The Prognostic Roles Of Sirtuins And Its Correlation With The Functional Impairment Of Infiltrating γδT Cells In Hepatocellular Carcinoma

Tumor microenviroment played an important role in cancer progression, the immune system plays a dual role in cancer:it can not only suppress tumor growth and invasion but also promote cancer progression either by immunoselection or establishing an immune inhibition network within the tumor microenviroment that facilitate cancer development. In the previous study, we analyzed the roles of y8T cells in hepatocellular carcinoma. The results showed that the infiltration of y8T cells in HCC tissue was significantly decreased compared with paired peritumor tissue, and an cytotoxic function impairment may exist in tumor infiltrating y8T cells. But we did not further study the prognostic value of y8T cells in HCC through a larger scale of HCC by the way of IHC.Recent studies showed that HDAC inhibitors could affect the progression of tumor cells through upregulating the expression of NKG2D, which was a remarkable effect way of γδT cells. Sirtuins family played critical roles in the progression of multiple cancer types. There would be some connection between Sirtuins and y8T cells in tumor environment, which was supposed to be the molecular mechanism of the functional impairment of y8T cells in HCC.In this study, firstly we figured out the prognostic role of y8T cells in HCC. Secondly, we analyzed the correlation between the functional markers expression of y8T cells with Sirtuins family members. Thirdly, we evaluated the expression and prognostic value of Sirtuins. Finally, we focused the role of Sirt3in the progression of HCC.PartⅠDecreased intratumoral γδT cells correlated with recurrence of hepatocellular carcinoma after resection Purpose:This part aimed to evaluate the infiltrating of yST cells in paired tumor and peritumor tissues, correlate it with clinicopathologic features and clinical outcome of HCC.Methods:We first analyzed the extent of infiltrating y8T cells in paired tumor and peritumor tissues by IHC. We also evaluated and correlated the expression pattern of yST cells with clinicopathologic features and clinical outcome of HCC.Results:IHC studies showed a decreased expression of y8T cells in tumoral tissues compared with peritumoral tissues (P<0:0001). Decreased expression of y8T cells peritumoral tissues was associated with decreased time to recurrence (TTR) and decreased overall survival (OS) rate by univariate analyses (P=0.010for TTR, P=0.036for OS), the prognostic value of TTR but not OS was strengthened by multivariate analyses (P=0.014for TTR, P=0.279for OS). Peritumorl γδT cells also showed a favorable prognostic value of TTR in patients with BCLC stage A (P=0.003). In addition, we found that intratumoral γδT cells had a prognostic value of OS after univariate anaylsis (P=0.003), but not strenghened by multivariate analyses. Conclusion:Down-regulation of peritumoral γδT cells was associated with poor recurrence outcome in HCC after resection, moreover, peritumoral y8T cells was a favorable prognostic predictor of recurrence in early stage patients.Part IIThe correlation of functional impairment of γδT cells with the expression of Sirtuins in HCCPurpose:We aimed to explore the molecular mechanism of functional impairment of y8T cells in tumor through analyzing the correlation of γδT cells with the expression patterns of Sirtuins.Methods:We evaluated the expression of TGFβ, IL-10, NKG2D, perforin, granzyme B, CD107a, IFN-y secretion in yST cells derived from both tumor and peritumor tissues of HCC. Furthermore, we also analyzed the expression pattern of Sirtuins in the same cohort of HCC patients through the way of WB (n=13).Results:After an overall statistical analysis, there existed a correlation of NKG2D and perforin of y8T cells with the expression of Sirt3in tumor. Furthermore, we also found a different expression of functional markers of y8T cells and Sirt3between tumor and peritumor tissues.Conclusion:There existed a correlation between infiltrating y8T cells with Sirt3in tumor, which could be a molecular modulation way of the functional impairment of γδT cells.Part ⅢDown-regulation of Sirt3is associated with poor prognosis in hepatocellular carcinoma after resectionPurpose:Sirtuin3(Sirt3), one of the seven Sirtuins family members, plays critical roles in the progression of multiple cancer types. However, its role in the prognosis of hepatocellular carcinoma (HCC) has not yet been investigated systematically.Methods:The correlation of Sirtuins expression with prognosis of HCC was determined by IHC in a large HCC patient cohort (n=342). Expression of Sirt3in tumoral and peritumoral tissues of HCC patients were further determined by WB.Results:IHC and WB studies both showed a decreased expression of Sirt3in tumoral tissues compared with peritumoral tissues (P=0.003for IHC, P=0.0042for WB). Decreased expression of Sirt3in both tumoral and peritumoral tissues was associated with increased recurrence probability and decreased overall survival rate by univariate analyses (intratumoral Sirt3:P=0.011for TTR, P=0.001for OS; peritumoral Sirt3: P=0.017for TTR, P=0.023for OS), the prognostic value was strengthened by multivariate analyses (intratumoral Sirt3:P=0.031for TTR, P=0.001for OS; peritumoral Sirt3:P=0.047for TTR, P=0.031for OS). Intratumoral Sirt3also showed a favorable prognostic value in patients with BCLC stage A (TTR, P=0.011; OS, P<0.001). In addition, we found that IHC studies of other sirtuin members showed a decreased expression of Sirt2, Sirt4and Sirt5and an increased expression of Sirtl, Sirt6and Sirt7in intratumoral tissues compared with peritumoral tissues. In contrast to Sirt3, other members did not showed a remarkable correlation with HCC prognosis.Conclusions:Down-regulation of intratumoral and peritumoral Sirt3were both associated with poor outcome in HCC, moreover, intratumoral Sirt3was a favorable prognostic predictor in early stage patients.Part IVSirt3induced apoptosis and suppressed proliferation, migration and invasion of SMMC-7721cell line in vitroPurpose:This part aimed to evaluate the impact of Sirt3expression on the activity of apoptosis, proliferation, migration and invasion of SMMC-7721cells in vitro.Methods:We employed lentivirus transfection system to specifically up-regulate Sirt3expression. We used TUNEL assay, CCK-8kit, scratch assay, transwell migration assay and transewell matrigel invasion assay to analyze the impact of Sirt3expression on the activity of apoptosis, proliferation, migration and invasion of SMMC-7721cells in vitro.Results:WB results demonstrated stable up-regulation of Sirt3protein in SMMC-7721cells transfected with over-expression vehicle. TUNEL assay demonstrated overexpression of Sirt3could promote apoptosis of SMMC-7721cells. CCK-8kit assay demonstrated declined proliferation activity of SMMC-7721cells with upregulation of Sirt3protein. Transwell migration assay confirmed this tendency: upregulation of Sirt3expression in SMMC-7721cells led to significantly less migrated cell counts. Transwell matrigel invasion assay demonstrated decreased invaded cell counts in SMMC-7721cells with Sirt3up-regulation.Conclusions:Results of this part demonstrated potent promotive effect for apoptosis and suppressive effect of Sirt3expression for the activity of proliferation, migration and invasion of SMMC-7721cells in vitro. Conclusions1The infiltration of y8T cells in HCC tissues was significantly decreased compared with paired peritumor tissue, and peritumor infiltrating y8T cells displayed the prognostic value of recurrence for HCC patients after resection.2Down-regulation of Sirt3was associated with poor prognosis in hepatocellular carcinoma after resection. Moreover, intratumoral Sirt3was a favorable prognostic predictor in early stage patients.3Sirt3induced apoptosis and suppressed proliferation, migration and invasion of SMMC-7721cell line in vitro.Novelty1We firstly demonstrated that the infiltration of peritumoral γδT cells was associated with the reccurence of HCC patients after resecion..2We firstly showed that down-regulation of Sirt3in tumor and peritumor was both associated with poor prognosis in hepatocellular carcinoma after resection. Moreover, intratumoral Sirt3was a favorable prognostic predictor in early stage patients3We firstly evaluated the prognostic value of Sirt3in hepatocellular carcinoma, and partly due to Sirt3induced apoptosis and suppressed proliferation, migration and invasion of SMMC-7721cell line in vitro.