The Effects And Molecular Mechanisms Of PBLD In The Proliferation,cell Cycle And Apoptosis Of Colorectal Cancer Cells

BACKGROUND AND OBJECTIVEColorectal Cancer is one of the most common gastrointestinal tumor,according to the world health organisation International Agency for Research on Cancer(IARC),there are about 1.36 million new cases of colorectal cancer in 2012 all over the world.And it is the third most popular male malignant tumor,the second most popular female malignant tumor,ranking behind the lung cancer and breast cancer.The death rate of CRC is behind the the lung cancer,liver cancer and stomach cancer,stands for the 4th place of malignant tumors,and has almost 690,000 cases died each year.CRC was not caused by only one risk factor,in addition to age and gender,there are many risk factors which is worth noticing,such as family history of colorectal cancer,inflammatory bowel isease,smoking,excessive drinking,obesity and diabetes,etc.At our country,colorectal cancer stands for the fourth place of malignant tumor,just after gastric cancer,lung cancer and esophageal cancer.And its incidence is rising year by year.Same as those most common cancer treatments,the treatment of colorectal cancer is primarily by surgical resection,but except for some patients with early tumors,about half of patients with colorectal cancer will suffer from cancer metastasis and recurrence after surgery.So those terminal stage patients who undergo surgical removal of tumors still need chemotherapy.But now those drugs use for colorectal cancer chemotherapy still have different degrees of adverse reactions,which has a shortcoming for poor effects in single-usage and can not be taken for long-term application.Therefore,the in-depth study of the etiology and mechanisms of colorectal cancer and new intervention target research shows its extremely vital significance.Nowadays,the specific molecular mechanism of colorectal cancer are not fully understood.It is generally acknowledged that colorectal cancer is a multi-step,multi-stage process,which involved many genes and processes,such as the activation of oncogene and deactivation of tumor-suppressor genes,disorder of cell cycle and apoptosis,malfunction of mismatch repair genes,and the increase of telomerase activity and so on.The biology of tumorgenisis is always thought to be lossing control of tumor growth.The main molecular mechanism includes the cell cycle disorder which leads to excessive cell proliferation and the reduction of regular cell apoptosis.The destruction of the mechanism of cell cycle regulation is an important reason of tumorgenisis.Research suggests that the cell cycle monitoring mechanism inactivation associated with malfunction of cell cycle driving mechanism are the most important reasons for tumorgenisis.The regulation of cell cycle are maintained by cell cycle protein and mutual adjustment cycle dependent kinase protein and its inhibitors.Among the life cycle of cells,the G1 to S phase shift plays an important role in the regulation of cell cycle,and the G1 stage of cell cycle related proteins are mainly cyclin D1 and cyclin E,associated with G1 phase of the cell cycle inhibitory proteins are respectively is P21 and P27.The study found that in about 30%of the patients with colorectal cancer had an elevated protein expression of cyclinDl;This excessive expression may be an early event of colorectal cancer;Other similar studies have also shown that the expression level of cyclin E in normal colorectal mucosa,hyperplastic polyps and adenomas to colon cancer is gradually rising,cyclin E also closely related to the occurrence and development of colorectal cancer.On the contrary,the expression of P21 and P27 in colorectal cancer were significantly lower than adjacent normal mucosa tissues,and significantly negative correlated with primary tumor infiltration depth,regional lymph node metastasis and TNM stages.Cell apoptosis also called programmed cell death,is an important part of cell life cycle,is a kind of strictly controlled cell death process.Cell apoptosis is an important link in maintaining the body’s internal environment stable.Cell apoptosis may be related to the abnormal expression of oncogenes and tumor suppressor genes.Cells rely on routine processes of apoptosis to refresh itself such as eliminating aged cells,and abnormal cells or cells not participating in the immune response of lymphocytes.PBLD also called MAWBP,positioning in 10 q21.1 area,have a total length of 50.268 bp and can transcribe two isoform subtypes,about 32 kd protein isoform a and about 31 kd protein isoform b.Amino acid sequence analysising of PBLD encoded protein found that from lower to higher degrees species,the genes are commonly expression,is a relatively conservative gene,presumably the genes may be associated with biological certain basic physiological functions.Related studies found that PBLD expression in gastric cancer and liver cancer was lower than untumorous tissues.Thinking that PBLD may be a new protein marker of gastric cancer.Dong-mei li found MAWBP worked with its ligands(MAWD)to achieve the purpose of inhibiting gastric cancer cell EMT through inhibition of TGF-beta pathway;Ai-min li found the PBLD such as lower the expression in liver cancer tissue,and through the in vivo experiment and found the PBLD inhibited hepatocellular carcinoma cells’ ability of proliferation,invasion and tumor formation,induced liver cancer cell cycle arrest in S and G2/M phase;Zhao xin mei,etc analyzed the differences of ulcerative colitis and normal tissue protein expression spectrum,found the PBLD expressed in ulcerative colitis tissues,and further confirmed by immunohistochemistry.Our preliminary analysis of the PBLD role in the proliferation and invasion of colon cancer,migration,express PBLD may inhibit the EMT process have been found to inhibit colorectal cancer cell proliferation,invasion ability of purpose.To sum up,we supposed that PBLD played a role in the development of tumor,cell cycle distribution,and the processing of inflammation.It might be a potential tumor suppressor gene.Based on the former.works of our team,further exploration of function of PBLD will be done.Such as observation of PBLD gene and protein expression in CRC clinical tissuses and analysis of the relationship between PBLD expression and clinical fetures and patients overall survive time.And explore the influences of PBLD overexpression on cell cycle and cell apoptosis in colorectal cancer cells and changes of some key molecules in cell cycle and apoptosis signaling pathways.METHODS1.The expression of PBLD in colorectal cancer tissues and its significance in predicting the prognosis of patients(1)Tissue collection:Fresh CRC tissue samples,together with matched adjacent non-tumorous tissues,were collected from a total of 56 patients,who underwent surgical resection at our institute between 2008 and 2009.All enrolled subjects gave their written informerd consent.(2)Detection of PBLD in colon cancer tissues:The expression of PBLD in colorectal cancer tissues and adjacent tissues was detected by real-time quantitative PCR.The differences of PBLD expression in colorectal cancer tissues with varied differentiation degree were also analyzed.(3)The relationship between the expression level of PBLD and the clinical characteristics and prognosis of colorectal cancer patients:Immunohistochemical staining was performed on 76 patients with colorectal cancer using tissue microarray(Shanghai core super).The correlation between PBLD and clinical indications of colorectal cancer was analyzed by X2 test.Regression analysis was performed to analyze the prognostic value of PBLD on colorectal cancer patients.Kaplan-Meier method was used to confirm the relationship between PBLD and patietns survival time.2.Effect of PBLD on proliferation,cell cycle and apoptosis of colon cancer cell line(1)Lentiviral vector was transfected with LoVo cell.Stable clones were screened with puromycin and verified using western blot and qPCR.(2)The effect of PBLD on the proliferation of colorectal cancer cells was detected by CCK8.The effect of PBLD on the colony forming ability of colorectal cancer cells was detected by plate cloning.Flow cytometry was used to detect the effect of PBLD on the cell cycle and apoptosis(3)qPCR and Western blot were used to detect the expression of cell cycle related genes and apoptosis-related genes in colorectal cancer cells with high expression of PBLD.RESULTS1.The expression of PBLD in colorectal carcinoma was significantly lower than that in adjacent normal tissues(1)Results of qPCR showed that the average expression level of PBLD mRNA of colorectal cancer was significantly lower than that in adjacent tissues,which was detected in 69.64%(39/56)of CRC patients.At the same time,the lower degree of differentiation comes along with the lower PBLD mRNA relative expression(2)Immunohistochemical staining showed that the positive rate of PBLD in colorectal cancer tissues was 73.68%(56/76),which was lower than that in adjacent normal tissues(76/76),the differentiation degree of cancer tissues were statistically significant(X 2=23.03,P<0.001).The strong positive rate of PBLD in colorectal cancer group was 9.21%(7/76),which was lower than that in adjacent normal tissues 48.68%(37/76),(X2=28.79,P<0.001).2.The expression level of PBLD was significantly correlated with the prognosis of colorectal cancer patients.(1)The expression level of PBLD is closely related to the clinical characteristics and prognosis of colorectal cancer.The expression level of PBLD is closely related to the degree of differentiation(P=0.006)of colorectal cancer.The higher the expression level of PBLD,The higher the degree of differentiation.(2)In addition,the overall survival time(OS)of colorectal cancer patients with negative PBLD expression were significantly lower than those of positive PBLD expression group.3.Overexpression of PBLD can inhibit the proliferation of colon cancer cells,block cell cycle and promote the early apoptosis of colon cancer cells.(1)Overexpression of PBLD inhibited the proliferation of colorectal cancer cells:CCK8 showed that the growth rate of colorectal cancer cells in PBLD overexpression group was significantly decreased(P<0.01);platelet cloning showed that colony forming ability of colon cancer cells in PBLD overexpression group was also decreased(P<0.001);(2)Overexpression of PBLD block the cell cycle of colorectal cancer:Flow cytometry showed that PBLD overexpression group showed G0/G1 phase arrest in cell cycle,the difference was statistically significant(P<0.05);(3)Overexpression of PBLD promote the early apoptosis of colon cancer cells:Hoechest33258 staining showed that apoptotic cells were increased in PBLD overexpression group;Annexin V-APC/7AAD flow detection showed that PBLD overexpression group had an increasion of early apoptotic cells.4.Western blot and qPCR were used to detect the expression of cell cycle and apoptosis related genes.We found the expression of PCNA,cyclin D1,cyclinE2,CDK4 and Bcl2 were down-regulated,and the expression of p21,Bax and cleaved-caspase3 were up-regulated in PBLD overexpression group.CONCLUSION1.PBLD in colon cancer tissue expression was significantly lower than the adjacent normal tissue.2.There was a correlation between the expression level of PBLD and the prognosis of patients with colon cancer.PBLD expression level correlated with colorectal cancer differentiation and patients AJCC stage,which means,cancer tissues with higher PBLD expression had a higher stage of differentiation and earlier clinical stage.PBLD can be used as a predictor of prognosis in patients with colorectal cancer.The Kaplan-Meier analysiss showed that the overall survival time of PBLD negative expression group was significantly lower than the positive expression group,and the same with the clinical prognosis.3.Overexpression of PBLD can inhibit the proliferation of colon cancer cells.4.The expression of PCNA,cyclin D1,cyclinE2,CDK4 and Bcl2 was down-regulated in colorectal cancer cells overexpressing PBLD,while the expression of p21,Bax and cleaved-caspase3 was up-regulated.