ADAMTS12 Promotes Colorectal Cancer Progression By Activating The Wnt/β-catenin Signaling Pathway

Research Objective:In this study,we analyze the relationship between ADAMTS12 and survival prognosis and clarify the role of ADAMTS12 on survival prognosis of CRC to provide an important theoretical basis for developing prognostic markers by detecting the differential expression of ADAMTS12 in colorectal cancer（CRC）and para carcinoma tissues at the RNA level and protein level.In order to provide an important theoretical basis for further elucidating the mechanism of colorectal cancer progression,we explore the biological function of ADAMTS12.Research methods:1.Expression analysis:We analyzed the m RNA expression of ADAMTS12 in 275 COAD,349 para carcinoma tissues,92 READ and 318 para carcinoma tissues from TCGA online database.We made use of a tissue microarray consisting of 41 colorectal tissue samples to analyze the ADAMTS12 protein expression through immunohistochemical staining.2.Prognostic analysis:In the TCGA online database,we divided patients into high expression and low expression group based on ADAMTS12 expression level in colorectal cancer tissues.Kaplan-Meier was used to analyze the correlation between ADAMTS12expression and prognosis of patients with colorectal cancer.3.Functional experiment:To obtain Flag-ADAMTS12,c DNA-encoding ADAMTS12was cloned into a vector.An ADAMTS12 knockout plasmid was established by CRISPR/Cas9 hit KO technology.ADAMTS12 overexpression and knockout cell lines were constructed by lipofectamine 2000 after transient transfection.CCK-8 experiment,clone formation experiment and transwell experiment were conducted to verify the effect of ADAMTS12 on the proliferation and migration of HCT116 cells.4.Mechanism research:HEK293T was transfected with ADAMTS12 over-expression plasmids or control plasmids and subsequently cotransfected with theβ-catenin luciferase reporter vectors and p RL-TK control vectors via lipofectamine 2000.The effect of ADAMTS12 on the transcriptional activity ofβ-catenin was verified by dual luciferase reporter gene.Western Blot and RT-q PCR were used to verify the effect of ADAMTS12 on Wnt/β-catenin signaling pathway downstream genes Cyclin D1 and myc.Research Results:1.The TCGA online database was used to analyze the data of colorectal adenocarcinoma and rectal adenocarcinoma.In the colorectal adenocarcinoma group,there were 275 tumor tissues and 349 para carcinoma tissues.And in the rectal adenocarcinoma group,there were92 tumor tissues and 318 para carcinoma tissues.It showed that the m RNA expression of ADAMTS12 was significantly increased in tumor tissues compared with para carcinoma tissues in both COAD and READ（P<0.05）.Immunohistochemical staining of colorectal cancer microarrays consisting 41 cancer tissues and 41 para carcinoma tissues showed that ADAMTS12 was significantly increased in tumor tissues compared with adjacent noncancerous tissues（P<0.01）.2.We divided the colon cancer patients into ADAMTS12 high expression group and low expression group according to their ADAMTS12 expression levels in the TCGA database,with 135 samples in each group.By Kaplan Meier plotter analysis of prognosis,the results showed that among 270 colon cancers,patients with high ADAMTS12 expression had a significantly worse DFS than those with low ADAMTS12 expression（P=0.029,HR=1.7）,and there was no statistical significance in OS（P=0.2,HR=1.4）.3.1 Overexpression of ADAMTS12:the clonogenic assay showed that the clonogenic capacity of the Flag-ADAMTS12 group was higher than that of the control group,P<0.001;CCK8 assay showed that cell proliferation ability of ADAMTS12 group was higher than that of control group at D3 and D5 when the baseline level of D1 was consistent;Cell migration assay showed that the migration ability of the Flag-ADAMTS12 group was higher than that of the control group.3.2 Knockout of ADAMTS12:the clonogenic assay showed that the clonogenic ability of groups ADAMTS12^-/-1#and ADAMTS12^-/-2#was significantly weaker than that of the control group（P<0.05 and P<0.01）;CCK8 experiments showed that groups ADAMTS12^-/-1#and ADAMTS12^-/-2#had a weaker proliferative capacity than the control group at D3 and D5 when the baseline level was consistent at D1;Cell migration assay showed that the migration ability of group ADAMTS12^-/-1#and ADAMTS12^-/-2#was weaker than that of the control group.4.Dual luciferase reporter gene experiments further confirmed that ADAMTS12overexpression enhanced the transcriptional activity ofβ-catenin in the Wnt/β-catenin signaling pathway（P<0.001）.In the absence of ADAMTS12 expression,the downstream genes of the Wnt/β-catenin signaling pathway,such as Cyclin D1 and myc were significantly reduced compared with wild-type cancer cells（P<0.05,P<0.01）.Research Conclusions1.The expression of ADAMTS12 in tumor tissues was significantly elevated compared with para carcinoma tissues.2.Patients with high expression of ADAMTS12 have shorter DFS and poor prognosis.3.The expression of ADAMTS12 was positively correlated with the proliferation and migration of tumor cells.4.ADAMTS12 overexpression enhanced the transcriptional activity ofβ-catenin in Wnt/β-catenin signaling pathway.Knockout of ADAMTS12 significantly reduced the expression of Wnt/β-catenin signaling pathway downstream genes myc and Cyclin D1.