p65 was also attenuated after MIR17HG ablation in 8 CRC cell lines?P<0.001?.However,this correlational regulation did not occur for LINC00460 and XLOC006495.2.2 The expression levels of RELA/MIR17HG in colorectal cancer tissues and its relationship with diagnosis of colorectal cancer patientsISH and IHC assay showed that relative expression levels of MIR17HG and RELA/p65 were dramatically accumulated in CRC tumor tissues compared with adjacent tissues?P<0.001?.Moreover,Kaplan-Meier?KM?analysis showed that patients with high expression of both RELA/p65 and MIR17HG gained a significantly shorter overall survival time than those with low levels?Log-Rank P<0.01?.3.Role of RELA/p65/MIR17HG positive feedback signaling in CRC development3.1 Role of RELA/p65 transcriptional regulation on MIR17HGChIP results showed that RELA/p65 could directly bind to the promoter region of MIR17HG in colorectal adenomas and cancer tissues;Spearman correlation analysis showed that RELA/p65 was positively correlated with MIR17HG expression in colorectal cancer samples?z=0.6004,P<0.001?.Colitics-associated cancer model results showed that Rela/p65 intestinal conditional knockout Rela-/-mice had longer colon length?P<0.01?,significantly increased survival time?Log-Rank P<0.05?,decreased colon tumor formation rate?P<0.01?and decreased pathological score?P<0.01?than wild Relafl/fl mice.In situ hybridization?ISH?demonstrated that MIR17HG exhibited an increasing trend from adjacent to adenoma,and CRC tissues in Relafl/fl mice?P<0.001?,but not in Rela-/-mice3.2 Effects of MIR17HG on the biological founction of colorectal cancer cellsIn vitro assays indicated that MIR17HG knockdown in SW620 and HCT116 cells resulted in significantly decreased colony formation,migration,and invasion abilities?P<0.001?MIR17HG knockdown markedly reduced tumor volumes,the lung and liver metastasis potential after cells were subcutaneously injected into the hind limb of nude mice?P<0.05?3.3 MIR17HG regulates RELA/p65 through sponging miR-375Two miRNAs,miR-375 and miR-2116-5p,were predicted to be sponged by MIR17HG via miRBase and TargetScan database analyses.MS2-based RIP assays results showed that MIR17HG was markedly enriched for miR-375?P<0.01?compared with miR-2116-5p,but not the MIR17HG with mutated miR-375 targeting sites.The luciferase reporter assay showed that miR-375 significantly reduced the luciferase activity of the cells transfected with wild-type RELA/p65 3'UTR reporter vector?P<0.001?,but not the mutant RELA/p65 3'UTR reporter vector?P>0.05?.qRT-RT-PCR results showed showed that RELA/p65 expression levels were elevated after MIR17HG overexpression?P<0.001?.Consistently,results were observed after MIR17HG overexpression+miR-375 suppression in cells?P<0.001?.Notably,compared with MIR17HG overexpression+miR-375 negative control,the expression level of RELA/p65 with MIR17HG overexpression+miR-375 overexpression was significantly inhibited in HCT116 and SW620 cells?P<0.001?,4.Role of MIR17HG/miR-17-5p/BLNK signaling axis in CRC development4.1 Effects of miR-17-5p on the biological founction of colorectal cancer cellsData retrieved from TCGA showed significantly higher expression levels of miR-17-5p in CRC tissues compared with normal ones?P<0.05?.Moreover,miR-17-5p was differentially expressed among adjacent,adenoma,and CRC tissue samples as quantified using qRT-PCR?P<0.05?.ISH results showed that the expression of miR-17-5p in colorectal cancer tissues was significantly higher than that in adjecent normal tissues?P<0.05?.Moreover,high expression of miR-17-5p was associated with shorter survival time of colorectal cancer patients?Log-Rank P<0.01?.SW620 and HCT116 cells lost colony formation,migration,and invasion capabilities,as well as tumor formation and metastasis lacking the miR-17-5p?P<0.05?4.2 MIR17HG regulated BLNK through cleaving into miR-17-5pThe results of DLR assay showed that overexpression of miR-17-5p resulted in the blunting of the luciferase activity of wild type?P<0.01?but not the mutated BLNK 3'UTR?P>0.05?.mRNA levels of BLNK were increased by miR-17-5p or MIR17HG individual knockdown?P<0.001?and blunted with either miR-17-5p or MIR17HG overexpression?P<0.001?.Furthermore,the co-overexpression of MIR17HG and miR-17-5p decreased BLNK expression?P<0.001?,and the double knockdown of MIR17HG and miR-17-5p showed the opposite effect?P<0.001?).Notably,BLNK was upregulated after MIR17HG overexpression while miR-17-5p was knocked down?P<0.001?.In contrast,BLNK was decreased after MIR17HG knockdown combined with miR-17-5p overexpression?P<0.01?.In addition,miR-17-5p expression was negatively correlated to BLNK mRNA levels in CRC,which was similar to the relationship between MIR17HG and BLNK?MIR17HG:z=-0.5807,P<0.0001;miR-17-5p:z=-0.4314,P<0.0001?4.3 Effects of BLNK on the biological founction of colorectal cancer cells and BLNK expression in colorectal cancer TMA constructionBlunt of BLNK strikingly accumulated colony formation,migration,and invasion abilities in both SW620 and HCT116 cells?P<0.01?.In agreement,in vivo data showed that increased tumor volumes were observed in nude mice lacking BLNK?P<0.01?.The same pattern was observed with liver and lung luciferase biochemical measurements,with markedly promoted liver and lung metastasis in mice carrying flank tumors expressing low levels of BLNK?P<0.05?.TMA analysis showed that BLNK expression levels were reduced dramatically in CRC tumor tissues compared with adjacent samples?P<0.01?.Consistently,KM analysis revealed that the mean overall survival time was significantly decreased in patients with relatively low BLNK expression?Log-Rank P<0.01?4.4 RELA/p65 regulates BLNK expression through MIR17HGISH results showed that miR-17-5p was up-regulated from normal,adenomas to cancer tissues of wild-type Relafl/fl mice?P<0.05?.Howover,no significant difference was found between normal-adenomas-cancer tissue of Rela-/-mice?P>0.05?.The expression of BLNK was decreased from normal,adenomas,to cancer tissues of wild-type mice?P<0.05?,but not the Rela-/-mice?P>0.05?.Both the BLNK augmentation of RELA/p65 knockdown?P<0.001?and BLNK attenuation of RELA/p65 overexpression?P<0.001?were clearly observed Notably,BLNK was upregulated after RELA/p65 overexpression while miR-17-5p or MIR17HG was knocked down?P<0.001?.In contrast,BLNK was decreased after RELA/p65 knockdown combined with miR-17-5p or MIR17HG overexpression?P<0.01?5 Preliminary study on MIR17HG in PD-L1 immunotherapy for colorectal cancer5.1 Regulation of PD-L1 by MIR17HGThe predicted results of RPIseq online software showed MIR17HG might bind with PD-L1 protein.RNA-pull down assay showed that the PD-L1 protein binds to the biotinylated MIR17HG probe.The mRNA expression levels of PD-L1 in HCT116 and SW620 cells were indistinguishable from control,following either MIR17HG overexpression or knockdown?P>0.05?.However,WB assay suggested increased PD-L1 expression following MIR17HG overexpression and decreased PD-L1 expression following MIR17HG knockdown in HCT116 and SW620 cells.IHC staining suggested significantly lower expression levels of PD-L1 in tumor tissues formed by subcutaneous injection of shMIR17HG treated SW620 or HCT116 cells,compared with NC-treated control?P<0.01?;PD-L1 expression levels were significantly higher in tumor tissue and adjacent normal tissue in Relafl/fl when compared with Rela-/-murine intestine?P<0.01?5.2 Expression of PD-L1 on colorectal cancer TMA constructionImmunohistochemistry results showed that the expression of PD-L1 in colorectal cancer tissues was significantly higher than that adjecent normal tissues?P<0.001?Conclusion1.MIR17HG Functions as a nocogene in CRC2.MIR17HG upregulates RELA/p65 expression through sponging miR-375;RELA/p65 activates MIR17HG transcription through directly binding to the promoter region of MIR17HG3.MIR17HG may cleavage into miR-17-5p,and then regulate the expression of BLNK gene in immune-related pathways,ultimately leading colorectal development4.MIR17HG increases stablity of PD-L1 protein through directly binding to PD-L1 protein. |
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