The Role And Mechanism Of ADAM12 In Colon Adenocarcinoma

Colon Cancer(CC)is a common malignant tumor.Among global tumors in 2020,colon cancer ranks 8th.New colon cancer cases account for 6.0%of new tumor cases.Colon cancer deaths are close to 6.0%of the total tumor deaths.And the American Anti-Cancer Association predicts that in 2021 there will be more than 104,000,will be close to 53,000 deaths.The diagnosis and treatment of colon cancer is currently the focus of medical research.With the continuous advancement and development of detection technology,some patients have achieved early detection,early diagnosis and early treatment,but a large number of patients still have recurrence and metastasis,and the prognosis is poor.Similarly,studies have shown that most colon cancers are sporadic and have a greater relationship with lifestyle habits;they are less affected by genetics or family history.More and more evidences show that the abnormal expression and activation of oncogenes,tumor suppressor genes and related molecular signaling pathways in colon cancer play an important role in the occurrence and progression of colon cancer.In this study,we cross-compared the TCGA database,GSE62321 and GSE146587 in the GEO database,and found genes that are co-expressed differently in colon cancer:Disintegrin and Metalloproteinase 12a(Disintegrin and Metalloproteinase 12,ADAM 12).The disintegrin-metalloproteinase family(A Disintegrin And Metalloproteinase,ADAM)is a class of protein molecules containing disintegrin and metalloproteinase domains.Proteins of this family are abnormally expressed in a variety of tumors,mainly by affecting tumor cells,cell-to-cell,cell-to-extracellular mechanism adhesion,proteolysis,and regulation of tumor cell signal transduction.Including the induction of tumor angiogenesis and other effects,mainly involved in the occurrence and development of tumors.Current research reports have confirmed that ADAM28 is related to the poor prognosis of pancreatic cancer,AD AM17 plays a role in the progression of colorectal cancer,and ADAM9 is related to cervical cancer.However,the role of AD AM 12 in colon cancer is rarely reported.The homeobox(HOX)gene encoding transcription factor is a key gene in biological development.HOXA1 is a member of the HOX family.Research in recent years has focused on its role in oral cancer and cervical cancer.The NF-κB signaling pathway is mainly involved in a variety of normal physiological processes in the human body,and it also includes the regulation of tumor proliferation,invasion and apoptosis.The transcription factor family of NF-κB plays a vital role as a stressor in the cellular environment and controls the expression of important regulatory genes such as immunity,inflammation,death,and cell proliferation.Therefore,in order to study the role and mechanism of AD AM 12 in colon cancer.This study intends to conduct research from the following four aspects,and further elaborate the impact and clinical significance of AD AM 12 on colon cancer.Part 1:The relationship between the expression of ADAM 12 in colon cancer and the clinical prognosis of patients.Part 2:ADAM 12 promotes the proliferation of colon cancer cells.Part 3:MEF2A positively regulates the expression of ADAM12.Part 4:ADAM12 upregulation activates HOXA1-dependent NF-κB signaling to promote the growth of colon cancer cells.Part Ⅰ The relationship between the expression of ADAM12 in colon cancer and the clinical prognosis of patientsMethods1.The transcriptome RNA-sequencing data of the colon adenocarcinoma(COAD)were obtained from TCGA database.Two microarray datasets(GSE62321 and GSE146587)were obtained from the GEO database.The differential expression genes(DEGs)were analyzed by using the R software with Linear Models for Microarray and RNA-Seq Data(Limma)package(http://bioconductororg/packages/Limma/)and a p<0.001&log2|fold change|>2.5 was set as the cutoff values.The up-regulated genes from the abovementioned three datasets were analyzed by R software and the Venn diagram was plotted to obtain the intersection genes.2.The UALCAN online tool(http://ualcan.path.uab.edu/index.html)was used to analyze the expression of ADAM 12 in COAD in terms of sample type,individual cancer stage and tissue subtype and the prognostic analysis of AD AM12 in COAD.The mRNA expressions of ADAM12 from GSE62321 and GSE146587 datasets were analyzed by R software.The immunohistochemical pictures of two colorectal cancer tissues and normal colon tissues were obtained from The Human Protein Atlas online website(https://www.proteinatlas.org/)and the prognostic analysis of AD AM12 in GSE38832 was determined by R software.3.IHC was used to detect the expression level of ADAM 12 in tissue chips.The patients’ information of colon cancer was gathered.The correlation between the expression of ADAM 12 and clinical information was conducted by chi-square test or Fisher’s exact test.The Kaplan Meier survival curve and log-rank test were used to evaluate and compare the survival time between the two different groups.Results1.By cross-comparing the differential genes in the three databases,the common differential gene ADAM 12 was screened for subsequent verification.2.ADAM12 is highly expressed in TCGA COAD,GSE62321 and GSE146587 cancer tissues.The expression of ADAM 12 in high stage is higher than that in low stage.The expression of ADAM 12 in colon cancer mucinous adenocarcinoma is higher than that in adenocarcinoma.3.For the total of 82 patients of colon cancer,there was 40 patients for the stronger positive group,while 42 patients for the weakly positive group.There was no difference between the expression level of ADAM 12 and patients’ age,gender and tumor size.While,there was difference between the expression of AD AM12 and clinical stage,tumor invasion depth(T),lymph node metastasis(N)and distance metastasis(M)with p values of 0.003,0.024,0.001 and 0.011.The rate of overall survival was higher in ADAM 12 stronger positive than weakly positive group for colon cancer patients.Conclusion1.The expression level of AD AM 12 is higher in colon cancer tissues than normal colon tissues,and higher in colon mucinous adenocarcinoma than adenocarcinoma.2.TCGA database and GSE38832 suggest that the high expression of ADAM12 is related to the poor prognosis of patients,suggesting that AD AM12 may be an oncogene of colon cancer.3.Colon cancer patients with high AD AM12 expression have a poor prognosis.The expression of ADAM 12 was significantly associated with clinical stage,tumor invasion depth(T),lymph node metastasis(N)and distance metastasis(M).Part Ⅱ ADAM12 promotes the proliferation of colon cancer cellsMethods1.Western blot and qRT-PCR were used to analyze the expression levels of AD AM12 in several colon cancer cells.2.We conducted ADAM 12 overexpression experiments to HCT116 and SW480,lowly ADAM 12 expression cells,by overexpression lentivirus.We also conducted AD AM12 knockdown experiments to SW48,highly AD AM12 expression cell,by lentivirus.All the three stably expressed cells,selected by puromycin,were verified by mRNA and protein expression levels.3.The proliferation,apoptosis,cell cycle and colon abilities were compared between ADAM 12 lower/over expression groups by conducted CCK8,EdU,the expression level of Ki-67,colon formation,Annexin V-APC/7-AAD,caspase-3/7 and cell cycle test.4.We conducted in vivo experiments to measure,weight and observe the tumor growth in different groups.For the tumor,we also carried out IHC experiments to check the positive proportion of Ki-67。Results1.The mRNA and protein expression level of ADAM 12 was relatively high in SW48 and SW1116,while relatively low in SW480 and SW620 and barely in HCT116.2.We successfully constructed two stably overexpressed ADAM 12 group of cells as HCT116 and SW480.We also successfully constructed stably knockdown ADAM12 group as SW48.3.When the expression of AD AM12 for SW48 was low,the value of CCK8,numbers of colon cells,positive rate of EdU cells,the expression of Ki-67 cells into stage S were lowly than the control group.As for the overexpression ADAM 12 group for HCT116 and SW480,the results were on the contrary.4.The results showed that the tumors,ADAM 12 overexpression group,grown faster,bigger and heavier than the control group.The positive rate of Ki67 for Lv-NC and Lv-AD AM 12 were(34.67±5.03)%and(79±8.54)%respectively.ConclusionADAM 12 showed the ability as oncogene in the colon cancer,which could promote the cell cycle,reduce cell death,enhance the colon formation abilities and tumor growth in vivo.Part Ⅲ MEF2A positively regulates the expression of ADAM12Methods1.In order to identify the upstream transcriptional factors that regulate the expression of AD AM 12 in COAD,we first analyzed the correlated genes in 308 cases of colorectal cancer(GSE147571).The correlation between ADAM 12 and MEF2A was also confirmed in TCGA COAD database.2.HCT116、SW480、SW48 were transfected with FLAG-MEF2A or MEF2A-siRNA,mRNA and protein levels were determined by real-time PCR assay and Western Blot.3.JASPAR、Ensembl website were used to analyze the ADAM 12 promoter region and the potential MEF2A response elements.4.The transcriptional activation of MEF2A on the ADAM 12 promoter region was detected by a dual-luciferase reporter assay.Results1.Found the upstream transcription factor of ADAM12:MEF2A through bioinformatics.Next,we analyzed the ADAM 12 promoter region through the JASPAR website,and found two potential MEF2A response elements,located at-618 to-608 bp and-908 to-898 bp relative to the Transcription Start Site(TSS).2.Knockdown or overexpression of MEF2A,the expression of AD AM12 will also change accordingly.We found that overexpression of MEF2A can directly up-regulate the expression of ADAM 12 at both mRNA and protein levels.Conversely,silencing MEF2A by siRNA inhibited ADAM 12 expression.3.The results of dual luciferase show that MEF2A has 2 promoters corresponding to ADAM 12.Through the luciferase reporter assay,we found that MEF2A can promote the luciferase activity of the ADAM 12 promoter containing both potential MEF2A response elements.Mutation of one putative element can reduce MEF2A-mediated luciferase activity,and mutation of both elements can completely block the luciferase activity.ConclusionMEF2A positively regulates the expression of ADAM 12.AD AM 12 is a direct target of the transcriptional factor MEF2A.Part IV ADAM12 activates the NF-κB pathway through HOXA1Methods1.To investigate the biological function of AD AM12 in colon cancer,we first searched for the regulatory pathways related to ADAM12 expression in 308 cases of colon cancer(GSE147571).Through the Gene Set Enrichment Analysis(GSEA).2.HCT116 cells were transfected with pNFκB-luc and pSV40-renilla plasmids and an increased dose of FLAG-ADAM12 plasmids for 36 hours.The luciferase activity was then measured.SW48 cells were transfected with pNFκB-luc and pSV40-renilla plasmids and two ADAM12-shRNA plasmids for 36 hours.The luciferase activity was then measured.The relative mRNA expression levels of TNF-α、IL-1β、c-MYC and Cyclin D1 were determined by real-time PCR assay.3.BioGRID database were used to search the interacting proteins of ADAM 12 Immunoprecipitation(IP)and GST pull-down were performed to verify the Interaction between ADAM 12 and HOXA.4.1n HCT116 cells,four groups(con、ADAM 12、HOXA1-shRNA、ADAM 12+HOXA1-shRNA)were transfected with pNFκB-luc and pSV40-renilla plasmids for 36 hours.The luciferase activity was then measured.In SW48 cells,Four groups(con、ADAM12-shRNA、HOXA 1、AD AM12-shRNA+HOXA1)were transfected with pNFκB-luc and pSV40-renilla plasmids for 36 hours.The luciferase activity was then measured.5.EdU,clone formation and CCK8 experiments were carried out to investigated whether HOXA 1-mediated the proliferation promoting effect of ADAM 12 in COAD.Results1.Through the Gene Set Enrichment Analysis(GSEA),we found that AD AM 12 was involved in a variety of cancer-promoting signal pathways,including MYC_TARGETS_V1,MTORC1_SIGNALING,TNFA_SIGNALING_VIA_NFKB and E2F_TARGETS.2.We found that ADAM 12 expression increased the activity of NF-κB in a dose-dependent manner.Moreover,ADAM 12 expression further induced the expression of NF-κB downstream target genes and inflammatory cytokines.On the contrary,silence of ADAM 12 in SW48 cells inhibited the basal NF-κB activity,decreased the expression of NF-κB downstream target genes and inflammatory cytokines,suggesting a critical role of AD AM 12 in the activation of NF-κB signaling in COAD.3.In order to further understand the carcinogenic mechanisms of ADAM 12 in COAD,we searched the interacting proteins of ADAM 12 in the BioGRID database.A total of 21 interacting proteins of ADAM 12 curated by both high throughput and low throughput were revealed.The transcriptional factor HOXA1 is listed as a high throughput associated protein of ADAM12.The cell lysates from HCT116 cells expressing FLAG-ADAM12 or FLAG-Con were immunoprecipitated by FLAG M2 beads and subjected to immunoblotting with anti-FLAG M2 or anti-HOXA1 antibodies,respectively.As expected,endogenous HOXA1 was readily detected in FLAG-AD AM 12 immunoprecipitated.Furthermore,the interaction between endogenous HOXA1 and ADAM12 was further confirmed in SW48 cell lysates.Moreover,HOXA1 directly bound to ADAM12 protein in a GST pull-down assay.In the presence of ADAM 12,the interaction between HOXA1 and RBCK1 was significantly reduced,without affecting the expression of both proteins.4.We found that in the presence of HOXA1,ADAM12-induced NF-κB activation was significantly increased.However,co-transfection of ADAM 12 with HOXAl-shRNA resulted in a decreased ability for ADAM 12 to trigger NF-κB activation,indicating the requirement of HOXA1 for ADAM12-induced NF-κB activation.5.As expected,overexpression of ADAM 12 significantly promoted the proliferation of HCT116 cells,which was largely inhibited by HOXA1-depletion.On the contrary,overexpression of HOXA1 promoted the growth of ADAM12-depleted SW48 cells.In summary,these results indicate that AD AM12’s promotion of COAD proliferation depends to a large extent on HOXA1.Conclusion1.ADAM 12 could activate the TNFA/NF-κB pathway in COAD.2.AD AM12 promotes NF-κB activation by modulating the protein-protein interaction between HOXA1 and RBCK1.3.AD AM12’s promotion of proliferation depends to a large extent on HOXA1.