| Hepatocellular carcinoma (HCC) is the common hepatic highly malignant tumor, usually with the later detection and faster development. The etiopathogenisis and developments of HCC are not well known. Programmed cell death, or apoptosis, of HCC cells is a process of self destruction directed by the activated genes. That may lead to the changes in cell morphology, DNA fragmentation, and protein cross linking. Less apoptosis than the normal may lead to carcinogenesis.Deregulation of cell proliferation and cell apoptosis underlies neoplastic initiation and development, which involves multiple gene alterations,and is regulated by complicated signal transduction pathways. The inhibitor of apoptosis protein (IAP) family is a sort of important factors of inhibiting apoptosis, which is characterized by the presence of one or more baculoviral IAP repeat (BIR) domains at the amino terminal, possessing or not a carboxyl terminal RING zinc-finger domain. FLIP is a novel member of IAP family, which has potential antiapoptosis activity. In addition, FLIP also plays an important role in maintaining normal cell mitosis, promoting cell proliferation and angiogenesis. FLIP (FLICE-inhibitory protein, also known as CASH, Casper, I-FLICE, CLARP, FLAME-1, MRIT) is one sort of the recently reported inhibitory proteins of apoptosis characterized with death effect domains(DEDs), which is present in virus, parasite, mammalian, and et al. FLIP belongs to the inhibitor of apoptosis (IAP) gene family. Its chromosomal location is 2q33-34. FLIP is generally expressed in embryonic tissuesm, but is not expressed in most normal adult tissues, whereas is overexpressed in the majority of human cancers. It indicates that FLIP may associate with the tumorigenesis and progress of most human cancers.In the case of cancer,an abnormally increased cellular lifespan as a consequence of reduced apoptosis is ideal to favor the insurgence of genetic mutations, as well as to shield transformed cells from death induced by chemotherapy or radiotherapy, and to promote their survival at distant sites. Therefore, it is not surprising that manipulation of apoptosis has emerged as a new therapeutic strategy to help eliminate cancer cells. FLIP is a bifunctional member of the inhibitor of apoptosis gene family that counteracts cell death and controls mitotic progression. FLIP possesses anti-apoptotic activity by binding and inhibiting the terminal effector, caspases 8. Accordingly, FLIP prevents apoptosis induced by Fas, Bax, caspases and anticancer drugs. FLIP is a natural inhibiting protein of caspases 8. It is lack of the essential 2-amino-3-thiopropionic acid structure which is important to catalytic activity. FIIP is a Fas mediated negative regulatory protein. Decreasing level of FLIP may induce the Fas and TRAIL mediated cell apoptosis. However,the mechanism of regulating the expressing of FLIP is still unknown, it is important to detect the nosogenesis of FLIP associated disease, and may find new ways for clinic therapy. RNA interference (RNAi) represents a phenomenon of double-stranded RNA (dsRNA)-mediated post-transcriptional gene silencing (PTGS). During this process, exogenous cellular dsRNA is cleaved by the ribonuclease, Dicer, to generate 19-23 bp small RNA fragments, referred to as small interfering RNA (siRNA). By joining an effector complex termed RISC (RNA-induced silencing complex) with Dicer and other special proteins, siRNA binds to the cellular RNA with homologous sequences, and contributes to the degradation of the corresponding RNA. At present, RNAi has been widely applied in the research of gene function and gene therapy as an efficient tool for specific gene silencing. Objective:To investigate the expression of FLIP in human hepatic tissue and HCC tissues with different stage,and the specific inhibition of FLIP gene expression by siRNA in Hepg2, 7721 cell lines and its relation with the cell apoptosis.Methods:1. The detection of the expression of FLIP.The expression of FLIP in 46 cases of HCC samples, and 27 cases of hepatic cirrhosis, 18 cases of hepatic hemangiomas, 12 cases of normal hepatic tissues, 10 cell slides of Hepg2 and 7721 were detected by immunohistochemistry methods. And statistic analysis was performed.2. The contrast analysis of FLIP expression in HCC tissues.Histopathologic findings of 86 HCC samples were divided into four grades according to Edmondson standard,including 18 Grade I, 25 Grade II, 21 Grade III, 22 Grade IV. The different expression levels of FLIP in different HCC tissues were compared by statistical methods.3. The construction and identification of RNAi vectors.According the sequence of the FLIP mRNA, the siRNA oligonucleotides were designed and synthesized to the targeted RNAi regions at 526～544, 1164～1182, 1305～1323 nt. After renaturation,double strands DNA was gained. The DNA fragment and pSUPER vector were cleaved with restriction enzyme Bgl II and Hind III,then linked overnight(16℃). After E.Coli,DH5α,transfected by the recombinant vectors,the positive clones were selected. With positive plasmids,the sequences were verified.4. The analysis of the interfering efficacy of the various recombinant vectors.7721,Hepg2(HCC) and Hela(UCC) cell lines were cultured in 1640 medium supplemented calf serum(10 g/L). The cells in exponential growth were trypsinized (2.5g/L),then inoculated in the 6-well plate with the invariable cell density. After 24 hours,the cultured cells were transfected with Lipofectamine2000 (Invitrogen). After another 24 hours,passage cells were screened with G418(400μg/ml). After 4 weeks,several single-collecting clones were given extended culture. The siRNA-induced silencing of targeted gene was determined by using RT-PCR at RNA level and Western Blot at protein level.After all,the tumorigenisis of nude mice was studied. Each six nude mice received injection of control cells and pSUPER-S1 transfected 7721and Hepg2 cells,respectively.Results: 1. The expression of FLIP in HCC tissues and cellsThe positive expression of FLIP was 39 cases (84.73%) in 46 cases of HCC tissues,and 4 cases in 27 cases of hepatic cirrhosis (14.81%). But the FLIP expression in the normal hepatic tissue,only 2 cases (6.67%) was positive (P<0.01). The expression of FLIP in 10 cell cultures was all positive (100%).2. The relation of the expression of FLIP with the HCC grade.The positive expression of FLIP in 86 cases of HCC was 72 cases(83.72%),with Grade I 13/18 (72.22%),Grade II 20/25 (80.00%),Grade III 18/21 (85.71%),IV class 21/22(95.45%)(P﹤0.05),and the positive rate was 100% in 10 cell samples (P<0.01).3. The identification of the recombinant vectors.After the recombinant plasmid were digested with BglⅡ和HindШ,the 60 bp targeted segment was got. But to the control plasmid(containing a DNA segment of EGFP,without homology with human DNA sequence),the two sites of BglⅡand HindШare close,so no corresponding segment was got. The target segments could be cut out in all the three recombinant siRNA vectors. DNA sequencing also verified the target segments were inserted in the vectors correctly. The 3 positive plasmids were termed as pSUPER-S1,pSUPER-S2,and pSUPER-S3,respectively.4. Interfering efficacy of RNAi.With pSilencer 3.0-H1 neo,the specific RNAi vector were constructed to transfect Hepg2 and 7721 cell,and then the stable transfected cell lines were screened with G418. To the positive and the control cell samples,the morphologic characteristics were studied with HE staining and transmission electron microscope(TEM),and cell apoptosis with TUNEL method,expression of FLIP protein and mRNA with RT-PCR, indirect immunofluorescence, or Western blot. The relation between the cell apoptosis and the expression of FLIP were studied.All of the 3 various recombinant vectors showed the interfering efficacy with different grade, in protein or mRNA level. The inhibitory efficacy of pSUPER-S1 was more than 70%, with the most distinct result. The HCC cells (7721, HepG2) transfected with pSUPER-S1 became enlarged and plate, and lose the Fusiform shape, but no other morphological changes were found. And the result of TUNEL showed that the ratio of apoptosis cells was 5 times of that of the control group. The fluorescence microscope and TAM showed the pSUPER-S1 transfected cells have typical apoptosis changes, such as nuclear condensation and fragmentation. The ratio of apoptosis cell could reach 17.3%, which was higher than that of the control group significantly.5. Tumor forming in vivo.Tumors developed to 740mm by week 4 in all of the animals of the control group. In contrast, 2 of 6 mice developed palpable tumor with 1.3mm in diameter that received injection of cells with pSUPER-S1 transfectants. FLIP siRNA apparently suppressed tumor formation in nude mice in vivo.CONCLUSION:1. The high expression of FLIP was found in human HCC tissue, compared with the benign disease and normal hepatic tissues.2. The expression of FLIP is closely related to the histologic differentiation of HCC. The positive rate of FLIP expression was higher in poorly differentiated HCC tissues.3. siRNA-mediated gene inhibition of FLIP suppressed the proliferation of tumor cells and increased cell apoptosis significantly. These findings may help to explore the mechanism of the genesis and development of HCC and find an effective therapeutic method for it. |
