Gene Function Research Of CDH20 In Colorectal Cancer

Background:Colorectal cancer(CRC) is one of the most common malignancies in the world. CRC is the third leading cause of cancer death in the world and still a major threat to human’s health. The morbidity and mortality of CRC tend to rise steadily for a long period of time. CRC treatment failure was mainly due to postoperative recurrence and metastasis and the liver is the most important sites of metastasis. The development of CRC is the accumulation of multi-gene and multi-event. A growing number of genes have been confirmed to participate in the carcinogenesis or metastasis of CRC.In our previous researeh, we performed the exome capture sequencing of the normal mucosa, adenoma and adenocarcinoma tissues from the same patient, we identified mutation of CDH20 in adenoma and lower expression in adenoma and adenocarcinoma. It also was reported previously that CDH20 was mutated or loss in breast cancer and esophageal adenocarcinoma. These indicated that CDH20 may play a unknown role in progress of CRC. CDH20 is a member of cadherin superfamily which is represented by E-cadherin, there are plenty of researches demonstrated that the cadherin superfamily are closely tied to cancer, E-cadherin, and N-cadherin are key proteins in epithelial-mesenchymal transition, which are involved in the invasion and metastasis of tumor cells. Presently, the relationship between CDH20 and cancer also is unclear, studies on CDH20 in CRC has not been reported. Preliminary experiments showed that the gene was likely to involved in the progression of CRC.In this study, we explore the function of CDH20 in the progression of CRC via clinical sample analysis and a series of cell biology research, illuminate its potential clinical implications, and the potential role of CDH20 in the intervention of metastasis, exploiting the potential metastatic and prognostic markers of CRC.MethodsⅠ Analysis of correlation between CDH20 and clinicopathological characteristics of colorectal cancerCollected 53 pairs of specimens of colorectal cancer including cancer and cancer adjacent tissues, extracted the total RNA from specimens. Use RT-qPCR technique to evaluate the relative expression of CDH20 in different specimens and analyse the correlation with clinicopathological features.Ⅱ Gene function research of CDH20In vitro experiments1. The transcript and expression level of ST13 in different CRC cell lines was evaluated by qRT-PCR and Western Blot.2. CDH20-overexpressed and CDH20-knockdown CRC cells were constructed respectively by lenti-viral transduction.3. Immunofluorescence was used to observe the position of CDH20 in cell. MTT assay, plate colony formation, migration and invation assays was used to evaluate the influence of CDH20 on proliferation and migration in vitro. In vivo experiments4. Mouse xenograft study was performed to test in vivo liver metastasis capaeity of CDH20-overexpression CRC cells.Mechanism research5. Change of EMT-related protein and transcript in CDH20-overexpressed and CDH20-knockdown CRC cells was evaluated by Western Blot and RT-qPCR, to research the influence of CDH20 on process of EMT.6. Co-Immunoprecipitation, immunofluorescence and Western Blot were used to observe the position of β-catenin and the possible relationship between CDH20 and β-catenin.ResultsⅠ Analysis of correlation between CDH20 and clinicopathological characteristics of colorectal cancer1. CDH20 expression was significantly decreased in lymphnode-positive and metastatic colorectal cancers. However, CDH20 expression level was not significant correlation to gender, age, tumor location, tumor size, degree of differentiation and T stage. The results suggest that CDH20 may play an important role in metastasis of colorectal cancer.Ⅱ Gene function research of CDH20In vitro experiments1.CDH20 located in the membrane, lentivirus-mediated overexpression of CDH20 in CRC cells has no effect on cell morphology and cell proliferation in vitro.2. CDH20 may enhance the adhesive ability between CRC cells and fibronectin, and lead to a significant reduction in migration and invasion. In vivo experiments3. In addition, overexpression of CDH20 significantly inhibit the liver metastasis capaeity of CRC cells in vivo. Mechanism research4. Epithelial markers including E-cadherin and p-catenin expression increased accompany with overexpression of CDH20. Contrarily, mesenchymal markers such as Vimentin and N-cadherin were reduced while CDH20 expression increased. The results suggested that CDH20 play an important role in colorectal cancer EMT process.5. β-catenin also located in the membrane. The increased β-catenin may bind with CDH20 and didn’t accumulate in the nuclei.Conclusions1.CDH20 expression was significantly decreased in lymph node-positive and metastatic colorectal cancers. However, which was not significant correlation to gender, age, tumor location, tumor size, degree of differentiation and T stage.2.CDH20 located in the membrane, has no effect on cell morphology and cell proliferation in vitro,3.CDH20 may enhance the adhesive ability between CRC cells and fibronectin, and lead to a significant reduction in migration, invasion and metastasis in vivo and in vitro.4.The possible mechanism is CDH20 can increase the expression of E-cadherin and β-catenin and reduce the expression of Vimentin and N-cadherin, inhibit the process of EMT in CRC cells.