Study On The Potential Activated Phenotype And Pathway Related To MACC1 On The Progression Of Pancreatic Cancer

BackgroundPancreatic cancer ranks 4th in the world among all malignant tumors,with high invasiveness,early metastasis,lack of specific symptoms and high mortality.The median survival time is only 3-6 months,with a 5-year survival rate of<5%.In China,the incidence of pancreatic cancer has increased 6-fold in the past 20 years.In 2012,the incidence of pancreatic cancer was 4.8/100,000,ranking 7th among all malignant tumors.Even when early diagnosed,small pancreatic cancer(<2 cm in diameter)can easily metastasize and lead quickly to the death of the patient.Surgical resection remains the only treatment for radical treatment.Nevertheless,only 15-20%of pancreatic cancer patients are suitable for surgical treatment.Even with multidisciplinary comprehensive treatment,the median survival time of patients with advanced pancreatic cancer is only 6 months.Furthermore,early postoperative recurrence and occult metastasis also reduce the efficacy of surgical treatment.Even after systemic therapy,liver metastasis occurs in 50%of patients.However,the main reasons for the high mortality rates of pancreatic cancer are the failure of an early diagnosis,before it moves to other organs,and the resistance of the cancer to existing therapies.MACC1(Metastasis-associated in colon cancer 1)gene,which has been identified in colorectal cancer as its name,is a novel regulator for tumor proliferation and metastasis3.It has been found that MACC1 encodes hepatocyte growth factor(HGF),regulates mTOR signaling pathway,HGF/Met signaling pathway and accelerates cancer cell to migrate,invade,and metastasize3-5.Moreover,some studies have elucidated that overexpression of MACC1 is significantly related to poor clinical outcomes in colorectal carcinoma,hepatocellular carcinoma,lung adenocarcinoma,gastric carcinoma,esophageal cancer,and ovarian Cancer6-11.However,the precise mRNA expression and survival analysis of MACC1 on pancreatic cancer patients remain unclear.With the development of high-throughput technologies,such as gene chip and RNA-seq,gene-related analyses have been able to identify the significance of target genes from overall gene expression data.To the best of our knowledge,bioinformatics methods has not been applied to explore the function of MACC1 in pancreatic cancer.PurposeTaken together,we hope to understand the prognostic value of MACC1 and pivotal role with targeted signal pathway for MACC1 in pancreatic carcinogenesis.These finding may offer new perspectives in the future research and clinical practice for pancreatic cancer patients.Materials and methods1.Twenty-five patients with pancreatic cancer who underwent pancreatico-duodenectomy in the First Affiliated Hospital of Zhengzhou University from March 2017 to November 2019 consent to donate their cancer tissues and corresponding adjacent tissue.All the patients had signed informed consent before surgery and all the cancer and adjacent tissues were stored in nitrogen canister.Then we used quantitative PCR to detect the expression of MACC 1 in 25 cases of thyroid papillary carcinoma and corresponding paired para-carcinoma tissues,and performed visual and statistical tests.2.Through bioinformatic methods,including the programming languages,data mining with pancreatic cancer data from Oncomine,GEO database and TCGA database,the mRNA expression of MACC 1 in pancreatic cancer tissue was identified.Then,the protein level of MACC 1 in pancreatic cancer tissue was verified through immunohistochemical result in the Human Protein Atlas.3.The expression of MACC1 in ASPC-1 and PANC-1 pancreatic cancer cell lines were obtained by extracting Next-Generation Sequencing gene expression data from CCLE cell line database.siRNA was designed and transferred into experiment group of ASPC-1 for knockout,while panc-1 cell lines with lower expression of MACC1 were designed to be transferred by corresponding MACC1 plasmids for overexpression.4.The Next-Generation Sequencing mRNA data of 178 pancreatic cancer tissues in TCGA were extracted and normalization.The possible role and pathway of MACC1 in pancreatic cancer were predicted by R language gene enrichment analysis(GSEA)algorithm.5.In the above treated cell lines and their respective control group,CCK 8 kits and clone formation experiment were done to test cell migration and proliferation,cell migration and invasion experiment with transwell chambers were done to test malignant ability,flow cytometry instrument was used to detect the changes of the apoptosis before and after transfection.6.In the above experimental group and control group,western blot was used to detect the key proteins in the relevant pathways predicted by GSEA and find the pathways with different protein expressions.7.R 3.6.1 and Prism 6.0 were used for statistical analysis of all data.One-way anova and two-sided t-test were used to compare quantitative data.Chi-square test is used to compare qualitative data.Result1.The expression of MACC1 significantly increased in pancreatic cancer tissue compared with normal tissue.2.Although only 1 pancreatic cancer analyses shows upregulation of MACC1,there was an outlier in 6 pancreatic cancer analyses,indicating that MACC1 mRNA serves as a decent biomarker for pancreatic cancer.Then,MACC1 is significantly upregulated in Pei pancreatic cancer cohort in Oncomine,with 36 pancreatic cancer samples and 15 normal pancreatic duct tissues(P<0.05).The consequences from TCGA analysis,paired comparison from GSE15471 and GSE28735 also confirmed this conclusion.The data of pancreatic cancer patients with different clinical stages,pathological grades and survival time were acquired from TCGA datasets.Based on these data,the high expression of MACC1 is strongly corelated with higher clinical stage,pathological grade and poorer prognosis.Immunohistochemical imagines from Human Protein Atlas confirmed the overexpression of MACC1 in pancreatic cancer tissue compared with normal tissue in protein level.3,The result from CCLE database indicated that the expression of MACC1 in ASPC-1 is higher than PANC-1.According to this finding,we chose ASPC-1 to be transfected with siRNA and PANC-1 to be transfected with MACC1 plasmid.Overexpression of MACC1 can promote the proliferation and downregulated MACC1 can restrain the proliferation of pancreatic cancer cell.Moreover,according to the result of GSEA,the related pathway including cell cycle and pancreatic cancer are also enriched in TCGA-PAAD cohort.The result of clone formation,transwell migration,invasion and flow cytometry indicated upregulated MACC1 can promote tumor migration,invasion and restrain apoptosis in pancreatic cancer cell.Downregulated MACC1 can restrain tumor migration,invasion and predispose to apoptosis in pancreatic cancer cell.Additionally,some related results of GSEA also demonstrated the potential function of MACC1 as a tumor promoter and apoptosis depressor.4.To evaluate the exact signaling pathway which MACC1 is participated in,we searched the potential signaling pathway in GSEA results above and some pathways were identified with high correlation(P<0.05),including p53 signaling pathway(P<0.001),Notch signaling pathway(P=0.014),and Rig I like receptor signaling pathway(P=0.029).After verification through western blot assay,we confirmed that Notch signaling pathway related proteins,including Notch 1,Heyl and Hesl were downregulated and p53 signaling pathway related protein,including p53 and MDM2 were significantly changed after knockdown of MACC1 expression level in pancreatic cancer cell.These results indicated that MACC1 plays an important role in pancreatic tumorigenesis through p53 and Notch signaling pathway:ConclusionOur study indicated that MACC1 are significantly high-expressed in pancreatic cancer tissue and distinctly relative to poorer AJCC stages,grades,OS and PFS.Overexpression of MACC1 could promote malignant phenotype of pancreatic cancer via activating P53 and Notch signaling pathway.