E2 Positively Regulates The P-ERK Signaling Pathway By Down-regulating MFN2 And Inhibits The Expression Of Apoptotic Genes In Breast Cancer Cells

Objective:Breast cancer is one of the most common malignant tumors in women.In 2018,breast cancer was the most common type of cancer among8.6 million new female cancer patients?24.2%,one fourth of all new female cancer cases worldwide were breast cancer?,and the cancer was the most common in 154 countries and regions.Breast cancer accounts for 15%of the4.2 million female cancer deaths,which is the main cause of cancer deaths.Among them,hormone receptor-positive breast cancer accounts for70%of all breast cancer.The study shows that ER??estrogen receptor??and ER??estrogen receptor??are the main related receptors.Er?,as the first estrogen receptor,is well known for its role in the occurrence and development of breast cancer,and has been used in the clinical diagnosis and endocrine therapy of breast cancer.However,up to now,there is no unified theory about the biological behavior of Er?in breast cancer.Some studies think that ER?is a oncogene that leads to the occurrence of tumor,and some studies think that ER?gene is a normal group Weaving plays a protective role.Many studies have shown that the expression of Mfn2?mitofusin 2?in breast cancer tissue is significantly lower than that in normal tissue,which has the effect of anti proliferation and inducing apoptosis,but the mechanism is not clear.In this study,we examined the expression of Mfn2 and related genes in MCF-7 cells cultured with different concentrations of estrogen,studied the effect of exogenous estrogen Er?on the expression of Mfn2 and related downstream genes in breast cancer cells,further clarified the mechanism of Mfn2 induced by Er?in the process of apoptosis,and explored potential targeted therapeutic sites.Methods:1. Western blot was used to detect the expression of MFN2 and p-ERK inbreast cancer cell MCF-7 after pretreatment with 10^-5mol/l,10^-6mol/l and10^-7mol/l E2 for 48 hours.2.Eukaryotic plasmids and no-load plasmids knocked down by MFN2were constructed and transfected into human breast cancer MCF-7 cells by liposome transfection,and blank controls?MCF-7-KD,MCF-7-NC and MCF-7-N,respectively?were set up.The expressions of MFN2,p-ERK,cleaved caspase3 and BAX were extracted by western blot.3.In MCF-7 cells transfected with MFN2 knockdown plasmid,p-ERK pathway inhibitor PD98095 was used to extract and extract cellular proteins.Western blot was used to detect the expression of MFN2,p-ERK,cleaved caspase3 and BAX.Results:1. In MCF-7 cells pretreated with 10^-5mol/L,10^-6mol/L and 10^-7mol/L estrogens for 48h,the expression of Mfn2 protein was lower t han that of untreated cells,and the expression of 10-6mol/l group wa s the lowest,while the expression of p-ERK was increased after treated with different concentrations of estrogens,and the expression of 10-6m ol/l group was the highest.2. We successfully constructed MFN2 knockdown stable cell lines inMc F-7 cells of breast cancer,and the p-ERK significantly increased wi th the expression of MFN2 knockdown protein.The proapoptotic genes cleaved caspase3 and BAX were decreased by knockdown of MFN2.3. In the knock-down MFN2 group,the p-ERK pathway inhibitor PD98095 was used.Western blot results showed that after knocking dow n the MFN2 p-ERK inhibitor PD98095 group,p-ERK was significantly inhibited,and the expression of cleaved caspase3 and BAX protein incre ased.High,that is,PD98095 reverses the increase in p-ERK after the MFN2 gene is knocked out,and at the same time promotes the expressi on of the pro-apoptotic genes cleaved caspase3 and BAX,which promot es the apoptosis of breast cancer cells.Conclusions:1.MFN2 and p-ERK were expressed in MCF-7 of breast cancer,both of which were influenced by estrogen concentration.The expression of MFN2and p-ERK was reversed.2.MFN2 knockdown stable cell lines could be constructed in human breast cancer cell line MCF-7.MFN2 knockdown activates the p-ERK pathway and reduces the expression of pro-apoptotic gene cleaved caspase3and BAX,thus inhibiting the apoptosis of breast cancer cells.3. PD98095 can inhibit the activation of the p-ERK pathway after MFN2knockdown,increase the pro-apoptotic effect of cells,and thus reverse the anti-apoptotic effect of breast cancer cells after MFN2 knockdown.4. MFN2 is a tumor suppressor gene,which negatively regulates theexpression of p-ERK and has a certain pro-apoptotic effect under the action of estrogen.