The Study Of The Mechanism Of LINC00460 Promoting The Growth And Metastasis Of Colorectal Cancer Through MYC/CD47

BackgroundWith the rapid development of gene sequencing technology,more and more long non- coding RNAs(Lnc RNAs)have been found to play a role in promoting or inhibiting the progress in CRC.Recent studies at home and abroad in the past decade have shown that the imbalance of Lnc RNAs has an impact on the proliferation,angiogenesis,metastasis,invasion,apoptosis,dryness,anti-tumor drug resistance and genomic instability of colorectal cancer,so it has clinical significance.Several recent studies have found that Lnc RNAs may have an effect on tumor immunity and immunotherapy,but the number of studies is small and the specific mechanism of the effect of Lnc RNAs dysfunction on the panoramic picture of cell types and distribution in CRC tumor immune microenvironment has not been clearly revealed.In previous studies,we screened out Lnc RNA LINC00460 that was significantly highly expressed in CRC tissues.Combined with our previous research results,we found that the expression level of LINC00460 was significantly positively correlated with the risk score of the stromal and immune score prognostic model,and LINC00460 may be related to the heterogeneity of immune-infiltration between metastatic tumors in different organs and their primary lesions.Therefore,we believe that LINC00460 may play an important role in CRC tumor immunity.Clarifying the biological function of LINC00460 in CRC and exploring the specific molecular mechanism of its influence on tumor immunity can help us further understand and expand the molecular network of the pathogenic mechanism of CRC,which is conducive to providing new ideas and possible targets for the diagnosis and treatment of CRC,and has positive and far-reaching clinical significance.ObjectiveTo study the role of LINC00460 in the growth and metastasis of CRC,clarify the influence of LINC00460 on CRC tumor immunity,and explore its specific molecular mechanism.MethodsLnc RNA LINC00460,which is differentially expressed in human CRC tumor and normal colon tissues and correlated with survival prognosis,was screened out by bioinformatics analysis using CRC transcriptome sequencing data of our research group and CRC gene expression profiles downloaded from GEO/TCGA database.The differential expression of LINC00460 was verified by q RT-PCR and in situ hybridization analysis of 102 pairs of matched CRC tumor tissues and adjacent normal colon tissues.The stable overexpression and knockdown cell lines of LINC00460 were constructed,and the effects of LINC00460 on the proliferation,migration and invasion of CRC cancer cells were studied by in vitro functional experiments,such as colony formation,Ed U,CCK-8,wound healing and transwell assays.The effects of LINC00460 dysfunction on CRC proliferation,metastasis and angiogenesis were studied in vivo by constructing mouse models of subcutaneous tumor,liver and lung metastasis.In terms of molecular mechanism,bioinformatics was used to analyze downstream targets and functional axes anchored by LINC00460.Direct binding of target molecules was determined by double luciferase reporter assay,RNA immunoprecipitation(RIP),RNA pull down and other experiments.The established stable cell lines and mouse CRC tumor models were used for q RT-PCR,WB and immunohistochemical experiments to verify the influence of LINC00460 dysregulation on downstream target molecules and cell infiltration in tumor microenvironment in vitro and in vivo,so as to clarify the specific molecular mechanism of LINC00460’s function on CRC.ResultsThe expression level of LINC00460 increased step by step in adjacent normal colon,primary CRC tumor and distant metastasis,and was significantly correlated with the characteristics of patients’TNM staging and CMS molecular typing.High expression of LINC00460 predicted poor survival prognosis of patients.In vitro and in vivo functional experiments showed that up-regulated or down-regulated LINC00460 expression could promote or inhibit the proliferation,metastasis and angiogenesis of CRC cells,respectively.Mechanism studies have found that LINC00460,as a molecular sponge of miR-186-3p,can release the inhibition of miR-186-3p on MYC/CD47 protein translation at the post-transcriptional level,significantly improve the protein expression level of downstream target molecule MYC/CD47&PD-L1,and create an inhibitory immune microenvironment.The infiltration of CD8~+T cells and M1 macrophages was reduced,and the infiltration of Treg,M2macrophages and CAF cell subgroups was promoted,thus promoting the growth,metastasis and immune escape of CRC.In addition,MYC can promote the expression level of LINC00460 at the transcriptional level,thus achieving a positive feedback loop of MYC/LINC00460/miR-186-3p/MYC/CD47&PD-L1 and continuously promoting the progression of CRC.Conclusion1.The expression of LINC00460 is significantly up-regulated in CRC tumor tissues,and is associated with poor clinicopathological manifestations and survival prognosis of patients.2.The high expression of LINC00460 promotes the proliferation,migration,invasion and angiogenesis of CRC cells in vivo and in vitro.3.In terms of mechanism,as a molecular sponge of miR-186-3p,LINC00460 can release the inhibition of miR-186-3p on MYC/CD47 protein translation at the post-transcriptional level,and significantly improve the protein expression level of MYC/CD47&PD-L1.Thus promoting the proliferation,metastasis,angiogenesis and immune escape of CRC cancer cells.4.The imbalance of LINC00460 affects the distribution of cell subsets in CRC tumor microenvironment.Overexpression of LINC00460 inhibited the infiltration of CD8~+T cells and M1 macrophages,and promoted the infiltration of Treg,M2 macrophages and CAF.5.As a transcription factor,MYC molecule promoted the expression level of LINC00460 at the transcription level,thus forming a positive feedback loop of MYC/LINC00460/miR-186-3p/MYC/CD47&PD-L1,which continuously promoted the progress of CRC.