Study On The Mechanism Of SENP3 In The Occurrence And Development Of Triple-negative Breast Cancer

Breast cancer is one of the most common malignancies in women,which seriously harms women's health.Triple negative breast cancer(TNBC)is a special type of breast cancer subtype,which is characterized by aggressiveness,high distant metastasis rate,and poor prognosis.Due to the lack of targets for endocrine and anti-Her-2 treatment,there is currently no targeted standard treatment plan.SUMOylation is a reversible post-translational modification of proteins,which is closely related to the occurrence and development of various diseases.It has been reported that SENP3 affects the proliferation,metastasis and occurrence of various malignant tumor cells,but the role of SENP3 in breast cancer is still unknown.In order to explore the role of SENP3 in breast cancer cells,we used lentiviral packaging and infection technology to knock down or overexpress SENP3 in TNBC cells to examine the effect of SENP3 on the proliferation and migration.The cell countting experiment results showed that SENP3 knocked down promoted the growth of TNBC cells and SENP3 overexpression inhibited it.And after SENP3 knockdown,the clone formation of TNBC cells increased,when SENP3 was overexpressed,it decreased.Western blottingting was used to detect the expression of cell cycle-related proteins.The results showed that CDK4 expression increased and p16 expression decreased after SENP3 knockdown.When SENP3 overexpression,CDK4 expression decreased and p16 expression increased.At the same time,the wounding healing test was used to observe the cell scratch healing ability,so as to detect the cell migration ability.The results showed that the migration of TNBC cells increased after SENP3 knockdown,when SENP3 was overexpressed,it slowed.The above results indicate that SENP3 inhibits the proliferation and migration of TNBC cells.We next examined the effect of SENP3 on the process of epithelial-mesenchymal transition(EMT).Western blottingting was used to detecte the expression of EMT-related proteins,the results showed that E-cadherin expression was reduced,N-cadherin and Vimentin expression were increased after SENP3 was knocked down,E-cadherin expression was increased,N-cadherin and Vimentin expression were decreased after SENP3 overexpression.We tested the activity of ?-galactosidase and explored the effect of SENP3 on senescence of TNBC cells,and found that SENP3 knockdown significantly accelerated the senescence process.Q-PCR was used to detect the expression of tumor-associated stem cell differentiation factors.The results showed that the expression of NANOG,SOX2 and OCT4 increased after knocking down SENP3,and overexpression of SENP3 decreased the expression of them.Immunofluorescence and Western blottingting were used to detecte the expression of LC3.The results showed that after knocking down SENP3,autophagy of TNBC cells increased,and overexpression of SENP3 weakened.In summary,SENP3 inhibit the EMT process,differentiation potential,senescence and autophagy of TNBC cells.Our previous results indicate that SENP3 plays a tumor suppressor role in TNBC cells,but the target protein regulated by it is still unclear.Earlier literature reported that Hippo signaling pathway regulates the occurrence and development of TNBC,but the role of SENP3 in regulating Hippo signaling pathway is still unknown.We detected the expression of Hippo signaling pathway-related proteins in SENP3 knockdown and overexpressed MDA-MB-231 and MDA-MB-468 cells to find the SUMOylation target protein regulated by SENP3 in TNBC cells.The results showed that knocking down SENP3 inhibits the expression of Lats1,and promotes the expression of Yapl,overexpression of SENP3 promotes the expression of Lats1,and suppresses the expression of Yap 1,because Yap 1 is a key input Nuclear factor,and SENP3 is located in the nucleolus.Therefore,we speculate that Yapl is a SUMO-modified target protein under the regulation of SENP3 TNBC cells.Co-IP and immunofluorescence results showed that Yap1 is indeed modified by SUMO and the main modified protein is SUMO1.SENP3 can remove the SUMOylation of Yap1,Yap1 and SUMO1 were co-localized in the nucleus,while Yap1 and SENP3 were co-localized in the nucleoli.We further found that SUMO1 can enhance the stability of Yapl protein,while SENP3 can suppress the expression of Yapl protein in the nucleus through SUMO modification,and further inhibit the transcription of the downstream factor genes of Yap1.In summary,we use a variety of biological experimental methods and techniques to study the function of SENP3 in TNBC cells.It is found that SENP3 plays a tumor suppressive role in TNBC cells.Yapl is modified by SUMO1 to enhance the stability,and SENP3 can specifically remove the SUMOylation of Yapl protein in TNBC cells,inhibit the expression of Yap1 in the nucleus,and then inhibit the transcription of downstream regulatory genes of Yapl.This paper reveals the important role of SENP3 in the occurrence and development of TNBC,providing a theoretical basis and potential targeting site for the treatment of TNBC.