Molecular Mechanism Of HOXC6 Promoting Growth,Metastasis And Metabolic Reprogramming Of MSS With Colon Cancer

Background:Colorectal cancer(CRC)is one of the most common malignant tumors in the world,ranking the third in the incidence of cancer and the second in the mortality,second only to lung cancer[1].In China,according to the latest data released by the National Cancer Institute,the incidence of colorectal cancer ranks the third and the mortality ranks the fifth among in the country[2].In recent years,the research on the difference between left and right-sided colon cancer has gradually become a hot topic in the field of colorectal cancer.A number of studies have shown that the prognosis of patients with microsatellite stable(MSS)is worse than that of patients with microsatellite instability(MSI).In addition,the prognosis of patients with MSS colorectal cancer with liver metastases originating from the right-sided colon is the worst among those with MSI with colorectal cancer with liver metastases and those with MSS with colorectal cancer with liver metastases from the left-sided colon.Our previous results showed that HOXC6 expression in the right-sided of MSS with colon cancer was significantly higher than that in the left-sided of MSS with colon cancer and was negatively correlated with the overall survival of CRC patients.Meanwhile,functional experiments showed that HOXC6 overexpression promoted the growth,invasion and migration of SW480 cells.Therefore,this study aims to explore the molecular mechanism of transcription factor HOXC6 in left and right-sided colon cancer and the role of distant metastasis and in order to provide a molecular target for precise treatment of patients with MSS of left and right-sided colon cancer.Methods:The MSS differential gene HOXC6 was screened out of MSS with left and right-sided colon cancer and the migration,invasion and drug-resistant cell phenotype experiments were conducted.On the basis of the previous study,CCK-8 method was used to detect the effect of HOXC6 on the proliferation ability of colorectal cancer cells,clone formation experiment was used to detect the effect of HOXC6 on the cloning ability of colorectal cancer cells,and flow cytometry was used to detect the effect of HOXC6 on the cell cycle of colorectal cancer cells.At the animal level,the effect of HOXC6 on the tumorigenic ability of colorectal cancer cells was detected by xenograft tumor experiments in nude mice,and the effect of HOXC6 on the metastasis ability of colorectal cancer cells was detected by spleen cultivation of liver metastasis method in nude mice.In order to explore the molecular mechanism of downstream regulation of HOXC6,ChIP-seq was used to find the downstream target genes of HOXC6,and the combined analysis of ChIP-seq and mRNA-seq was conducted to explore the downstream signal pathway regulated by HOXC6,and Western blot was used to verify the results.According to the results of the combined analysis,the metabolic pathways specifically regulated by HOXC6 were studied through untargeted metabolomics in the direction of metabolism.Results:In vitro cell experiments,it showed that HOXC6 could promote the proliferation and clone formation of colorectal cancer cells,and regulate the cell cycle of colorectal cancer to G2 phase.In vivo animal experiments,it had shown that HOXC6 can promote the tumorigenic ability of colorectal cancer cells in nude mice and promote liver metastasis.Through analysis of RNA-seq data,we found that HOXC6 overexpression could induce significant differential expression of 407 genes,of which 343 genes were up-regulated and 64 genes were down-regulated.KEGG enrichment analysis of the significantly differential genes showed that the pathways were mainly enriched in MAPK,PI3K-Akt,Ras and the metabolism.At the same time,GO_BP indicated that HOXC6 was significantly enriched in L-glutamate import across plasma membrane,D-aspartate import across plasma membrane and D-aspartate transport.According to the analysis of ChIP-Seq data,KEGG results showed that the downstream target genes regulated by HOXC6 were mainly enriched in HIPPO,stem cells,Wnt and metabolic pathways related to microflora.At the same time,GO_BP indicated that target genes were significantly enriched in cell metabolism and nitrogen metabolism pathways.We further analyzed RNA-seq and ChIP-seq combined using BETA method.The results showed that a total of 1610 downstream genes were directly regulated by HOXC6,including 634 up-regulated genes and 976 down-regulated genes.Including MYC,NFATC4,FGFR4,LPIN1,HMGCR and other genes,among which MYC had a higher Peak score of 29.17.KEGG pathway enrichment of target genes showed that HOXC6-regulated genes were mainly enriched in stem cells,Wnt,AMPK,and metabolic pathways.It also suggested that HOXC6 plays an important role in the regulation of metabolic reprogramming of colorectal cancer and stem cells pathways.We further verified the important genes in the significantly enriched pathway by RT-qPCR,and the results showed that HOXC6 had a positive regulation effect on MYC,FGFR4,LPIN and other genes.Dual-luciferase reporter assay confirmed that HOXC6 could bind to the promoter region of MYC and activate the MYC transcription.Four binding sites of HOXC6 and MYC were screened through the Jaspar website,and it was found that the specific binding sites of HOXC6 and MYC were in the MYC promoter region 1178-1794,with negative sense chain and the biding sequence is CGCATGAATTAACTACG.Western blot analysis showed that HOXC6 expression could up-regulate the expression of downstream target gene MYC,and also up-regulate the expression of GSK-3β,β-catenin,c-myc and c-jun in Wnt pathway.Through the analysis of untargeted metabolomics,the results showed that KEGG pathway was mainly enriched in amino acids and lipid metabolic pathways.HMDB metabolites were concentrated in lipids,lipids molecules and organic acids,and LIPID MAPS metabolites were mainly concentrated in glycerolipids and fatty acids.In addition,we analyzed the metabolites of 263 colorectal cancer patients at the time of first diagnosis and found that there were significant differences in albumin,aspartate aminotransferase,low-density lipoprotein,lactate dehydrogenase,alkaline phosphatase,and glutamine transferase between colon cancer and rectum cancer patients.Significant differences were found in albumin,indirect bilirubin,aspartate aminotransferase,creatinine,uric acid,low density lipoprotein,andprotein/globulin in left and right-sided colon cancer patients.Conclusion:above all,the high expression of HOXC6 gene in MSS with left and rightsided colon cancer indicates the increased tumor malignancy.HOXC6 can promote the growth of colorectal cancer cells and the formation of liver metastasis and can target MYC gene activating Wnt pathway.It also affects lipid metabolism,and promotes tumor occurrence and development by regulating tumor metabolic reprogramming.