Lentiviral-mediated Smad4 RNAi induced anti-proliferation by p16 up-regulation and apoptosis by caspase 3 down-regulation in hepatoma SMMC-7721 cells
Institute of Digestive Endoscopy and Medical Center for Digestive Diseases, Second Affiliated Hospital of Nanjing Medical University, Nanjing, P.R. China.Oncology Reports (Impact Factor: 2.3). 12/2008; 20(5):1053-9. DOI: 10.3892/or_00000109
Transforming growth factor-beta (TGF-beta)-Smad signaling pathway participates in the regulation of a variety of cellular activities. Unlike the high incidences of Smad4 mutation or deletion in pancreatic cancer and gastrointestinal cancers, Smad4 gene is seldom mutated or deleted in hepatocellular carcinoma (HCC). The role of TGF-beta-Smad4 signaling pathway in leading to carcinogenesis of liver cells remains unknown. In this study, we succeeded in silencing Smad4 using lentiviral-mediated Smad4 RNA interference (RNAi). We investigated the role of Smad4 in TGF-beta1-induced cell proliferation and apoptosis of HCC cell line SMMC-7721. We determined cell proliferation, apoptosis, and expression of p21, p16, p53 and caspase 3. Results showed that TGF-beta1 not only had a significant anti-proliferation effect but also induced cellular apoptosis in SMMC-7721 cells. These effects induced by TGF-beta1 were almost completely blocked by the knockdown of Smad4. Western blot analysis revealed that p16 was up-regulated and caspase 3 was activated by silencing of Smad4, and the expression of p21 and wild-type p53 were not affected. These results suggest that TGF-beta1-induced cell growth inhibition by up-regulating p16 expression and cellular apoptosis by activating caspase 3 was Smad4-dependent. Additionally, the knock down of a specific gene using lentiviral-mediated RNAi appears to be a promising tool and strategy for analyzing endogenous gene function.
- [Show abstract] [Hide abstract]
ABSTRACT: The genesis and development of hepatocellular carcinoma (HCC) is related to the abnormity of signaling pathway, telomerase, cell cycle, apoptosis, angiogenesis, and others, in which STAT3 signaling pathway plays a key role. The HCC cell line HepG2 was transfected with small interfering RNA (siRNA) directed against STAT3. After 72 h, cell growth and cycle were analysed by MTT and Flow cytometry. Then, the protein was extracted and the protein expression of STAT3, Smad3, p44/42, TERT, caspase-3, XIAP, Grp-78, HSP-27, MMP-2, MMP-9, VEGF-A, cyclin A, and cyclin E was detected by Western blot. After the transfection, HCC cell growth was inhibited during the 24-72 h time period and the cell cycle was arrested in G0/G1. STAT3 protein expression was inhibited at 72 h after the transfection. Interestingly, Smad3, p-caspase-3, p-p44/42, Grp78, cyclin A, and cyclin E protein expression was increased at 72 h, while TERT, caspase-3, XIAP, MMP-2, MMP-9, and VEGF-A protein expression decreased at 72 h. However, P44/42, and HSP27 protein expression showed no change following transfection. The results demonstrated that STAT3 signaling pathway may participate in HCC genesis and development through regulating the protein expression of other signaling pathway, telomerase, apoptosis, cell cycle and angiogenesis; thereby, blockade of the Stat3 pathway represents a potential strategy for future treatment. Keywords: STAT3, signaling pathway, telomerase, cell cycle, apoptosis, angiogenesis.Neoplasma 01/2011; 58(2):158-71. DOI:10.4149/neo_2011_02_158 · 1.87 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Prohibitin (PHB), a ubiquitous protein, is involved in a variety of molecular functions. Renal interstitial fibrosis (RIF) is a hallmark of common progressive chronic diseases that lead to renal failure. This study was performed to investigate whether PHB was associated with caspase-3 expression/cell apoptosis in RIF rats. Twenty-four male Wistar rats were randomly divided into two groups: sham operation group (SHO) and model group subjected to unilateral ureteral obstruction (GU), n = 12, respectively. The model was established by left ureteral ligation. Renal tissues were collected at 14 days and 28 days after surgery. RIF index, cell apoptosis index, protein expression of PHB, transforming growth factor-βl (TGF-β1), collagen-IV (Col-IV), fibronectin (FN) or caspase-3 in renal interstitium, and mRNA expression of PHB in renal tissue were detected. Compared with that in the SHO group, the PHB expression (mRNA and protein) was significantly reduced (P < 0.01). Protein expressions of TGF-β1, Col-IV, FN and caspase-3, and RIF index or cell apoptosis index in GU group were markedly elevated compared with those in SHO group (all P < 0.01). The protein expression of PHB had a negative correlation with the protein expression of TGF-β1, Col-IV, FN or caspase-3, and RIF index or cell apoptosis index (each P < 0.01). Less expression of PHB is associated with increased caspase-3 expression/cell apoptosis in RIF rats. However, further research is needed to determine the effect of PHB on caspase-3 expression/cell apoptosis and to determine the potential of PHB as a therapeutic target.Nephrology 09/2011; 17(2):189-96. DOI:10.1111/j.1440-1797.2011.01522.x · 2.08 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Interferon-based (IFN-based) therapy is effective in the treatment of advanced hepatocellular carcinoma (HCC). However, the issue of resistance to this therapy remains to be solved. The aim of this study was to identify microRNAs (miRNAs) that govern the sensitivity to IFN-α in HCC cells. miRNA microarray analysis using IFN-α-resistant clones of PLC/PRF/5 (PLC-Rs) and their parental cells (PLC-P) was conducted. Changes in the anti-cancer effects of IFN-α were studied after gain-of-function and loss-of-function of the candidate miRNA. miR-146a expression was significantly higher in PLC-Rs than in PLC-P. miR-146a decreased the sensitivity to IFN-α through the suppression of apoptosis. Further experiments showed that miR-146a-related resistance to IFN-α was mediated through SMAD4. The results indicated that miR-146a regulated the sensitivity of HCC cells to the cytotoxic effects of IFN-α through SMAD4, suggesting that this miRNA could be suitable for prediction of the clinical response and potential therapeutic target in HCC patients on IFN-based therapy.Biochemical and Biophysical Research Communications 11/2011; 414(4):675-80. DOI:10.1016/j.bbrc.2011.09.124 · 2.30 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.