| Hepatocellular carcinoma (HCC) is reported to be the fifth most common cancer in the world . Moreover, the mortality of HCC is the third highest among all cancers,only behind lung and colon cancer. The incidence rates of HCC have increased in recent years in China and is over that in Africa, becoming the highest in the world. The mortality rates of HCC in China have also increased since 1990s and is now the second highest among all cancers. Liver resection is the potentially curative therapy. However, the recurrence rates are as high as 60%-80%. Systemic therapies such as standard chemotherapy and radiotherapy do not have significant efficacy. Therefore, the five-year survival rates of HCC is as low as 3%-6%. The current situation in HCC therapy has urged clinical scientists to conduct extensive research on the genomics and signaling pathways implicated in hepatocarcinogenesis, with the hope that a better understanding of HCC molecular pathogenesis will shed light on the new strategies of conquering this devastating disease.Hepatocarcinogenesis involves the mutation of multiple genes and the alteration of the relevant signaling pathways. Critical genes include c-myc, Cyclin A2, Cyclin D1, Rb1, AXIN1, p53, IGFR-II/M6PR , VEGF, p16, E-Cadherin, SOCS , PTEN and STAT3. Signaling pathways include Ras/MAPK, PI3K/Akt, and Jak/STAT. In recent years, STAT3 and related signaling pathways in liver carcinogenesis have drawn increasing attention. STAT3 is an important member of the family of signal transducers and activators of transcription ( STAT), a group of constitutionally expressed proteins that have dual roles as both cytoplasmic mediator of cytokine signaling and nuclear transcription factor. The STAT3 involved pathway is conservative in many species and plays a central role in the regulation of multiple biological processes, such as fetal development, cell growth, differentiation, apoptosis, the immune response, inflammation, cellular transformation and carcinogenesis. To play its physiological function, STAT3 need to be activated by tyrosine phosphorylation, which is strictly regulated under normal physiologic conditions. Over-activation of STAT3 may lead to enhanced cell survival and proliferation, resistance to apoptosis, and neoplastic transformation . Constant activation of STAT3 is very common (50% to 90%) in human cancers. Many studies reported that constant activation of STAT3 can promote tumor cell proliferation by regulating genes associated with cell growth and proliferation, enhance tumor cell metastasis and angiogenesis by inducing expression of metastatic genes such as MMP-2(matrix metallo-proteinases-2) and VEGF (vascular endothelial growth factor) , facilitate tumor immune evasion by inhibiting tumor cell-produced proinflammatory mediators and chemokines. Thereby, targeting STAT3 can attack tumors through multiple pathes. Besides, STAT3 has a highly conserved serine residue in addition to the conserved tyrosine residue, therefore,can be regulated by both the tyrosine kinase and serine kinase pathways, and thus sit at a key convergence point to integrate the effects from diverse stimuli. Based upon the findings that STAT3 is a critical mediator of oncogenic signaling, STAT3 is considerated as an important target for anti-cancer drug design.Studies of STAT3 have been focused on a variety of tumour cell lines, including leukemias, multiple myeloma, breast cancer, protaste cancer, melanoma tumor, lung cancer, etc, while heptaocellulor carcinoma has been rarely investigated. Current reasearches shows that constantitutive activated STAT3 is detected in many HCC cell lines and HCC tissues. The abnormal activation of STAT3 may change the signaling pathway among hepatic cells, promote cell proliferation, inhibit cell apoptosis, and therefore is closely related to the initiation and development of liver cancer. This suggests that STAT3 is a promising molecular target for HCC gene therapy. Inhibition of abnormal expression of STAT3 may be an effective strategy of HCC biotherapy. Some anti-tumor drugs, such as STAT3 inhibitor, tyrosine kinase inhibitor AG-490, micromolecule peptide and antisense oligodeoxynucleotide (ODN), have been tried to target STAT3 in vitro and in vivo for HCC, however the efficacies are not satisfactory due to their poor specificity, rapid degradation, unsuitable molecule size, immunogenicity, poor ability to cross cell membranes, and need of special drug delivery. Strategies of targeting STAT3 include decreasing STAT3 levels, reducing tyrosine phosphorylation of STAT3 and receptor complexes, reducing STAT3 recruitment to receptor complexes and its dimerization, and reducing STAT3 binding to promoters. While progress has been made to pursue each strategy, no anti-tumor drug that directly target STAT3 has yet reached clinical stage.RNA interference (RNAi) is a regulatory mechanism existing in most of eukaryotic cells that uses small double stranded RNA (dsRNA) molecules to direct homology-dependent control of gene activity. Known as small interfering RNAs (siRNA), these 21–23 bp dsRNA molecules can be recognized by the enzymatic machinery of RNAi and eventually leads to homology-dependent degradation of the target mRNA. Both artificially synthesized and plasmid-transcribed siRNAs can target complementary mRNAs for degradation. Because of the potency and specificity of RNAi, it has become an important tool to control gene expression through the introduction of short interfering RNAs. It has been used in studies of genomic function, cell signaling pathway, antivirus and antitumor. Recent progress in in vivo siRNA delivery raised expectations for siRNA drugs as the up-and-coming 'magic bullet' for cancer, autoimmune diseases, dominant genetic disorders and viral infections. Despite of the exciting results from a large number of animal model studies, there are a number of hurdles and concerns that must be overcome before RNAi can be applied as a new therapeutic model. These include off-target effects, triggering of type I interferon responses, competition with cellular RNAi components and effective delivery in vivo. As all biological discoveries, understanding of the mechanism is critical to effective applications in human disease. Given the pace of new findings and discoveries of applications, we anticipate that RNAi will be a major therapeutic model for human cancer and viral disease within the next several years.Studies of targeting in vitro and in vivo overexpressed genes in HCC by RNAi, including c-myc,survivin,CCR1 and SMYD3, have been reported. There is still no report about targeting STAT3 by RNAi in HCC. In this study, we used DNA-vector-based RNAi approach to block STAT3 expression in HCC cells and HCC tumor-bearing nude mice to determine the role of constitutively activated STAT3 during HCC pathogenesis and to explore the role and molecular mechanism of targeting STAT3 in HCC therapy. Objective:To investigate the effect of silencing STAT3 gene expression by RNAi on the growth suppression and apoptosis of human hepatocellular carcinoma(HCC)cells and the growth inhibition of HCC in tumor-bearing nude mice, in order to explore an effective technique for liver cancer gene therapy. Methods:(1)Construct the recombinant plasmid of pSilencer 3.0-H1-siRNA-STAT3(p-Si-STAT3)using gene recombination technique.(2)Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemical method to detect mRNA and protein expressions of STAT3 gene in HCC tissues and HCC cell lines SMMC7721, Bel7402 and HepG2. (3) In vitro studies: The HCC cell lines SMMC7721, Bel7402 and HepG2 were transfected with plasmids p-Si-STAT3. Using MTT assay to investigate the proliferation of transfected cells, the STAT3 protein expression level was determined by Western blotting and immunohistochemical staining. Transcription of STAT3 gene was detected by semi-quantitative RT-PCR. STAT3-related genes such as survivin, c-myc, VEGF, p53 and caspase3 mRNA and protein expression were detected in transfected cells as the same time. Flow cytometry (FCM) and AO/EB double dyeing were used to examine cell cycle phase distribution, apoptosis rate, and apoptosis cells in transfected cells. Stably-transfected cell lines, which could maintain p-Si-STAT3 plasmid in transfected cells, were screened and established. Cytoactivity of the stable transfection cells were determined and growth curves were draw.(4) In vivo studies: To establish the tumor-bearing nude mice model of HCC cell line SMMC7721, we use the intratumoral injection together with electroblot method to transfect the recombinant plasmid pSilencer 3.0-H1-siRNA-STAT3 into the transplanted tumor in the tumor-bearing nude mice. The weight of nude mice and the volume of tumors were recored. STAT3 gene transcription was detected by semi-quantitative RT-PCR. Level of protein expression and location of STAT3 was determined by Western blotting and immunohistochemical staining. STAT3-related genes such as survivin, c-myc, VEGF, p53 and caspase3 mRNA and protein expression were detected in tumor tissues at the same time. The TUNEL assay was used to detect the apoptosis of tumor cells.Results:(1) The plasmids p-Si-STAT3 containing siRNA-STAT3 was constructed and confirmed by restriction enzyme digestion.(2) The mRNA and protein expression levels of STAT3 in HCC tissues and tumour-surrounding tissues were significantly higher than that in normal liver tissues(P<0.01). There is no difference between HCC tissues and tumour- surrounding tissues. STAT3 was also overexpressed in HCC cell lines SMMC7721, Bel7402 and HepG2.(3) The stable transfection cell lines were established, and cytoactivity was detected to be significantly lower than that of scramble plasmid transfected cells and mock control cells(P<0.01). MTT assay demonstrated that cell growth was inhibited in transfected cells, and 48h and 72h growth inhibition rates of reconstruct plasmid-transfected group were significantly lower than that of the scramble plasmid-transfected group and the mock control group(P<0.01). There is no difference among three HCC cell lines. The results of RT-PCR and Western blotting showed that mRNA and protein level of STAT3 declined markedly in transfected cells. The change of STAT3-related genes expression in transfected cells at mRNA and protein level varied, with the expression of survivin and VEGF reduced significantly, the expression of p53 and caspase3 increased ( P<0.01), while the expression of c-myc did not change. The results of FCM and AO/EB double dyeing indicated that there was apparent cell apoptosis in the reconstructed plasmid transfected group. The apoptosis rates in SMMC7721,HepG2 and Bel7402 cell lines were 20.8±1.77,21.18±1.62 and 11.84±1.47,respectively. The apoptosis rates in reconstructed plasmid-transfected group were significantly higher than that in scramble plasmid transfected group and mock control group(P<0.01). Among three HCC cell lines, the apoptosis rates in Bel7402 cell line was significant lower than that in SMMC7721 and HepG2 cell line(P<0.05,P<0.01). Analysis of cell cycle showed that HepG2 cells were inhibited in G2 stage, while SMMC7721 and Bel7402 cells were inhibited in G0/G1 stage.(4)The weights of treated nude mice group increased, and the tumor volumes of treated nude mice group decreased dramatically, compared to that of mock group and negative group(P<0.01). The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in treated group. The change of STAT3-related genes expression in tumor tissues at mRNA and protein level also varied, with the expression of survivin, VEGF and c-myc reduced obviously and expression of p53 and caspase3 increased(P<0.01). Most of the tumor tissue cells developed apoptosis that was detected by TUNEL.Conclusions:The persistent activation of STAT3 gene may occur at early stage of HCC pathogenesis and plays an important promoting role in the carcinogenesis of HCC. The synthetic recombinant plasmid p-Si-STAT3 significantly suppressed HCC cells growth and induced HCC cells apoptosis in vitro, and inhibited the growth of HCC in vivo in tumor-bearing nude mice. The mechanism may be related to downregulation of survivin, VEGF and c-myc expression and upregulation of p53 and caspase3 expression. Accordingly, STAT3 gene may act as an important and effective target in gene therapy of HCC. The RNAi technique of silencing STAT3 gene expression in human hepatocellular carcinoma therapy has prominent therapeutic effect. Our study provides a new approach and strategy for HCC biological therapy by targeting STAT3, and offers theoratical and experimental foundation for applying the synthetic dsRNA-based RNAi to clinic. |
