The Expression And Functional Role Of ZFYVE26in Hepatocellular Carcinoma

Background&ObjectiveHepatocellular carcinoma (HCC) is one of the most common cancer, which ranked third cancer killer in the world. This phenomenon is worse in China, which with the highest incidence, and has been ranked as the second cancer killer in China.300.000people died in HCC every year in our country, whcih is up to50%of total number of people who died in HCC all over the world. HCC is also a high malignancy with an extremely poor prognosis and with no effective therapy for the vast majority of HCC patients. Currently the main method of treatment in early HCC is surgical resection and liver transplantation, but50%of patients who treat with surgery will be recurrence after two years. Chemotherapy and radiotherapy are minimally effective, can have significant toxicity, and have not been shown to improve patient survival. So molecular targeted therapy for HCC will lead to future advances in the treatment of HCC. HCC is a very heterogeneous disease in terms of its etiology, molecular carcinogenic mechanisms, and biological behavior, which complicate our ability to identify rational molecular therapeutic "targets". Therefore, it is very important to investigate the molecular mechanism of the occurrence and development of HCC.The etiology of HCC is contributed to a few of factors including hepatitis viruses infection, contamination of aflatoxin B, chemical carcinogen and hereditary alteration and so on, which may lead to the oncogene overexpression and the lost expression and mutation of tumor suppressor and finally caused the carcinogenesis of HCC. But the precise molecular mechanism of HCC is not well understood up to now. It is already accepted that HCC is a polygenic and a multiple-step disease where tumor progression carries a seemingly endless combination of genetic and epigenetic alterations.The activation of oncogene comes from gain-of-function mutations. Whereas, the significance of loss-of-function mutations of tumor suppressor gene in carcinogenesis has become increasingly clear. Most studies found that there are more tumor suppressor genes loss-of-function mutations contribute to each step of HCC. But none of them affect more than50%of cases of HCC. So, it is possible that other tumor suppressors exist and play a important role in the occurrence and development of HCC. Identification and functional characterization of tumor suppressor genes which play a important role in HCC carcinogenesis. It would be a significant contribution toward not only the definition of the molecular pathogenesis of HCC but would also broaden the potential for developing effective treatment of HCC.The ZFYVE26locus to a2.64Mb interval on chromosome14q23.3-q24.2, which encodes a zinc-finger protein with a FYVE domain. ZFYVE26, a protein that is often found mutated in patients with hereditary spastic paraplegia, has been also been shown to localize to the midbody and to control cytokinesis. It also can be recruit to membranes for signal transduction, and play critical role in membrane trafficking, cytoskeletal functions, and apoptosis. In addition, the stronger and wider expression of ZFYVE26in embryos which suggests a critical role of this gene during embryonic development. Recently, it was shown that an additional mechanism for the tumor suppressor functions of Beclin1, namely its ability to bind ZFYVE26and participate in the regulation of cytokinesis. These findings reveal a novel regulatory role of the tumor suppressor Beclin1and its binding partner ZFYVE26that has potential implications for carcinogenesis. Similarly to tumor suppressor Beclin1, the average expression of ZFYVE26was significantly lower in high vs. low grade breast cancers. All these results suggest that ZFYVE26gene might play a critical role in development of human cancer, although the molecular mechanism of ZFYVE26in cancer is not well known. Failure to complete cytokinesis has been implicated in carcinogenesis, Sagona’s studies demonstrate that ZFYVE26play important roles in controlling this process. So it will be interesting to investigate whether this is relevant to carcinogenesis of HCC. Up to date, there is rare published data concerning expression levels of ZFYVE26in HCC. The principal aim of this study was to clarify whether functional inactivation of ZFYVE26gene by mutation or down-regulation contribute to pathpgenesis of HCC, and to explore the implication of ZFYVE26as a possible therapeutic way to treat HCC and other human cancers. Part I Expression of ZFYVE26in human hepatocellular carcinoma and its clinical significanceObjective:To study the expression of ZFYVE26in human hepatoma cell line and hepatocellular carcinoma and to learn the corelation between its expression and cell proliferation index Ki-67, which could be used to provide basis for further study on the functions of ZFYVE26in the occurrence and development of HCC.Methods:1. Quantitative real-time PCR were applied to detect the expression of ZFYVE26in normal liver cell line L-02and hepatoma cell lines HepG2and MHCC-LM3.2. We used quantitative real-time PCR and western blot to detect the expression of ZFYVE26in seven cases of HCC and their paraneoplastic tissues.3. The immunohistochemistry was used to examine the difference expression of ZFYVE26in45cases of HCC and their corresponding adjacent tissues, and8cases of normal tissues. And then analyse the corelation between ZFYVE26expression with the proliferative index Ki-67.4. Statistical analysis was done using SPSS13.0. The measurement data of each subgroup data are expressed as mean±standard deviation (x±s). qRT-PCR results using single factor analysis of variance, multiple comparisons using the LSD test in the cell lines’study. Western blot and qRT-PCR were analyzed by paired t test in detection of the expression in hepatocellular carcinoma and adjacent tissues. Using the χ2test and Spearman correlation analysis to analyse the expression of ZFYVE26and Ki-67by immunohistochemistry.Results:1. Expression of ZFYVE26mRNA in hepatocellular carcinoma cell line MHCC-LM3and HepG2was significantly decreased compare to the expression of normal liver cell line L-02(P<0.05). Compare to HepG2cells with low metastatic potential, the expression of ZFYVE26in hepatocellular carcinoma cell line MHCC-LM3of high metastatic potential was decreased significantly (P<0.05).2. In seven cases of HCC tissues and its corresponding adjacent tissues, the relative expression of ZFYVE26mRNA were (0.574±0.348) and (0.956±0.316) respectively. And its encoded protein relative expression level were (0.081±0.126) and (0.203±0.169).By statistical analysis, the expression of ZFYVE26mRNA and protein in HCC tissues were significantly lower than that in adjacent tissues, and the difference was statistically significant (P<0.05).3. ZFYVE26protein was expressed in24(53.33%) of the45cases of HCC, in36(80.00%) of the45cases of the adjacent tissues, and in8(100%) of the normal tissues in hemangioma respectively by immunohistochemistry. The difference between HCC and adjacent tissues and the difference between HCC and normal tissues in hemangioma both had statistical significances (P<0.0167), but the difference between adjacent tissues and normal tissues had not statistical significances (P>0.0167).4. Ki-67protein, detected by immunohistochemistry, was expressed in31(68.89%) of the45cases of HCC, in10(24.39%) of the45cases of the adjacent tissues respectively. And none of hemangioma tissues express the protein of ZFYVE26. The difference between HCC and adjacent tissues and the difference between HCC and normal tissues in hemangioma both had statistical significances(P<0.0167), but it was not statistical significances between adjacent tissues and normal tissues(P>0.0167).5. In HCC, the cases of ZFYVE26protein positive and negative expression were respectively13and18in Ki-67protein positive expression group; The cases of ZFYVE26protein positive and negtive expression were11and3in Ki-67protein negtive expression group. The value of rs was-0.416by Spearman rank correlation analysis, and P<0.05. All above proved that the expression levels of ZEYVE26and Ki-67showed negative correlation in HCC.Conclusion:1. In the human hepatoma cell lines ZFYVE26mRNA expression was significantly lower than that which is express in the normal liver cell lines. The expression of ZFYVE26in MHCC-LM3which is more high metastatic potential decrease most apparent. While in seven cases of HCC tissues the expression of ZFYVE26mRNA and protein were significantly decreased compared to adjacent tissues. The ZFYVE26protein expression detected by IHC in HCC tissues which also found that the expression was significantly reduced. Therefore, it can be able to get the preliminary conclusion, the low expression of ZFYVE26may be associated with the occurence and development of hepatocellular carcinoma.2. It is negative correlation between the expression of ZFYVE26protein and Ki-67protein in primary hepatocellular carcinoma by Spearman correlation analysis. It is indicate that with the liver cancer tissue of Ki-67expression rate increasing, the trend of ZFYVE26expression was lower expression.Part Ⅱ Experimental study of suppressing effect of shRNA targeting ZFYVE26gene on growth of HepG2in vitroObjective:In preliminary studies, we found that ZFYVE26mRNA expression in HepG2cells which is the low-metastatic potential carcinoma cell line were significantly higher than the high metastatic potential cell line MHCC-LM3, suggesting that it may be involved in the development of hepatocellular carcinomaprocess. To further validate its role in the process of development of HCC, we have taken the method of RNAi research to study the function of ZFYVE26that is to observe the interference affect the proliferation of HepG2cells and invasion of metastatic ability. So it can provide new ideas for further research of HCC development mechanism.Methods:1. Search the human ZFYVE26mRNA sequence from NCBI and analyse bioinformatics sequence. Three chemical synthesis siRNAs targeting human ZFYVE26-mRNA were designed by siRNA web which is from the company of Ambion. At the same time, we also designed negative siRNA for control in this way. Transfect them into HepG2cell with Lipo2000and extract total RNA from HepG2cell after transfection about48hours. And then use qRT-PCR to detect the expression of ZFYVE26in different groups. So it can filter the most effective siRNA.2. According to the most effective siRNA sequence design short hairpin RNA (shRNA) oligonucleotides, and to build pGPU6/GFP/Neo recombinant plasmid expression vector. Recombinant plasmid was transfected into the cell line HepG2by liposomes, and extracted total cellular RNA and protein from cells after48hours and60hours respectively, qRT-PCR and western blot were used to analyse the changes of ZFYVE26gene and protein. 3.CCK8assay was applied to detect the proliferation of HepG2cells after transfect with shRNA plasmid at24hours,48hours and72hours.4.Transwell assay was used to evaluate the changes of metastasis and invasion after transfect shRNA vector into HepG2at48hours and60hours respectively.Results:1. At48hours after transfection, three siRNAs had their best interfering results and ZFYVE26-mRNA inhibition ratio were all over50%. The siRNA-2had the highest inhibition ratio which was79.42%.2. pGPU6/GFP/Neo-ZFYVE26-shRNA-1was positive recombinant vectors and sequences is correct. The inhibition ratio of ZFYVE26-mRNA and protein in shRNA-1HepG2cells were abviously higher than those in shRNA-0and shRNA-NC group (P<0.05)3. In order to detect the proliferation of different time points after transfection by CCK-8assay. At24hours after transfection OD value of the different groups was not statistically significant (P>0.05). The OD value of independent of the sequence group and HepG2cells group also was not significant difference (P>0.05) at48hours and72hours after transfection. While the OD value of the targeted interfere with the expression of ZFYVE26in shRNA-1cells was significantly higher which was significant differences (P<0.05) compare to HepG2group (shRNA-0) and unrelated sequence group (shRNA-NC).4. Transwell assay was used to assess the changes of metastasis and invasion capacity after transfection. We found that targeting interfere with ZFYVE26group compared to shRNA-0and shRNA-NC groups, metastasis and invasion capacity was significantly enhanced (P<0.05).Conclusion:we constructed the human ZFYVE26specific shRNA plasmid successfully, it can be provided the experimental basis for the study of ZFYVE26gene in the pathogenesis of HCC. Use this shRNA vector to interfere ZFYVE26 expression, the proliferation, metastasis and invasion was significantly enhanced which suggests that ZFYVE26play a pivotal role in the occurrence and development of HCC, ZFYVE26may be one of HCC tumor suppressor gene.Our study is only a preliminary study in the role of ZFYVE26in HCC, this study can still be more thorough and comprehensive.