Micropeptide MP48 Encoded By An Upstream Open Reading Frame In SOX4 Promotes Progression Of Intrahepatic Cholangiocarcinoma

Background and objectiveIntrahepatic cholangiocarcinoma(ICC)is one of the most common primary liver cancers,second only to hepatocellular carcinoma(HCC).It accounts for about 20% of all primary liver cancers,with the incidence continuously increasing over the past few decades.ICC is characterized by complex etiology,insidious onset,and a high degree of malignancy.Common risk factors include intrahepatic bile duct stones,primary sclerosing cholangitis,viral hepatitis,cirrhosis,parasitic infections,and non-alcoholic fatty liver disease.The vast majority of patients are diagnosed at late stages,with no opportunity for surgical treatment.Curative liver resection remains the mainstay of curative treatments for ICC patients,but the prognosis is poor,with a 5-year overall survival rate of no more than 35%,Ribosome profiling(Ribo-Seq),a method based on high-throughput sequencing to detect and analyze short RNA fragments bound by ribosomes,is one of the main approaches in the study of the translatome.Traditional views suggest that non-coding RNAs(nc RNAs)are usually lack of protein-coding capability.However,with the deepening of research on translatome,an increasing number of nc RNA coding products have been discovered.These nc RNAs may contain short open reading frames(s ORFs)that are typically less than 300 nucleotides in length,and capable of encoding peptides usually shorter than 100 amino acids.These micropeptides are involved in regulating various physiological and pathological processes in humans,and play a critical role in the development and progression of cancer.Some micropeptides have been proven to be related to patients’ treatment responses and prognosis,potentially serving as therapeutic targets and prognostic biomarkers.To date,there have been no reports on micropeptides related to ICC.Based on Ribo-Seq,this study aims to explore ICC-related micropeptides,investigate their correlation with clinical prognosis in patients,and delve into their functions and molecular mechanisms in regulating the progression of ICC.Methods1.Based on Ribo-Seq,the translational abundance of coding genes in ICC was determined and differential analysis was performed.Combined with transcriptional abundance,the translational efficiency of each gene was calculated to explore the translation characteristics of ICC.2.Utilizing multiple s ORF encoding capacity scoring systems like ORFscore and RRS,differentially expressed s ORFs in ICC cancer and adjacent tissues were subjected to multidimensional screening to identify target s ORFs and their coding micropeptides.3.Antibodies against the MP48 micropeptide were developed to validate its endogenous expression in various ICC cell lines and tissues.4.Immunohistochemistry was used to score the expression levels in 90 patients with ICC,which were then divided into high and low expression groups.Kaplan-Meier survival analysis and Cox regression analysis were conducted to investigate the correlation between MP48 expression levels and the clinical prognosis of ICC patients.5.Stable low-expression and high-expression ICC cell lines were established,and in vitro cell functional experiments(clonogenic assay,CCK-8 assay,Transwell assay)were conducted to explore the impact of MP48 on the proliferation and invasion abilities of ICC tumor cells.6.Subcutaneous tumor models and lung metastasis models were established in BALB/c nude mice to investigate the effects of MP48 on the growth and metastasis of ICC in vivo.7.Transcriptome sequencing was performed on ICC cell lines with stably low expression of MP48.Significantly differentially expressed gene sets were identified by differential analysis,followed by GO and KEGG enrichment analyses to explore biological processes and key molecules potentially affected by MP48.8.For the key pathways identified through enrichment analysis,western blotting experiments were conducted to verify their expression levels.For key molecules that may interact with each other,co-immunoprecipitation(Co-IP)experiments were performed to validate their interaction with MP48.ResultsThe first part of the study involves Ribo-seq and transcriptome sequencing analyses of nine pairs of ICC and adjacent non-cancerous tissues.A total of 12,124 significantly differentially expressed genes were identified at the translation level,which is considerably higher than the number of differentially expressed genes identified at the transcription level,suggesting complex translational regulation in the development of ICC.There were no significant differences in overall translational efficiency between ICC and adjacent non-cancerous tissues,and no significant correlation between the translational efficiency of coding genes and their transcription abundance.Enrichment analyses were performed on genes that were significantly differentially expressed at the translational level,co-directionally changed at the translational-transcriptional level,and in terms of translation efficiency between cancerous and non-cancerous tissues.Significant enrichment was observed in biological processes such as immunity,inflammation,and metabolism,indicating significant changes in these aspects during the development and progression of ICC.Subsequently,s ORFs in the non-coding regions were searched using Ribo-seq,and their coding potential was scored from multiple dimensions using tools like ORFscore and RRS,combined with differential analysis.Ultimately,SOX4-u ORF with the highest fold difference was selected from the 15 u ORFs with the most coding potential,and it may encode a micropeptide with a length of 48 amino acids,which was named MP48.In the second part,we first prepared monoclonal antibodies against MP48.The expression of MP48 was validated in ICC cell lines(RBE,HCCC-9810,QBC939,Hu CCT1)and normal intrahepatic biliary epithelial cells(HIBEC)through western blotting experiments.The endogenous expression of MP48 was also tested in twelve pairs of ICC cancerous and adjacent non-cancerous tissues,revealing that MP48 is indeed endogenously expressed in ICC cells and tissues,with the highest expression in HCCC-9810 cells.The expression of MP48 varied significantly in different ICC cancerous tissues,but was significantly higher than in adjacent non-cancerous tissues.Immunohistochemistry(IHC)staining was performed on tissues from all ninety ICC patients,and the IHC score of MP48 in cancerous tissues was significantly higher than in para-cancerous liver tissues.Patients were divided into high and low expression groups based on the median total score of MP48 expression in cancerous tissues.Baseline clinical and pathological data analysis indicated significant differences between high and low MP48 expression groups in terms of hepatitis B virus infection and tumor diameter.Kaplan-Meier survival analysis showed that the postoperative OS and RFS were significantly better in the low MP48 expression group than in the high expression group.Univariate and multivariate Cox analyses identified high MP48 expression and tumor diameter >5cm as independent risk factors affecting postoperative OS in ICC patients,while high MP48 expression,microvascular invasion,and lymph node metastasis were independent risk factors affecting postoperative RFS.The third part explores the functional role and specific molecular mechanisms of MP48 in promoting the malignant progression of ICC.In terms of cell function,HCCC-9810 and RBE cells were chosen to construct stable cell lines with low and high MP48 expression,respectively.The increased expression of MP48 significantly enhanced RBE cells’ proliferation and migration capabilities,while the reduced expression of MP48 resulted in the inhibition of the proliferation and migration of HCCC-9810 cells.In vivo experiments using subcutaneous tumor and lung metastasis models in nude mice confirmed that the downregulation of MP48 significantly decreased the growth and metastasis of ICC.Mechanistically,after sequencing HCCC-9810 cell lines with three knockdowns,a common set of 1,053 significantly differentially expressed genes was identified.Ranking these genes by differential multiples,we found that CD34,which was related to tumor angiogenesis,was one of the most significantly altered genes following MP48 knockdown.GO and KEGG enrichment analyses were utilized to screen for key pathways and molecules potentially affected by MP48.GO enrichment analysis showed significant enrichment in angiogenesis terms,while KEGG enrichment analysis indicated significant changes in the PI3K/AKT signaling pathway.western blotting experiments confirmed significant changes in p-PI3 K and p-AKT after MP48 knockdown and overexpression.Silver staining mass spectrometry analysis and CO-IP experiments confirmed the interaction between MP48 and CD34.Therefore,MP48 may promote the growth and metastasis of ICC by acting on the CD34 molecule and the PI3K/AKT pathway.ConclusionThis study conducted Ribo-seq and transcriptome sequencing on cancerous and non-cancerous tissues from multiple ICC patients,combined with bioinformatics analysis,to explore the translational features of ICC.Through various nc RNA coding potential scoring systems like ORFscore and RRS,the target micropeptide MP48,potentially encoded by SOX4-u ORF,was selected.Subsequently,the endogenous expression of MP48 in ICC cells and tissues was confirmed by developing monoclonal antibodies against MP48.IHC analysis of cancerous and non-cancerous tissues from ninety ICC patients demonstrated that high MP48 expression is an independent risk factor for postoperative OS and RFS in patients.Finally,in vitro experiments using ICC cell lines,as well as in vivo experiments using subcutaneous tumor models and lung metastasis models in nude mice,confirmed that MP48 promotes ICC cell proliferation and migration.Mechanistically,transcriptome sequencing analysis combined with western blotting and CO-IP experiments indicated that MP48 may contribute to ICC progression and metastasis by acting on CD34 and the PI3K/AKT pathway.