| Objective: Exosomes are messengers of intercellular signal transduction,transmitting genetic information materials such as mi RNAs through endocytosis and exocytosis,thereby regulating the tumor microenvironment and participating in the occurrence and development of tumors.The mi RNAs encapsulated in the exosome vesicles play an important role in tumor proliferation,colony formation,invasion,and migration.In order to study the diagnostic value and biological mechanism of mi RNAs secreted in breast cancer,based on the previous gene sequencing results and database information of the research group,the bioinformatics analysis was used to screen the mi RNAs with significant differential expression in breast cancer: mi R-146a-5p,mi R-192-5p.Firstly,the differential expression of mi R-146a-5p and mi R-192-5p was verified at the supernatant exosomes level in cell culture.Secondly,the expression levels of mi R-146a-5p and mi R-192-5p in the peripheral serum of healthy people and breast cancer patients were detected,and the difference between healthy people and breast cancer patients was compared.The receiver operating characteristic curve(ROC)was drawn to evaluate the clinical diagnostic value of mi R-146a-5p and mi R-192-5p in the serum secretion for breast cancer.According to the pathological detection results,breast cancer patients were divided into invasive breast cancer and non-invasive breast cancer.The expression levels of mi R-146a-5p and mi R-192-5in the peripheral serum of healthy people were compared to evaluate the clinical diagnostic value of invasive breast cancer and non-invasive breast cancer.Thirdly,mi R-146a-5p and mi R-192-5p in the exosomes body were analyzed jointly with the commonly used clinical tumor markers CEA,CA125 and CA15-3 of breast cancer to evaluate the diagnostic ability.Finally,by inhibiting/overexpressing mi R-146a-5p and mi R-192-5p,we studied the effects on the proliferation,colony formation,invasion and migration of breast cancer cell MCF-7 and triple negative breast cancer cell MDA-MB-231,and predicted the possible signal pathways and downstream target genes involved in regulation.This study provides ideas for the application of mi R-146a-5p and mi R-192-5p in the clinical diagnosis and treatment of breast cancer.Methods: First,based on the previous gene sequencing results,using the DESeq2 analysis based on binomial distribution,we screened out the mi RNAs with different expressions in the supernatant exosomes of three-dimensional cultured breast cancer cells and normal breast cells,and further screened out the mi RNAs with the same differences in the db DEMC database.Secondly,normal breast epithelial cell MCF-10 A,breast cancer cell MCF-7 and triple negative breast cancer cell MDA-MB-231 were cultured,and the expression of mi R-146a-5p and mi R-192-5p in the supernatant exosomes of three cell lines was detected by Real time Quantitative PCR(q RT-PCR).Thirdly,we collected 47 peripheral blood samples from healthy people during physical examination from September 2021 to August 2022,and 55 peripheral blood samples from breast cancer patients during the same period(according to the pathological test results,they were divided into two categories: 34 invasive breast cancer samples and 21 non-invasive breast cancer samples),of which 3 were triple negative breast cancer peripheral blood samples.To detect the relative expression of mi R-146a-5p and mi R-192-5p in serum exosomes body,and compare and analyze their expression differences between healthy group and breast cancer group,invasive breast cancer group and non-invasive group.The ROC curve was drawn to evaluate the clinical diagnostic value of mi R-146a-5p and mi R-192-5p in the exocrine body for breast cancer,invasive breast cancer patients and non-invasive breast cancer.The ROC curve of the secretions mi R-146a-5p,mi R-192-5p and clinical common tumor markers CEA,CA125,CA15-3 were analyzed jointly,and the Area Under Curve(AUC)was compared to evaluate the diagnostic ability of the secretions mi R-146a-5p,mi R-192-5p and tumor markers in combination for high breast cancer,infiltrating breast cancer,and non-invasive breast cancer.Finally,mi R-146a-5p,mi R-192-5p corresponding mimics,inhibitors and corresponding control sequences were designed and synthesized,and then transfected into logarithmically growing breast cancer cell MCF-7 and triple negative breast cancer cell MDA-MB-231.This experiment was divided into an overexpression control group(transfection independent mimics-NC),overexpression group(transfection independent mimics-mi R-146a-5p/mimics-mi R-192-5p),inhibition control group(transfection independent interference inhibitors NC),and inhibition group(transfection inhibitors mi R-146a-5p/inhibitors mi R-192-5p).After 24 hours of transfection,RNA was extracted from the cells,and the expression of mi R-146a-5p,mi R-192-5p,and predicted target gene m RNA was detected using q RT-PCR to verify successful overexpression/inhibition,and to compare and predict changes in target gene m RNA expression.Ed U experiment,CCK8 experiment,colony experiment,Transwell experiment,and scratch experiment were conducted to compare the differences in cell proliferation,colony formation,invasion,and migration abilities.Results:(1)On the basis of previous gene sequencing results,two mi RNAs differentially expressed in breast cancer were screened by DESeq2 analysis: mi R-146a-5p,mi R-192-5p.(2)At the cell level,q RT-PCR was used to verify that mi R-146a-5p and mi R-192-5p were highly expressed in the supernatant of breast cancer cell MCF-7 and triple negative breast cancer cell MDA-MB-231,and the supernatant of normal breast epithelial cell MCF-10 A was moderately low expressed(p<0.01).(3)Serum exosomes body level detection: mi R-146a-5p and mi R-192-5p were highly expressed in breast cancer group and invasive breast cancer group,and low expression in healthy group(p<0.05);There was no statistical difference in the expression level between the non-invasive breast cancer group and the healthy group.(4)The ROC curve AUC of mi R-146a-5p in serum exosomes body for diagnosis of breast cancer,invasive breast cancer and non-invasive breast cancer was 0.7564,0.8544 and 0.6349 respectively;The ROC curve AUC of mi R-192-5p in serum exosomes body for diagnosis of breast cancer,invasive breast cancer and non-invasive breast cancer was 0.6288,0.6683 and 0.6171 respectively;ROC curve AUC of CEA,CA125 and CA15-3 in diagnosis of breast cancer were 0.6013,0.7025 and0.7251 respectively;ROC curve AUC of CEA,CA125 and CA15-3 in diagnosis of invasive breast cancer was 0.5906,0.6899 and 0.7092 respectively;ROC curve AUC of CEA,CA125 and CA15-3 in the diagnosis of non-invasive breast cancer was 0.616,0.7245 and 0.7541respectively;The ROC curve AUC of breast cancer,invasive breast cancer and non-invasive breast cancer diagnosed by CEA combined with CA125 was 0.8414,0.715 and 0.7772respectively;The ROC curve AUC of CEA combined with CA15-3 in the diagnosis of breast cancer,invasive breast cancer and non-invasive breast cancer were 0.7345,0.7487 and 0.7459respectively;The ROC curve AUC of CA125 combined with CA15-3 in the diagnosis of breast cancer,invasive breast cancer and non-invasive breast cancer were 0.7812,0.657 and 0.8148respectively;The ROC curve AUC of CEA,CA125 and CA15-3 in the simultaneous diagnosis of breast cancer,invasive breast cancer and non-invasive breast cancer were 0.789,0.6681 and0.8123 respectively;The ROC curve AUC of mi R-146a-5p in serum exosomes combined with CEA,CA125,CA15-3 for breast cancer was 0.8322,0.8666,0.884;The ROC curve AUC of mi R-146a-5p in serum exosomes combined with CEA,CA125,CA15-3 for invasive breast cancer was 0.864,0.8814,0.9078;The ROC curve AUC of mi R-146a-5p in serum exosomes combined with CEA,CA125,CA15-3 for non-invasive breast cancer was 0.6239,0.8399,0.8381;The ROC curve AUC of mi R-192-5p in serum exosomes combined with CEA,CA125,CA15-3 for breast cancer was 0.6939,0.7791,0.7772 respectively;The ROC curve AUC of mi R-192-5p in serum exosomes combined with CEA,CA125 and CA15-3 for invasive breast cancer was 0.7112,0.7621 and 0.7847 respectively;The ROC curve AUC of mi R-192-5p in serum exosomes combined with CEA,CA125 and CA15-3 for non-stop invasive breast cancer was 0.6468,0.8028 and 0.7518 respectively;The ROC curve AUC of mi R-146a-5p and mi R-192-5p in serum exosomes body for breast cancer invasive breast cancer and non-invasive breast cancer was 0.8414,0.8761,0.7842;The AUC of ROC curve of mi R-146a-5p in serum exosomes combined with three tumor markers for diagnosis of breast cancer,invasive breast cancer,and non-invasive breast cancer were 0.911,0.9195,and 0.9086,respectively;The ROC curve AUC of mi R-192-5p in serum exosomes combined with three tumor markers for diagnosis of breast cancer,invasive breast cancer and non-invasive breast cancer were 0.8367,0.8431 and 0.8398 respectively;The ROC curve AUC of mi R-146a-5p,mi R-192-5p and three tumor markers in serum exocrine body for diagnosis of breast cancer,invasive breast cancer and non-invasive breast cancer were 0.9407,0.9481 and 0.9249 respectively.The mi R-146a-5p and mi R-192-5p in serum exosomes body have certain clinical diagnostic value for breast cancer,and have higher clinical diagnostic value for invasive breast cancer.The combination of tumor markers can effectively improve the diagnostic efficiency for breast cancer,invasive breast cancer,and non-invasive breast cancer.(5)After 24 hours of transfection with mimicsmi R-146a-5p/mimics-mi R-192-5p,the expressions of mi R-146a-5p and mi R-192-5p were significantly upregulated(p<0.001).After 24 hours of transfection with inhibitors-mi R-146a-5p/inhibitors-mi R-192-5p,the expressions of mi R-146a-5p and mi R-192-5p were significantly downregulated(p<0.001),verifying the success of expression/inhibition.(6)The results of Ed U experiment,CCK8 experiment,colony formation,scratch experiment and Transwell experiment suggest that overexpression of mi R-146a-5p and mi R-192-5p can improve the proliferation,colony formation,invasion and migration ability of breast cancer cell MCF-7 and triple negative breast cancer cell MDA-MB-231,while overexpression of mi R-146a-5p and mi R-192-5p can reduce the proliferation,colony formation,invasion and migration ability of cells.(7)When overexpressing mi R-146a-5p,P53,BAX,TRAF6,NF-κ B1 expression decreased,while MMP9,VEGF,EREG,E-cadherin,N-cadherin expression increased;P53,BAX,TRAF6,when suppressing mi R-146a-5p the expression of NF-κ B1 increased,while the expression of MMP9,VEGF,EREG,E-cadherin,N-cadherin decreased.Overexpression of mi R-192-5p decreased the expression of P53 and BAX,and increased the expression of MMP9,VEGF,EREG,E-cadherin,and N-cadherin;During inhibition,the expression of P53 and BAX increased,while the expression of MMP9,VEGF,EREG,and E-cadherin decreased.Conclusion:(1)mi R-146a-5p and mi R-192-5p were highly expressed in the supernatant exosomes of breast cancer cell MCF-7 and triple negative breast cancer cell MDA-MB-231,and moderately low expressed in normal breast cell MCF-10A;(2)The expression levels of mi R-146a-5p and mi R-192-5p in serum exosomes bodies of breast cancer and invasive breast cancer were higher than those of healthy people;(3)The secretion mi R-146a-5p,mi R-192-5p have certain value in the clinical diagnosis of breast cancer and invasive breast cancer.The combined analysis with breast cancer tumor markers CEA,CA125,CA15-3 can effectively improve the diagnostic ability of breast cancer,invasive breast cancer,non-invasive breast cancer;(4)Overexpression of mi R-146a-5p and mi R-192-5p can improve the ability of proliferation,colony formation,invasion and migration of breast cancer cell MCF-7 and triple negative breast cancer cell MDA-MB-231,suggesting that mi R-146a-5p and mi R-192-5p are oncogenes of breast cancer;(5)mi R-146a-5p regulatory the expression of genes P53,BAX,TRAF6,NF-κB1,MMP9,VEGF,EREG,E-cadherin,N-cadherin;mi R-192-5p regulates the expression of genes P53,BAX,MMP9,VEGF,EREG,E-cadherin,and N-cadherin. |
PDF Full Text Request