The Mechanism Of MYG1 Promoting The Proliferation,invasion And Metastasis Of Colorectal Cancer Through PKM2

Background and purposeColorectal cancer(CRC)is one of digestive system carcinomas with high morbidity and mortality worldwide and one of the most common malignant tumors in China.Its incidence rate and mortality rate are increasing,and CRC patients tend to be younger and younger year by year.Although we have made some achievements in the treatment of CRC by operation,chemoradiotherapy and other means,the overall prognosis of CRC patients is still not optimistic.As a result,it is significant for CRC diagnosis,intervention,treatment and its effeciacy evaluation by elucidating the molecular mechanism of CRC formation and development.In our previous study,we found that melanocyte proliferation gene 1(MYG1)was up-regulated in CRC and survival analysis revealed that high MYG1 expression was related to poor prognosis of CRC patients.In the nucleus,MYG1 is involved in dealing with ribosomal preRNA of ribosome assembly and regulates cytoplasmic translation.In the mitochondrial matrix,MYG1 binds to mitochondria ribosome and the 3’-ends of messenger RNA,and regulates the translation of mitochondrial proteins.In recent years,MYG1 was reported to serve as a 3’-5’RNA exonuclease,and high MYG1 expression was associated with the sensitivity of vitiligo and it acted as a dual localization protein in regulating mitochondrial.Recently,the expression of MYG1 protein in colorectal cancer tissue was found to be significantly higher than that in normal intestinal mucosa tissue,but the biological function and molecular mechanism of its abnormal expression is not further elucidate.Therefore,exploring the biological role and molecular mechanism of MYG1 in development and progression of CRC will provide a new theoretical basis for the prevention and treatment of colorectal cancer.MethodsThe expression of MYG1 was detected by real-time quantitative PCR and western blot in fresh human CRC tissue and matched adjacent normal tissues,human CRC cell lines and colorectal epithelial cell line.And the expression of MYG1 was determined by immunohistochemistry in 149 paraffin slides of human CRC tissues and matched normal tissues.The correlation between the expression level of MYG1 and the clinicopathological parameters of CRC patients was analyzed.The stable CRC cell lines with MYG1 overexpression and knockdown were established by using the lentiviral vector.After the lentivirus vectors were established,the proliferative ability of CRC cells in vitro was measured by CCK-8 and cloning formation assays.The migrative and invasive ability of MYG1 of CRC cells in vitro was detected by scratch healing assay and transwell chamber migration assay,as well as matrigel invasion assay.Flow cytometry was employed to analyze the changes of cell cycle distribution and apoptosis rate.Subcutaneous tumor model in nude mice and spleen subcapsular injection assays were utilized to observe the tumor formation and metastasis ability of CRC cells in vivo.Then,the transcriptome sequencing of MYG1 overexpressed cells,gene set enrichment and western blotting assays suggested that MYG1 overexpression could activate the JAK/STAT3 signaling pathway.Subsequently,the nucleoprotein in the MYG1-overexpressed cells were extracted by nucleoplasm separation assay.In order to identify the target protein of MYG1,IP and mass spectrometry assay were employed and the interacting protein pyruvate kinase 2(PKM2)was screened.The expression of intracellular PKM2 protein was detected by western blot after changing MYG1 expression.The protein synthesis inhibitor Cycloheximide was used to detect whether overexpression of MYG1 prolonged the half-life of PKM2 protein,which thereby caused upregulation of PKM2 protein in cells.Subsequently,the expression level of phosphorylated STAT3 and MYG1-mediated biological behaviors of CRC cells after downregulating PKM2 in MYG1-overexpressed CRC cells or overexpressing PKM2 in MYG1-silenced CRC cells were detected to explore the impacts of PKM2 on the JAK/STAT3 signaling pathway regulated by MYG1.Additionally,we tested pyruvate kinase activity to assess whether overexpression of MYG1 increased pyruvate kinase activity.And the glucose metabolism and lactose level were detected to evaluate the effects of MYGl on the aerobic glycolysis of CRC cells.Spearman and other statistical methods were used to analyze the relationship between MYG1 and PKM2 protein in 100 colorectal cancer samples.ResultsThe mRNA expression level of MYG1 in human CRC cell lines was significantly higher than that in normal colorectal epithelial cells.The expression level of MYG1 in human CRC tissues was significantly higher than that in normal adjacent normal tissues.Moerover,high MYG1 expression was related to lymph node metastasis,distant metastasis and clinical stage.Moreover,upregulation of MYG1 predicts poor prognosis of CRC patients.Overexpression of MYG1 significantly promoted proliferation,migration and invasion of CRC cells in vitro.Downregulation of MYGl achieved the opposite results.The results of Co-IP assay and immunofluorescence assay confirmed the interaction between MYG1 and PKM2.PKM2 was up-regulated in the cytoplasm and nucleus after overexpressing MYG1,but the mRNA level of PKM2 didn’t change.Further mechanism studies highlighted that MYG1 inhibited the degradation of PKM2 through the ubiquitin-proteasome pathway after overexpressing MYG1.Importantly,silencing PKM2 in MYG1-overexpressed CRC cells could effectively inhibit the activation of MYG1-mediated JAK/STAT3 signaling pathway,followed by downregulation of the downstream target genes CCND1,MMP2,MMP9,BCL-2L1 mRNA and upregulation of P21 and P27 mRNA,thereby suppressing CRC cell proliferation and invasion.But overexpression of PKM2 in MYGl-silenced CRC cells could partially reversed the inhibitory effects of JAK/STAT3 signaling pathway by interference with MYG1,thereby saving this pathway-meidiated cell proliferation and invasion.These results indicate that stability of PKM2 protein is essential for to activation of MYG1-mediated the JAK/STAT3 signaling pathway.In addition,MYG1 also promoted aerobic glycolysis of CRC cells by enhancing the pyruvate kinase activity of PKM2.In 100 cases of CRC paraffin specimens,the expression level of MYG1 protein was positively associated with the expression level of PKM2 protein.Moreover,high expression of MYG1 and PKM2 were associated with lymph node metastasis,distant metastasis,and clinical stage in CRC patients.ConclusionOur study found that MYG1 was up-regulated in CRC and associated with lymph node metastasis,distant metastasis and clinical stage of CRC patients,and high MYG1 expression predicted poor prognosis.MYG1 could promote proliferation,invasion,and metastasis in vivo and in vitro experiments.Furthermore,MYG1 increased the aerobic glycolysis of CRC cells by enhancing the pyruvate kinase activity of PKM2.MYG1 promoted CRC cell proliferation,invasion and metastasis by activating JAK/STAT3 signaling pathway through interacting with PKM2.These results suggest MYG1 will be a potential molecular marker for colorectal cancer detection and reflecting the aggressiveness and prognosis of CRC,and it will become a promising target for clinical diagnosis and treatment of CRC.