The Mechanism By Which PEG10 Exerts Its Biological Effects During Hepatocarcinogenesis

Backgroud & Aims: HCC is a global highly malignant tumor. According to the WHO statistics in 2002, the incidence of HCC ranks 4th position in male human tumors and 7th position in female human tumors. Each year nearly 50 million people died of the disease. In China, over the past two decades, HCC ranks 2nd from 3rd among the cause of death, Clinical characteristics of early stage HCC is a symptomatic, occult and difficult to detect. 70% - 80% patients are already at the advance stage (III or IV) when they are go to see their doctors. HCC is very often characterized by recurence and metastasis, which contribute to the poor outcome and high matality. Notwithstanding a variety of novel therapeutics strategies for HCC emerged in recent years, the prognosis of HCC has not been noticeably improved. Nowadays gene therapy for cancers tumors has become the hot topics, molecular target relevant to HCC, such as p53,p16,c-myc,PTEN etc were explored, but the study shows that they play roles only in the part of tumors and lack high specificity. Hunting for specific molecular target which play an important role in the development of majority of HCC is the hot interests in the field of molelar oncology at home and abroad.PEG10, paternally expressed gene 10, a new genetic imprinting gene, was found by Ryuichi in 2001 in hepatocellular carcinoma tissue by means of cDNA microarray, originated from retrotranscription transposon, is located on human chromosome 7q21. So-called genetic imprinting, also named genome imprinting, refers to the genetic information according to parental origin to determine the expression of allele, while another'silent'. Genetic imprinting genes affect mammalian cell growth, development and behavior, and closely relate to human genetic diseases, hypoplasia and tumors. Tsou Ap and Okabe H is results that PEG10 was highly expressed in the regenerative liver of mice and in human HCC tissue but not surrouding adjacent tissue and normal hepatic tissue. PEG10 amino acid sequence was detected to contain a CCHC type zinc finger which can be found in most retrovirus and retrotransponsons. Therefore it can be inferred that PEG10 may be involved in transcriptional regulation. Most interesting is the first discovery that PEG10 was lightly expressed in human hepatoma cell line and other cancer cell line derived from gastrointestinal carcinoma, and the expressive quantity of PEG10 has the positive correlation with the metastatic potential. In view of the high specificity of the expression of PEG10 in HCC tissue and hepatoma cell lines, PEG10 was chosen as the molecular target for the evaluation of the relationship between the expression of PEG10 and malignant phenotype and metastasis of the tumor. In this study, PEG10 as a molecular target was investigated intensively and extensively by using gene silencing and genetic modification. As expected, the results obtained were very exciting: PEG10 may be a potential promising molecular target for gene therapy of HCC.Methods: the mRNA of PEG10 was examined in different HCC lineages by real-time quantitative fluorescence PCR and RT-PCR. Total RNA was extracted in hepatoma cell line HepG2 and was amplified the full-length of PEG10 by RT-PCR; Then we recombinant the plasmid with the eukaryotic expression vector pcDNA3.1hisC, normal liver cell line LO2 was transfected PEG10 and screened the clone cells use of G418; The PEG10 gene's mRNA and protein expression level were detected in LO2-PEG10 cells by RT-PCR and Western blot; the expression change of p16, p21 gene in LO2 cells by fluorescence quantitative PCR, The protein expression of p16, p21, Cyclin D1, Cyclin E, CDK4, CDK2, Rb, pRB (ser780) and E2F1 were detected by Western blot; the proliferate and adhere affection, migrate and invasive ability of PEG10 gene in LO2 cells were measured by MTT,TUNEL,CFSE flow cytometry,Transwell tests etc. Laser scanning confocal microscope imaging cytoskeleton in LO2 cells which over expressed PEG10; At the same time the changes of cells ultrastructure in LO2-PEG10 cells was observed by TEM; and the ability of tumor-growing in vitro was measured with soft agar colony formation. nude mice model observation PEG10 expression of liver cancer invasion and metastasis phenotypic effects Finally we also evaluated the invasion and metastasis influence on over expression due to PEG10 in use of HCC nude mice model.Results:1. The expression of PEG10 mRNA in five HCC lineages with different metastasis potential was detected by FQ-PCR, we found that the expression levels of PEG10 gene in liver cancer cells were positively correlated with their different metastatic potentials.2. PEG10 stable expression of the LO2 cell clone and the control cell line 293. Recombinant plasmid contains the full length PEG10 was constructed successfully. LO2 cells which treated with PEG10 was stable screened, at the same time human renal cell line 293 was used as control group.3. The recombinant vector was stably transfected into the normal liver cells LO2. It was found that exogenous PEG10 promotes cell proliferation which changes the acceleration from G1 to S phase and inhabits cells apoptosis. The expression of P16, p21 protein was down-regulated, however, the expression of Cyclin D1, Cyclin E, CDK4, CDK2, Rb, pRB (ser780), E2F1 protein was up-regulated. Doubtless the above findings clearly suggests us PEG10 maybe educe carcinogenic action by influencing the hepatic cells cell cycle.4. Exogenous PEG10 assign human liver cells LO2 the phenotype of invasion andmetastasis by changing cell polarity and ultrastructure.5. Animal experiments showed that exogenous PEG10 makes the LO2 cells heve the ability of tumor-growing in vivo and promotes cells invasion and migration. Conclusions: PEG10 mRNA expression was detected in different metastatic potential hepatoma cell lines by Real-time PCR technology analysis, recombinant plasmid was constructed with molecular cloning technology, normal liver cells LO2 and the non-liver derived cells 293 were stably transfected PEG10 gene, screening cells which stably expressed PEG10gene, and take PEG10 gene as a target for further study.By way of a series of experiments, the expression level of PEG10 was verified that it occurs positive correlation to the metastasis ability of Hepatoma cell lines. The LO2-PEG10 cells changes the cell cycle protein expression through p16, p21 two tumor suppressor gene signal pathways downstream, PEG10 gene makes LO2 cells have malignant phenotype: proliferation, invasion and metastasis, however, the non-liver-derived cell line 293 will not be affected.During the time in studying the molecular mechanism how PEG10 to participate in HCC, it was found that exogenous PEG10 gene can down regulated E-cadherin/catenin intracellular signal, promote human liver cells LO2 proliferation, movement and tumor growing in vitro; change the ability of E-cadherin/catenin complex to maintain cell polarity and ultrastructural functions, assign human liver cells LO2 the metastatic phenotype. It was testify doubtless that the over expression of PEG10 can promote the following abilities of tumor-growing of HCC cells in vivo and metastasis to other organs.From all the above, we may reach the conclusion that PEG10 gene change the malignant biological phenotype of LO2 cells and down-regulating the cells signal pathway. Therefore, it also indicates that PEG10 will be an effective molecular target for liver cancer gene therapy.