Study About The Relationship Between Overexpression Of COL10A1 And Poor Prognosis In Colorectal Cancer

Colorectal cancer(CRC)is one of the most common malignant cancers in the world,with about 1.4 million new cases and more than 690000 deaths per year.Many patients with CRC were diagnosed at an advanced stage because of a lack of mass screening.Distant metastasis,especially peritoneal and liver metastasis,is the main cause of death in CRC.CRC metastasis directly leads to poor prognosis in patients and the median survival time was only 5-9 months.Because of early screening and treatment,decreasing CRC mortality rates have been observed in some countries worldwide.Therefore,if we can fully apply tumor molecular biological research,select some novel molecular markers for early screening,the positive predictive value for early diagnosis and the clinical prognosis of CRC will obviously be improved.In CRC,there are a large number of differentially expressed genes between primary tumors and metastases.Further clarifying the molecular mechanism of CRC distant metastasis is of great significance.Therefore,we used gene chip technology to compare the difference of genome-wide expression levels in four pairs of CRC primary tumor tissues and corresponding peritoneal metastasis tissue,and determined the differentially expressed genes through multiple methods and performed functional annotation cluster analysis for these genes.Ultimately,217 differentially expressed genes were selected(Figure S1).Through bioinformatics analysis,QPCR and IHC technology verification,we found two genes,CTHRC1 and COL10A1,as the metastasis relevant candidate genes of CRC.In the previous studies,we had confirmed that CTHRC1 was abnormally high express in CRC and it was an independent risk factor for CRC prognosis.COL10A1,a protein in humans,is a member of the collagen family.The COL10A1 gene "encodes the alpha chain of type X collagen,a short chain collagen expressed by hypertrophic chondrocytes during endochondral ossification".COL10A1 gene mutation is associated with Japanese type spondylometaphyseal dysplasia(SMD)and Schmid type metaphyseal chondrodysplasia(SMCD).High COL10A1 expression was observed in various solid human tumor tissues and it was likely to be associated with tumor angiogenesis.But the relationship between COL10A1 and CRC remains unknown,and the specific role of COL10A1 in cancer is unclear.Our aim was to further research the expression characteristics of COL10A1,to explore its mechanism to promote proliferation and invasion in CRC and to preliminarily clarify the clinical interrelation between COL10A1 and CRC.The study was divided into five parts,as follows:Chapter 1 Expression of COL10A1 in colorectal cancer tissue and cellsThe expression level of COL10A1 was detected in 40 paired CRC tissues and adjacent normal tissues by QPCR and Western blot assays.The results of Western blot analysis showed that COL10A1 was frequently up-regulated(33 of 40,82.5%)in CRC tissues(T)compared to adjacent normal tissues(N)(P<0.001).The QPCR results showed that COL10A1 expression was up-regulated in 25 of 40(62.5%)CRC tissues compared with corresponding nontumorous tissues.Moreover,we performed the further verification through IHC assay with 30 CRC patient specimens.We scored them according to the intensity of the staining(negative:0;weak:1;moderate:2;intense:3).The same result was observed that CRC tissues staining were obviously deeper(higher scores)than the corresponding normal tissues,indicating that COL10A1 was stably high expressed in CRC tissues(P<0.001).The expression of COL10A1 in six cell lines-HCT116,Caco2,SW480,SW620,LoVo and FHC was detected by QPCR and Western blot assays.The results were accordant that COL10A1 was high expressed in SW480,SW620 cells and relatively low expressed in the others cell lines.Chapter 2 The function of COL10A1 in colorectal cancer cellsWe chose HCT116 and LoVo cells to structure HCT116-COL10A1-OE and LoVo-COL10A1-OE cell lines,while SW480 and SW620 cells were used to generate SW480-COL10A1-KD,SW620-COL10A1-KD cell lines by transfecting with COL10A1 siRNA lentivirus.The transfection efficiency was detected by QPCR and Western blot assays(all P<0.01).Cell migration rate was measured by wound healing assay.The migration rate and ability was markedly increased in HCT116-COL10A1-OE(P<0.01)and LoVo-COL10A1-OE(P<0.001)cells,and inversely,reduced in SW480(P<0.01),SW620(P<0.001)cells when COL10A1 was knockdown compared with respective NC groups.The same results were observed by transwell chamber migration assay and invasion assay(all P<0.001).Consequently,it was shown that COL10A1 dramatically increased the ability of migration and invasion ability in CRC cells.Cell counting kit 8(CCK-8)assay and clone formation assay were performed to investigate the effect of COL10A1 on CRC cells proliferation.In CCK-8 assay,the proliferation ability was markedly reduced in SW480-COL10A1-KD cells compared with SW480-NC cells(P<0.01).Moreover,the number of colonies was significantly reduced in SW480 cells when COL10A1 was knocked down compared with the NC groups(P<0.001).Therefore,Knockdown of COL10A1 inhibited CRC cells proliferation.Chapter 3 COL10A1 promotes tumor cells growth in vivoFor exploring the effect of COL10A1 on CRC cells growth in vivo,the stable transfection cells SW480-COL10A1-KD and SW480-NC cells were injected subcutaneously into the right flank of nude mice.After 30 days' repeated observation and measure,we found that the tumor growth rate of SW480-COL10A1-KD group was evidently slower than that of SW480-NC group(P<0.001).The results showed that knockdown of COL10A1 repressed the CRC cells growth in vivo.Chapter 4 COL10A1 accelerates EMT in CRCWestern blotting was performed to explore the potential relation between COL10A1 and EMT phenomenon.EMT indicators were detected included E-cadherin,N-cadherin,Beta-catenin,Slug and Snail.Western blot analysis showed that COL10A1 overexpression promoted EMT in LoVo and HCT116 cell lines,and,on the contrary,EMT was suppressed in SW480,SW620 cell lines when COL10A1 was knocked down.Therefore,COL10A1 was a positive regulator for the EMT process.Chapter 5 COL10A1 overexpression relates to adverse prognosis in CRCThe tissue microarray(TMA)involving a total of 197 CRC patients with clinical baseline data was used to research the clinical prognostic relevance of COL10A1 expression in CRC by IHC analysis.The results showed that the expression of COL10A1 in CRC had a high relevance with prognosis and overall survival.According to the Kaplan-Meier Log-rank test,abnormal up-regulated expression of COL10A1(P = 0.004)related to poor overall survival.Univariate Cox regression model analysis showed that poor survival was significantly correlated with tumor size(hazard ratio,HR:2.10,95%confidence interval,95%CI:1.18-3.76;P = 0.012),differentiation(HR:2.22,95%CI:1.13-4.38;P= 0.021),invasion degree(HR:3.20,95%CI:1.26-8.09;P = 0.014),nodal metastasis(HR:3.25,95%CI:1.77-5.96;P<0.001),M(metastasis)stage(HR:7.20,95%CI:3.92-13.21;P<0.001),AJCC(American Joint Commitee on Cancer)stage(HR:5.13,95%CI:2.60-10.11;P<0.001),and COL10A1 expression(HR:2.28,95%CI:1.28-4.06;P =0.005).Multivariate survival analysis was performed based on the results of the univariate survival analysis.Through adjustment,tumor size,differentiation,AJCC stage and COL10A1 expression were confirmed as covariates in multivariate analyses.Because of the interactions with AJCC stage,colorectal wall invasion degree,nodal metastasis and M stage were excluded from the multivariate survival analysis.According to the results of multivariate analysis,AJCC stage(HR:4.71,95%CI:2.36-9.39;P<0.001)and COL10A1 expression(HR:2.04,95%CI:1.11-3.74;P = 0.022)were considered to be independent risk predictors for poor overall survival.The analysis results of clinical samples and TMA showed that COL10A1 was associated with prognosis and survival of patients with CRC.Our study demonstrates that COL10A1 is over-expressed in CRC tissues and involves in proliferation,migration,invasion and tumorigenesis process of CRC cells.Meanwhile,COL10A1 promotes the EMT process.High-level expression of COL10A1 can be considered as an independent risk factor of prognosis and overall survival in CRC.COL10A1 is likely to be a potential target gene for CRC therapy.