Role And Mechanism Of Acetyltransferase ARD1 In Metastasis Of Colorectal Cancer

Background: Metastasis is the leading death cause of colorectal cancer(CRC)patients,but its exact regulatory mechanism remains unclear.Therefore,there is a need to explore the underlying molecular mechanisms of CRC metastasis.Arrest Defective Protein 1(ARD1)is an acetyltransferase involved in post-translational acetylation of proteins,and has attracted much attention for its controversial role in tumors.Based on bioinformatics analysis we found that ARD1 is related to CRC metastasis,but the function and regulatory mechanism of ARD1 in CRC metastasis are still unclear.Objectives: This study aims to reveal the role of ARD1 in CRC metastasis,and its molecular mechanism of regulating CRC metastasis.It also aims to provide a basis for clarifying the role of ARD1 in CRC metastasis,and to provide new clues for finding intervention targets for CRC metastasis as well as to explore the possibility of ARD1 as a potential biomarker to predict CRC metastasis.Methods: This study employed clinical data and specimens,cells,and nude mouse models of CRC to study the role of ARD1 in CRC metastasis and its molecular mechanism,which were divided into four parts.The first part screened the key proteins of CRC metastasis and identified ARD1 as the target protein,and predicted the signaling pathway of ARD1 regulating CRC metastasis.The second part verified the relationship between ARD1 and the prognosis of CRC patients at the clinical level.The third part studied the effect of ARD1 on the biological function of CRC cells and animal model,and the fourth part explored the specific molecular mechanism of ARD1 regulating CRC metastasis.The study design and methods employed for each part are as follows.Part I: Using the nested case-control study design,this part identified the target protein ARD1 and predicated its signaling pathway that regulates CRC metastasis.The data of CRC patients who visited the Yunnan Cancer Hospital from January 2016 to December 2020 were retrospectively collected and those patients were divided into two groups: metastatic group and non-metastatic group based on whether metastasis occurred in their follow-up.After that,the propensity score was used to match the confounding factors of the two group CRC patients and quantitative proteomic sequencing was performed on paraffin sections of tumor tissues collected during their first therapeutic surgery of the tumor,including paracancerous tissues and cancer tissues of the successfully matched CRC patients.Based on bioinformatics analysis of key mediator proteins of CRC metastasis,ARD1 was identified as the target protein to be studied.Meanwhile,ARD1 was used as the gene of interest to conduct a singlegene GSEA enrichment analysis,which was combined with KEGG and GO analysis to differentially expressed genes of metastasis,so as to screen out the signaling pathway of ARD1 regulating CRC metastasis,which will be verified in the fourth part.Part II: A retrospective cohort study was used to analyze the relationship between ARD1 and CRC prognosis.In order to exclude the influence of potential related genotypes in different geographical populations on the experimental effect,another location,the Sixth Affiliated Hospital of Sun Yat-sen University,was selected where the data of patients with non-metastatic primary CRC who visited this hospital from January 2013 to December 2015 and the paraffin sections of primary tumor tissue of those patients made during their first therapeutic surgery were collected.The level of ARD1 in these paraffin sections was evaluated by immunohistochemistry,and the relationship between ARD1 and recurrence,metastasis,death,and disease-free survival of those patients was analyzed by COX proportional hazards model combined with sensitivity and trend tests.Part III: Examining the effects of ARD1 on the biological function of CRC cells and tumor metastasis markers in vivo and in vitro,respectively.The corresponding sh RNA and plasmid DNA were transfected to construct cell models of knockdown and overexpression of ARD1,respectively,and to study its effects on CRC cell cycle,proliferation,migration and invasion.Meanwhile,cell models of knockdown and overexpression of ARD1 were used to exam its effects on xenograft tumors and lung metastasis.Western blot experiments were used to detect the effects of knockdown and overexpression of ARD1 on the expression level of epithelial-mesenchymal transformation molecular markers.Part IV: Exploring the molecular mechanism of ARD1 regulates CRC metastasis.This part verified the signaling pathway of ARD1 regulating CRC metastasis screened out by the first part of this research,and the effect of ARD1 on the expression of molecular markers of the signaling pathway was forwardly verified by using the constructed cell model of knockdown and overexpression of ARD1 while the effect of ARD1 on the expression of tumor metastasis markers was reverse-verified by the addition of relevant pathway inhibitors in the cell model of over-expressing ARD1.Results: Of 1025 CRC patients in the Yunnan Cancer Hospital were included in the nested case-control analysis,and 12 pairs of paraffin sections of primary CRC patients with the smallest propensity score difference were finally selected for quantitative proteomic analysis according to the minimum sample size required by proteomics.A total of 786 differentially-expressed proteins of paracancerous and cancer tissues and156 differentially-expressed proteins of metastatic and non-metastatic group were obtained from the paraffin sections of the 24 patients,and 82 key proteins of CRC metastasis were obtained by cross-sectional analysis.The top 20 proteins with the smallest P value between the metastatic and non-metastatic group were selected for literature research,and finally,ARD1 was selected as the target protein.We found that the expression of ARD1 in cancer tissues was higher than that in paracancerous tissues,and higher in the primary tumor tissues of metastatic group than in that of nonmetastatic group.Furthermore,functional annotations of differentially-expressed genes that regulate CRC metastasis show that ARD1 may regulate CRC metastasis through the MAPK signaling pathway,HIF-1 signaling pathway,and m TOR signaling pathway.In total,146 CRC patients attending the Sixth Affiliated Hospital of Sun Yat-sen University were included in the retrospective cohort analysis,and the results showed that ARD1 was strongly associated with high CEA and high clinical pathological stage.High ARD1 expression is a risk factor for recurrence of non-metastatic CRC after surgery(HR: 3.774,95% CI: 1.463-9.732,P=0.006),metastasis(HR: 3.417,95% CI:1.323-8.823,P=0.006),death(HR: 3.350,95% CI: 1.390-8.017,P=0.007),and diseasefree survival(HR: 2.058,95% CI: 1.023-4.140,P=0.043).By studying the effect of ARD1 on CRC in vitro and vivo,we found that ARD1 can promote the cycle progression,proliferation,migration and invasion of CRC cells in vitro,and promote the growth of CRC cell xenograft tumors and lung metastasis in vivo.Further studies found that ARD1 regulates CRC metastasis through the RAF/MEK/ERK MAPK signaling pathway.By adding MEK pathway inhibitors to the cell models of overexpressing ARD1,we found that ARD1 regulates the epithelialmesenchymal transformation process through the RAF/MEK/ERK MAPK signaling pathway to facilitate metastasis of CRC.Conclusion: ARD1 is a prognostic biomarker to predict outcomes for patients with non-metastatic CRC.ARD1,regulating the epithelial-mesenchymal transition through the RAF/MEK/ERK MAPK signaling pathway,promotes the metastasis of CRC.It is a potential therapeutic target for CRC.