The Study Of RBM8A Promoted Tumorgenesis And Regulated Proliferation And Apoptosis In Hepatocellular Carcinoma

Part I Overepression of RBM8 A promotes tumorgenesis and predicts unfavorable prognosis in Hepatocellular Carcinoma patientsAim:RNA-binding motif protein 8A(RBM8A) has been speculated to play important roles in the development of several kinds of cancers. However, the role of RBM8 A in hepatocellular carcinoma(HCC) is not clear. In this study, we aimed to investigate the role and molecular mechanism of RBM8 A in promoting tumorgenesis and predicting prognosis in HCC patients.Methods:105 pairs of snap-frozen primary human HCC samples, adjacent noncancerous tissues from adjacent regions, and 67 hepatic hemangioma samples were provided by the Affiliated Tumor Hospital of Guangxi Medical University from Match 2012 to December 2014, from patients treated surgically. Firstly, Real-time quantitative polymerase chain reaction(q RT-PCR) assay was performed to assess RBM8 A m RNA level in different tissues. Secondly, Immunohistochemical(IHC) and western blot were performed to investigate the expression of RBM8 A in different tissues. In addition, on the basis of q RT-PCR and western blot results, HCC patients were divided into high expression and relationship between RBM8 A expression and clinicopathological characteristics was investigated. Besides, on the basis of IHC analysis, HCC patients were divided into high expression and low expression groups as well and Kaplan-Meier survival analysis was performed to investigate the correlation between RBM8 A expression and prognosis of primary HCC patients. Further, enzyme-linked immuno sorbent(ELISA) assay was performed to investigate the difference of RBM8 A and AFP levels in serum among 110 HCC patients, 110 liver cirrhosis patients and 110 normal persons and explore the possibility to consider RBM8 A as a potential diagnostic biomarker combined with AFP to enhance the sensibility of diagnosis of HCC.Results:(1)q RT-PCR showed that the expression rates of RBM8 A m RNA was significantly higher in primary human HCC tissue(△△CT= 7.99595±5.49764) compared to adjacent cancer tissues(△△CT= 4.6667±3.78338) and hepatic hemangioma tissue(△△CT= 1.6460±1.53852)(P<0.0001);(2)Western blot analysis showed that expression of RBM8 A protein was significantly elevated in human primary HCC tissue compared to adjacent noncancerous hepatic tissues or hepatic hemangioma tissue;(3)IHC analysis confirmed that RBM8 A expression as positive was higher in primary HCC tissue(92/105, 85%) than adjacent noncancerous tissues(64/105, 1161.67%) or hepatic hemangioma tissue(3/67, 5.71%)(P<0.0001);(4) Chisquare analysis revealed that the expression of RBM8 A in HCC tissues was highly correlated with HBV(P <0.0001) and histological grade(P =0.003), but not with vascular invasion(P = 0.276), serum AFP(P = 0.433), liver cirrhosis(P = 0.58), tumor size(P = 0.013) or TNM stage(P =0.153); Chi-square analysis revealed that the expression of RBM8 A in HCC tissues was highly correlated with HBV(P <0.0001), tumor size(P = 0.013), TNM staging(P <0.0001) and histological grade(P =0.003), but not with vascular invasion(P = 0.276), serum AFP(P = 0.433) or liver cirrhosis(P = 0.58);(5) On the basis IHC analysis, 64 HCC patients(60.95%)was considered as high expression of RBM8A(strong positive/moderate positive) while 41 patients(39.05%)was considered as high expression of RBM8A(weak positive/negative). KaplanMeier survival analysis revealed that HCC patients with high levels of RBM8 A expression had significantly poorer overall survival(OS) and progression free survival(PFS) than those with low expression levels; and multivariate Cox regression analysis indicated that RBM8 A expression was an independent predicator of OS and PFS for HCC.(6) ELISA tests domenstrated that the serum RBM8 A level was significantly higher in HCC patients compared with liver cirrhosis patients and normal patients. Besides, the sensibility of single test of serum AFP level to diagnosis of HCC was 70.0%. Whereas the sensibility of diagnosis of HCC was enhanced to 86.3% when alliantly tested of serum RBM8 A and AFP level.Conclusion:Our findings suggest that RBM8 A might have an oncogenic role in human primary HCC, overepression of RBM8 A promotes tumorgenesis and predicts unfavorable prognosis in hepatocellular carcinoma patients. RBM8 A could be considered as a pontential novel diagnostic and prognostic biomarker for HCC.Part II Down-regulation of RBM8 A gene inhibited proliferation and migration and induced apoptosis and S cell cycle arrest in Hepatocellular Carcinoma cellsAim:RNA-binding motif protein 8A(RBM8A) has been speculated to play a positive role in the development and progression of HCC probably through inhibiting HCC apoptosis. In this study, the role of RBM8 A in apoptosis and cell cycle arrest of HCC were investigated in vitro.Methods:Western blot was performed in serial of HCC cell lines in vitro. Then the cell line with the highest expression of RBM8 A was selected to generate RBM8 A knock down(RBM8A-KD) cell line and the cell line with the lowest expression of RBM8 A was selected to generate RBM8 A over expression(RBM8A-OE) cell line. Then CCK8 assays were performed to evaluate the proliferation capabilities of RBM8 A overepression and RBM8 A knock down HCC cell lines. FCM assays were performed to investigate the apoptosis capabilities and cell cycle arrest of RBM8 A overepression and RBM8 A knock down HCC cell lines HCC cell lines. Wound healing test and Transwell invasion assay were performed to investigate the migration and invasion capabilities of RBM8 A knock down and RBM8 A re-supply HCC cell lines.Results:(1)western blot comfirmed that the expression of RBM8 A was highest in Bel-7404 cell line and lowest in HL-7702 cell line. Then Bel-7404 cell line was selected to generate RBM8A-KD cell line and HL-7702 cell line was selected to generate RBM8A-OE cell line.(2) CCK8 assay domenstarted that up-regulation of RBM8 A promoted proliferation in HL7702 cell line(P=0.00159), whereas down-regulation of RBM8 A inhibited proliferation in Bel-7404 cell line(P=0.00325);(3)FCM assays revealed that up-regulation of RBM8 A inhibited apoptosis(P=0.000358)and promoted HL7702 cell line, whereas down-regulation of RBM8 A promoted apoptosis(P=0.0004) and S phase cell cycle arrest(P=0.0065)in Bel-7404 cell line;(4)Wound healing test and transwell invasion assay showed that downregulation of RBM8 A inhibited migration(P=0.04383) and invasion(P=0.0089)in Bel-7404 cell line. Wehrea re-upregulaition of RBM8 A promoted migration(P=0.020727) and invasion(P=0.0016)in Bel-7404 cell line.Conclusion:Our study demonstrate that RBM8 A plays a positive role in the development and progression of HCC probably through inhibiting HCC apoptosis, downregulation of RBM8 A gene inhibited HCC cells proliferation and migration and invasion and induced apoptosis and cell cycle progression to arrest in S phase in vitro. Our study showed that RBM8 A could be considered as a novel potential therapeutic target for HCC patients.