| Objective:Breast cancer is a common malignancy cancer among women worldwide,with a rate of about 23% of all malignancies and a tumor-related mortality rate of up to 14%.More than 90% types of breast cancer is atheter and lobular breast cancer.The origin and developing process of breast cancer are complex and involve multiple genes and procedures.Lots of reports show that altered expression or activity of specific genes involve in the pathogenesis of breast cancer cells,such as microRNA(miRNA).MiRNA is a kind of(~22 nt)non-coding RNAs that play important roles in the negative regulation of gene expression through sequence-specific translational repression or mRNA degression involved in cell growth,proliferation,differentiation and apoptosis.Some researches indicate that a lot of miRNAs regulate the cell biology of cancer via joining in the cell signal pathway,exerting functions as up-regulation or down-regulation in breast cancer.Thus,exploring the targeting mechanism of miRNAs could lay the foundation for the diagnosis and therapy of malignant transformation for breast cancer.ERLIN1 belongs to a larger family of proteins that share an evolutionarily conserved stomatin/prohibitin/flotillin/HflK/C(SPFH)domain.SPFH-containing proteins localize to different membranes,but have common characteristics.ERLIN1 and its homologue ERLIN2 were originally identified as components of lipid rafts that localize to the ER.ERLIN1 and ERLIN2 interact with each other to form a functional complex.Recent high-resolution genomic analysis of copy number in human breast cancer specimens demonstrated that in 28 % cases of breast cancer patient,the expression of ERLIN1 increased.More recently,human genetic studies identified theERLIN1 gene mutations are associated with childhood neuronal diseases characterized by progressive weakness and spasticity of the lower extremities and intellectual disability.However,the role and mechanism of ERLIN1 in pathophysiology remain poorly understood.In this paper,we focus on looking for a miRNA and its host gene,which participate in ERLIN1 signal pathway,and explore their function in breast cancer.Methods:1.We investigated the effects of ERLIN1 on cell proliferation,migration and invasion in CCF-7 and T47 D cells by CCK-8 assay,colony formation assay,Transwell migration assay,Transwell invasion assay and Flow cytometry.2.We screened miR-410,which can target ERLIN1 by bioinformatics method and validated that mi R-410 regulates ERLIN1 directly by Real-time PCR and Western Blot.3.Real-time PCR and Western Blot were used to detect the expression of miR-410 and ERLIN1 in five breast cancer cells,20 pairs of breast cancer tissues and adjacent normal tissues.4.We investigated the effects of miR-410 on cell proliferation,migration and invasion in CCF-7 and T47 D cells by CCK-8 assay,colony formation assay,Transwell migration assay,Transwell invasion assay and Flow cytometry.Results:1.Knocking down of ERLIN1 inhibited the growth,migration and invasion of MCF-7 and T47 D cells,and promoted cell apoptosis.In MCF-7 and T47 D cells,the OD450 nm value of NC groups was 0.875±0.053 vs 0.961±0.042,respectively,and the value of OD450 nm in shR-ERLIN1 groups was 0.325±0.050(p =0.0013)vs 0.417±0.049(p=0.0015)by CCK-8 assay.The number of clones in NC groups was 672±29 vs 771±32,respectively,and in shR-ERLIN1 groups was 436±17(p=0.023)vs 414±17(p=0.019)by colony formation assay.The number of migratory cell in NC groups was 327±12 vs 178±24,respectively,and in shR-ERLIN1 groups was 128±25(p=0.015)vs 80±9(p=0.014),respectively.The number of invasive cell in NC groups was 420±21 vs 262±22,and in shR-ERLIN1 groups was 262±11(p=0.014)vs 126±12(p=0.015),respectively.In MCF-7 cells,Flow cytometry confirmed that ERLIN1 could significantly inhibit the apoptosis of breast cancer cells(p=0.0014).2.miR-410 could directly target ERLIN1.We predicted potential mi RNAs which could target of ERLIN1 according to biological information software: targetscan,microrna,pictar.The results of Real-time PCR and Western Blot showed that mi R-410 could significantly inhibit the expression of ERLIN1 in MCF-7 cells(p=0.0110;p=0.0090).Dual fluorescence reporter assay confirmed that ERLIN1 was a direct target gene for miR-410.3.Expression of miR-410 and ERLIN1 in breast cancer cells and clinical tissues.MiR-410 expressed highest and ERLIN1 expressed lowest in MCF-7 cells among the five breast cancer cells.MiR-410 expressed lowest and ERLIN1 expressed highest in T47 D cells among the five breast cancer cells.In the clinical tissues,the expression of miR-410 mRNA was significantly lower than that in the adjacent normal tissues,and the mRNA expression of ERLIN1 was significantly higher.4.mi R-410 inhibited the growth,migration and invasion in MCF-7 and T47 D cells and promoted apoptosis in MCF-7 cells.In MCF-7 cells,the OD450 nm value of ASO-NC group was 0.901±0.013,and in ASO-mi R-410 group was 1.450±0.034(p = 0.0014).The clone formation number of ASO-NC group was 178±18,and in ASO-mi R-410 group was 452±28(p=0.020).The number of migratory cell in ASO-NC group was 120±1 and in ASO-miR-410 group was 262±1(p=0.012).The number of invasive cell in ASO-NC group was 420±21,and in ASO-miR-410 group was 262±11(p=0.014).The result of Flow cytometry showed that ERLIN1 could significantly inhibit the apoptosis of breast cancer cells(p=0.0014).The OD450 nm value of miRNA-NC mimics group was 0.948±0.061,and in miR-410 mimics group was 0.489±0.017(p=0.0043).The clone formation number of miRNA-NC mimics group was 414±17,and in miR-410 mimics group was 171±12(p=0.021).The number of migratory cell in miRNA-NC mimics group was 146±1 and in miR-410 mimics group was 80±2(p=0.011).The number of invasive cell in miRNA-NC mimics group was 126±1,and in miR-410 mimics group was 62±2(p=0.0082).Conclusions:1.ERLIN1 promoted proliferation,migration and invasion and inhibited cell apoptosis in breast cancer cells.2.miR-410 regulated expression of ERLIN1 directly.3.In the five breast cancer cells,the expression of ERLIN1 was the lowest in MCF-7 cells and the expression of mi R-410 was highest.In the five breast cancer cells,the expression of ERLIN1 was the highest in T47 D cells and the expression of miR-410 was lowest.In the clinical tissues,the expression of miR-410 mRNA was significantly decreased than that of adjacent normal tissues and the expression of ERLIN1 mRNA was significantly increased4.miR-410 inhibited proliferation,migration,invasion and promoted cell apoptosis in breast cancer cells.Collectively,miR-410 act as a tumor suppressive miRNA in breast cancer by inhibiting cell proliferation,migration and invasion in vitro by targeting ERLIN1.These findings enriched our understanding of the pathogenesis of breast carcinoma,potentially providing new biomarkers for clinical treatment and new drug development. |
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