| Background:cinoma (HCC) is one of the most common digestive system malignant tumors in China.Due to the heavy load of HBV infection, the new cases and deaths of HCC patients are highest in the world. Because of its difficulty to diagnose at the onset, quick progression, high grade and poor prognosis,HCC threatens human health seriously, also brings heavy burden to the family and the society.Nowdays, with the developmnt of new treatment concept and medical technology in HCC, the operation based comprehensive treatment strategies greatly improve the patients survival rate.Especially, the new emerging liver transplantation (LT) provides a way to radical treatment for HCC.However, frequent metastasis and recurrence imposes a major obstaclefor long-and medium-term survival of HCC patients after LT. For prolonging the survival of HCC patients with LT, it is enssential to find more effective prognosis related biomarker for LT, and explore its potential role in HCC.Objectives:By2D electrophoresis, a proteomic technology, the aim of this study was to screen differentially expressed proteins between HCC patient with LT who suffers HCC recurrence and those without recurrence. Next, we explored the potential molecular biomarkers which were related with HCC recuurence after LT and HCC development through bioinformatics analysis and literature search.For the selected potential molecular biomarker RBBP4, we firstly verified its differentially expressionon in more HCC patients’tissues who underwent liver resection or LT.Then, we analyzed the relationship between RBBP4expression and clinical pathological parameters and the prognosis of HCC patients with LT. Finally, we further verified the biology function of RBBP4on HCC cell lines and explored its possible underlying molecular mechanism in HCC development.Methods:1. Screening and identifying of differentially expressed proteins between1pairs of tissues gained from HCC patients with LT who underwent recurrence or not by the proteomic technology of two-dimensional gel electrophoresis combined mass spectrum (2D-MS).2.30pairs of HCC and matched adjacent frozen tissues from HCC patients with liver resection, and70pairs of HCC and matched adjacent paraffin embedded tissues from HCC patients with LT were collected to further verify the mRNA and protein expression of RBBP4by real-time fluorescent quantitative PCR (qRT-PCR), western blotting and immunohistochemical staining (IHC). Meanwhile, the expression of RBBP4was also examined in normal, cirrhosis, metastatic liver cancer and metastatic lymph node tissue by IHC.3. According to the IHC results, we analyzed the relationship between RBBP4expression and clinical parameters and prognosis of HCC patients with LT.4. We examined the expression of RBBP4in several types of cancer and matched adjacent tissues to verify whether the expression of RBBP4was universally abnormal in tumors. HCC cell line BEL7402and HepG2were stably transfected with RBBP4overexpression or knockdown lentivirus. And then, the effects of RBBP4on cell proliferation, cell apoptosis, cell invasion, cell migration ability and tumorigenicity were detected using cell counting kit-8(CCK8) assay, Flow cytometryapoptosis assay, transwell tumor invasion and migration assay. Meanwhile, nude mouse tumorigenicity assay and clone formation assay were used to evaluate the effect of RBBP4on tumorigenesis capability of HCC cells.5. miRNA target prediction websites and dual-luciferase reporter assey were used to find the upstream miRNA which caused the dysregulated expression of RBBP4. qRT-PCR and western blotting were use to find the downstream gene regulated by RBBP4, and to explore the potential molecular mechanism of RBBP4in HCC development.Results:1.2D-MS analysis showed RBBP4was high expressed in HCC patient with LT who underwent recurrence compared those who had no recurrence (fold change≥2.0, p<0.05).2. The result of western-blot confirmed that RBBP4had a higher expression in HCC tissues than in matched adjacent tissues. qRT-PCR showed that RBBP4mRNA expressed higher in HCC tissues than that in matched adjacent tissues (p=0.014). IHC result showed the expression trend of RBBP4was gradually increased in normal liver tissue, cirrhosis liver tissue and liver cancer tissue. Meanwhile, RBBP4also had a high expression in metastatic liver cancer tissue and metastatic lymph node.3. According to the IHC results, we observed significant correlation between high RBBP4expression and vascular invasion (P=0.027), tumor size (P=0.01), pTNM stage (P=0.002) and tumor recurrence (.P=0.000). No correlation was observed in patients’ age, gender, preoperative AFP, tumor differentiation, tumor number and HBV infection.Kaplan-Meier survival analysis indicated that recurrence-free survival (RFS) and5-year overall survival (OS) of HCC patients with high RBBP4expression was significantly lower than HCC patients with low RBBP4expression (P<0.01). Cox multivariate analysis showed that high RBBP4expression may be a novel independent prognostic factor for recurrence-free survival of HCC patients after LT (Hazards ratio,5.425;95%confidence interval,1.803-16.324, P=0.003, Table3)4. The further IHC results showed that RBBP4was found up-regulated in several other kinds of cancers, such as esophagus cancer, pancreatic cancer, colorectal cancer, extrahepatic cholangiocarcinoma and lung cancer. Cytology experiments in vitro revealed that RBBP4knockdown inhibited the cell viability, cell invasion and migration and anti-apoptosis ability of HCC cells. In contrast, up-regulation of RBBP4markedly increased cell viability, cell invasion and migration and anti-apoptosis ability of HCC cells. In vivo animal experiment revealed that overexpression of RBBP4could promote tumorigenicity of BEL7402cells in vivo.5. The further study on molecular mechanism proved that RBBP4was the target gene of miR-199a-5p, the high expression of RBBP4may due to the downregulation of miR-199a-5p.The high expressed RBBP4could activate AKT protein by suppress PTEN, while the activated AKT protein could further downregulate the expression of miR-199a-5p.In conclusion, we found a miR-199a-5p/RBBP4/AKT positive loop in HCC, this loop may play an important role in HCC development.Conclusion:1. RBBP4was expressed higher in HCC patient with LT who suffered recurrence compared with HCC patient with LT who had no recurrence.It indicated that RBBP4may be closely relate to HCC recurrence after LT.2. Prognosis of HCC patients with LT who had a high RBBP4expression was poorer than those who had a low RBBP4expression. Multivariate analysis indicated that RBBP4was a predictive biomarker for recurrence of HCC patients with LT.3. Expression trend of RBBP4was gradually increased in normal liver tissue, cirrhosis liver tissue and liver cancer tissue.Meanwhile,RBBP4was found up-regulated in several other kinds of cancers.These results indicated that RBBP4may play an essential role in tumorigenesis.4. There existed a miR-199a-5p/RBBP4/AKT positive loop in HCC development. |
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