The Function And Mechanisms Of SIRT7 In Pathological Mammary Cells

As a member of acetylated protein family,SIRT7 is a kind of NAD+-depended deacetylase,which localizes in the cell nucleolus and participates in various of biological processes,including ribosomal RNA and protein synthesis,DNA damage response,cell stress and the occurrence of tumors.Recently,it has been reported that SIRT7 was down-regulated in LPS-induced THP-1 cells,and up-regulated in the tissues of thyroid cancer,bladder cancer,liver cancer,colon cancer and breast cancer,which is considered as a potential cancer factor.However,there are few studies of SIRT7 in mastitis and breast cancer and the specific regulatory mechanism is still not clear.In this study,we built up the knockdown and overexpression of SIRT7 cell model to study the functional mechanism of SIRT7 in LPS-induced inflammatory response in dairy cow mammary epithelial cells(DCMECs);And the regulatory mechanism of SIRT7 in the proliferation of breast cancer cells,which could provide new targets and clues for dairy cow mastitis and breast cancer diagnosis.The results of this study consist of the following aspects:1.SIRT7 reduces the LPS-induced inflammatory injury in DCMECsThe health of cattle breast directly affects the diary quality and economic efficiency.Although prevention and treatment methods have been implemented for decades,cattle mastitis is still an intractable disease.LPS is the key cytotoxic factor to induce mastitis.Therefore,we need to develop new drugs and vaccines to effective control LPS-induced cow mastitis.In this study,we collected the breast tissues from five different mastitic cattles and normal cattles,and we found that SIRT7 expression was significantly decreased in mastitic cattles by western blot.In the meanwhile,we cultured DCMECs in vitro and used LPS(1?g/mL)to induce DCMECs inflammation for 6 h and then we collected the cells for qRT-PCR,western blot.We found that the expression of SIRT7 was also decreased after LPS treatment.Meanwhile,depletion of SIRT7 significantly increased the expression of inflammatory cytokine TNF-?,IL-1?,and IL-6 in DCMECs;And SIRT7 knockdown also promoted LPS-induced inflammatory mediator ROS and NO generation;whereas overexpression of SIRT7 significantly inhibited the expression of TNF-?,IL-1?and IL-6 in LPS-induced DCMECs,and SIRT7 also significantly attenuated LPS-induced ROS and NO generation.Therefore,we conclude that SIRT7 reduce inflammatory injury through regulating the secretion of inflammatory cytokines(TNF-?,IL-1? and IL-6)and inflammatory mediators(ROS and NO)in LPS-induced DCMECs.2.SIRT7 inhibits the activation of NF-?B signaling pathway and reduces cell apoptosis in LPS-induced DCMECsMammals of the inflammatory response is a complex process of biology,physiology and pathology to defense against invading pathogens.Excessive inflammation can lead to cell apoptosis,host tissue injury and disease.Usually,LPS activates NF-?B p65 signaling pathways and promotes cell apoptosis.To detect the effect of SIRT7 on NF-?B signaling pathway activation in LPS-inudced DCMECs,we first detected the expression of TLR4,TAB1 and p-TAK1 which are the upstream factor of NF-?B pathways.The results showed that SIRT7 knockdown significantly increased the expression of TLR4,TAB1 and p-TAK1 in LPS-induced DCMECs;but SIRT7 overexpression significantly attenuated the LPS-induced the expression levels of TLR4,TAB 1 and p-TAK1.In addition,our results also showed that LPS could activate NF-?B signaling pathway,which increase the phosphorylation of NF-?B p65;and knockdown of SIRT7 could further increased the phosphorylation of NF-?B p65,whereas SIRT7 overexpression blocked NF-?B p65 activation and decreased the phosphorylation of NF-?B p65 in LPS-induced DCMECs.Immunofluorescence staining showed that SIRT7 knockdown promoted nuclear translocation of NF-?B p65,whereas SIRT7 overexpression inhibited nuclear translocation of NF-?B p65 in LPS-induced DCMECs.In the meantime,we also examined the protein level of NF-?B p65 in cell nucleus and cytoplasm by western blot,which was consiste with immunofluorescence staining results.LPS can induce severe inflammation and cause breast tissue and breast epithelial cell injury.Therefore,we detect the effect of SIRT7 on LPS-induced apoptosis by flow cytometry.Our reuslt showed that SIRT7 knockdown significantly increased LPS-induced DCMECs apoptosis;whereas SIRT7 overexpression significantly suppressed LPS-induced DCMECs apoptosis.Meanwhile,SIRT7 knockdown significantly increased the ratio of Bax/Bcl-2 and increased the cleaved-caspase-3 levels.Whereas SIRT7 overexpression significantly reduced the ratio of Bax/Bcl-2 and the level of cleaved-caspase-3.Above all,our results showed that SIRT7 could suppressed the LPS-induced the activaiton of NF-?B pathway,which reduced the release of inflammatory factors and cell apoptosis.Therefore,SIRT7 plays a key role in inflammatory reaction-induced breast tissues and DCMECs injury.3.The function of SIRT7 in the proliferation,migration and apoptosis of MCF-7 and MDA-MB-231 cellsBreast cancer is the main cause of cancer death in women all over the world.It is significance to explore a new type of mechanism can effectively prevent breast cancer.To explore the biological function of SIRT7 in breast cancer cell.The siSIRT7 and pcDNA3.0-SIRT7 plamid were transfected into MDA-MB-231 and MCF-7 cells by Lipofection 2000.CCK8 and EDU results showed that SIRT7 knockdown significantly reduced cells proliferation,whereas SIRT7 overexpression significantly increased cells proliferation in both MDA-MB-231 and MCF-7 cells.In the meantime,we also used scratch wound healing assay and transwell to examine cell migration,the results showed that SIRT7 knockdown significantly inhibited cell migration,wherease SIRT7 overexpression obviously promoted cell migration.According to these results,we hypothesized that knockdown and overexpression of SIRT7 could cause the change of cell cycle and cell apoptosis,which further affected cell proliferation and migration.First,we examined the effect of SIRT7 on cell cycle and apoptosis by flow cytometry(FCM);the cells exhibited a significantly accumulation in G1 phase and a remarkable decrease in S phases after SIRT7 depletion.Whereas SIRT7 overexpression significantly reduced the percent of cells in G1 phase and increased the stage of S phase cells,suggesting that SIRT7 promoted G0/G1 to S phase transition in MCF-7 and MDA-MB-231 cells.Meanwhile,the percent of apoptosis cells were significantly increased in SIRT7 deficient cells;whereas there was no significantly effect after SIRT7 overexpression.To further validate the mechanism of SIRT7 in cell apoptosis and cell cycle,we first determine cell cycle related genes by qRT-PCR.Our results showed that the mRNA levels of CDK4/6 and CyclinDl/2 were significantly decreased after silencing SIRT7.Whereas after SIRT7 overexpression,the CDK4/6 and CyclinDl/2 mRNA expression was significantly increased,which indicated that SIRT7 might regulate cell cycle progression by monitoring CDK4/6 activation,and CyclinDl/2 expression,which further regualted G0/G1 to S phase transition.We also investigated some apoptotic regualted genes.Our results showed that the mRNA expression of Bax/Bcl-2 was significantly increased,and the protein level of Bax/Bcl-2 and cleaved-caspase-3 were also increased after SIRT7 depletion both in MCF-7 and MDA-MB-231 cells,which further validated the occurrence of apoptosis after SIRT7 depletion.In the meantime,the increased protein level of cleaved-Parpl and p-p38MAPK further demonstrated that SIRT7 could participate in different apoptotic related signal pathways that induced the occurance of apoptosis.Above all,these results showed that SIRT7 played a key role in breast cancer cell proliferation,migration and apoptosis through regulating the cell cycle and apoptosis related gene and protein expression,and p38MAPK signaling pathway activation.4.Stable silencing of SIRT7 in MDA-MB-231 decreased tumors growth and increased the sensitivity of DOXEstablishing a tumor model in vitro is an important method to exmine the clinic preventation research in basic medical research.In the present study,a stable SIRT7 knockdown cell line was established by lentiviral infection in MDA-MB-231 cells,which could be used for subcutaneous tumorigenesis in nude mice.MDA-MB-231 cells from two groups(shCon and shSIRT7)were subcutaneously injected into the armpit of the nude mice.Results show that the speed of tumor formation of in shSIRT7 group was slower than shCon group.After tumors had been stripped from nude mice,HE staining resulted showed that the tissue was more loose and more lacuna.Tunel assay result showed that the rate of apoptosis was significant increased in the shSIRT7 group,which means there were more apoptotic cell during the tumor formation after SIRT7 knockdown.The side effects of DOX greatly limits the application of DOX in breast cancer therapy.When the cells were subcutaneous injected to the nude mice,the tumor started to growth.After 7 days,the nude mice were injected DOX to inhibit tumor growth every 3 days.After 25 days,the tumor volume and weight were significantly decreased in the shSIRT7 group compared with shCon group.Then the tumors were analyzed by HE staining and Tunel staining.The HE staining result showed that the tumor tissue had more lacuna;and the tunel staining showed that there were more apoptotic cells after DOX treatment,which indicated that SIRT7 depletion could significantly increase the efficiency of DOX that promoted the effect of DOX on MDA-MB-231.According to the above results,we think that SIRT7 depletion can inhibit tumor formation and raise the sensitivity of MDA-MB-231 cells to DOX.