Downstream Gene Expression Of Mir-210 In Breast Cancer Cell Control Pathway

Objective: The incidence and mortality of breast cancer in the world ranks first among women with malignant tumors.The annual incidence of new cases is about 1.7 million(25% of new female cancer cases).It is the most common cancer among women.In recent years,the incidence and death of breast cancer have become more common.The rates have shown a clear upward trend,and there has been a trend of youthfulness,which seriously jeopardizes women's life safety.Mir-210 is located in the human genome coding site(chr11:568089-568198)and is closely related to the hypoxic pathway.It is a classic hypoxia-responsive microRNA and cancer cells can maintain rapid proliferation under hypoxic conditions.mir-210 is strongly and ubiquitously upregulated in hypoxic cells,plays an important role in the differentiation of breast cancer cells,and participates in the proliferation,invasion,metastasis,and poor prognosis of breast cancer tumors.Therefore,by exploring mir-210 in the breast Up-regulated expression and related mechanisms in cancer cells are expected to achieve certain results in the treatment of breast cancer,efficacy evaluation,and prognosis prediction.In this study,the effect of mir-210 expression on the expression of downstream gene proteins in breast cancer was studied and the relationship between mir-210 expression and downstream genes of breast cancer cell control pathways was explored to further explore mir.-210 possible intervention mechanisms such as breast cancer cell proliferation,migration and invasion provide ideas and references for subsequent basic and clinical research.Methods :(1)Screening microRNAs that are aberrantly expressed in breast cancer and are statistically significant.Using the Perl(http://www.perl.org/)software and R(https://www.r-project.org/)software on the breast in the TCGA(https://cancergenome.nih.gov/)database Cancer data were screened and differentially expressed and statistically significant microRNAs were detected in TCGA database breast cancer samples.By downloading the high-throughput breast cancer high-throughput sequencing-microRNA expression profile data online,the microRNA expression profile data of breast cancer samples were obtained.(2)Screening the pubmed database for downstream genes that have been confirmed by other studies to be antagonistic to the target microRNA,and searching for cell lines that highly express the gene by The Human Protein Atlas(https://www.proteinatlas.org/)for Western blot experiments.Positive reference.(3)The human breast cancer MDA-MB-231 cell line was stored in liquid nitrogen from the cell bank.After cell resuscitation,adherent culture,and passage,a uniform cell suspension was prepared and counted and diluted.(4)The candidate microRNAs were transiently transfected into target breast cancer cell lines by Lipofection,and the transfection effect was detected by Real-time qPCR.Trizol reagent extracted RNA cell line,TOYOBO reverse transcription kit reverse transcribes the collected RNA,and reverse transcription makes the upstream and downstream primers of microRNA used as gene-specific primers designed by Stem-Loop Method.U6 is used as an internal reference.,Then use qPCR reaction system for amplification,amplification with primers,PCR instrument fluorescence quantitative determination.(5)Use RIPA lysate reagent to extract total protein,and use Microplate Reader to analyze and calculate the sample protein concentration.(6)The sample to be tested is taken out,Western-blot technique is used,sample loading,transfer membrane,anti-MNT,anti-GAPDH,etc.,and the enhanced chamber illumination(Enhanced Chemiluminecence,ECL)is used to develop the film on the reaction substrate in a dark room.Fixing and gel imaging scans.The transcription level of candidate microRNAs and the expression level of downstream gene proteins in breast cancer cell lines after transfection were examined.Through comparison,the levels of candidate microRNAs in breast cancer cell lines and their changes in downstream gene expression levels were examined.(7)Genes differentially expressed in the transfected breast cancer cell line were analyzed by comparison of gene expression profiles based on cDNA sequencing.Results:(1)After R software analysis,1103 cases from TCGA.The microRNAs in the breast cancer tissue micro RNA expression profile samples were screened out for statistically significant microRNAs.Among them,301 were highly expressed and 92 were lowly expressed.(2)mir-210 ranked 13 th in the high expression group,FDR(False Discovery Rate)was 2.81E-51,and log FC(log2 Fold Change)was about 3.2176,which was statistically significant.Track the mirBase(http://www.mirbase.org)database information on the mirBase Tracker(http://www.mirbasetracker.org/)website and update the mir-210 gene name to mir-210-3p and select from it.The mir-210 downstream gene MNT was screened by The Human Protein Atlas website as a positive reference group for HeLa cell line with high expression of MNT.(3)The growth of MDA-MB-231 breast cancer cells and Hela cells was good.The cell lines rapidly entered the logarithmic phase of cell growth after resuscitation.The passage or cryopreservation cycle was approximately 1 day apart.Total RNA was extracted from each group with a concentration of more than 1.5 ?g/?l and the A260/280 values were between 1.8 and 2.0.The expression of mir-210-3p in mir-210-3p Inhibitors transfected group and mir-210-3p Inhibitors Negative Control transfected group was lower than that in control group by Real-time qPCR assay.Inhibitors group The expression level of mir-210-3p was decreased to 0.037-fold in the control group.(4)Total protein of MDA-MB-231 breast cancer cells in the control group,the mir-210-3p Inhibitors Negative Control transfection group,and the mir-210-3p Inhibitors transfection group,which were cultured for 48 hours after transfection,were extracted.After stained by Coomassie Blue Staining(CBS),the sample concentrations measured by the Microplate Reader were: 5.286/5.479/3.645 ?g/?l.The positive reference group Hela cell total protein sample measured a concentration of 7.184 ?g/?l.40 ?g of total protein was quantified in each sample.The expression of MNT protein was detected by Western-blot,the expression of MNT protein was low in the control group,and the expression of MNT protein in the mir-210-3p Inhibitors Negative Control transfection group and the mir-210-3p Inhibitors transfection group.The average level of water increased,and the Inhibitors group was more pronounced than the other two groups,which was consistent with the Real-time qPCR results.(5)Sequencing RNA sample number 1(control group),number 2(Inhibitors transfection group)quantification and purity A260/A280 were 1.9 and 1.92,respectively,A260/A230 were 2.34 and 2.15,respectively.The RIN values were 10.50 and 9.90,respectively.The quality test results passed and the sample integrity was good enough for the chip experiment.(6)Through the comparison of cDNA chip expression profiles,a total of 316 differentially expressed genes were screened,of which 176 were up-regulated and 140 were down-regulated.(7)Signal pathway enrichment analysis of KEGG: Among the MDA-MB-231 breast cancer cell lines transfected with mir-210-3p Inhibitors,the differentially expressed genes were mainly in Transcriptional misregulation in cancer,MAPK(Enrichment occurs in the Mitogen-Activated Protein Kinase(MAPK)signaling pathway and the Chemokine Signaling Pathway signaling pathway.(8)Gene Ontology(GO)Enrichment Analysis: Differentially Expressed Genes Affect Chemokine Activity,Transcription Factor Binding,Transcription Regulatory Region DNA Binding,Transcriptional Activator Activity,and RNA Polymerase II Core Promoter Proximity The specific binding of the region sequence,differential gene products are located in the nuclear chromomatin,plasma membrane,plasma membrane components,anchored component of the membrane,CHOP-ATF3 complexes and protein complexes.Conclusion:(1)The overall expression of mir-210 in breast cancer was obtained by bioinformatics analysis;(2)316 differentially expressed genes were screened in breast cancer cells by mir-210 expression level analysis through gene expression profiling.(3)Enrichment analysis showed that there are chemokine signaling pathways,MAPK signaling pathways,and oncogenic error transcription pathways in the differentially expressed gene-related signaling pathways;differential gene products are located in the nuclear chromomatin,plasma membrane,plasma membrane module,and membrane anchoring module.,CHOP-ATF3 complexes,protein complexes;(4)mir-210-3p level changes affect breast cancer cell multiple signaling pathways and downstream gene expression levels.