A Study Of The Roles Of CUL4B In Gastric Cancer

Gastric cancer(GC)is the fifth most prevalent type of malignancy and the third-leading cause of cancer-related death worldwide.The incidence rates are distinct in different geographic regions.Of these cases,over 73.6%occurred in Asia,and over 42.7%of the world total occurred in China.It was reported that gastric cancer is the second leading cause of cancer-related mortality in male and the third in femal in China in 2015.The main therapeutic methods of GC include surgery,chemotherapy,immunotherapy and targeted therapy.Despite recent advances in diagnosis and treatment,the median overall survival of patients with advanced GC is still below 1 year.That is mainly because early diagnosis is very difficult in clinics and the prognosis for advanced-stage patients is still very poor.Therefore,understanding the molecular mechanisms underlying GC is crucial for development of novel diagnostic and treatment strategies for patients with GC.CUL4B acts as a scaffold protein in Cullin4B-RING-based E3 ubiquitin ligases(CRL4Bs),a member of the largest known of E3 ubiquitin ligases.CRL4B has been shown 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,CRL4B regulates cell proliferation and senescene.Moreover,CRL4B could also epigenetically repress transcription of genes including p16,p21 PTGDS and miRNA-371/372 through catalyzing H2AK119 ubiquitination(H2AK119ub1)at promoters of these genes.Accumulating evidences have shown that CUL4B plays an important role in tumor.Results from the analysis in lung cancer,glioma,esophageal and cervical carcinoma indicated that CUL4B was much higher in these tumor tissues.It was showed that CRL4B is capable of repressing a number of tumor suppressors including p16,Wnt antagonists,PTEN,IGFBP3 and miR-194 through catalyzing H2AK119 monoubiquitination(H2AK119ubl)at their promoters.So CUL4B possesses oncogenic properties by repressing these tumor suppressors in a variety of human cancers including cervical carcinoma,hepatocellular carcinoma,and non-small cell lung cancer.Interestingly,a recent research found that CUL4B could inhibit tumorigenesis through regulating differentiation of myeloid-derived suppressor cells in tumor microenvironment,suggesting CUL4B may have different roles in different contents of cancer.However,the roles of CUL4B in gastric cancer are unknown.In this study,we investigated the expression of CUL4B in GC tissues and analyzed its functions in proliferation,migration,invasion and drug sensitivity of GC cells.We first examined CUL4B protein expression in paired cases of GC specimens by immunohistochemistry using paraffin-embedded GC tissue array.The results showed that CUL4B was mainly localized in the nucleus of cancer cells and expression of CUL4B in GC tissues was significantly increased compared with that in the non-cancerous tissues.The correlation between CUL4B expression and clinicopathological parameters of patients with GC were further analyzed and we found that high expression levels of CUL4B was significantly correlated with pathological grading.To explore the biological significance of CUL4B in tumorgenesis of GC cells,CUL4B was stably knocnkdown or overexpressed by lentivirus-mediated way in MKN45,BGC823 and SGC7901 cells and expression of CUL4B was verified.MTT assays showed that stably knockdown of CUL4B 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 CUL4B did not affect cell cycle progression of G1-S and M-G1,and DNA replication in these GC cells,suggesting CUL4B regulates cell growth of GC cells independent of these processes.Next,we investigated the roles of CUL4B in cell migration and invasion of GC cells.The results of wound healing assay showed that the speed of healing in CUL4B 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 CUL4B knockdown cells than that of control cells.Further,matrigel invasion assay was employed to evaluate the effects of CUL4B knockdown on cell invasion.Our results showed that the number of invading cells was considerably reduced in the CUL4B knockdown GC cells when compared with control cells.Together,these results indicate that inhibition of CUL4B significantly attenuates migration and invasion of GC cells.We then analysis the role of CUL4B in drug sensitivity of GC cells.MTT results showed that the viability of GC cells is much lower in CUL4B knockdown cells than that in control cells after the treatment of cisplatin and pirarbicin.TUNEL assay also revealed that knockdown of CUL4B significantly increased apoptosis in drug treated GC cells.These data suggests that lower expression of CUL4B could decrease chemo-drug sensitivity of GC cells.Collectively,we found that CUL4B is up-regulated in gastric cancer and is correlated with pathological grading.We also showed that CUL4B could promote the growth,migration,invasion and drug resistance of gastric cancer cells.Thus,CUL4B may function as an important oncogene in gastric cancer.Our work provides new evidence to understand the potential role of CUL4B in gastric cancer,which may be helpful for exploring new therapeutic method and understanding function of CUL4B in cancer.