The Role And Mechanism Of CENPK In Proliferation,Invasion And Migration Of Hepatocellular Carcinoma

?Background?Hepatocellular carcinoma(HCC)the sixth most prevalent cancer and third leading cause of cancer-related mortality worldwide.Although some progress has been achieved in clinical diagnosis and treatments,due to the complex pathogenic factors of HCC,frequent metastasis,insensitivity to chemotherapy and recurrence after surgical resection,the overall survival of patients with HCC still remains poor.Therefore,it is of great practical significance to investigate the molecular mechanisms involved in the development of HCC and explore new potential therapeutic targets for HCC.Kinetochore is a protein structure on chromatids,and plays a critical role in the process of chromosome segregation.Kinetochore dysfunction or dysregulation leads to aneuploidy and promotes carcinogenesis.Kinetochore is consists of at least 80 different proteins,many of these proteins play an important role in tumor malignant progression,including CENPA,CENPB,CENPC,CENPI,CENPK and CENPM.Centromere protein K(CENPK),a kinetochore centromere protein,is critical for proper kinetochore function and mitotic progression.Studies have found that CENPK is up-regulated in a variety of tumors and is associated with the malignant progression of tumor,may be act as an oncogene.However,the expression and clinical significance of CENPK in HCC remains unclear,and its biological functions and related mechanisms remain to be further studied.Therefore,the main purpose of this study was to investigate the expression of CENPK in HCC and its related functions and mechanisms.?Aim?1.To investigate the expression and clinical significance of CENPK in HCC2.To study the effect of CENPK on the malignant biological function of HCC cells.3.To explore the underlying mechanism of CENPK on regulating malignant phenotype of HCC cells ?Methods?1.Firstly,the expression and clinical significance of CENPK in HCC tissues were statistically analyzed in the TCGA database.Then,the expression of CENPK in clinical HCC samples was detected by qRT-PCR and Western Blot for further verification.2.A CENPK interfering lentiviral plasmid(shCENPK)and negative control plasmid(NC)was transfected into HCC cells to stably knockdown CENPK or negative control,respectively.The Celigo cell counting,CCK-8,colony formation,wound healing and Transwell assay analyzed the effects of CENPK on cell proliferation,migration and invasion ability in vitro.The effect of CENPK on HCC cell proliferation was detected by subcutaneous tumorigenesis in nude mice in vivo.3.Using mRNA microarray to gain different gene expression profiles in BEL-7404-shCENPK and BEL-7404-NC cells.We used the Ingenuity Pathway Analysis(IPA)database to select the crucial pathway molecules regulated by CENPK.Then,qRT-PCR,Western Blot and immunofluorescence assays were used to further confirm.Using lentivirus interference technology to knockdown the expression of YAP1 in HCC cells,and investigated its effect on HCC cell proliferation,migration and invasion ability.Recovery experiments were also used to verify the function of YAP1.We detected the relationship between CENPK/YAP1 and EMT markers protein levels by Western Blot.?Results?1.The TCGA database showed that the expression of CENPK was up-regulated in HCC tissues,and was related to clinical grading,T stage,pathological stage and predicted a poorer prognosis in HCC patients.Further study confirmed that the mRNA and protein expression of CENPK were highly expressed in our department clinical collected HCC tissues.The increased proportion of CENPK mRNA and protein were 86.7%(28/30)and 75%(6/8),respectively.2.The HCC cell lines BEL-7404 and SMMC-7721 transfected with shCENPK lentiviral plasmid was significantly down-regulated the expression of CENPK compare to NC.The Celigo cell counting,CCK-8,colony formation and subcutaneous tumorigenesis assays showed that silencing CENPK inhibited the cell proliferation,colony formation and tumor growth of HCC cells in vitro and in vivo.The wound healing and Transwell assay implied that silencing CENPK suppressed HCC cell migration and invasion in vitro.3.According to the analysis of IPA database,we found that the expression of CENPK was related to growth of tumor,proliferation of cancer cells,proliferation of stem cells,invasion of tumor cell lines,invasion of cells and development of epithelial tissues,and YAP1 may be a crucial pathway molecule regulated by CENPK in HCC malignant progression.Then,we confirmed that silencing CENPK significantly down-regulated the mRNA and protein level of YAP1.4.Down-regulation of YAP1 inhibited the cell proliferation,colony formation,migration and invasion in HCC,and in a manner similar to silencing CENPK in these cells.Furthermore,the inhibitory effects of CENPK knockdown on HCC cells were partially eliminated by restoring YAP1 expression.5.Down-regulation of CENPK and YAP1 significantly enhanced the expression of epithelial marker E-cadherin and reduced the expression of mesenchymal maker N-cadherin,implying that silencing CENPK and YAP1 inhibited EMT progress in HCC cells.However,the EMT inhibitory effect of CENPK knockdown on HCC cells was partially counteracted by overexpression of YAP1.?Conclusions?1.CENPK was highly expressed in HCC tissues,and was associated with tumour malignant phenotype and predicted a poor prognosis in HCC patients.2.Down-regulation of CENPK inhibited cell proliferation,migration and invasion of HCC cells in vitro and in vivo.3.Down-regulation of CENPK suppressed EMT progress in HCC cells,and this inhibitory effect can partially counteract by overexpression of YAP1.4.CENPK influenced the malignant progression of HCC by regulating the expression of YAP1.Targeting CENPK/YAP1 may provide a new strategy for the treatment of HCC.