Study Of Diagnostic Markers In Exosomes Of Three-Adimensional Breast Cancer Cell Model

Objective Breast cancer is the most prevalent malignancy in women.Due to the high morbidity and mortality rate and the ease of metastasis,breast cancer poses a threat to the life and health of women.With the continuous development of modern medical technology,the treatment of breast cancer has achieved remarkable results.The 5-year survival rate for patients with early-stage breast cancer is more than ninety percent,and the 5-year survival rate for patients with stage II and III breast cancer can be greater than fifty percent,but the 5-year survival rate for patients with advanced breast cancer is less than thirty percent^[1].Although the diagnosis and treatment of breast cancer is constantly improving and updating,early detection,early diagnosis and early treatment are still the most effective and convenient strategies to prolong the survival time and improve the prognosis of patients.In the past decades,tumor markers such as CEA（carcinoembryonic antigen）,AFP（alpha fetoprotein）,CA-125、CA-155（carbohydrate antigen）,have been discovered and have been used in clinic practice.However,these tumor markers lack sensitivity and specificity.Therefore,it is urgent to find more biomarkers that can help to detect diseases in the early stage.Exosomes are small extracel ular vesicles（30～120 nm）released by cels^[2].The vesicles contain cel-derived substances such as protein,lipids,DNA,m RNA and nc RNA^[3].They can participate in intercellular communication and participate in pathophysiological processes such as immune response,cell proliferation and differentiation in vivo^[4].Micro RNAs（miRNAs）are a group of endogenous and evolutionarily conserved single stranded noncoding micro RNAs.They are key regulators in a variety of biologica l processes of cels and play a role in the occurrence,development and metastasis of tumors^[5,6].The secrete vesicles released by cells contain different miRNAs,which can play a role in the regulation of human gene expression.They have high value in tumor diagnosis and can be used as potential tumor markers.The traditional two-dimensional cell culture system has been widely used in experimental studies such as cell proliferation,apoptosis and cytotoxicity.It is considered to be one of the most effective methods to study tumors at the cellular level.The research of tumor markers in vitro is mostly carried out under the traditional two-dimensional culture conditions,but there are some recognized shortcomings in two-dimensional monolayer cell culture:1.Lack of physiological characteristics of real tissue,2.The conditions for cell growth are simplified.Compared with the three-dimensional cell culture system,the conclusions drawn from the two-dimensional cell culture experiment may be different from the in vivo environment,which can not provide tumor cels with a physiological microenvironment similar to that in vivo,and can not accurately reflect the complexity of cell-cell interaction.Therefore,three-dimensional cell culture,which is closer to the in vivo environment,is gradually being applied to tumor research.The purpose of this study is to explore the difference of miRNA expression in breast cancer cell exudate by culturing breast cancer cells,and to investigate the difference in expression of exocrine miRNA between breast cancer patients and normal subjects,and explore the possibility of exocrine miRNA as a new biomarker for diagnosing breast cancer.Methods The normal breast cell（MCF-10A）,human breast cancer cell line（MCF-7）,drug-resistant breast cancer cell line（MCF-7-Taxo）and human triple negative breast cancer cell line（MDA-MB-231）were selected as experimental subjects.Firstly,cell culture was performed using different cell culture methods to observe the differences.Cellular exosomes from cell culture supernatants were extracted for miRNA sequencing analysis,and miRNAs with differential expression were selected after statistical analysis of the sequencing results.Next,breast cancer cels were cultured,and exosomal miRNAs were extracted from different cell culture supernatants,reverse transcribed into c DNA and then verified the expression levels using q RT-PCR technique.The differences in the expression of target miRNAs were compared between cels in three-dimensional culture and normal two-dimensional culture conditions.Finally,exosomal miRNAs were extracted separately from blood samples of clinical breast cancer patients and normal humans to verify whether the expression differences of cellular exosomal miRNAs had the same expression differences and trends in clinical specimens.Results Cellular exosomes from cell culture supernatants were extracted for miRNA sequencing analysis,and statistical analysis of the sequencing results identified four differentially expressed miRNAs:mi R-193-5p,mi R-196-5p,let-7c-5p,and mi R-10a-5p,and they were studied as target miRNAs.The expression of the target miRNAs in breast cancer cels MCF-7,MCF-7-Taxo,and MDA-MB-231 was significantly different from that of MCF-10A in two-dimensional culture with an increasing trend.The target miRNAs in MCF-7,MCF-7-Taxo,and MDA-MB-231 were expressed in a more significant upward trend in three-dimensional culture than in two-dimensional culture.The expression of the four miRNAs in peripheral blood of clinical breast cancer patients was significantly higher than that in normal human peripheral blood and statistically different（P<0.05）,which was the same trend as that in cell culture.Taken together,the results of this study suggest that all four miRNAs have potential as clinic al diagnostic markers for breast cancer.Conclusion 1.The 3D culture model is closer to the in vivo environment and has potential as a new tool for breast cancer research.2.miRNAs differentially expressed in breast cancer cell lines and normal breast cells:mi R-196a-5p,mi R-193a-5p,let-7c-5p,mi R-10a-5p were screened by statistical analysis of the sequencing results for study.3.Objectives miRNA expression in cell culture showed the same trend as in the sequencing results and was significantly higher in three-dimensional culture than in two-dimensional culture（P<0.05）.4.The expression of target miRNAs in peripheral blood of both normal subjects and breast cancer patients was statistically different（P<0.05）,and four miRNAs were found to have some diagnostic ability in breast cancer.