Musashi-2 Promotes Hepatitis B Virus Related Hepatocellular Carcinoma Progression VIA The WNT/β-Catenin Pathway

ObjectiveHepatocellular carcinoma is a highly malignant tumor, and there is still a lack of diversification of common tumor treatment options. Hepatocellular carcinoma (Hepatocellular Carcinoma, HCC) is the main type of primary liver cancer, and it is also one of the highest malignant tumor in the world. Our country is a high incidence of liver cancer area, especially caused by hepatitis B virus in hepatocellular carcinoma patients, the potential mechanism of hepatitis B virus related HCC is of great significance to be explored.At present, radical excision is still considered as the leading method in the treatment of HCC, but the overall survival is unsatisfactory. Telomere shortening, the change of environment, genetic epidemiological effect, changes in cellular functions such as leading malignant cell clone selection, eventually led to the occurrence of liver cancer. Therefore, to explore a new gene activity in HCC cells and its role in the signal transduction pathway, will help to elucidate the mechanism of occurrence and development of tumor biology and find effective target therapy.Musashi-2 (MSI2) belongs to the RNA binding protein Musashi family, is located on human chromosome 17q22, encoding a 328 amino acid polypeptide. The high expression of MSI2 can induce malignant transformation of hematopoietic cells, promote leukemia. Foreign related report showed that there is a correlation between MSI2 and several solid tumors. With the deepening study of the biology of liver, the importance of Wnt/β-catenin signaling pathway in liver cancer progression is confirmed by more and more research, and β-catenin is one of the most important downstream effector molecules of Wnt/β-catenin signaling pathway. Our early experimental results found that the up regulation of MSI2 expression in HBV related hepatocellular carcinoma tissues were correlated with the expression pattern of β-catenin; thus, we hypothesized that, MSI2 may be involved in the carcinogenesis, it is necessary to carry on deep foundation and clinical research in the mechanism of liver cancer cells which are illustrated.This paper will further search for the relationship of MSI2 and Wnt/β-catenin signaling pathway in hepatocellular carcinoma, in order to provide a new theoretical basis for clarifying mechanism of hepatocellular carcinoma.MethodsⅠ The significance of MSI2 expression in HCC1 MSI2 and β-catenin gene expression in 10 cases of patients with hepatocellular carcinoma was detected by Realtime PCR at the molecular level, and further analysis of the correlation between MSI2 and β-catenin was performed.2 Immunohistochemistry was used to detect the expression of MSI2 and β-catenin in 106 cases of HBV related hepatocellular carcinoma and noncancerous cirrhotic liver tissue microarray, analyzing MSI2 expression and clinicopathological features of HCC. The correlation between MSI2 and β-catenin was analyzed. COX proportional hazard regression model is established, and the survival of patients was analyzed using the Kplan-meier method. The relationship between MSI2 expression and the prognosis of patients with hepatocellular carcinoma was investigated.Ⅱ Study of the effect of MSI2 on the malignant biological behavior of hepatocellular carcinoma cells and its mechanism in vitro1 To detect the expression of MSI2 in three kinds of HCC cell line SMMC-7721, HepG2, and Hep3B by realtime PCR, screening MSI2 high expression cell line.2 To construct shRNA interference expression vector targeting MSI2 gene. Interference ability in HCC cell proliferation activity and cell viability was detected by MTT method.3 Application of soft agar cloning experiments testing hepatocellular carcinoma cells clone formation ability; application of wound healing experiment in the migration distance of different groups at different time points and dynamic detection of hepatocellular carcinoma cells; the number of transmembrane cell detection in different groups of liver cancer cell by Transwell method; cell apoptosis and cell cycle detection in different groups of hepatoma cells by flow cytometery.4 The activity of important molecular switch of β-catenin, LEF-1, TCF-4 was detected by Western blot method and Realtime PCR method after High expression of recombinant plasmid carrying the RNAi expression vector of MSI2 gene was transfected into hepatocellular carcinoma cell lines.Ⅲ Study of the effect of MSI2 on liver cancer progression and its potential regulation mechanism in vivo1 Select the SPF nude mice for in vivo experimental animal model; establish subcutaneous transplanted hepatoma model; observe the tumor size and volume changes at different time points; draw tumor growth curve.2 The above established liver transplanted tumor model was used; the tumor volume and weight were measured after the mice executed; the important molecules β-catenin, TCF-4, and LEF-1 were examined in transplanted tumor by western blot and realtime PCR method.ResultsI The significance of MSI2 expression in HCC1 The expression of MSI2 was closely related to the progression and recurrence after resection of hepatocellular carcinomaOur previous study showed the upregulation of the expression of MSI2 in hepatocellular carcinoma tissues using realtime PCR method. In order to study the further details, we selected 106 cases of hepatitis B virus related hepatocellular carcinoma patients to construct tissue microarray. The relationship between MSI2 expression and clinicopathological features of HCC patients was further studied by immunohistochemical method at the protein level, which showed MSI2 had no correlation with patient age, gender, tumor capsule, tumor thrombus and AFP level in hepatocellular carcinoma tissues; on the contrary, there is correlation with differentiation degree, tumor size, tumor stage, postoperative recurrence, and vascular invasion; this suggests that MSI2 expression is closely related to the progression and recurrence after operation of hepatocellular carcinoma.2 The correlation between the expression of MSI2 and P-catenin in hepatocellular carcinomaThe upregulation of P-catenin expression was detected simultaneously in the study of tissue specimens at the molecular level, then its expression was further detected at the protein level by immunohistochemical method in 106 cases of tissue chip, the results showed that the expression of β-catenin in hepatocellular carcinoma tissues was higher than that in paracancerous tissues, and correlated with the expression of MSI2. Therefore, we speculate that MSI2 may be involved in the progression of HCC through the Wnt/β-catenin signaling pathway, which laid the foundation for the next step of our further research.3 The upregulation of MSI2 expression predicts poor prognosis of HCC patients after operationIn order to further study the expression of MSI2 with the prognosis of patients with hepatocellular carcinoma after operation, the clinical pathological parameters of patients was used as risk factors affecting the prognosis of HCC patients, establishing the COX proportional hazards regression model; the results showed that tumor size, tumor differentiation and tumor embolus were considered as independent risk factors influencing patient disease-free survival rate; whereas tumor size, tumor differentiation, tumor embolus and upregulation of MSI2 were independent risk factors affecting overall survival in patients with hepatocellular carcinoma after operation; this suggests that MSI2 expression may predict poor prognosis of HCC patients, might be considered as biomarkers for HCC patients to assess overall survival.II Study of the effect of MSI2 on the malignant biological behavior of hepatocellular carcinoma cells and its mechanism in vitro1 High expression of MSI2 in HCC cell linesIn order to further study the mechanism of MSI2 and the malignant biological behavior of HCC cells, MSI2 expression was detected in SMMC-7721, Hep3B and HepG2 cell lines using realtime PCR method; the results showed that high expression of MSI2 were in the three cell lines, the SMMC-7721 was the highest, followed by Hep3B and HepG2; SMMC-7721 will be choosed to study the function and mechanism in the next step, and Hep3B and HepG2 will be selected to verify.2 MSI2 can increase the proliferation of HCCIn order to clarify the effect of MSI2 expression in the growth of HCC cells, RNA interference technology was used to silence MSI2, and the method of MTT was used to record the expression through dynamic observation in hepatoma cells of different groups. The results showed there was significant difference in proliferation of HCC between Mock group, Control group and MSI2 si-RNA group at 96h point; this shows that MSI2 can increase the proliferation of HCC, and provides a theoretical basis for the future possible gene therapy.3 MSI2 can promote hepatocellular carcinoma cell clone formationIn order to explore the effect of expression of MSI2 on the growth of liver cancer cells, the activity changes of cell proliferation were detected by soft agar cloning experiments, we found that cell proliferation activity of MSI2 si-RNA group decreased significantly compared with Mock group and Control group; the results further verify the aforementioned conclusions, the expression of MSI2 knockdown can reduce the proliferation of HCC.4 MSI2 can improve the liver cancer cell migration and invasion abilityIn order to further elucidate the role of MSI2 on liver cancer progression, hepatoma cell migration ability was detected by wound healing assay in SMMC-7721 cell lines, which showed that cell migration distance was significantly decreased in MSI2 si-RNA group compared with Mock group and Control group; at the same time, the invasion ability of SMMC-7721 was detected by Transwell assay in different groups, which showed that transmembrane cell number of MSI2 si-RNA group hepatocellular carcinoma cells was significantly less than Mock group and Control group; this shows that MSI2 can improve the liver cancer cell migration and invasion ability and promote HCC progression.5 MSI2 can inhibit the early apoptosis of hepatoma cellsIn order to reveal the relationship between MSI2 and apoptosis of hepatoma cells, apoptosis in different groups of hepatocellular carcinoma cell line SMMC-7721 was detected by flow cytometery after Annexin V and PI staining of liver cancer cells. The results showed that the number of hepatoma cells in MSI2 si-RNA group maintaining the vitality was significantly higher than that in Mock group and Control group; while the number of hepatoma cell with early apoptosis in MSI2 si-RNA group was less than that in Mock group and Control group. This shows that the expression of MSI2 can inhibit the early apoptosis of hepatoma cells6 MSI2 can affect HCC cell cycle distribution and promote cell proliferationIn order to investigate the effect of MSI2 on cell cycle of liver cancer, the distribution changes of cell cycle were detected in different groups of SMMC-7721 cell lines by flow cytometery. We found that the cell number of phase G1 in MSI2 si-RNA group was significantly higher than that in Mock group and Control group; on the contrary, the cell number of phase S in MSI2 si-RNA group was significantly lower than that in Mock group and Control group; this shows that the expression of MSI2 can accelerate the DNA reproduction of hepatocellular carcinoma cells and promote cell mitosis, thereby improving the hepatocellular carcinoma cell proliferation activity.7 MSI2 can upregulate β-catenin, LEF-1 and TCF-4 activity of Wnt/β-catenin signaling pathway in vitroIn order to clarify the regulation mechanism of MSI2 in HCC invasion and metastasis, and disclose the effect of MSI2 on the downstream important effector molecules of P-catenin signaling pathway; β-catenin, LEF-1 and TCF-4 activity in hepatocellular carcinoma cell line SMMC-7721 were detected by western blot technique at the protein level; the results showed that MSI2 silencing can inhibit expression of β-catenin, LEF-1 and TCF-4. In order to further verify the above expression pattern, we further detect MSI2, β-catenin, LEF-1 and TCF-4 expression applying realtime PCR method at the transcriptional level in different groups of SMMC-7721, the results was in accordance with that previously done; this shows that MSI2 can regulate the biological behavior and promote progression of hepatocellular carcinoma via the Wnt/β-catenin signaling pathway in vitro.Ⅲ Study of the effect of MSI2 on liver cancer progression and its potential regulation mechanism in vivo1 MSI2 promotes tumor growth in vivoIn order to further verify the effect and mechanism of MSI2 on human hepatocellular carcinoma, the subcutaneous transplanted tumor model of HCC was constructed using SPF level 4-6 week old nude mice. The tumor volume and weight were significantly less in MSI2 si-RNA group than that in Control group; this indicated that MSI2 can promote tumor growth in vivo.2 MSI2 can increase the liver cancer cell atypiaIn order to observe the morphological change of the cells of subcutaneously transplanted tumor in MSI2 si-RNA group and Control group, HE staining was adopted; we found hepatocellular carcinoma cells with small volume, relatively high degree of cell differentiation, the small cell atypia, and visible tissue necrosis in MSI2 si-RNA group; while nests, no obvious interstitial component of visible nucleus, large volume, poor differentiation, and cell atypia degree were presented in Control group hepatoma cells; this indicated that MSI2 can increase the liver cancer cell atypia, improve liver malignant behavior tendency.3 MSI2 can upregulate p-catenin, LEF-1 and TCF-4 activity of Wnt/β-catenin signaling pathway in vivoIn order to validate the effect of MSI2 on hepatocellular carcinoma growth in vivo, and to further clarify the effect of MSI2 on the downstream important effector molecules β-catenin, LEF-1, and TCF-4 in Wnt/β-catenin signaling pathway, the activity of MSI2, P-catenin, LEF-1, and TCF-4 was detected in nude mice subcutaneous transplanted tumor by western blot technology in MSI2 si-RNA group and Control group, the results showed that the expression of P-catenin, LEF-1 and TCF-4 was obviously down regulated after MSI2 knockdown; all the above experiments were further performed by realtime PCR method; the results were in accordance with the previously done. This indicated that MSI2 can promote liver cancer progression via the Wnt/β-catenin signaling pathway in vivo.Conclusions1 The upregulation of the expression of MSI2 in hepatitis B virus related HCC, associated with the clinicopathological features of HCC and postoperative recurrence, predicts poor prognosis in hepatocellular carcinoma.2 MSI2 can promote the proliferation, migration and invasion of HCC cells, inhibit the early apoptosis and alter cell cycle distribution of HCC cells, leading to regulating changes of malignant biological behavior of HCC.3 MSI2 can upregulate the expression of β-catenin, LEF-1 and TCF-4, could promote HCC progression through the Wnt/β-catenin signaling pathway.Originality and Significance1 For the first time, the correlation between MSI2 and hepatitis B virus related HCC was disclosed; the upregulation of MSI2 expression is closely related with the recurrence after resection of hepatocellular carcinoma; we confirme that the expression of MSI2 may promote the progression of hepatocellular carcinoma in vitro and in vivo. The results may provide the theoretical support and experimental basis for gene therapy targeting at MSI22 More importantly, we found that MSI2 can regulate malignant biological behavior of hepatocellular carcinoma by different mode of action. The results laid a theoretical foundation in the advanced research on the MSI2 function and mechanism in liver cancer.3 We confirmed that MSI2 could upregulate the activity of β-catenin, LEF-1 and TCF-4 in vitro and in vivo, proved that MSI2 could promote HCC progression through the Wnt/β-catenin signaling pathway, pointed out a new direction for the further study on the function and molecular mechanism of MSI2 in the malignant tumor.Limitations1 The correlation between MSI2 and clinical characteristics need to be examined more fully by increasing the sample data to avoid single center bias.2 Further studies need to be done between MSI2 and apoptosis and cell cycle and the related mechanism need to be elucidated.