SIRT7 Low Expression Down-regulating LAP2α-mediated Chromosomal Instability Promoting Breast Cancer Cell Metastasis

[Objective]Breast cancer is one of the most common malignant tumors,and its morbidity and mortality rate are among the top of female tumors,while metastasis is the main cause of death and poor prognosis of patients.Chromosomal instability(CIN)has been found to play an important driving role in cancer development and is strongly associated with increased tumor metastasis,but its specific molecular mechanisms remain unclear.SIRT7 as a member of the class III histone deacetylase family,is involved in regulating a variety of cellular physiological functions,including DNA damage repair,maintenance of genomic stability and tumor development.In previous studies we have identified the relevance of SIRT7 to breast cancer metastasis,but the specific mechanism of its regulation of breast cancer metastasis remains to be further clarified.The aim of this study was to investigate: the role of knockdown of SIRT7 in causing cellular CIN and thus promoting breast cancer metastasis.[Methods]1.In the CIN cells constructed after pretreatment of breast cancer cells with low dose of doxorubicin for 7 days,and the cellular SIRT7 expression level was detected using Western blot and RT-PCR techniques,the metastatic ability of the cells was analyzed using a scratch assay and a Transwell assay.2.A model of SIRT7 knockdown breast cancer cells was constructed,and the relevant indicators of chromosome stability in knockdown SIRT7 and negative control breast cancer cells were detected using Western blot,RT-PCR and laser confocal imaging techniques.3.Protein profiling of protein glue strips after IP experiments was performed to find Lap2α(Lamina-associated polypeptide 2α),a downstream molecule of SIRT7-regulated cellular CIN,and the relationship between SIRT7 and Lap2α was verified by CO-IP,Western blot,RT-PCR and ubiquitination level assay.4.Immunohistochemistry was performed on the collected pathological tissues of breast cancer to examine the correlation of SIRT7 and Lap2α protein expression in breast cancer tissues.5.Kaplan Meier plotter website was used to analyze the correlation between the mRNA expression level of Lap2α in public database and the survival time of breast cancer patients.6.Lap2α knockdown cell lines and SIRT7 knockdown cell lines with back-complemented Lap2α were constructed,respectively.Western blot and RT-PCR were used to detect chromosome stability related indexes.The role of Lap2α in SIRT7-mediated breast cancer cell metastasis was also analyzed by cytological experiments such as scratch assay and Transwell assay.[Results]1.CIN increases the migration ability and invasion ability of SK-BR-3 and MCF-7 cells.2.There is a correlation between CIN and SIRT7.SIRT7 expression is significantly reduced in CIN cell.Knockdown of SIRT7 in wild-type breast cancer cells resulted in the development of CIN in the cells.3.SIRT7 binds directly to Lap2α protein intracellularly,and knockdown of SIRT7 leads to downregulation of Lap2α expression at the protein level,while knockdown of Lap2α has no effect on SIRT7 expression,SIRT7 is an upstream molecule of Lap2α.4.The protein expressions of SIRT7 and Lap2α were positively correlated in pathological tissues of breast cancer patients.5.Knockdown of SIRT7 in breast cancer cells could increase protein degradation by affecting the ubiquitination level of Lap2α protein,leading to down-regulation of Lap2αexpression.6.Some of the chromosome stability-related indicators were partially restored after back-supplementation of Lap2α in SIRT7 knockdown cells,indicating that SIRT7 could lead to cellular CIN through down-regulation the expression of Lap2α.Knockdown of either SIRT7 or Lap2α alone resulted in enhanced metastatic ability of breast cancer cells,and back-supplementation of Lap2α in cells with knockdown of SIRT7 attenuated the metastatic ability of the cells.Low expression of Lap2α mRNA is a poor prognostic factor for breast cancer patients.It was shown that knockdown of SIRT7 could improve the metastatic ability of breast cancer cells SK-BR-3 and MCF-7 by down-regulating the expression of Lap2α.[Conclusion]1.Knockdown of SIRT7 leads to increased CIN in breast cancer cells.2.SIRT7 interacts with Lap2α intracellularly,and knockdown of SIRT7 increases the level of ubiquitinated degradation of Lap2α protein,causing the downregulation of Lap2αexpression at the protein level.3.Down-regulation of SIRT7 increased cellular CIN and metastatic ability of breast cancer cells by regulating Lap2α,suggesting that the SIRT7/Lap2α pathway could be a potential therapeutic target for chromosomally unstable breast cancer cells.