| Purpose:This study was based on the second-generation sequencing results of Small RNA in wild-type human breast cancer cell line(MCF-7)and adriamycin-resistant(ADR)human breast cancer cell line(MCF-7/ADR).The literature screened Hsa-mi R-1284(mi R-1284)and Hsa-mi R-1247-5p(mi R-1247-5p)which were differentially expressed in MCF-7/ADR cells as subjects.Synthesis of mi R-1284 and mi R-1247-5p mimics(mimics)transfected into MCF-7/ADR to investigate whether mi R-1284 and mi R-1247-5p can reverse MCF-7/ADR resistance and MCF-7 /ADR cell function has an effect.At the same time,the molecular mechanism of mi R-1284 and mi R-1247-5p to reverse MCF-7/ADR resistance was studied.This study aims to provide a theoretical basis and reference for mi RNA treatment of drug-resistant breast cancer.Method:1.Detection of MCF-7/ADR resistance MTT assay was used to compare the drug resistance differences between MCF-7 and MCF-7/ADR cells.2.To verify the expression levels of mi R-1284 and mi R-1247-5p in MCF-7/ADR cells The expression of mi R-1284 and mi R-1247-5p in MCF-7 and MCF-7/ADR was detected by q PCR assay.3.Bioinformatics analysis of mi R-1284 and mi R-1247-5p The mi R-1284 and mi R-1247-5p target genes were predicted by RNAhybrid,Miranda and Target Scan online tools,and the intersection of the three prediction results was taken as the final prediction result.The biological processes and enriched signaling pathways of two small molecule target genes were analyzed using the GO ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)database in the context of the human genome.4.Effects of mi R-1284 and mi R-1247-5p on proliferation and cell function of MCF-7/ADR The effects of mi R-1284 and mi R-1247-5p on the proliferation and cell function of MCF-7/ADR cells were detected by WST-1 method,flow cytometry and transwell chamber assay.5.c Bio Portal website analyzes gene burst rate,inter-gene correlation,and impact on overall survival of breast cancer patients The c Bio Portal website was used to analyze the burst rate,inter-gene correlation,and overall survival of eight breast cancer patients with SMAD2,SMAD3,SMAD4,ZEB1,SNAI1,SNAI2,CDH2,and VIM in the TGF-bsignaling pathway.6.Effects of mi R-1284 and mi R-1247-5p on the expression of 8 genes including SMAD2 in MCF-7/ADR cells The expression of SMAD2,SMAD3,SMAD4,ZEB1,SNAI1,SNAI2,CDH2 and VIM in MCF-7/ADR after transfection of mi R-1284 and mi R-1247-5p was detected by q PCR.Results:1.The results of MTT showed that the resistance of MCF-7/ADR cells to ADR was significantly higher than that of wild MCF-7 cells.2.q PCR assay was used to verify the expression of mi R-1284 and mi R-1247-5p in MCF-7 and MCF-7/ADR.The results of q PCR assay were consistent with the results of second-generation sequencing.mi R-1284 and mi R-1247-5p were lower expression MCF-7/ADR cells.3.The online tool predicted that the number of mi R-1284 and mi R-1247-5p target genes was 1918 and 581,respectively.The biological process of mi R-1284 target genes GO mainly focuses on biological process regulation,cell differentiation,regulation of cell death and cell cycle.The target genes of mi R-1247-5p mainly concentrate on negative regulation of biological processes,cell differentiation,regulation of cell proliferation,and assembly of SMAD protein complexes.The mi R-1284 target genes enrichment pathway mainly includes transcriptional misregulation in cancers and MAPK,TGF-b signaling pathway,while mi R-1247-5p target genes is mainly enriched in pathways in cancer,transcriptional misregulation in cancers and ABC transporter pathway.4.After 48 h of transfection,mi R-1284 and mi R-1247-5p significantly inhibited the proliferation of MCF-7/ADR cells and increased the sensitivity of MCF-7/ADR to ADR;mi R-1284 and mi R-1247-5p significantly increased apoptosis rate of MCF-7/ADR cells;compared with NC group,mi R-1284 and mi R-1247-5p significantly reduced the S phase distribution rate of MCF-7/ADR cells,significantly increased the distribution rate of G0/G1 phase;The number of cells penetrating was reduced in the experiments of transwell chamber invasion and migration.5.c Bio Portal website analysis results showed that there was a positive correlation between mi R-1284,mi R-1247-5p target genes SMAD2,SMAD3,SMAD4,SNAI2 and key genes ZEB1,SNAI1,CDH2 and VIM during EMT.At the same time,SMAD2,SMAD3,SMAD4,ZEB1,SNAI1 SNAI2,CDH2,and VIM all have highmutation rates in breast cancer patients.In addition,the results of analysis showed that inhibition of these 8 genes mutations is conducive to the survival of breast cancer patients.6.mi R-1284 reduced the expression levels of SMAD3,ZEB1 and VIM in MCF-7/ADR cells,while mi R-1247-5p down-regulated the expression levels of SMAD2,SMAD3,SMAD4,ZEB1,SNAI1,CDH2 and VIM.Conclusion:1.MCF-7/ADR had high drug resistance.2.mi R-1284,mi R-1247-5p are involved in the ADR tolerance of MCF-7/ADR cells and are lower expressed in MCF-7/ADR.3.Through the analysis of bioinformatics of target genes,it was found that mi R-1284 and mi R-1247-5p may play an important role in the growth anddrug resistance of MCF-7/ADR tumor cells.4.mi R-1284 and mi R-1247-5p inhibited the proliferation of MCF-7/ADR cells,increased the sensitivity of MCF-7/ADR to ADR,promoted the apoptosis of MCF-7/ADR cells,made the MCF-7/ADR cell cycle by blocking G0/G1 phase and affecting cell DNA synthesis;reduced invasion and migration of MCF-7/ADR cells.5.The results of c Bio Portal website indicated that mi R-1284,mi R-1247-5p regulates the target genes SMAD2,SMAD3,SMAD4,SNAI2 in TGF-b /SMADs signaling pathway may further affect the expression of downstream genes ZEB1,SNAI1,CDH2,VIM,Influencing the EMT process to participate in the drug resistance process of MCF-7/ADR cells.6.It was preliminarily shown that mi R-1284 and mi R-1247-5p participated in the ADR-resistant process of breast cancer by regulating TGF-b/SMADs signaling pathway. |
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