Effect Of SIRT1 Gene Silencing On Liver Cancer Stem Cells And Its Potential Mechanism

Background:Liver cancer is one of the common malignant tumors of the digestive system,with the high incidence rate and mortality ranking sixth and fourth respectively in the worldwide.Hepatocellular carcinoma?HCC?is a primary liver cancer with high degree of malignancy and cellular heterogeneity.The traditional treatment methods for HCC mainly include surgical resection,chemotherapy and radiotherapy.Because of its high recurrence,HCC is difficult to be cured fundamentally.Live cancer stem cells?CSCs?are a group of tumor cells with self-renewal,multi-differentiation and tumor.The ability of CSCs to initiate the drug resistance and low sensitivity to the radiotherapy and chemotherapy drugs may contribute to the high metastasis and recurrence of liver cancer.Therefore,to search for new and effective methods for targeting liver CSCs is essential for the long-term stability and cure of liver cancer.Pro-senescence therapy for cancer is a novel tumor treatment strategy,and cellular senescence is considered to be an effective tumor suppressor mechanism.In contrast to normal cells cleared by the immune system after aging,cancer cells have the capability to escape senescence,thus acquiring a limitless replicative potential.Neverthless,if the cancer cells can be returned to the senescence state to trigger the body's immune clearance system,it will effectively alleviate the bottleneck problem of current liver cancer treatment.In recent year,the studies have reported that SIRT1 is highly expressed in a variety of CSCs and is closely related to the patient's poor prognosis.In addition,some studies also have illustrated that SIRT1 plays an important role in the aging and aging reversal of adult stem cells such as hematopoietic stem cells and mesenchymal stem cells.Therefore,our study hypothesized that the induction of senescence of liver CSCs by regulating the expression level of SIRT1 could effectively inhibit the development and progress of liver cancer.Methods:?1?Immunohistochemistry?IHC?and western blot were used to detect the expression level of SIRT1 in liver cancer tissue samples and liver CSCs and non-CSCs;?2?shRNAs were conducted to knockdown the expression level of SIRT1,and then the changes of cancer stem cell genes expression in liver CSCs were analyzed;and EdU assay was used to detect the proliferation of CSCs;?3?self-renewal and tumorigenic potential of liver cancer stem cells were detected by clone-,sphere-and tumor-formation assay;IHC staining was performed to detect the expression of SIRT1 and EpCAM in the forming tumor tissues;?4?relevant indicators of cell senescence such as senescence-associated?-galactosidase?SA-?-Gal?activity,cell cycle inhibitors P53,P21,P16 and the expression of CDK2 and CDK4,which are related to the p53-p21 and p16 signaling pathway,was evaluated to interfere with the senescence mechanism of SIRT1 inhibiting the proliferation of HCC CSCs,and the expression of P21 and Ki67 in tumorigenic tissues were detected by IHC staining.Results:?1?The expression of SIRT1 in clinical liver cancer tissues was much higher than that in adjacent tissues;the expression of SIRT1 in CD13-positive and EpCAM-positive liver CSCs was significantly higher than that in CD13 or EpCAM-negative cells;SIRT1 is at a high expression level in HCC cell lines,particularly in Nanog^Pos liver CSCs.?2?The expression levels of CSCs markers such as Nanog,SOX2,OCT4,CD13,CD44 and EpCAM were significantly decreased in liver CSCs after knockdown of SIRT1 in liver CSCs,and the proliferation level of liver CSCs decreased with the inhibition of SIRT1 expression.?3?knockdown of SIRT1 inhibited the efficiency of clone formation,sphere formation and tumorigenic ability of liver CSCs.And the proportion of EpCAM-positive stem cells in the control group with high SIRT1 expression was significantly higher than that in low SIRT1 expression group.?4?After inhibiting the expression of SIRT1,the SA-?-Gal activity in liver CSCs elevated and the expression of cell cycle inhibitors P53,P21 and P16 increased.At the same time,the expression of p53-p21 and p16 signaling pathway-related proteins CDK2 and CDK4 decreased.Moreover,the expression of Ki67 was significantly increased but the expression of P21was declined in the control group with high SIRT1 expression.In the contrast,the expression of P21 was significantly increased and the number of Ki67-positive cells was markely decreased in the SIRT1 expression-suppressed group.It is further illustrated that down-regulation of SIRT1 induced the senescence of liver CSCs,thereby inhibiting the self-renewal ability and inhibiting the development of liver cancer.Conclusion:SIRT1 is highly expressed in liver CSCs,and decreasing the expression of SIRT1 can reduce the stemness,self-renewal and tumorigenic ability of CSCs.Knockdown of SIRT1 promotes the development of liver cancer by regulating the senescence of CSCs through p53-p21 and p16 signaling pathways.This research will be a new breakthrough in the treatment of HCC.It inhibits liver cancer through inducing senescence of liver CSCs,which provides new ideas for the treatment of clinical liver cancer.