Biological Function Of CBX4and Its Prognostic Significance And Corresponding Molecular Mechanisms In Hepatocellular Carcinoma

Objective:CBX4(Chromobox homolog4) initially proved to be a kind of E3ligase, involved in chromatin remodeling and transcription repression, as well as maintain sternness of epithelium stem cells and prevent senescence. CBX4relationship with tumor research so far is less. This study aimed to explore CBX4expression in primary hepatocellular carcinoma and its relationship with the prognosis, to reveal the role of CBX4in hepatocellular carcinoma.To investigate influence of CBX4on malignant phenotypes of liver cancer cells and reveal corresponding molecular mechanisms.Methods:1) qRT PCR and Western Blot used to detect CBX4mRNA and protein expression level on specimens of surgical primary hepatocellular carcinoma, including adjacent normal liver tissue and HCC tissues, respectively;2) CBX4expression was detected via immunohistochemical staining in246cases of paraffin embedding primary hepatocellular carcinoma specimens, which with clinical outcome data completely;3) Pearson Chi-Square and Kruskal Wallis tests were used to determine relationship between clinicopathological features and CBX4expression. Kaplan-Meier method, log-rank test were applied to do univariate survival analysis to confirm charicteristics related to survival. Multivariable Cox regression model to screen variates as independent prognosis factors in primary HCC.4) In primary hepatocellular carcinoma cells, QGY7703and BEL7402, transient transfection with small double strain RNA interference for knocking down CBX4, In vitro, to detect cell proliferation and clone formation capacity by MTT and planet clone formation assay.5) After knocking down CBX4, double thymidine method makes BEL7402and QGY7703synchronization in the G1/S junction and release into S phase, proportion of S phase cells was tested via the flow cytometry instrument.6) After knockdown of CBX4, Western blot detect changes of the downstream target genes. Results:1) CBX4expression in primary HCC tissue is upregulated. Real-time PCR detect8pair of samples, CBX4mRNA expression in HCC tissue is higher than tissue adjacent to carcinoma; Western blot test8pair of specimens on the corresponding matching, CBX4protein expression in cancer tissue is also higher than tissue adjacent to carcinoma;2) After evaluating IHC staining scores on246surgical specimen with primary HCC, we found that CBX4express disorders exist in patients with primary HCC. Compared with the tissue adjacent to carcinoma, CBX4protein expression in carcinoma tissues significantly increase, mainly located in the cytoplasm of HCC cells.3) Clinical significance of CBX4expression in primary HCC. Immunohistochemical results show that the CBX4expression is closely related to the clinicalpathologic features of patients with primary HCC:serum AFP level (P=0.036), tumor size (P=0.029), pathological differentiation (P=0.033), the clinical TNM stage (P=0.032). High expression of cytoplasmic CBX4in patients with hepatocellular carcinoma predicts poor prognosis, overall survival and disease-free survival time shorter than those patients with low CBX4expression. Multiple variables regression analysis showed that’CBX4cytoplasmic expression’ and ’tumor number’ in patients with hepatocellular carcinoma are independent prognostic factors for postoperative overall survival and recurrence free survival. Knockdown of CBX4reduce primary HCC cell proliferation.4) After knockingdown of CBX4, QGY7703and BEL7402cells at slower pace of proliferation and fewer foci of clony formation.5) After knockdown of CBX4, hepatocellular carcinoma cells, including BEL7402and QGY7703cells, cell proporation through G1/S border into S phase decreased significantly, cells progress from G1/S point to S phase at a slower pace.6) CBX4can affect the primary HCC cell proliferation and G1/S transition by inhibiting expression of PCNA and cyclinE2, accompanied by upregulate p16.Conclusion:1) CBX4expression in primary HCC is upregulated.2) CBX4gene mainly located in cytoplasm of HCC tissue.3) CBX4cytoplasmic expression and prognosis of patients with primary HCC is inversely correlated, cytoplasmic CBX4could be as one of the independent predictors in postoperative HCC.4) CBX4is closely related to primary HCC cell malignant proliferation and colony formation capacity.5) CBX4can regulate PCNA expression to maintain rapid proliferation of primary HCC cells.6) CBX4can affect cell cycle progression through regulating cyclinE2and p16.