| 【Background】According to the latest epidemiological survey data of the American Cancer Society,the incidence of breast cancer ranks first among women malignant tumors.Overexpression of human epidermal growth factor receptor-2(HER2)occurs in 20% to 25% of breast cancer cases,which is closely related to disease progression and prognosis.Overexpression of HER2 is one of the important targets for the treatment of breast cancer.Trastuzumab(also called Herceptin),a monoclonal antibody against HER2,brings a large clinical benefit in the treatment of breast cancer.Recent studies have found that non-coding RNA plays an important role in regulating tumor drug resistance.MicroRNAs(miRNAs)regulate gene expression through degradation of target mRNAs.Our previous studies shown that miR-200c(Bai et al,Int J Cancer,2014),miR-221(Ye et al,BMB Rep,2014)and miR-375(Ye et al,BMC Cancer,2014)are involved in trastuzumab resistance of HER2-positive breast cancer.These results suggest that miRNAs play an important role in HER2-targeted therapy.However,miRNA only exerts post-transcriptional regulation and can not completely explain the abnormal gene expression in HER2-targeted therapy.Recent studies reveal that long noncoding RNA(lncRNA)regulates tumor proliferation,migration,metastasis and drug resistance.However,are lncRNAs associated with trastuzumab resistance and do they regulate malignant transformation of breast cancer cells? How does lncRNA interact with miRNA and target genes? How does the non-coding RNA regulatory network function in breast cancer resistance? These issues have not yet been elucidated.Keloids are cutaneous benign tumors characterized by overgrowth of fibroblasts and excessive deposition of collagen in the damaged extracellular matrix of the skin.Many studies have shown that PPAR-γ agonists have antifibrotic effects in the kidney,liver,pancreas,and lungs,suggesting that PPAR-γ agonists may be a promising drug for anti-fibrotic diseases.Troglitazone is a synthetic PPAR-γ agonist.PPAR-γ is a nuclear receptor that binds to the peroxisome proliferative hormone response element(PPRE)on target gene and regulates a variety of physiological phenomena.However,the molecular mechanisms underlying the anti-fibrotic effects of PPAR-γ agonists in keloid fibroblasts remain unknown.Studies have shown that some microRNAs(miRNAs)have the effect of regulating fibrosis.Our continuous studies show that miR-21 and miR-145 are widely involved in the process of skin fibrosis.However,are there any miRNAs involved in the antifibrotic effects of PPAR-γ agonists in scar formation? How do PPAR-γ agonists regulate miRNAs in keloids? How do miRNAs and target genes participate in PPAR-γ agonists impairment of collagen production in keloid fibroblasts? These questions need to answer.【Aims】The aim of this study was to identify long-chain non-coding RNAs associated with drug resistance in trastuzumab-resistant cells of breast cancer,and to screen for miRNAs and target genes regulated by the long-chain non-coding RNA.We also attempt to explore the function of long non-coding RNAs as potential miRNA "sponges" in breast cancer resistance,with the aim of finding new target molecules and strategies for the treatment of breast cancer.We aim to verified that PPAR-γ agonists inhibit collagen synthesis in keloids,and screen for miRNA molecules and target genes regulated by PPAR-γ agonists.We are also dedicated to investigate the effect of target gene Egr1 on collagen production,with the aim of providing new targets for the treatment of keloids.【Methods】1.UCA1,miR-18 a,and YAP1 expression was accessed by qRT-PCR;2.Analysis of the effect of UCA1 on proliferation of trastuzumab-resistant breast cancer SKBR3 cells was performed by cell proliferation assay,and half lethal concentration of trastuzumab was measured by cell proliferation assay;3.The effect of UCA1 on the apoptosis of trastuzumab-resistant breast cancer SKBR3 cells was detected by flow cytometry apoptosis assay;4.The effect of the invasive ability of breast cancer SKBR3 cells was confirmed by transwell assay;5.Prediction of the binding sites of UCA1 and miR-18 a,miR-18 a and YAP1 was performed via bioinformatics method;6.UCA1 binding to miR-18 a was analyzed by luciferase reporter gene assay.7.Binding of Ago2 by UCA1 and miR-18 a was detected by biotin pull-down test;8.Transient transfection was performed to analyze the effect of altering UCA1,miR-18 a and YAP1 expression on upstream and downstream gene expression.1.Egr1,miRNA,and Col1 expression was analyzed by qRT-PCR;2.Egr1 and Col1 protein levels was detected by Western blotting and enzyme-linked immunosorbent assays;3.Expression of Egr1 in keloids and normal skin tissues was confirmed by immunohistochemistry;4.miRNA expression profile of keloid fibroblasts treated with PPAR-γ agonists was examined by high-throughput screening of miRNA arrays;5.Transient transfection was carried out to evaluate the effect of miRNAs on collagen secretion;6.PPAR-γ binding on miR-543 promoter region was assayed by chromatin immunoprecipitation assay;7.Bioinformatics prediction of peroxisome proliferator response element sequences on miR-543 promoter region,and miR-543 binding site on the 3’UTR of Egr1 were analyzed using online tools;8.miR-543 specific binding to Egr1 mRNA was examined by luciferase reporter gene assay.【Results】1.qRT-PCR results revealed that the long-chain non-coding RNA UCA1 was upregulated in trastuzumab-resistant breast cancer SKBR3 cells;2.UCA1 knockdown results showed that UCA1 expression is significantly reduced by siRNAs in trastuzumab-resistant breast cancer SKBR3 cells;3.Cell proliferation and cell invasion experiments showed that UCA1 knockdown significantly suppressed trastuzumab-resistant breast cancer SKBR3 cell proliferation and invasion.4.Biotin RNA pull-down results revealed that UCA1 and miR-18 a bind to each other in trastuzumab-resistant breast cancer SKBR3 cells;5.Bioinformatics analysis and luciferase reporter assay confirmed that UCA1 can "sponge" miR-18a;6.qRT-PCR results showed that reducing UCA1 can significantly increase the expression level of miR-18 a,and elevated miR-18 a inhibits YAP1 expression;7.Bioinformatics analysis and luciferase reporter assays confirmed that miR-18 a can specifically inhibit YAP1,and this inhibition is dependent on the predicted binding sites;8.qRT-PCR and western blotting results revealed that downregulation of UCA1 and YAP1 sensitizes breast cancer cells to trastuzumab;9.UCA1 regulates YAP1 via miR-18 a since miR-18 a inhibiton abrogated the regulatory of UCA1 on YAP1.1.qRT-PCR results revealed that in keloid fibroblasts,Egr1 and Col1 were downregulated upon stimulation of PPAR-γ,while miR-543 expression was increased;2.Western blot and enzyme-linked immunosorbent assay showed the same results;3.miRNA high-throughput microarray results revealed that miR-543 and other miRNA were abnormally expressed in keloid fibroblasts,which was confirmed by qRT-PCR assay;4.Bioinformatics results revealed that miR-543 has peroxisomal proliferative response element sequences in the promoter region,and the 3’UTR of Egr1 has potential miR-543 binding site;5.Luciferase reporter assay results showed that miR-543 can bind to Egr1 3’UTR;6.qRT-PCR assays showed that Egr1 mRNA level was increased after combined treatment with troglitazone and miR-543 inhibitor,whereas it was decreased when treated with PPAR-γ inhibitor and miR-543 mimic.7.Enzyme-linked immunosorbent assays showed that Col1 protein level was changed similar to Egr1 mRNA level. |
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