The Function And Mechanism Of PDRG1 In Proliferation And Metastasis Of Colorectal Cancer

BackgroundColorectal cancer(CRC)is the third most common cancer in the world.The incidence and mortality of colorectal cancer are on the rise in China.At present,the incidence of colorectal cancer is the third most common malignant tumor in China,and the mortality is the fifth.The pathogenesis of colorectal cancer is complex and highly heterogeneous.There are no specific symptoms of colorectal cancer in the early stage,and most patients are already in the middle and late stage when they are found.At present,surgical excision is the preferred comprehensive treatment for colorectal cancer,in which targeted therapy is an important auxiliary means.However,the effectiveness of targeted drugs is not satisfactory,and the median survival of patients is not effectively prolonged.Therefore,exploring the molecular mechanism of the occurrence and development of colorectal cancer and finding more effective molecular targets will contribute to the development of new targeted drugs.P53 gene is one of the most common mutated genes in human tumors.It is involved in a variety of cell biological behaviors,and its role in cell cycle regulation and apoptosis has been widely confirmed.Studies have shown that p53 can cause the activation of some genes such as p27,and the downstream effects of these cascade reactions are related to the induction of cell cycle arrest or apoptosis.DNA damage widely exists in the process of cell growth,division and proliferation,and is often caused by a variety of internal and external factors,such as ionizing radiation,ultraviolet radiation and other physical factors,alkylation agents,nitrosamine salts and other chemical factors,virus infection and other biological factors.There are two ways of cell response to DNA damage,namely DNA damage repair and AUTOPhagy and apoptosis induced by DNA damage.If repair is defective,DNA damage can result in two things:cell death;The second is the occurrence of gene mutation,or malignant transformation into tumor cells.P53 and DNA damage-regulated gene 1(PDRG1),the newly discovered oncogenes,encode small proteins of 133 amino acids and bear no resemblance to any known proteins in the database.Studies have shown that PDRG1 expression is abnormally high in a variety of human tumors,such as lung,stomach,breast,ovarian,colon,rectal and uterine cancers.It can affect a variety of cell life activities,including promoting cell proliferation and metastasis,inhibiting cell apoptosis and blocking cell cycle,thus regulating the occurrence and development of a variety of tumors.Therefore,PDRG1 is considered as a potential biomarker for these tumors.However,the effects and mechanisms of PDRG1 on the biological behavior of colorectal cancer cells remain unclear.Therefore,to explore the role and molecular mechanism of PDRG1 in the proliferation and metastasis of colorectal cancer will be of great significance for biomarker screening,molecular diagnosis and targeted therapy of colorectal cancer.This study is divided into three parts:(1)Bioinformatics analysis and immunohistochemistry were used to detect the expression level of PDRG1 in colorectal cancer tissues.At the same time,the correlation between PDRG1 and clinicopathological features and survival of colorectal cancer patients was explored by combining clinicopathological data and survival data.(2)The protein levels of PDRG 1 in human normal colorectal epithelial cell lines and various colorectal cancer cell lines were detected by western blot assay.Stably silenced PDRG1 lentivirus cell lines were constructed in Colorectal cancer cells SW620 and HT29,and the effects of PDRG 1 silencing on proliferation,metastasis,apoptosis and cycle of colorectal cancer cells were detected by CCK-8,colony formation,Transwell,striation and flow cytometry experiments.(3)The downstream signaling pathway of PDRG 1 was screened by bioinformatics analysis and verified by western blot experiment and analyze its mechanism.Further confirmation was made by restoration experiments.The tumor-bearing model and lung metastasis model of nude mice were established to detect the effects of PDRG1 on the growth and metastasis of colorectal cancer and animal survival in vivo.Part Ⅰ:Expression and clinical significance of PDRG1 in colorectal cancerObjective:To detect the expression level of PDRG 1 in colorectal cancer tissues and its correlation with clinicopathological features and survival of colorectal cancer patients.Methods:The mRNA levels of PDRG 1 in colorectal and normal colorectal tissues were analyzed using GEPIA and TCGA databases.TCGA database was used to analyze the correlation between PDRG 1 and clinicopathological stage and survival of colorectal cancer patients.Colorectal cancer tissues of patients undergoing radical rectal cancer surgery in The Affiliated Hospital of Xuzhou Medical University from January 2015 to December 2015 and their paired paracancer non-cancer tissues were collected to prepare tissue chips,and the samples had complete clinical information.Including gender,age,tumor differentiation,tumor size,depth of tumor invasion,lymph node metastasis,pTNM stage and other clinicopathological parameters,as well as marriage,place of residence and date of surgery.Immunohistochemistry was used to detect the protein level of PDRG1 in colorectal cancer tissues and corresponding adjacent tissues,and its correlation with clinicopathological features and survival of patients was analyzed.Results:Bioinformatics analysis showed that the mRNA level of PDRG1 was significantly increased in colon cancer tissues,and correlated with clinicopathological stage(P=0.003)and patient survival(P=0.011).The results of colorectal cancer tissue microarray combined with clinicopathological characteristics showed that:The high expression of PDRG1 protein in colorectal cancer tissues was positively correlated with tumor size(P<0.001),TNM stage(P<0.003),distant metastasis(P=0.039)and tumor invasion depth(P=0.015).It was associated with overall survival(P<0.001)and disease-free survival(P<0.05).Conclusion:Our study found that PDRG1 was highly expressed in colorectal cancer and correlated with clinicopathological stage and patient survival,suggesting that PDRG1 may play an oncogene role in this tumor.Further study on the possible clinical significance of PDRG1 in patients with colorectal cancer showed that the expression level of PDRG1 was correlated with tumor size,TNM stage,distant metastasis,tumor invasion depth,overall survival and disease-free survival.These results suggest that PDRG1 may be a potential biomarker involved in and promoting the development of colorectal cancer.Part Ⅱ:The effect of PDRG1 silencing on the biological behavior of colorectal cancer cellsObjective:To investigate the effects of PDRG1 silencing on proliferation,metastasis,apoptosis and cell cycle of colorectal cancer cells.Methods:1.The protein levels of PDRG1 in human normal colorectal epithelial cell lines and various colorectal cancer cell lines were detected by western blot assay.2.Construct lentivirus cell lines stably silencing PDRG1 in colorectal cancer cells SW620 and HT29.3.CCK-8 and colony formation assay were used to detect the effect of PDRG1 silencing on colorectal cancer cell proliferation.4.Transwell and scratch test were used to detect the effect of PDRG1 silencing on colorectal cancer cell metastasis.5.Flow cytometry was used to detect the effects of PDRG1 silencing on apoptosis and cycle of colorectal cancer cells.Results:1.The protein level of PDRG1 in colorectal cancer cells SW480,SW620,HT29,HCT116,DLD1 and LoVo was significantly higher than that of normal colorectal epithelial cells FHC,and the protein expression of PDRG1 in SW620 and HT29 cells was the highest,so cell lines were selected for this study.2.Knock down of PDRG1 can significantly reduce the expression level of PDRG1 in SW620 and HT29 colorectal cancer cells.3.Knock down of PDRG1 can significantly inhibit the proliferation of SW620 and HT29 colorectal cancer cells.4.Knock down of PDRG1 could significantly inhibit the metastasis of colorectal cancer cells SW620 and HT29.5.Knock down of PDRG1 can induce apoptosis and G1/S phase cell cycle arrest of SW620 and HT29 colorectal cancer cells.Conclusion:1.The protein level of PDRG1 in colorectal cancer cells was significantly higher than that in normal colorectal epithelial cells.2.As an oncogene in colorectal cancer cells,PDRG1 silencing can inhibit cell proliferation and metastasis,induce apoptosis and G1/S phase cell cycle arrest.Part Ⅲ:Studies on the mechanism of PDRG1 effect on biological behavior of colorectal cancer cellsObjective:To investigate the molecular mechanism of PDRG1 regulating the proliferation,metastasis,apoptosis and cycle of colorectal cancer cells.Methods:1.PDRG1 was enriched by bioinformatics analysis,and downstream signaling pathways that might be regulated by PDRG1 were screened.2.Western blot assay was used to detect the expression levels of apoptosis and cycle-related proteins in colorectal cancer cells after silencing PDRG1.3.Luciferase reporter gene and chromatin immunoprecipitation assay were used to detect the regulatory mechanism of PDRG1 on p21.4.PDRG1-silenced colorectal cancer cells were further knocked out,and western blot,CCK-8 and Transwell assay were used to detect whether PDRG1 regulation of colorectal cancer cell proliferation and metastasis was related to cell cycle.4.The tumor-bearing model of nude mice was established to analyze the effect of PDRG1 silencing on tumor growth in vivo.5.A nude mouse lung metastasis model was constructed to analyze the effects of PDRG1 silencing on lung metastasis and animal survival of colorectal cancer cells.Results:1.PDRG 1 is mainly enriched in cycle and apoptosis-related signaling pathways.2.Cleaved-caspase 3,cleaved-caspase 7,and cleaved-caspase 9 protein expression were significantly promoted by PDRG 1 silencing.It also inhibited the expression of cyclin B1,cyclin E2,cyclin D1 and CDK2,and promoted the expression of p21,but did not significantly regulate p27.3.PDRG1 can bind to the p21 promoter to inhibit transcription.4.Knock down of p21 significantly restored the proliferation and metastasis capacity of colorectal cancer cells weakened by PDRG1 silencing.4.Silencing PDRG1 can significantly inhibit tumor growth in animals.5.Silencing PDRG1 can inhibit lung metastasis of colorectal cancer cells and prolong the survival time of animals.Conclusion:1.PDRG 1 silencing can inhibit the proliferation and metastasis of colorectal cancer cells by regulating cell cycle.2.Silencing PDRG1 can inhibit tumor growth and metastasis in vivo.