Long-stranded Non-coding RNA LOC645166 Promotes Doxorubicin Resistance In Breast Cancer By Regulating GATA3

Background and objective:Malignant tumor is a public problem that needs to be solved urgently in the world.It has a great impact on people's health,the national economy and social development.The prevention and control of cancer has gradually gained attention,and it has become the focus of the global health strategy.Breast cancer is the most common malignancy in women.So far,surgery,radiotherapy,and chemotherapy are still the main treatments for breast cancer.Chemotherapy plays an irreplaceable role in the comprehensive treatment of breast cancer because of its special effects of controlling and reducing the lesion before surgery and preventing recurrence and metastasis after surgery.But almost inevitable chemotherapy resistance has been the bottleneck for effective breast cancer control.Breast cancer resistance is associated with recurrence,invasion and metastasis of breast cancer patients,but the mechanism is not clear.Therefore,studying the mechanism of breast cancer resistance and finding new drug resistance markers have important theoretical significance and potential clinical application value for effectively reversing breast cancer resistance and improving breast cancer treatment.Long-non-coding RNA?LncRNA?is a type of non-coding RNA with a length of more than 200 bp.Many studies have shown that LncRNA may be involved in a variety of pathological processes such as tumor development and therapeutic tolerance.This study used LncRNA gene chip to detect the difference in the expression of doxorubicin-induced breast cancer multidrug resistance cell line?MCF-7/ADR?and parental cells?MCF-7?,which was established in the previous stage.Differential expression profiles of cells and sensitive cells,screening and identification of breast cancer resistance-related LncRNA,provide a new theoretical basis for the description of breast cancer resistance mechanisms.Methods:1.Analysis of long non-coding RNA expression between doxorubicin-induced breast cancer multidrug resistance cells?MCF-7/ADR?and parental breast cancer cells?MCF-7?by long non-coding RNA high-throughput microarray difference.2.LncRNA with significant expression difference was screened from MCF-7/ADR,231/ADR and amphiphilic cells MCF-7,231 cells by real-time PCR.3.The LOC645166 lentiviral interference plasmid was constructed and stably transfected into cells.The interference efficiency was verified by RT-qPCR,and the cell proliferation ability of different treatment groups was determined by CCK8 and clone formation assay.4.To establish a subcutaneous xenograft model of MCF-7/ADR-resistant nude mice,observe the tumor formation of nude mice in different treatment groups,and verify the expression level of LOC645166 in subcutaneous tumors by qPCR.5.MCF-7/ADR and LOC645166 overexpressing MCF cells stably transfected with LOC645166 were selected.After ADR treatment,the changes of STAT3 and AKT signaling pathways were confirmed by western blotting.6.Co-transfection of LOC645166 overexpression and STAT3 interference sequence,CCK-8 detection of cell sensitivity to epirubicin7.After overexpression or interference with LOC645166,qPCR and Western Blotting were used to detect the expression of GATA3 gene in different treatment groups,and the dual luciferase reporter gene was used to detect the effect of LOC645166 on GATA3 transcriptional activity.Co-transfection of LOC645166 overexpression and GATA3 interference sequences,CCK-8 detection of changes in sensitivity of epirubicin in different treatment groups8.RNA pulldown and RIP assay to detect the binding of LOC6451667 to NF-?B.After co-transfection of LOC645166 overexpression and NF-?B interference sequences,the expression level of GATA3 was detected by PCR,and CCK-8 was used to detect the sensitivity of cells in different treatment groups to epirubicin.ChIP experiments verified that LOC6451667 promotes the transcriptional activation of GATA3 by NF-?B.Results:1.Analysis of IncRNA and mRNA expression profiles in patients with breast cancer epirubicin resistance and parental cellsThe expression of IncRNA and mRNA in MCF-7 and MCF-7/ADR cell lines were analyzed by gene chip.The differentially expressed genes were analyzed by software,and the intersection gene was searched by GEO database.The images were identified by GO and KEGG pathway enrichment analysis.The formation of epirubicin resistance may be related to transcription factors,anaerobic glycolysis,kinase activation,intercellular calcium adhesion and other biological functions as well as apoptosis,PI3K/AKT,FOX,AMPK,ErbB and other signaling pathways.2.Breast cancer epirubicin resistance and parental cell differences IncRNA verificationTen IncRNAs with the highest expression level in drug-resistant cells were selected,and their expression levels were verified by PCR in breast cancer resistant cells MCF-7/ADR,231/ADR and corresponding parental cells MCF-7 and 231.The results showed that compared with the two parental cells,the two resistant strains had significantly up-regulated IncRNA,and the expression of LOC645166?NR₁30928?was extremely significantly increased.Therefore,the related mechanism of LOC645166 was discussed.3.In vitro experiments confirmed that the expression of LOC645166 interfered with the sensitivity of breast cancer resistant cells to epirubicin.To validate the role of LOC645166 in the expression of epirubicin in breast cancer,we constructed a LOC645166 lentiviral interference plasmid and stably transfected two resistant cell lines,MCF-7/ADR and 231/ADR.The interference efficiency was verified by RT-qPCR,and both interference sequences significantly decreased the expression level of LOC645166?p<0.05?.CCK8 assayed cell viability in different treatment groups,and the results showed that the tolerance of epirubicin was significantly lower in the LOC645166 interference group compared with the control group.The clonal formation assay was used to detect the cell proliferation ability of different groups after epirubicin treatment.The results showed that the interference of LOC645166 did not affect the clonal formation ability of cells without drug,but after epirubicin The interference with the expression of LOC645166 significantly inhibited the clonality of MCF-7/ADR and 231/ADR cells.4.In vivo experiments confirmed that the expression of interfering LOC645166enhances the chemosensitivity of subcutaneous tumors to epirubicinIn the nude mouse subcutaneous xenograft model established by MCF-7/ADR drug-resistant cells,the subcutaneous tumors of the LOC645166 interference group and the control group did not show statistical difference without the administration of epirubicin,but in the table soft ratio In the star injection group,the expression of the subcutaneous tumor was significantly inhibited by interfering with the expression of LOC645166.The PCR results showed that the expression level of LOC645166 was significantly decreased in subcutaneous tumors.5.Overexpression of LOC645166 promotes chemotherapy tolerance of epirubicin in breast cancer via STAT3 and AKT signaling pathwaysMCF-7/ADR and LOC645166 overexpressing MCF cells stably transfected with LOC645166 were selected and treated with ADR.Western blotting showed that LOC645166 overexpression could promote STAT3,AKT phosphorylation and activate STAT3 and AKT in breast cancer parental cells.Signaling pathways that promote the tolerance of breast cancer to doxorubicin.However,the expression of p-STAT3 and p-AKT was significantly decreased in LOC645166,a drug-resistant cell line MCF-7/ADR.6.Overexpression of LOC645166 promotes chemotherapy tolerance of epirubicin in breast cancer by regulating downstream GATA3After MCF-7/ADR and MCF-7 interfered with or overexpressed LOC645166,respectively,RT-PCR and western blotting showed that the expression of GATA3 was significantly increased in the overexpressed group and the expression of GATA3 in the interference group was compared with the control group.Significant elevation was reduced;luciferase reporter assay results showed that overexpression of LOC645166 promoted the transcriptional activity of GATA3,while interference with LOC645166 inhibited its transcription.CCK-8 results showed that interference with GATA3 expression reversed the drug resistance caused by overexpression of LOC645166.7.LOC645166 regulates the transcription of GATA3 by adsorbing NF-?BBy synthesizing biotinylated LOC645166 and its antisense strand,RNA pull down and RIP experiments showed that LOC645166 can bind to NF-?B.PCR results showed that interference with NF-?B could reverse the promotion of GATA3 expression by LOC645166.The CCK-8 assay showed that interference with NF-?B expression reversed the effect of LOC645166 on drug resistance to doxorubicin in breast cancer.The results of CHIP assay showed that LOC6451667 promoted the transcriptional regulation of GATA3 by NF-?B.Conclusion:1.The expression level of LOC645166 is positively correlated with doxorubicin resistance in breast cancer cells.2.Overexpression of LOC645166 promotes chemotherapy tolerance of epirubicin in breast cancer through STAT3 and AKT signaling pathways.3.LOC645166 promotes the transcription of GATA3 by NF-?B,thereb enhancing the tolerance of breast cancer to epirubicin.