Decreased MiR-124-3p Promoted Breast Cancer Proliferation And Metastasis By Targeting MGAT5

Breast cancer(BC)is one of the malignant tumors that threaten women's health and quality of life worldwide.Its incidence is increasing,and proportion in various cancer-related deaths is increasing.Through the in-depth understanding of breast cancer development and drug resistance mechanisms,the development of new therapeutic targets,down-regulation of drug resistance,increase the coverage of treatment,to improve the disease-free survival rate and overall survival rate of breast cancer population,will still be the key research direction of breast cancer treatment.MGAT5 is an important polysaccharide chain processing enzyme distributed in the Golgi apparatus.It can change the sugar chain structure of cell surface glycoproteins such as cadherin,integrin and cell surface growth factor receptor,affecting malignant transformation and tumor metastasis of cells.In our previous study,tissue microarray technology was used to construct 12×10breast cancer tissue array blocks,and LPA-labeled MGAT5 activity was detected by lectin blotting.The results showed that the expression of MGAT5 in breast cancer tissues was significantly higher than that of benign lesions.Normal breast tissue(P<0.01),MGAT5 expression was increased in breast cancer,highly correlated with poor prognosis,and high expression in various pathological types of breast cancer,no statistically significant difference.Studies have shown that miRNA is closely related to the occurrence of various tumors,which can be used as a tumor suppressor gene to down-regulate the activity of proto-oncogenes.It can also be used as an oncogene to down-regulate the activity of tumor suppressor genes.This will also make miRNA possible as a new biological marker for disease diagnosis,and may also make this molecule a drug target,or simulate this molecule for new drug development,which may provide a new means for the treatment of diseases.In this study,we used bioinformatics analysis and classical molecular biology techniques to study the MGAT5 gene that promotes the development of breast cancer in clinical samples,molecular levels,cell levels,and animal in vivo experiments.The expression of miRNA molecules,and in-depth study of the mechanism of interaction between them,as well as the impact on the proliferation and metastasis of breast cancer cells,the impact on the prognosis of breast cancer patients.?.Breast cancer tissue samples confirmed that miR-124 was negativelycorrelated with MGAT5 expression,and low expression of miR-124 was associated with poor prognosis in breast cancer patients.Methods:(1)Collect breast cancer and normal breast tissue samples from patients with breast cancer surgery in Changhai hospital,extract total RNA from tissue samples collected,obtain cDNA by reverse transcription,and quantitatively detect MGAT5 in tissue samples by qRT-PCR.mRNA expression level;2)extraction of total protein from tissue samples,Western Blot assay for expression of MGAT5 protein;(3)bioinformatics analysis predicts possible binding site microRNAs with MGAT5-3'-UTR Synthetic miRNAs mimics and interfering agents;(4)construction of a double luciferase plasmid(WT)and a mutant site plasmid(MUT)comprising a MGAT5 3'-UTR region comprising a miRNAs binding site sequence,dual fluorescence The enzyme assay confirmed the binding efficiency of MGAT5 to miRNAs;(5)mimics,inhibitor and negative control molecules of miRNAs transiently transfected breast cancer cell lines MDA-MB-231,MCF-7,extracted total RNA and total protein,detected MGAT5 Changes in mRNA and protein expression levels;(6)cDNA obtained from reverse transcription of total RNA in tissue samples,qRT-PCR quantitative detection of miRNAs expression in tissue samples;(7)Bioinformatics analysis of miRNAs and breast cancer Prognosis;(8)Coefficient of correlation MGAT5 correlation with the expression level of miRNAs in clinical specimens.Results:(1)We collected 20 pairs of breast cancer and normal breast tissue samples.qRT-PCR quantitative detection showed that the expression of MGAT5 mRNA in breast cancer tissues was significantly higher than that in normal breast tissues(P<0.001);(2)Extracted tissues The total RNA of the sample,Western Blot experiment,found that the expression level of MGAT5 protein was significantly higher than that of normal breast tissue;(3)we predicted the presence of microRNAs at the Targetscan website that may be associated with the presence of MGAT5-3'-UTR,and found that has been searched by the literature.-miR-124-3p(miR-124)and has-miR-101-3p(miR-101)and MGAT5 3'-UTR may have a highly conserved binding site and negatively correlated with the expression of MGAT5,constructing synthetic Simulated mimics and interfering agents;(4)Construction of a double luciferase plasmid(WT)and a mutant site plasmid comprising a MGAT53'-UTR region comprising a miR-124,miR-101 binding site sequence(MUT),293 T cells were co-transfected with miR-124,miR-101 mimics,and negative control NC,respectively,and fluorescence values ??were measured 48 hours later.The results showed that the fluorescence value of miR-124 cells was significantly lower than that of the control group(P<0.001),but there was no significant difference between miR-101;(5)mimic,inhibitor and negative control molecules of miR-124 transiently turned into mammary gland After the cancer cell line,the total RNA and total protein were extracted,and the results of RT-PCR and Western Blot showed that the expression of MGAT5 mRNA and protein was significantly decreased after overexpression of miR-124,and the expression of miR-124 was inhibited.The expression of MGAT5 mRNA and protein increased significantly.(6)The expression of miR-124 was significantly lower in breast cancer tissues than in normal breast tissues(P<0.001).(7)The relationship between the expression of miR-124 and the overall survival rate(OS)of 1262 breast cancer patients in the METABRIC database was analyzed by the online bioinformatics tool Kaplan-Meier plotter,and the expression level of miR-124 was found to be high.Patients had better survival benefit compared with patients with low miR-124 expression levels,P<0.05;(8)20 pairs of breast cancer and normal breast tissue miR-124 and MGAT5 mRNA expression levels were performed.Correlation analysis revealed a significant negative correlation between the two,r =-0.5815,P <0.01.Conclusion: In clinical tissue samples,miR-124 is less expressed in breast cancer than in normal breast tissue,and MGAT5 is highly expressed in breast cancer,which is negatively correlated.Biological informatics analysis and cell experiments confirmed that there is an effective binding site between miR-124 and MGAT5,and there is a negative phase regulation relationship between them.At the same time,miR-124 is positively correlated with the survival benefit of breast cancer patients.?.miR-124 inhibits proliferation and migration of breast cancer cellsMethods:(1)Transient transfection of miR-124 mimetic,inhibitor and corresponding negative control molecule(miR-124 mimic,mimic NC,miR-124 inhibitor,Inhibitor NC)to MDA-MB-231 and MCF-7(2)CCK-8 cell viability assay to detect the viability of each group of cells;(3)EDU cell proliferation assay to detect the proliferation of breast cancer cells in each group;(4)clone formation assay to detect cells of different transfection groups Clonal formation ability;(5)Transwell assay to detect the migration ability of cells in different transfection groups;(6)Scratch test to evaluate the ability of each group to scratch the cells.Results:(1)CCK-8 results: overexpression of miR-124 significantly reduced the viability of breast cancer cells compared to cells in the mimic NC group.In contrast,inhibition of miR-124 compared to cells in the Inhibitor NC group the expressionsignificantly increased the cell viability of breast cancer cell lines;(2)EDU experiments were counted by fluorescence microscopy,and the statistical results showed that overexpression of miR-124 significantly reduced MDA-compared with cells of the negative control group.Proliferative capacity of MB-231 and MCF-7 breast cancer cells.In contrast,inhibition of miR-124 expression significantly increased the proliferative capacity of breast cancer cell lines MDA-MB-231 and MCF-7 cells;(3)clone formation experiments demonstrated that overexpression of miR-compared to the control group The number of clones formed in 124 groups was significantly reduced,and the expression of breast cancer cells was significantly more than that of the control group after inhibiting the expression of miR-124.(4)Transwell results suggest that cells overexpressing miR-124 group and negative control Compared with the group,the number of cells passing through the small holes decreased significantly,while the number of cells interfering with the cells in the miR-124 group increased significantly compared with the control group;(5)The results of the scratch test indicated that: Compared with the over-expressed miR-124 group,the cell scratch healing ability was significantly decreased.On the contrary,the expression of miR-124 was inhibited,and the cell scratch healing ability was significantly increased.Conclusion: miR-124 has an inhibitory effect on the proliferation and migration of breast cancer cells.?.miR-124 inhibits the proliferation and migration of breast cancer cells by targeting MGAT5 expression.Methods:(1)construct lentiviral vector for miR-124,MGAT5 overexpression and interference plasmid;(2)transfect the lentiviral vector into MDA-MB-231 and MCF-7cells to utilize ampicillin resistance,screening stable cell lines;(3)CCK-8 cell viability assay to detect the viability of each group of cells;(4)EDU cell proliferation assay to detect the proliferation of breast cancer cells in each group;(5)clone formation test detection different Cloning ability of the cells in the staining group;(6)Transwell assay to detect the migration ability of cells in different transfection groups;(7)Scratch test to evaluate the ability of each group to scratch the cells.Results:(1)We co-transfected and screened eight stable cell lines: miR-124,MGAT5,miR-124 + MGAT5,NC,miR-124 inhibitor,sh-MGAT5,miR-124 inhibitor + sh-MGAT5,and sh-NC;(2)CCK-8,EDU,and colony formation experiments demonstrated that the proliferation of breast cancer cells was significantly decreased after overexpression ofmiR-124 alone,but at the same time overexpressing miR-124 and MGAT5,mammary gland The proliferation ability of cancer cells was significantly higher than that of miR-124 alone.(3)CCK-8,EDU,and colony formation experiments confirmed that miR-124 and MGAT5 were interfered at the same time,compared with cells that interfered with miR-124 alone.The proliferation ability of cancer cells decreased significantly.(4)Transwell assay and scratch test results indicated that the migration ability of breast cancer cells decreased significantly after overexpression of miR-124,but at the same time overexpressed miR-124 and MGAT5,breast cancer the migration ability of cells was significantly higher than that of miR-124 alone.(5)Transwell assay and scratch test results indicated that miR-124 and MGAT5 were interfered at the same time,and migration of breast cancer cells was compared with cells interfering with miR-124 alone.Significant decline in capacityConclusion: These cellular functional remediation experiments fully demonstrate that upregulation of MGAT5 expression can counteract the inhibitory effect of overexpression of miR-124 on proliferation and migration of breast cancer cells;down-regulation of MGAT5 can counteract the interference of miR-124 expression on breast cancer cell proliferation and migration the promotion of ability.In other words,miR-124 can effectively inhibit the proliferation and migration of breast cancer cells,and this effect is achieved by inhibiting the expression of the oncogene MGAT5.MGAT5 is a target of miR-124 inhibiting the proliferation and migration of breast cancer cells,that is,the down-regulation of miR-124 expression,which leads to the high expression of the oncogene MGAT5,which promotes the proliferation and migration of breast cancer cells and ultimately leads to breast cancer.The patient's poor prognosis.IV.Subcutaneous tumor-bearing experiments in nude mice demonstrate that miR-124 can also inhibit the proliferation of breast cancer cells by targeting MGAT5 in animals.Methods:(1)4-6 weeks old BALB/C female nude mice were purchased as tumor-bearing carriers for breast cancer cells;(2)8 groups of stable transgenic cells were subcultured,subcultured;(3)second pair of mammary glands in mice The tumor cell suspension was injected into the fat pad;(4)the tumor size was measured every 3 days,and the mice were sacrificed by cervical dislocation after 4-6 weeks to dissect the tumor;(5)the tumor growth curve was calculated after measuring the long and short diameter of the tumor.Results:(1)The overexpression group showed that the tumor growth rate was the fastest after overexpression of MGAT5 compared with the NC group,and the tumor growth rate was the slowest and the smallest in the miR-124 group.The tumor growth rate of miR-124+MGAT5 group was significantly faster than that of miR-124 group,and there was no significant difference with NC group.(2)The interference group showed that the sh-MGAT5 group had the smallest tumor volume and the slowest growth rate,while the miR-124 inhibitor group was completely opposite,with the fastest tumor growth and the largest volume.The growth rate of tumors in miR-124 inhibitor+sh-MGAT5 group was significantly slower than that in miR-124 inhibitor group,and there was no significant difference between shR group and sh-NC group.Conclusion: In vivo experiments in mice also confirmed that miR-124 affects the proliferation and migration of breast cancer cells by regulating the expression of MGAT5.This study validated the targeted regulation of miR-124 on MGAT5 from clinical tissue samples,bioinformatics analysis,cell level and animal level multi-level and multi-discharge,and the molecular biobiological aspects of breast cancer development and development.The relationship will provide new molecular targets for the diagnosis and treatment of breast cancer,and provide new ideas for the development of new targeted drugs in the future,thus helping to improve the prognosis and quality of life of breast cancer patients.