| Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and HCC-associated annual mortality ranks the third among all tumors. Despite the advancement of different therapeutic approaches, improvements in patients’ outcomes are still quite limited. Therefore, there is an urgent need to develop novel therapies that effectively target the pathological alterations underlying HCCCells communicate through intertwined regulatory signaling networks. More evidence demonstrates that abnormalities in JAK/STAT3 signaling pathway are associated with the pathogenesis of HCC.Signal transducers and activators of transcription 3(STAT3) mediates signal transduction from the extracellular environment to the nucleus.STAT3 regulates the expression of target genes involved in cell-cycle progression and the regulation of apoptosis and promotes cellular transformation and abnormal cell proliferation. The deregulation and constitutively activated of STAT3 has been frequently found in a large number of primary tumors and cancer-derived cells including hematologic malignancies, breast cancer, prostate cancer and Hepatocelluar Carcinoma. The greatest bulk of evidence implicated that STAT3 play a crucial role in oncogenesis, carrying that signal to the nucleus and binding to specific DNA response elements that regulate gene transcription. After a ligand binding with a receptor. JAK is activated, and then phosphorylates STAT3. Dimerization of STAT3 translocates to the nucleus and controls the transcription of many genes, such as, Cyclin D, myc,Bcl-xl and Mcl-1. It is well known that STAT3 is strongly involved in the proliferation and anti-apoptosis of cancer cells.RNA interference (RNAi) is an evolutionarily conserved genetic surveillance mechanism that permits the sequence-specific post-transcriptional down-regulation of target genes and RNAi technologies have been applied to study gene function and validate drug targets.To explore how JAK/STAT3 signaling pathway influences the HCC cell apoptosis, in this study, we performed DNA-vector-based RNAi approach silence STAT3 expression in Bel-7402 cells. According to Genebank’s STAT3 cDNA, the plasmid pGCsi.U6/neoRFP-STAT3 which was designed for expression of STAT3 siRNA was constructed and synthesized, and then transfected into Bel-7402 cells through Lipofectamine 2000. Cells with or without siRNA transfection were treated in wells. The apoptotic rate was detected by flow cytometry (FCM) and AnnexinV/PI apoptosis detection kit staining. Simultaneously, the mitochondrial membrane potential(△φm) was visualized by the JC-1 fluorescence staining and the inverted fluorescence microscope. Furthermore, the expression of Caspase-3 protein was analyzed by Western blotting.Previous studies provided strong evidence that siRNA specific to the STAT3 gene can significantly suppress STAT3 protein expression. We transfected Bel-7402 cells with pGC-siRNA. As a control, pGC-siRNA-scramble plasmid was transfected. Compared to the non-transfected cells, scramble-siRNA cells showed no reduction of STAT3 level, while STAT3-siRNA cells exhibited approximately 50% reduction in STAT3 level (P< 0.05).The results showed that cells with or without siRNA transfection were harvested and then stained by PI and Annexin V, cell apoptosis was detected by FCM. The percentage of apoptotic cells were(9.26±0.42)%(control), (17.53±0.98)%(scramble-siRNA), and (45.24±0.79)%(STAT3-siRNA), respectively. The results indicated that the treament of siRNA-STAT3 displayed effects in the Bel-7402 cells, causing significantly increased apoptotic ratio(P< 0.05).Much effort has been made at therapeutically inactivating specific transcription factors in cancer cells, both in vitro and in vivo.Inhibition of STAT3 activity may occur at the level of activation, translocation, or DNA binding. RNA interference(RNAi)is a sequence-specific posttranscriptional gene silencing mechanism,which is triggered by double-stranded RNA(dsRNA)and causes degradation of mRNAs homologous in sequence to the dsRNA.Apoptosis, or programmed cell death, which is a complex, genetically-determined process involved in the development and maintenance of homeostasis in multicellular organisms, has proven to be critical for the development and sustained growth of many, perhaps all, cancers and induction of apoptosis has now been considered as an important method for antitumor. In this study, we found that treatment of cells with STAT3-siRNA displayed effects in the Bel-7402 cells, causing significantly increased apoptotic ratio by FCM.This study observed that targeting STAT3 significantly reduced the percentage of cells emitting red fluorescence (58.87±1.90% for siRNA-STAT3), as compared to non-treated control or siRNA-scramble-transfected cells (90.73±1.75% and 90.03± 1.68%, P< 0.05), suggesting that disruption of mitochondria is, at least partially, responsible for the apoptotic effects induced by targeting STAT3. Apoptosis of the Bel-7402 cells induced by STAT3 gene silencing may be mainly through the Mitochondrial-caspase-mediated apoptosis pathway.The △ψm depolarization would trigger the release of cytochrome c that activates caspases in the cytosol and subsequently initiates the apoptotic cascade. Western blot showed that the highest level of cleaved caspase 3(17,19 kDa) was detected in cells targeted, followed by those targeted for STAT3 molecule and the lowest level was observed in non-treated control (control) or siRNA-scramble-transfected (siRNA-scramble) cells. There was no difference between groups for protein abundance of the full-length (35 kDa) caspase3.In conclusion, the present work demonstrates that treatment by silencing STAT3 gene blocks JAK/STAT3 pathway significantly promotes apoptosis in Bel-7402 cells. The research of singaling pathway networks associated with apoptosis may be lay a foundation to human Hepatocarcinoma adjuvant therapy... |
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