RhebL1 Regulates Biological Characteristics Of Breast Cancer Cells Based On PI3K/Akt Signaling Pathway

Background and Objective:Breast cancer is the most common malignant tumor in women all over the world,accounting for 25% of all cancer incidences in women and 15% of all cancer deaths.Despite advances in surgery and chemotherapy,the long-term survival rate of patients with advanced breast cancer is still very low,mainly due to tumor recurrence or distant metastasis.Although a large number of oncogenes and tumor suppressor genes have been found to be involved in the development of breast cancer,in order to clarify the potential targets of breast cancer treatment,it is still necessary to explore the potential molecular mechanisms in the development and development of breast cancer.Current studies have shown that the pathogenesis of breast cancer is complex,mediated by a variety of cell signaling molecules and cross-talk,such as mitogen-activated protein kinase(MAPK),phosphoinositide3-kinase(PI3K)and protein kinase B(Akt)are all related to the occurrence and development of breast cancer.In many human tumor studies,the PI3K/Akt signaling pathway is usually expressed abnormally.Mutations in certain signaling molecules in this pathway can also lead to changes in pathway function and cell biological behavior.It can not only regulate the survival and proliferation of tumor cells,but is also closely related to tumor invasion,metastasis,and drug resistance.With the continuous research on breast cancer and other tumors expanding,people have eventually be aware of the role of the Ras superfamily in tumorigenesis.The Ras superfamily proteins mediate various cellular effects,such as cell proliferation and apoptosis.Researches on the correlation between ras superfamily proteins and breast cancer have illustrated that ras superfamily proteins are closely related to the pathological data and prognosis of patients,among which.RhebL1 is a member of the Ras superfamily.The association of RhebL1 with breast cancer is the research priority.The objective of this study is to clarify the correlation between RhebL1 expression level and breast cancer,to distinguish the effect of RhebL1 on breast cancer cell proliferation,invasion and apoptosis,and to explore the influence of RhebL1 on breast cancer cell biological behaviors and the mechanism of occurrence and development in breast cancer,in the interest of providing new targets for the diagnosis and treatment of breast cancer.Methods:1 Expression of RhebL1 in human breast cancer cells1.1 RT-q PCR technology was used to detect the expression of RhebL1 in breast cancer cell lines MCF-7,MDA-MB-231 and normal breast epithelial cell line MCF-10 A at the m RNA level.1.2 Western Blot technology was used to detect the expression of RhebL1 in breast cancer cell lines MCF-7,MDA-MB-231 and normal breast epithelial cell line MCF-10 A at the protein level.2 The Effect of RhebL1 on the Malignant Biological Behavior of Breast Cancer Cells and Its Mechanism2.1 Establish a stable RhebL1 knockdown breast cancer cell line.The CCK8 method is used to detect breast cancer cell proliferation.By subcutaneously injecting breast cancer cells into nude mice,the effect of RhebL1 on the proliferation of breast cancer cells in vivo was detected.Flow cytometry is used to detect changes in the level of apoptosis.Transwell methodand scarification was used to evaluate influence of RhebL1 expression on breast cancer cell invasion and metastasis.2.2 Western Blot experimental technology is used to detect important proteins p21 and p27 that play an important role in tumor biological functions.2.3 Proteins with changing trends are closely related to the PI3K/Akt signaling pathway.We use Western Blot to detect PI3K/Akt pathway-related proteins2.4 We use the protein kinase inhibitor LY294002 of the PI3K/Akt signaling pathway to intervene,after which,CCK8 and Transwell are carried out to detect its effects on breast cancer cell proliferation and migration,with Western Blot performed to detect the changes of related proteins in the purpose of clarifying the mechanism of RhebL1 in breast cancer.2.5 Co-IP was performed using Akt antibody and RhebL1 antibody to reveal the interaction between RhebL1 and Akt.Results:1 Expression of RhebL1 in human breast cancer cells RhebL1 is expressed in both normal breast cells and breast cancer cells,with the expression in breast cancer cells significantly surpassing that in normal breast cells(P<0.05).2 The Effect of RhebL1 on the Malignant Biological Behavior of Breast Cancer Cells and Its Mechanism2.1 Down-regulating the expression of RhebL1 inhibits the proliferation,migration and invasion of breast cancer cells Knockdown of RhebL1 can inhibit markedly the viability of MCF-7 and MDA-MB-231 cells;knockdown of RhebL1 considerably increases the apoptosis rate of breast cancer cells;knockdown of RhebL1 substantially reduces breast cancer’s capabilities in migration and invasion.(P<0.05)2.2 Down-regulating the expression of RhebL1 inhibits the activity of the PI3K/Akt pathway Down-regulating RhebL1 in breast cancer cells can significantly regulate the activity of PI3K/Akt pathway related proteins.The expression levels of Bcl2,PI3 K and p-Akt protein are significantly reduced(P<0.05).And the expression level of Bax protein is significantly increased(P<0.05).There is no significant difference in Akt protein expression(P >0.05).2.3 RhebL1 regulates PI3K/Akt signaling pathway through Akt PI3K/Akt signaling pathway protease inhibitor LY294002 inhibits the proliferation and invasion of breast cancer cells.After application of PI3K/Akt signaling pathway protease inhibitor LY294002,the expression levels of Bcl2,PI3 K and p-Akt proteins in breast cancer cells were significantly reduced.And the expression levels of Bax,p21 and p27 proteins were significantly increased(P <0.05).There is no significant difference in Akt protein expression(P >0.05).Co-IP shows that RhebL1 interacts with Akt.Conclusion:1.RhebL1 is highly expressed in breast cancer cells.2.Down-regulation of RhebL1 can affect the biological behaviors of breast cancer cell proliferation,apoptosis and invasion.3.RhebL1 promotes the proliferation and migration of breast cancer through the PI3K/Akt signaling pathway.