| CUL4B is the skeleton protein of Cullin 4B-Ring ubiquitin ligases(CRL4B).CRL4B can regulate many important substrate proteins through ubiquitination modification,and participate in the regulation of cell cycle,DNA damage repair,signal transduction and other important physiological processes.Our previous studies have shown that CUL4B can also participate in the transcriptional regulation of tumor suppressor genes' by catalyzing the mono-ubiquitination of H2AK119 and recruiting a multicomb inhibitory complex PRC2.We conducted a series of studies on the expression of CUL4B in different solid tumors' and related molecular mechanisms.The results showed that CUL4B was highly expressed in hepatocellular carcinoma and promoted the proliferation and migration of hepatocellular carcinoma by activating Wnt/?-catenin signaling pathway;In gastric cancer,CUL4B could up-regulate the expression of HER2 and promote the infiltration of gastric cancer cells and epithelial-mesenchymal transformation(EMT);In prostate cancer,CUL4B and SOX4 form a positive regulatory feedback loop to promote the proliferation,EMT and metastasis of prostate cancer cells;In cholangiocarcinoma,CUL4B inhibits the expression of tumor suppressor gene P16 and phosphatase and tensin homologues,and promotes the proliferation,migration and EMT of cancer cells.The role of CUL4B in the carcinogenesis and development of malignant tumors and the related molecular mechanisms are still at the initial stage,and need further exploration.As an important transcription factor,C-MYC plays an important role in prostatic intraepithelial neoplasia(PIN)transformation and in maintaining the characteristics of prostate cancer stem cells.C-MYC plays an important role in survival,proliferation,metastasis and recurrence of cancer cells.Studies have shown that C-MYC promotes androgen receptor transcription and can be used as an adjuvant target for AR targeted therapy in castration-resistant prostate cancer(CRPC)patients.The level of C-MYC protein is affected by complex post-transcriptional and post-translational epigenetic regulation,which is inconsistent with its mRNA expression.The epigenetic regulation mechanism of C-MYC protein expression in prostate cancer is one of the hotspots of current research.MiRNAs(microRNAs)is a class of non-coding single-stranded RNA molecules encoded by endogenous genes.In cytoplasm,miRNAs can guide Argonaute(AGO)protein to bind to target gene 3'untranslated region(3'UTR),which is a post-transcriptional epigenetic regulation.Studies have shown that the expression of miRNAs is aberrant in many human malignant tumors,and plays an important role in obtaining sustained proliferation signals,avoiding growth inhibition,anti-apoptosis,promoting immortalization,inducing angiogenesis and promoting invasion and metastasis of malignant tumors.In view of the key role of C-MYC in the carcinogenesis and development of prostate cancer,this study explored the regulatory relationship between CUL4B and C-MYC in prostate cancer,and analyzed the molecular mechanism of their regulation from the perspective of epigenetic regulation of miRNAs.Part ? CUL4B is overexpressed in prostate cancer and is associated with poor prognosisProstate cancer is the most common malignant tumor among men in the United States,Britain and other Western countries.The incidence of prostate cancer in China is on the rise,and most patients are in the middle and late stages of treatment,which is one of the common tumors affecting the health of men.Our previous studies have shown that CUL4B is highly expressed in hepatocellular carcinoma,cholangiocarcinoma,non-small cell lung cancer and gastric cancer,and is associated with clinicopathological parameters in some tumors.The following results have been achieved in the expression of prostate cancer and its correlation with prognosis:1.Upregulation of CUL4B expression is correlated with different Gleason scores in prostate cancer:Immunohistochemical staining showed that the expression of CUL4B in prostate cancer was significantly higher than that in benign prostatic hyperplasia(P<0.001).The high expression of CUL4B was correlated with different Gleason scores(P=0.03).2.Data analysis of CUL4B in the public bioinformatics database(TCGA/GEO):The results of the public database(TCGA/GEO)analysis showed that the expression of CUL4B in prostate cancer tissues was significantly higher than that in normal prostate tissues around cancer.The expression of CUL4B in metastatic tumors was higher than that in primary tumors.The oyerexpression of CUL4B was associated with Gleason score and pathological Stage,distant metastasis and biochemical recurrence.Part ? CUL4B post-transcriptionally regulates the C-MYC protein level in prostate cancerIn this part,we use cell experiments and clinical case tissues to explore the regulatory relationship between CUL4B and C-MYC at the level of mRNA and protein.1.CUL4B positively regulates the expression of C-MYC protein:Western blot showed that the expression of C-MYC protein was significantly decreased in CUL4B siRNA knocked-down PC3 cells,and significantly increased in Flag4B plasmid transfected 22RV1 cells which overexpressed CUL4B.However,the expression of CUL4B protein did not change after C-MYC knock-down.These results suggest that CUL4B is a positive regulator of C-MYC protein and belongs to one-way regulation.IHC staining showed that CUL4B was highly correlated with C-MYC protein expression in prostate cancer tissues(P=0.0014).2.The aberrant expression of CUL4B did not affect the expression of C-MYC gene:RT-qPCR showed that there was no significant change in C-MYC mRNA expression after CUL4B knock-down in PC3 cells or after transfection of Flag4B overexpression plasmid in 22RV1 cells.It is suggested that the positive regulation of CUL4B on C-MYC protein in prostate cancer belongs to post-transcriptional regulation.Part ? CUL4B-MiR-33b-5p-C-MYC axis promotes prostate cancerprogressionSince the regulation of CUL4B on C-MYC is post-transcriptional positive,we speculate that the regulation of CUL4B on C-MYC may be mediated by miRNAs.This study provides a new entry point for the molecular mechanism of CUL4B promoting the carcinogenesis and progression of prostate cancer.1.CUL4B transcriptionally inhibits the expression of miR-33b:We extracted the total RNAs of PC3 cells after CUL4B knock-down and performed microarray analysis,and identified the miRNAs profile regulated by CUL4B.Next,we use bioinformatics technology to obtain the miRNAs data sets targeting C-MYC,and intersect it with the miRNAs up-regulated by more than two times after siCUL4B knock-down.The results showed that miR-33b met the above conditions,and the large sample analysis of TCGA data set showed that the expression of miR-33b-5p in prostate cancer was lower than that in tormal prostate tissue.Therefore,miR-33b-5p becomes the target of our next experiment.RT-qPCR assay showed that the expression levels of pri-miR-33 and miR-33b-5p were increased in PC3 cells with CUL4B depletion,while the expression levels of pri-miR-33 and miR-33b-5p were decreased in the CUL4B over-expression plasmid transfection group of 22RV1 cells.ChIP test showed that CUL4B could directly bind to the promoter region of miR-33b.These results suggest us CUL4B could regress miR-33b transcriptionally.2.MiR-33b-5p directly targeting C-MYC:Double luciferase reporter gene assay showed that over-expression of miR-33b-5p significantly inhibited the activity of pmiR-GLO-C-MYC 3'UTR-Wt,while co-transfection of miR-33b-5p mimics and pmiR-GLO-C-MYC 3'UTR-Mut plasmids showed no significant change in luciferase activity.Western blot analysis showed that over-expression of miR-33b in PC3 and 22RV1 cells significantly decreased the expression level of C-MYC,while down-regulation of miR-33b leaded to up-regulation of C-MYC.Rescue experiments showed that after co-transfection of Flag4B/Flaga and miR-33b-5p mimics/NC in 22RV1 cells,miR-33b-5p mimics partially inhibited the over-expression of C-MYC.After co-transfection of siCUL4B/siNC and miR-33b-5p inhibitor/inhibitor NC in PC3 cells,the inhibition of C-MYC expression was partially reversed by miR-33b-5p inhibitor.3.MiR-33b-5p inhibits the proliferation of prostate cancer cells induced by CUL4B:Cloning,MTS and EdU experiments showed that CUL4B could significantly promote the proliferation of 22RV1 cells,and this effect was partially inhibited after co-transfection of the mimics/NC and Flag4B expression plasmids.4.MiR-33b-5p inhibits the migration and invasion of prostate cancer cells induced by CUL4B:Scratch and Trans well experim nts showed that the over-expression of CUL4B could significantly enhance the migration and invasion ability of 22RV1 cells,and miR-33b-5p mimics could partially reverse the above effects of CUL4B.5.MiR-33b-5p inhibits the expression of stem cell-related molecules in prostate cancer cells induced by CUL4B:Flow cytometry sorting test showed that the number of CD 133+ cells in CUL4B over-expression 22RV1 cells increased significantly compared with negative control group,while the percentage of positive cells in Flag4B+ mimics group decreased compared with negative control group.Western blot showed that over-expression of CUL4B could up-regulate the expression of CD133,CD44,Nanog and BMI proteins,while co-transfection of miR-33b-5p mimimics could significantly reduce the effect of enhanced expression.To sum up,CUL4B is highly expressed in prostate cancer and can regulate the expression of C-MYC through transcriptional inhibition of miR-33b-5p.CUL4B is considered to be an important oncogene that promotes the proliferation and progression of prostate cancer.MiR-33b-5p can partially reverse the abilities of promoting proliferation,migration,invasion and the expression of stem cell-related molecular markers of prostate cancer induced by CUL4B.Our study elucidates the molecular mechanism of CUL4B in promoting the carcinogenesis and progression of prostate cancer,and provides a new potential target and theoretical basis for targeted therapy of prostate cancer. |
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