| Objective:To explore the effect of different expression level of ER-?36 and adriamycin resistance on miRNAs expression profile in triple negative breast cancer MDA-MB-231 cell lines.By screening out differentially expressed miRNAs and analyzing the target genes and signaling pathways regulated by them,to provide more experimental basis for further revealing the possible molecular regulatory mechanism of the effect of ER-?36 expression on the biological behavior of triple negative breast cancer and adriamycin resistance mechanism.It also provides new candidate detection indicators and new clinical detection methods for molecular diagnosis,clinical typing,prognosis judgment,and drug resistance monitoring of breast cancer patients with high expression level of ER-?36.Methods:1.A recombinant lentivirus carrying ER-?36 specific short hairpin RNA was transfected into triple negative breast cancer MDA-MB-231 cell line by lentiviral transfection method,and screened with puromycin to construct ER-?36 gene silenced cell line that stably transfected,and used the cell line to establish nude mouse xenograft model.At the same time,the adriamycin resistant triple negative breast cancer MDAMB-231/ADR cell line was used to establish the corresponding nude mouse xenograft model.2.The expression of ER-?36 m RNA and ER-?36 protein in MDA-MB-231 cell line and transplanted tumor after ER-?36 gene silenced and adriamycin resistance were detected by qRT-PCR and Western blot respectively.3.Setting up lentivirus-infected ER-?36 silent group(silent experimental group),lentivirus-infected ER-?36 non-silent group(negative control group),uninfected blank control group(blank control group),and adriamycin drug-resistant group and non drugresistant groups.Using next generation sequencing technology to analyze the miRNAs expression profile changes of each group of cells,according to the screening criteria of FC>1.5,P<0.05 and the average CPM of the group?1,the miRNAs with significant differences among the cells of each group were selected as candidate detection indicators.4.Using qRT-PCR to verify the expression level of candidate miRNAs in cell lines,transplanted tumor cells,clinical breast cancer pathological tissues and serum samples.5.Using miRDB and Target Scan databases to predict the function of related differentially expressed miRNAs.Results:1.Successfully constructed ER-?36 gene silenced cell line and nude mouse xenograft animal model.Compared with the negative control group and the blank control group,the expression of ER-?36 m RNA and protein were significantly reduced in the silent experiment group,and the difference was statistically significant(P<0.05).2.Successfully obtained adriamycin resistant cell line and nude mouse xenograft animal model;Compared with the non drug-resistant group,the drug-resistant group cells can grow stably in the culture medium that containing 2 ?g/ml adriamycin,the sensitivity to adriamycin decreased and the cell proliferation activity increased,the difference was statistically significant(P<0.05).3.Next generation sequencing analysis of miRNAs in cells of the silent experiment group,the negative control group and the blank control group showed that the silent experiment group had 45 miRNAs up-regulated and 28 down-regulated compared with the blank control group;32 miRNAs up-regulated and 24 down-regulated compared with the negative control group.By eliminating the differentially expressed miRNAs that may be caused by unrelated sequence sh RNA lentiviral vectors and combining with relevant literature reports,6 significantly differentially expressed miRNAs were finally selected as candidate indicators.The candidate indicators were verified by qRT-PCR in different cell lines and transplanted tumor cells.The results showed that hsa-miR-200a-3p,hsa-miR-146a-5p,hsa-miR-181a-5p were all up-regulated,hsa-miR-3940-3p,hsamiR-15b-5p,hsa-miR-197-3p were all down-regulated,and the difference was statistically significant compared with the negative control group and the blank control group(all P<0.05),and consistent with next generation sequencing results.4.The 6 miRNAs candidate indicators selected above were verified by qRT-PCR in clinical breast cancer pathological tissues and patient serum samples.The results showed that the expressions of hsa-miR-200a-3p,hsa-miR-146a-5p,hsa-miR-181a-5p in breast cancer tissues were reduced to different degrees,hsa-miR-3940-3p,hsa-miR-15b-5p increased to varying degrees compared with the adjacent tissues;Further analysis of ER(+)and ER(-)breast cancer tissues,found hsa-miR-200a-3p,hsa-miR-146a-5p,hsa-miR-181a-5p expression in ER(-)breast cancer tissue is significantly lower than that of ER(+)breast cancer tissue;hsa-miR-3940-3p,hsa-miR-15b-5p expression in ER(-)breast cancer tissue is significantly higher than that of ER(+)breast cancer tissue,and the difference was statistically significant(all P<0.05).The expressions of hsa-miR-200a-3p,hsa-miR-146a-5p,hsa-miR-181a-5p and hsa-miR-3940-3p in the serum of breast cancer patients are consistent with the expression results in breast cancer tissues,there was a statistically significant difference between healthy people(all P<0.05).5.Next generation sequencing analysis of miRNAs in cells of the drug-resistant and non drug-resistant group showed that the drug-resistant group had 197 miRNAs up-regulated and 200 down-regulated compared with the non drug-resistant group.To selecte 6 miRNAs with significant differential expression as candidate indicators,and qRT-PCR verification in drug-resistant cell lines and corresponding transplanted tumor cells.The results showed that hsa-miR-20b-5p,hsa-miR-9-5p and hsa-miR-155-5p were up-regulated,hsa-miR-584-5p,hsa-miR-34c-3p,hsa-miR-27b-3p were all down-regulated in the drug-resistant group,compared with the non drug-resistant groups statistical significance(all P<0.05),and consistent with next generation sequencing results.6.Perform functional prediction analysis on differentially expressed related miRNAs.Compared with the blank control group,the GO analysis showed that the differentially expressed miRNAs were mainly involved in cell metabolism and cell localization process;KEGG analysis showed that the up-regulated miRNAs were mainly related to Fox O and c AMP signaling pathways,and the down-regulated miRNAs were mainly related to Hippo signaling pathways.Compared with the non drug-resistant group,GO analysis showed that the differentially expressed miRNAs were mainly involved in cell metabolism and protein localization;KEGG analysis showed that the up-regulated miRNAs were mainly related to the Rap1 and Hippo signaling pathways,and the down-regulated miRNAs mainly related to p53 and Ras signaling pathways.A single miRNA has matching binding sites with multiple target genes,and there is a common target gene among multiple miRNAs.Conclusion:1.Different expression levels of ER-?36 had a significant effect on the change of miRNAs expression profile in triple negative breast cancer cells.The 6 miRNAs that significantly differentially expressed reflected the characteristic miRNAs expression profile in triple negative breast cancer with high expression of ER-?36,and could be used as a reference marker for breast cancer diagnosis and typing,also provided new monitoring indicators for clinical drug selection and prognosis judgment.At the same time,it suggested that differentially expressed miRNAs may be involved in the regulation of ER-?36 mediated signaling pathways in triple negative breast cancer.2.Adriamycin resistance also had a significant effect on the changes in the miRNAs expression profile of triple negative breast cancer cells.The 6 miRNAs that significantly differentially expressed reflected the characteristic miRNAs expression profile in adriamycin-resistant triple negative breast cancer,provided potential markers for drug resistance monitoring,and also laid the foundation for the further study of miRNA resistance mechanism in triple negative breast cancer.3.Circulating serum miRNAs had good consistency with breast cancer cells,suggesting that 4 miRNAs that significantly differentially expressed could be used as clinical detection indicators for the identification of breast cancer with different expression levels of ER-?36.At the same time,it provided a non-invasive and simpler detection method for the clinic.4.miRNA participated in the signal pathways related to the development of triple negative breast cancer,which can provide a new experimental basis for further research on the pathogenesis and drug resistance mechanism of triple negative breast cancer. |
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