Mechanism Study Of Tumor Promoting Properties Of Enolase Phosphatase 1 In Breast Cancer By Activating NF-κB Signaling Pathway

Breast cancer has surpassed lung cancer as the most common malignancy in women worldwide and the leading cause of most cancer-related deaths.An estimated 2.3 million new cases of breast cancer,accounting for 12% of all new cancers,and 680,000 deaths in 2020.In our country,breast cancer is the most common malignant tumor among women,with an estimated 430,000 new cases and 120,000 deaths in 2022.At present,there is still a gap between my country’s breast cancer mortality rate and Western countries.The main reason is that breast cancer patients in my country tend to be younger.The average age of diagnosis in Australia is 60 years old,while that in the United States is 61 years old.Only about a quarter of breast cancer patients Diagnosed before the age of 50,and less than 5% of patients diagnosed before the age of 35;the average age of breast cancer diagnosis in China is 49 years old,and about two-thirds of patients are between 40-59 years old.Generally speaking,compared with Western countries,there is still a huge room for improvement in the prevention and treatment of breast cancer in my country,and exploring the pathogenic mechanism of breast cancer will provide a theoretical basis for the treatment of breast cancer.Enolase-phosphatase 1(Enolase-phosphatase 1,ENOPH1)belongs to the Mas A family in the HAD(L-2-halo-acid dehalogenase)-like hydrolase superfamily.ENOPH1 is a bifunctional enzyme with phosphatase and atypical Enolase activity,which is not only involved in the synthesis of polyamines,but also necessary for the synthesis of methionine repair.ENOPH1 plays an important role in the occurrence and development of tumors.Many literatures have shown that high expression of ENOPH1 can promote the migration and invasion of glioma and hepatocellular carcinoma cells,and is associated with the prognosis of glioma and liver cancer patients.In addition,ENOPH1 has also been used to predict the survival of colorectal cancer patients.Therefore,ENOPH1 may play an important role in breast cancer progression.NF-κB regulates more than 500 genes involved in inflammation,cell transformation,survival,proliferation,angiogenesis,invasion and metastasis,and abnormal activation of NF-κB can lead to the occurrence of various diseases.Studies in breast cancer cell lines,breast cancer animal models and breast cancer patient samples showed that:(i)the expression levels of Rel A/p65,c-Rel and NF-κB2/p100 were all increased;NF-κB transcriptional activity was significantly enhanced before transformation;(iii)NF-κB regulates angiogenesis/lymphangiogenesis by upregulating NOS,COX-2 and VEGF during breast cancer cell metastasis.Polyamines can affect the activity of NF-κB transcription factor in breast tumor cells,and ENOPH1 is involved in the synthesis of polyamines,so the NF-κB pathway may interact with ENOPH1.This study will make full use of The Cancer Genome Atlas(TCGA)database,use bioinformatics to collect sequencing data of breast cancer samples,analyze and integrate and compare the expression levels of ENOPH1 m RNA in normal breast tissue and breast cancer tissue;in addition,this study will also The specific mechanism of ENOPH1 in the occurrence and development of breast cancer was studied at the cellular level by combining RNA interference,overexpression vectors,q PCR and western blotting.This study consists of three parts,as follows:Part one The study on the correlation between ENOPH1 level and clinical features of breast cancer patientsObjective: Bioinformatics method was used to collect the sequencing data of normal breast tissue and breast cancer tissue in the TCGA database,analyze the ENOPH1 m RNA level in normal breast tissue and breast cancer tissue,explore the relationship between the expression level of ENOPH1 m RNA and the stage and survival rate of breast cancer tissue,and explore the expression of ENOPH1 protein in breast cancer tissue.Methods:1.The TCGA database obtained the sequencing data of normal breast tissue and breast cancer tissue.Analyze and integrate and compare the expression levels of ENOPH1 m RNA in normal breast tissue and breast cancer tissue.2.The breast cancer tissues obtained from the TCGA database were staged(stage 1,stage 2,stage 3,stage 4),and the expression of ENOPH1 gene in normal breast tissues and breast cancer tissues of different stages were analyzed;the breast cancer tissues obtained from the TCGA database Classify according to the state of lymph node metastasis(N0 state,N1 state,N2 state and N3 state),and analyze the expression of ENOPH1 gene in normal breast tissue and breast cancer tissue of each lymph node metastasis state.3.The breast cancer tissues obtained from the TCGA database were divided into two groups(ENOPH1 gene low expression group and ENOPH1 gene high expression group).overall survival rate.4.In order to further verify the expression of ENOPH1 in breast cancer tissues,Western blotting was used to detect the ENOPH1 protein level in breast cancer tissues and matched adjacent normal tissues.The ratio of the gray value of the protein band(ENOPH1/β-actin value)to compare the relative content of ENOPH1 protein.Result:1.The sequencing data of 290 normal breast tissues and 769 breast cancer tissues were obtained using the TCGA database.Analyze and integrate and compare the expression levels of ENOPH1 m RNA in normal breast tissue and breast cancer tissue.The results showed that the expression level of ENOPH1 gene in breast cancer tissue was significantly higher than that in normal breast tissue(P < 0.001).2.The expression of ENOPH1 gene in stage 1(P < 0.001),stage 2(P <0.001),stage 3(P < 0.001)and stage 4(P < 0.01)breast cancer tissue was significantly higher than that in normal breast tissue;Cancer tissues were classified according to the status of lymph node metastasis.The results showed that the expression of ENOPH1 gene in breast cancer tissues of N0state(P < 0.001),N1 state(P < 0.001),N2 state(P < 0.001)and N3 state(P <0.001)The expressions were significantly higher than those in normal breast tissue.3.Kaplan-Meier results showed that the expression level of ENOPH1 was correlated with the overall survival rate of breast cancer patients(HR =1.36),and the survival rate of breast cancer patients with low ENOPH1 gene expression was significantly higher than that of breast cancer patients with high ENOPH1 gene expression(Log-rank P = 0.0096),indicating that the higher the expression level of ENOPH1,the lower the overall survival rate of breast cancer patients.4.The results of Western blot showed that except for the 6 pairs of samples N15/BC15,N28/BC28,N31/BC31,N32/BC32,N33/BC33,and N36/BC36,the expression of ENOPH1 protein in breast cancer tissues in the other 34 pairs of samples The levels were higher than that of adjacent normal tissues.Summary:Analysis of breast cancer tissue sequencing results in TCGA data found that the expression level of ENOPH1 gene in breast cancer tissue was significantly higher than that in normal breast tissue,and it was generally highly expressed in breast cancer tissue of different stages and lymph node metastasis status.In addition,the level of ENOPH1 gene It was negatively correlated with the survival rate of breast cancer patients,and the sequencing results were further verified by western blotting.Part two Effects of ENOPH1 Gene on Biological Behavior of Breast Cancer CellsObjective: Detect the level of ENOPH1 in breast cancer cell lines and normal breast ductal epithelial cells,knock down and overexpress ENOPH1 in breast cancer cell lines,explore the relationship between ENOPH1 expression level and breast cancer cell proliferation,migration,invasion and cell cycle,and clarify the relationship between ENOPH1 and breast cancer cancer cell behavior.Methods:1.The mRNA and protein levels of ENOPH1 in normal mammary ductal epithelial cells(MCF-10A)and 5 different breast cancer cell lines were detected by real-time fluorescent quantitative PCR(q PCR)and Western Blotting.2.In two breast cancer cell lines(BT474 and MCF-7),ENOPH1 gene was knocked down by RNA interference technology,ENOPH1 gene was overexpressed by overexpression vector,ENOPH1 m RNA and protein levels were detected by q PCR and Western blot,and ENOPH1 was evaluated been knocked down and overexpressed.3.CCK-8 was used to detect the proliferation ability of two breast cancer cells after ENOPH1 knockdown and overexpression.4.The proliferation ability of two breast cancer cells after ENOPH1 knockdown and overexpression was detected by EdU incorporation and flow cytometry.5.Western blotting was used to detect the levels of cycle-related proteins in two breast cancer cells after knockdown and overexpression of ENOPH1.6.Transwell assay was used to detect the migration ability of two breast cancer cells after ENOPH1 knockdown and overexpression.7.Transwell assay was used to detect the invasion ability of two breast cancer cells after ENOPH1 knockdown and overexpression.8.Western blotting was used to detect the effects of ENOPH1 knockdown and overexpression on two breast cancer cell migration-related proteins.Result:1.Compared with normal breast ductal epithelial cells,the m RNA and protein levels of ENOPH1 in 5 different breast cancer cells were significantly increased(P < 0.05),indicating that ENOPH1 is highly expressed in breast cancer.2.The results of qPCR and Western blot showed that,compared with the control group,the levels of ENOPH1 in the si RNA-pool group of the two cells were significantly down-regulated(P < 0.001),indicating that ENOPH1 was successfully knocked down;compared with the control group,the OE(overexpression The levels of ENOPH1 in the ENOPH1)group were significantly increased(P < 0.001),indicating that ENOPH1 was successfully overexpressed.3.The results of CCK-8 showed that compared with the control group,the cell proliferation ability of the two kinds of cells after ENOPH1 knockdown was significantly decreased at different time points(P < 0.01);on the contrary,when ENOPH1 was overexpressed,the proliferation ability of the two kinds of cells was significantly increased(P < 0.01).4.The results of EdU incorporation and flow cytometry showed that,compared with the control group,the cell proliferation activity of the two cells was significantly decreased after ENOPH1 knockdown,and the proportion of the S phase of the cells was down-regulated;on the contrary,when ENOPH1 was overexpressed,the two cells Cell proliferation activity was significantly increased,and the proportion of cells in S phase was increased.5.Western blot results showed that,compared with the control group,the levels of two breast cancer cell cycle-related proteins decreased after ENOPH1 knockdown;on the contrary,when ENOPH1 was overexpressed,the levels of two breast cancer cell cycle-related proteins increased.6.The results of Transwell migration assay showed that,compared with the control group,the migration ability of the two breast cancer cells was significantly decreased after ENOPH1 knockdown(P < 0.05);on the contrary,when ENOPH1 was overexpressed,the migration ability of the two breast cancer cells was significantly increased(P < 0.001).7.Compared with the control group,the results of Transwell invasion assay showed that the invasion ability of the two breast cancer cells was significantly decreased after ENOPH1 knockdown(P < 0.001);on the contrary,when ENOPH1 was overexpressed,the invasion ability of the two breast cancer cells was significantly increased(P < 0.001).8.Western blotting results showed that,compared with the control group,the levels of the two breast cancer cell migration-related proteins MMP9 and MMP2 decreased after ENOPH1 knockdown;on the contrary,when ENOPH1 was overexpressed,the levels of the two breast cancer cell migration-related proteins increased.Summary: ENOPH1 is abundantly expressed in a variety of breast cancer cell lines.When the expression of ENOPH1 is reduced,it can hinder the progression of breast cancer cell cycle,reduce the proliferation,migration and invasion of breast cancer cells;when the expression of ENOPH1 is increased,it can promote G1/S transition,Accelerates cell cycle process,promotes proliferation,migration and invasion of breast cancer cells.In general,the expression of ENOPH1 is positively correlated with the viability of breast cancer cells,and ENOPH1 can be used as a target for diagnosis and treatment of breast cancer.Part three Study on the mechanism of ENOPH1’s influence on the biological behavior of breast cancer cellsObjective: To explore the regulatory effect of ENOPH1 on the NF-κB signaling pathway,after ENOPH1 gene silencing or overexpression,investigate the changes in the activity of the NF-κB signaling pathway,and clarify the regulatory relationship between ENOPH1 and NF-κB;study the effect of ENOPH1 on breast cancer through the NF-κB signaling pathway Regulatory mechanism of cancer cell proliferation,migration and invasion,investigate the effect of NF-κB signaling pathway on breast cancer cell behavior,and clarify the mechanism of ENOPH1 regulating breast cancer cell behavior through NF-κB signaling pathwayMethods:1.RNA interference technology was used to knock down the ENOPH1 gene in two breast cancer cell lines BT474 and MCF-7,and Western blot was used to detect the levels of phosphorylated IκBα protein,total IκBα protein,phosphorylated p65 protein and total p65 protein,to assess the effect of ENOPH1 knockdown on the phosphorylation of cascade proteins in the NF-κB signaling pathway.2.Using RNA interference technology to knock down the ENOPH1 gene in two breast cancer cell lines BT474 and MCF-7,and using immunofluorescence staining to observe the nuclear translocation of p65 protein,and evaluate the regulation of ENOPH1 knockdown on p65 protein nuclear translocation effect.3.In two breast cancer cell lines BT474 and MCF-7,the ENOPH1 gene was overexpressed with an overexpression vector,and the levels of phosphorylated IκBα protein,total IκBα protein,phosphorylated p65 protein,and total p65 protein were detected by Western blot,to assess the effect of ENOPH1 overexpression on the phosphorylation of cascade proteins in the NF-κB signaling pathway.4.In two breast cancer cell lines BT474 and MCF-7,the overexpression vector was used to overexpress the ENOPH1 gene,and the nuclear translocation of p65 protein was observed by immunofluorescence staining technique,and the effect of ENOPH1 overexpression on the nuclear translocation of p65 protein was evaluated.Regulation.5.In the breast cancer cell line BT474,the overexpression vector was used to overexpress the ENOPH1 gene,and the NF-κB signaling pathway inhibitor BAY11-7082 was added,and CCK-8 was used to detect the effect of NF-κB signaling pathway inhibition on the proliferation of breast cancer cells.6.In the breast cancer cell line BT474,the overexpression vector was used to overexpress the ENOPH1 gene,and the NF-κB signaling pathway inhibitor BAY11-7082 was added.Transwell assay was used to detect the changes in the migration ability of breast cancer cells after the inhibition of the NF-κB signaling pathway.7.In the breast cancer cell line BT474,the overexpression vector was used to overexpress the ENOPH1 gene,and the NF-κB signaling pathway inhibitor BAY11-7082 was added.Transwell assay was used to detect the changes in the invasion ability of breast cancer cells after the inhibition of the NF-κB signaling pathway.Results:1.Western blot results showed that,compared with the control group,the phosphorylated IκBα and p65 protein levels of the si RNA-pool group in the two breast cancer cell lines BT474 and MCF-7 were significantly decreased(P< 0.05),indicating that ENOPH1 Silencing reduced the phosphorylation efficiency of IκBα protein and p65 protein in breast cancer cells.2.The results of immunofluorescence staining showed that,compared with the control group,the p65 protein in the si RNA-pool group in the two breast cancer cell lines BT474 and MCF-7 stained lighter in the nucleus,indicating that ENOPH1 silencing inhibited p65 in breast cancer cells Nuclear translocation of proteins.3.Western blot results showed that compared with the control group,the phosphorylated IκBα and p65 protein levels in the OE group were significantly increased in the two breast cancer cell lines BT474 and MCF-7(P < 0.05),indicating that ENOPH1 was overexpressed Promoted the phosphorylation of IκBα protein and p65 protein in breast cancer cells.4.The results of immunofluorescence staining showed that compared with the control group,the p65 protein in the OE group stained darker in the nucleus in the two breast cancer cell lines BT474 and MCF-7,indicating that the overexpression of ENOPH1 promoted the p65 protein in breast cancer cells.nuclear translocation.5.The results of CCK-8 showed that the proliferation ability of BT474 cell line in the empty vehicle(EV)+ BAY11-7082 group was significantly lower than that in the EV group,and the proliferation ability of BT474 cell line in the OE + BAY11-7082 group was significantly lower than that in the OE group,indicating that ENOPH1 Overexpression enhanced the proliferation ability of BT474 cell line,and inhibited NF-κB signaling pathway weakened the proliferation ability of BT474 cell line.6.The results of Transwell migration test showed that the cell migration ability of BT474 cell line in EV + BAY11-7082 group was significantly lower than that in EV group,and the cell migration ability of BT474 cell line in OE+ BAY11-7082 group was significantly lower than that in OE group,indicating that ENOPH1 overexpression enhanced BT474 Cell line migration ability,inhibition of NF-κB signaling pathway weakens the migration ability of BT474 cell line.7.The results of Transwell migration test showed that the cell invasion ability of BT474 cell line in the EV + BAY11-7082 group was significantly lower than that in the EV group,and the cell invasion ability in the OE +BAY11-7082 group was significantly lower than that in the OE group,indicating that ENOPH1 overexpression enhanced BT474 Cell line invasion ability,inhibiting NF-κB signaling pathway weakens the invasion ability of BT474 cell line.Summary: ENOPH1 promotes the phosphorylation of IκBα protein and p65 protein in NF-κB signaling pathway in BT474 and MCF-7 cell lines,and promotes the nuclear translocation of p65 protein,thereby promoting the activation of NF-κB signaling pathway.Inhibition of NF-κB signaling pathway can reduce the proliferation,migration and invasion ability of breast cancer cells.Therefore,ENOPH1 regulates the progression of breast cancer by mediating the activation of NF-κB signaling pathway.Conclusions:1.The expression level of ENOPH1 gene in breast cancer tissue was significantly higher than that in normal breast tissue.2.The level of ENOPH1 gene is negatively correlated with the survival rate of breast cancer patients.3.When the expression of ENOPH1 is reduced,it can hinder the progress of the breast cancer cell cycle,and the proliferation,migration and invasion of breast cancer cells are reduced;when the expression of ENOPH1 is increased,it can promote the G1/S transition,accelerate the cell cycle process,and promote the proliferation of breast cancer cells,migration and invasion.4.ENOPH1 promotes the phosphorylation of IκBα protein and p65 protein in NF-κB signaling pathway in BT474 and MCF7 cell lines,and promotes the nuclear translocation of p-p65 protein,thereby promoting the activation of NF-κB signaling pathway.