| Purpose: Statistics of breast cancer in 2021 shows that the global incidence of cancer in women is 8.6 million and the number of cancer deaths is 4.2 million.Of these,breast cancer is the most common malignancy in women,with approximately20% experiencing distant organ metastases.The most aggressive triple-negative breast cancer(TNBC)is a heterogeneous group of tumors characterized by aggressive behavior,high risk of distant recurrence,and low survival rates.Chemotherapy remains the primary treatment for this subset of patients,and triple-negative breast cancer is a clinical challenge due to the lack of expression of validated predictive markers estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor-2(HER-2),and therefore the lack of clear therapeutic targets.patients with TNBC do not benefit from endocrine or HER-2 targeted therapy,with a worse prognosis after chemotherapy compared to patients with other breast cancer subtypes.As a potential solution,mi RNAs have been proposed as a promising biomarker for breast cancer.Their role as an emerging group regulating gene expression has been recognized,playing an important role in several biological processes(e.g.,cell proliferation,apoptosis,proliferation,cell cycle progression,and organ development).In order to find new biomarkers with potential clinical application,this study intends to collect TNBC clinical samples,identify and screen differentially expressed mi RNAs in cancer tissues by combining sequencing and bioinformatics analysis,to search for breast cancer specific mi RNAs,providing theoretical basis and new research ideas for the development of breast cancer diagnosis and treatment.Materials and methods: 1.By collecting TNBC samples,the cancer tissues and adjacent tissues of TNBC cases collected clinically were detected by mi RNA sequencing,and the genes with significant differential expression were screened and enriched by bioinformatics technology;2.Analysis of differences in mi RNA expression abundance in cancer and adjacent tissues were screened out for further functional verification;3.The effect of target mi RNA on cell proliferation was detected by CCK-8 assay;4.The effect of target mi RNA on the size of the scratch area was verified by scratch assay;5.Detect the regulation of the target mi RNA on cell invasion and migration ability by transwell assay;6.Detect the regulation of the target mi RNA on apoptosis and cell cycle by flow cytometry;7.Observing the effect of target mi RNA on the expression of key AKT-m TOR proteins by Western Blot;8.Using RNA co-precipitation technology to enrich the effector target RNA of target mi RNA by magnetic beads for functional analysis;9.Constructing TNBC tumor-bearing mice model to study the effect of target mi RNA on tumor proliferation in experimental animals;10.Record the effect of tumor size and body weight in experimental animals under the regulation of target mi RNA for providing the regulation pattern of target mi RNA;11.Use immunohistochemical staining to analyze expression of cell proliferation-related gene Ki-67 in tumors.Results: In the first chapter,the author collected clinical samples of TNBC,examined cancerous tissues of clinically collected TNBC cases and adjacent tissues by mi RNA sequencing,screened and enriched genes with significant differential expression using bioinformatics techniques,and categorized potential signaling regulatory pathways and regulated life activities of differential genes using KEGG database and GO database,in order to find genes with The KEGG database and GO database were used to categorize the potential signaling pathways and regulated life activities of the differential genes to find biomarkers potentially involved in and regulating TNBC progression.Among the sequenced samples,the lysosomal function pathway,calcium-mediated signaling,epithelial signaling pathway,galactose metabolic pathway and platelet activation signaling pathway were enriched at the top of each life activity by KEGG analysis.Among the enrichment results of signaling pathways,calcium signaling pathway showed the highest relevance.The c AMP pathway and the c AMP-PKG pathway downstream of this pathway were also in the top,indicating that these pathways are highly differentially regulated.In the GO enrichment results,the aberrant calcium flow signaling pathway was once again shown to be related to the involvement of calcium in multiple signaling pathways that regulate cell cycle,migration,proliferation and differentiation,a result that suggests the involvement of calcium downstream pathways in the progression of TNBC.Analysis of the differences in mi RNA expression abundance in cancer and adjacent tissues identified 1540 mi RNAs that underwent expression differences,including 32 mi RNAs that underwent significant downregulation and 33 mi RNAs that were significantly upregulated.Based on the clustering results and a comprehensive consideration of the literature survey,hsa-mi R-18a-5p,hsa-mi R-135a-5p,and hsa-mi R-204-5p and hsa-mi R-934 were screened out and fluorescent real-time PCR was implemented in clinical samples to further narrow down the study.The results demonstrated that the expression of hsa-mi R-18a-5p showed a significant down-regulated region in the cancer and adjacent tissues of 10 patients,while the expression of the other three screened hsa-mi R-135a-5p,hsa-mi R-204-5p and hsa-mi R-934 did not show significant differences in the adjacent and cancer tissues,therefore,this paper selected Therefore,hsa-mi R-18a-5p was selected as the main topic of this paper,and the regulatory role of hsa-mi R-18a-5p and its potential mechanisms will be explored through in vitro and in vivo experiments.In the second chapter,the author mainly uses virous biological technologies,such as viability assay,cell scratch assay,transwell assay,clone formation assay,flow cytometry assay for apoptosis and cell cycle and immunoprecipitation,as well as animal models of tumor-bearing mice to verify the regulation mode of candidate mi RNAs on cellular activities.In the CCK-8 assay for cell viability,transfection of mimics will show a slowdown of proliferation,while transfection of inhibitor will lead to an acceleration of cell proliferation.The results of this assay suggest that when cells are stimulated,increasing the expression of hsa-mi R-18a-5p will inhibit cell proliferation;conversely,when deletion of hsa-mi R-18a-5p occurs,the cells will lose the regulation of proliferation and thus exhibit stronger viability.In the cell scratch assay,high expression of hsa-mi R-18a-5p showed inhibition of proliferation and viability;conversely,MDA-MB-231 cells showed higher cell activity when hsa-mi R-18a-5p expression was reduced,as evidenced by accelerated cell proliferation and enhanced migration ability.The results of transwell shown that hsa-mi R-18a-5p could negatively regulate the migration and invasion ability of MDA-MB-231 cells,and the results of this assay strongly demonstrated that hsa-mi R-18a-5p expression was inversely correlated with the capacities of migration and invasion of cancer cells.Apoptotic results conducted by flow cytometry showed that the incidence of apoptosis in MDA-MB-231 cells transfected with mimics was significantly higher compared with the control group,initiating the cellular death pathway and leading to an increase in the rate of apoptotic cells.Conversely,inhibitor inhibition would reduce the rate of apoptotic cells,indicating that the signaling pathway of programmed cell death could be terminated by down-regulated hsa-mi R-18a-5p.In cell cycle assays,the cell ratio in G1 phase of MDA-MB-231 cells was significantly upregulated after transfection with mimics compared to the control group,and the cell ratio in S phase and G2 underwent downregulation,demonstrating that the mitotic process of MDA-MB-231 cells was regulated and the cell proliferation rate was downregulated.Using protein immunoblotting to detect the cell cycle-related regulatory pathway PI3K-AKT-m TOR,it was found that AKT and m TOR were dephosphorylated in response to cellular stimulation after transfection with mimics;E-calmodulin expression was significantly up-regulated compared to the control,while N-calmodulin,which was highly expressed in the cytoplasm,was down-regulated by transfection with mimics;the agonist of PI3 K,740Y-P was able to reverse the cellular activity brought about by mimics transfection;Inhibitor and PI3 K inhibitor LY294002 showed completely opposite functions.RNA immunoprecipitation experiments showed that hsa-mi R-18a-5p and SLC12A6 were effectively enriched,indicating that they regulated cell cycle signaling pathways through binding to effector proteins,mainly in the form of negative feedback.This is mainly in the form of negative feedback to control cell proliferation,migration,apoptosis and other life activities.To complete the functional study of hsa-mi R-18a-5p,a tumor-bearing mouse model based on MDA-MB-231 cells was used to further investigate the regulatory function of this gene in vivo.In the tumor size observation experiments of the tumor-bearing mice,the intratumorally injection of mimics could effectively inhibit the growth of tumors afterwards,however,inhibitor played the opposite role,and its growth rate was much larger than that of the positive control group after the tumors were treated with the intratumorally injection of inhibitor,demonstrating that the high expression of hsa-mi R-18a-5p could significantly hinder the growth of tumor cells,while The deletion or inhibition of hsa-mi R-18a-5p expression would cause rapid proliferation of cancer cells.In immunochemical staining results,mimics-treated tumors expressed less Ki-67,indicating that mimics intratumoral injection treatment effectively attenuated the proliferation of MDA-MB-231 cellsConclusion: In this study,the potential biomarker hsa-mi R-18a-5p was screened from clinical needs by collecting and analyzing TNBC samples using RNA sequencing technology and bioinformatics research methods.hsa-mi R-18a-5p was subsequently validated from in vitro and ex vivo experiments to demonstrate the regulatory function of hsa-mi R-18a-5p on tumor progression,and the expression of hsa-mi R-18a-5p was shown to The regulatory pattern was that high expression of hsa-mi R-18a-5p significantly hindered the growth of tumor cells,while deletion or inhibition of hsa-mi R-18a-5p expression caused rapid proliferation of cancer cells,and this regulatory pattern was achieved by regulating PI3K-AKT-m TOR pathway. |
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