Explore The Effect Of CUL4B Expression On Ovarian Cancer Cells

Ovarian cancer is one of the most common malignant type of cancer among all gynecological tumors.The incidence rate is third in gynecological malignancy,and the mortality rate is first in female reproductive system.In 2012 there were 529,800 new cases of ovarian cancer in the world.The incidence of new cases of ovarian cancer is about 1,315,000 yearly in China,which is six times more than the developed countries.Ovarian cancer presents therapeutic challenges due to its cryptogenic,without early signs of clinical symptoms and specific molecular markers,and about 70% of patients are typically diagnosed late.The disease is easily invaded adjoining parts of the body and spread to other organs.It is too difficult to eradicate by surgery.The standard treatment for ovarian cancer is resection of carcinoma and postoperative with chemotherapy using cisplatin combined with paclitaxel.But chemotherapy is prone not only to therapeutic resistance,but also to bring severe side effects.Moreover,it leads to poor effect in clinical treatment and poor prognosis of patients.However,it is not clear the occurrence and development mechanism of ovarian cancer and how ovarian cancer cells spread to distant organs.Therefore,how to inhibit tumor cell proliferation,infiltration and metastasis.How to enhance the sensitivity of tumor cells to chemotherapy drugs and improve the effect of treatment of ovarian cancer.How to improve survival and quality of life.CUL4B acts as a scaffold protein in Cullin4B··RING·-based E3 ubiquitin ligases(CRL4Bs),a member of the largest known of E3 ubiquitin ligases.CRL4 B has beenshown to function in different physiologically and developmentally processes including cell proliferation,signal transduction,development and cell differention.Through catalyzing polyubiquitination of targeted proteins such as cyclin E and p53 for proteasomal degradation,CRL4 B regulates cell proliferation and senescene.Moreover,CRL4 B could also epigenetically repress transcription of genes includingp1 6,p2 1 PTGDS and miRNA ? 37 1/372 through catalyzing H2 AK 1 1 9 ubiquitination(H2AK I 1 9ub1)at promoters of these genesAccumulating evidences have shown that CUL4 B plays an important role intumor,Results from the analysis in lung cancer,glioma,esophageal and cervical carcinoma indicated that CUL4 B was much higher in these tumor tissues.It was showed that CRL4 B is capable of repressing a number of tumor suppressors including p16,Wnt antagonists:PTEN,IGFBP3 and miR - 1 94 through catalyzing H2 AK 1 1 9 monoubiquitination(H2AKll9ubl)at their promoters.So CUL4 B possesses oncogenic properties by repressing these tumor suppressors in a variety of humancancers including cervical carcinoma,hepatocellular carcinoma,and non—small celt lung cancer.Interestingly,a recent research found that CUL4 B could inhibit tumorigenesis through regulating differentiation of myeloid—derived suppressor cells in tumor mi croenvironment,suggesting CUL4 B may have different roles in different contents of cancer.However,the roles of CUL4 B in gastric cancer are unknown.In this study,we investigated the expression of CUL4 B in GC tissues and analyzed its functions in proliferation,migration,invasion and drug sensitivity of GC cells.We first examined CUL4 B protein expression in paired cases of GC specimens by immunohistochemistry using paraffin—embedded GC tissue array.The results showed that CUL4 B was mainly localized in the nucleus of cancer cells and expression of CUL4 B in GC tissues was significantly increased compared with that in the non—cancerous tissues.The correlation between CUL4 B expression and clinicopathological parameters of patients with GC were further analyzed and we found that high expression levels of CUL4 B was significantly correlated with pathological grading.To explore the biological significance of CUL4 B in tumorgenesis of GC cells,CUL4 B was stably knocnkdown or overexpressed by lentivirus-mediated way inMKN45,BGC823 and SGC7901 cells and expression of CUL4 B was verified.MTT assays showed that stably knockdown of CUL4 B in these GC cell lines resulted in significantly slower growth rate compared to that of the NC—transfected cells However,analysis of cell cycle and EdU incorporation showed that CUL4 B did not affect cell cycle progression of G 1 ? S and M—G I,and DNA replication in these GC cells,suggesting CUL4 B regulates cell growth of GC cells independent of these processes.Next,we investigated the roles of CU L4 B in cell migration and invasion of GC cells.The results of wound healing assay showed that the speed of healing in CUL4 B knockdown GC cells was much slower than that in control cells.Consistent with these results,the number of migrating cells penetrating the transwell membrane was significantly lesser in CUL4 B knockdown cells than that of control cells.Further,matrigel invasion assay was employed tO evaluate the effects of CUL4 B knockdown on cell invasion.This study aims to explore the effect of CUL4 B on ovarian cancer cells.First,immunohistochemistry was used to detect the expression of CUL4 B in human ovarian cancer tissues and adjacent tissues,suggesting that the expression of CUL4 B in human ovarian cancer tissues was significantly up-regulated.Subsequently,human ovarian cancer cell line HEY cells were selected and transfected with si-RNA by liposomes to construct low-expression CUL4 B cell lines in the experimental group to compare with the control cells.Cck-8 assay was used to detect the effect of CUL4 B on the proliferation of ovarian cancer cells,and the results suggested that inhibition of CUL4 B could obstruct the proliferation of ovarian cancer cells.The effect of CUL4 B on the migration and proliferation of ovarian cancer cells was detected by scratch test,showed that CUL4 B knockdown could inhibit the migration and invasion ability of ovarian cancer cells.Western blot assay was performed to detect the effect of CUL4 B on epithelial-mesenchymal transformation in ovarian cancer cells,suggested that CUL4 B knockdown inhibits epithelial mesenchymal transformation in ovarian cancer cells.In a word,through this experiment,we verified the increase in CUL4 B in human ovarian cancer tissue.The cell experiment showed that CUL4 B could stimulate the proliferation,migration,invasion ability and epithelial mesenchymal transformation of human ovarian cell.All of these results suggest that CUL4 B play the role of the proto-oncogene in human ovarian cancer cells.This result provides new ideas for further exploration of the function of CUL4 B,and presents new prospects for research of the pathogenesis of ovarian cancer.