| Part 1: Expression of SLFN5 in human hepatocellular carcinoma and its clinical significanceObjective: As a subgroup of IFN-stimulated genes(ISGs), Schlafen(Slfn) gene family is involved in interferon function, such as antiviral, anti-tumor and immune regulation. Schlafen-5(SLFN5) has been identified as a special tumor suppressor in the growth and invasion of human malignant melanoma and renal cell carcinoma. However, the association between SLFN5 expression and the pathological characteristics of human hepatocellular carcinoma(HCC) is still unknown. We conducted this study to examine the expression of SLFN5 in human HCC and to investigate the association between SLFN5 expression and the pathological manifestations of HCC patients.Methods: Real time-quantitative PCR, Western blotting and immunohistochemistry(IHC) analyses were used to evaluate the expression of SLFN5 in HCC cell lines and human tissues. Kaplan-Meier survival and Cox regression analyses were applied to evaluate the prognosis of ninety HCC patients.Results: The data showed significantly decreased SLFN5 expression in HCC tissues compared to corresponding non-cancerous tissues. Moreover, we compared the HCC and self-paired non-tumor tissue for SLFN5 expression by measuring the decrease in SLFN5 expression(denoted ΔSLFN5). Results showed that a higher ΔSLFN5 correlates with malignant behavior of HCC, including higher pathological grade(p=0.003), higher TNM stage(p=0.014) and bigger tumor diameter(p=0.017). Cox regression analysis further revealed that ΔSLFN5≥150 is an independent prognostic factor for overall survival in HCC patients(p=0.031).Conclusion: These data are the first to indicate that SLFN5 is significantly decreased in HCC and higher ΔSLFN5 expression correlates with higher TNM stage, pathological grade and tumor diameter in HCC patients. The higher ΔSLFN5 expression may predict poor survival outcomes in HCC patientsPart 2: SLFN5 induces growth arrest in hepatocellular carcinoma cellsObjective: We found SLFN5 expression was significantly lower in HCC tissue than the corresponding non-tumor tissue in the first part. And the higher decrease of SLFN5 correlates with malignant behavior and poor prognosis of HCC. This part was to study the potential role of SLFN5 in cell proliferation, cell cycle and apoptosis of HCC cell lines and explored the underlying mechanisms.Methods: Real time PCR and Western blot analysis were performed to detect the mRNA and protein expression of SLFN5 in HCC cells(SMMC-7721、Huh-7、Hep G2 and BEL7402) and normal liver cells(L02) respectively, and the subcellular localization of SLFN5 protein in HCC cells was confirmed by immunofluorescence. We down-regulated the expression of SLFN5 by the technique of small RNA interference(siRNA) and up-regulated the expression of SLFN5 using Lentivirus-mediated over-expression. Then the influence of SLFN5 expression level on growth inhibition was detected by CCK8 assay. SLFN5 effects on cell cycle and apoptosis were examined by flow cytometry.Results: The mRNA and protein expression of SLFN5 was significantly lower in HCC cells than normal human liver cells(L02).The nuclear SLFN5 localization in HCC cells is consistent with that of previous studies. Down-regulation of SLFN5 by siRNA increased cells proliferation of SMMC-7721 cells by accelerating cell cycle from G1 phase to S phase rather than decreasing apoptosis. Meanwhile, overexpression of SLFN5 inhibited cell proliferation by inducing cell cycle arrest in S-pahse without notable apoptosis. SLFN5 had no significant effects on the cell growth, cell cycle and apoptosis of Huh-7 cells. Additionally, down-regulation or overexpression of SLFN5 in SMMC-7721 cells led to up-regulated or down-regulated expression of cyclin D1, cyclin E, cyclin A and p-Cdk2(Thr 160) correspondly, which ultimately affected cell cycle.Conclusion: SLFN5 expression in normal human liver cells was significantly higher than in HCC cell. SLFN5 localized to the nuclear compartment in HCC cells. Down-regulation of SLFN5 promoted proliferation and cell cycle progression by regulating the expression of cell cycle proteins and cyclin-dependent kinases. SLFN5 overexpression inhibited proliferation and induced cell cycle S-phase arrest by down-regulating the expression of cell cycle proteins and cyclin-dependent kinases. However, SLFN5 had no significant effects on the cell proliferation, cell cycle and apoptosis of Huh-7 cells.Part 3. The preliminary study of relationship between SLFN5 and IFN-alpha signal pathwayObjective: In previous study of Katsoulidis it was certified that Stat1, Stat3 and p38 were required for induction of IFN-inducible mouse Slfn genes to varying degrees. But it is striking that the induction of mouse Slfn5 by IFN α was not only independent on Stat3 molecule, but also IFN α-induced mouse Slfn5 mRNA expression was significantly upregulated in the Stat3-/- knockout mouse embryonic fibroblast(MEF) cell line compared to wild-type MEF cells. However, the human SLFN5 and mouse Slfn5 had been verified as a pair of orthologous genes. Hence, we supposed that there may be an interaction between SLFN5 and Stat3. Therefore, in this part of the study, we want to confirm whether IFN α can induce the expression of SLFN5 in HCC cells firstly; Secondly, we intended to investigate the correlation of SLFN5 and signaling molecules such as Akt and Stat3 and explored the underlying mechanisms.Methods: Real-time PCR, Western blot analysis were applied to detect SLFN5 mRNA and protein expression of HCC cell lines with IFN α treatment and the influence of SLFN5 expression level on Stat3 and Akt signaling molecules. Gene-co-transfected and dual-luciferase reporter gene assay were applied to evaluate the transcriptional activity of Stat3.We also detected expression of p53, which is an upstream regulator of constitutively activated Stat3.Results: Except for BEL-7402 cells, IFN α can induce mRNA and protein expression of SLFN5 in SMMC-7721, HepG2, Huh-7 and L02 cells. Consistent with our conjecture, the expression of p-Stat3 and SLFN5 in HepG2 and SMMC-7721 appears to regulate each other negatively. Knock down of SLFN5 in SMMC-7721 cells lead to increased expression of p-Stat3. On the contrary, SLFN5 overexpression inhibited the expression of p-Stat3. Both of them didn’t affect the expression of total Stat3 levels. The SMMC-7721 cells were co-transfected with the corresponding SLFN5-siRNA, pCMV6-SLFN5-GFP and SIE Stat3 luciferase reporter plasmid, then the transcriptional activity of Stat were detected. The results further confirmed that, SLFN5 can negatively regulate transcriptional activity of Stat3. We found that overexpression of SLFN5 forward regulate the expression of p53 and its downstream target genes p21 in p53 wild-type SMMC-7721 cells. Conversely, SLFN5 overexpression had no effect on the expression of p53 and p21 in p53 mutant Huh-7 cells.Conclusion: IFNα could induce mRNA and protein expression of SLFN5 in normal human hepatocytes and HCC cell lines. SLFN5 can negatively regulate the constitutively activated of Stat3 by inhibiting phosphorylation of tyrosine residues of 705 points. In addition, negatively regulated the transcriptional activity of Stat3 and down-regulated the expression of downstream target genes cyclin D1, which inhibited the proliferation of HCC cells. The regulation of Stat3 phosphorylation by SLFN5 may be wild-type p53 dependent. Inhibition of Akt phosphorylation by SLFN5 may be also involved in the anti-proliferative effect of SLFN5. |
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