MiRNA-148a-3p Silences CANX/MHC-I Pathway And Impairs CD8+T Cell-mediated Immune Attack In Colorectal Cancer

Backgroud:Calnexin(CANX)is one of the important components in the antigen presentation process.Aberrant expressions of CANX prevent successful assembly of major histocompatibility complex I(MHC-I),antigen peptide processing,presentation on the tumor cell surface,and thus may contribute to escape immunosurveillance.In our previous work,we identified that low CANX expression was associated with poor survival rate and malignant progression in patients with colorectal cancer(CRC)by CANX-regulated expression of MHC-I and the infiltration of immunostimulatory T cell subsets.However,the underlying miRNAs controlling CANX expression and the consequent impact on the human immune system in CRC are not fulling understood.Objective:To screen and validate the miRNA that regulates the expression of CANX;to investigate functional impacts of miRNA-mediated CANX/MHC-I expression changes on cytotoxic T lymphocytes(CTL)-mediated tumor killing in vivo and in vitro.Methods:1.Screening and verification of miRNA that modulates CANX expression.In order to investigate expression profiles of miRNAs in LoVo cell lines with poor expression of CANX and CANX proficient cell lines DLD-1,we used a miRNA microarray platform covering 88 miRNAs involved in the regulation of immunopathology pathways.We further validated the array data by single-targeted qRT-PCR.Bioinformatics was used to predict whether upregulated miRNAs in Lovo cells could target CANX and its binding sites.Mi RNA-148a-3p targeting CANX was further validated using a dual luciferase reporting system.The effect of miR-148a-3p overexpression/knockdown on the expression of CANX and MHC-I was detected by western blot.2.Effect of miR-148a-3p induced CANX and MHC-I alteration on infiltration of immune cells.We constructed C57BL/6 mouse xenograft models and obtained CD8+T cells from mouse spleens by magnetic bead sorting.The CD8+T cells were co-cultured with miR-148a-3p knockdown MC-38 cells.The cytotoxic effect of CD8+T cells on MC-38 was detected by CCK8.ELISA was used to detect the secretion of TNF-a.Equal numbers of wide type(WT)or miR-148a-3p knockdown MC38 cells were transplanted subcutaneously into the right flanks of C57BL/6 mice,and monitored tumor growth.The infiltrating immune cells in the transplanted tumor were detected by flow cytometry and multi color immunohistochemistry.Results:1.There are fourteen miRNAs showing differential expression patterns between the two cell lines.It was predicted and confirmed that miRNA-148a-3p could regulate CANX expression.2.Knockdown of miR-148a-3p can enhance the killing effect of CD8+T cells on tumor cells in vitro,attenuate tumor growth,and increase the infiltration of CD8+T cells in the immune microenvironment of transplanted tumor through targeting CANX/MHC-I pathway.Conclusion:MiR-148a-3p is a potential regulator of CANX.Inhibition of miR-148a-3p restores surface levels of MHC-I and significantly enhanced the effects of CD8+T cell-mediated immune attack in vitro and in vivo by promoting CANX expression.