Fig 1: miR-140-3p directly targets DACH1. (A) The effect of miRNAs on endogenous DACH1 expression. HMCs were transfected with 10 miRNA mimics (20 nM) and NC, western blotting was performed at 48 h after transfection. (B) Inhibition of DACH1 3'UTR reporter activity by miRNAs. Luciferase activity was detected in 293T cells co-transfected with 10 miRNA mimics (20 nM) or NC together with the pGL3-DACH1 3'UTR luciferase reporter (***P<0.001 NC vs. miR-140-3p). (C) miR-140-3p inhibited expression of DACH1 in a dose-dependent manner. HMCs transfected with an increasing amount (5–20 nM) of miR-140-3p mimic or NC for 48 h. The transfected cells were collected for examined by western blotting and RT-qPCR (**P<0.01 NC vs. 5 nM miR-140-3p, ***P<0.001 NC vs. 10 nM miR-140-3p, ****P<0.0001 NC vs. 20 nM miR-140-3p). (D) Predicted binding sequences of miR-140-3p in the 3'UTR of DACH1 (miRTar, http://mirtar.mbc.nctu.edu.tw/human/). (E and F) Effect of mutation of miR-140-3p on endogenous DACH1 expression. miR-140-3p mimics and mutant were transfected into HMCs for 48 h, while the expression of DACH1 was detected by western blotting and RT-qPCR (****P<0.0001). (G) miR-140-3p mimics/mutant or NC were co-transfected with pGL3-DACH1 3'UTR luciferase reporter into 293T cells for 48 h and the luciferase activity detected (****P<0.0001). Data are expressed as mean ± standard deviation. CK, control check; miR, microRNA; DACH1, dachshund family transcription factor 1; HMCs, human mesangial cells; NC, negative control; 3'UTR, 3'-untranslated region; RT-qPCR, reverse transcription-quantitative PCR; mut, mutant.
Fig 2: Overexpression of DACH1 reduces HMCs proliferation and inhibits cell cycle progression. (A) The expression of DACH1 in HMCs cultured with pIgA-IgAN or pIgA-control was detected by western blotting. (B) Western blotting for DACH1 protein in HMCs infected with DACH1 or control lentivirus (pCDH). (C) HMCs stably transduced with lentiviral DACH1, as well as the control empty vector (pCDH) were seeded and examined for cell proliferation with CCK-8 test on days 1, 3 and 5 (****P<0.0001). Cell cycle assay was determined by flow cytometry. Cell cycle distribution by flow cytometry (E) and statistical results of cell cycle progression analysis (D) (*P<0.05 and **P<0.01). Protein expression of (F) Cyclin D1 and Cyclin A and (G) p21 and p53 in HMCs transduced with DACH1 or pCDH was determined by western blotting. Data are expressed as mean ± standard deviation. DACH1, dachshund family transcription factor 1; HMCs, human mesangial cells; pIgA, polymeric IgA; IgAN, immunoglobulin A nephropathy; pCDH, pCDH-CMV-MCS-puro; ns, not significant; pDACH1, pCDH-CMV-DACH1-puro.
Fig 3: Correlation between SNPs at rs9285274, rs9529895, rs17088351, and rs59352399 of DACH1 gene and progression-free survival (PFS) in EC patients A. Comparison of PFS of EC patients with different genotypes of rs9285274 at DACH1. B, Comparison of PFS of EC patients with different genotypes of rs9529895 at DACH1. C, Comparison of PFS of EC patients with different genotypes of rs17088351 at DACH1. D, Comparison of PFS of EC patients with different genotypes of rs59352399 at DACH1.
Fig 4: Multifactor dimensionality reduction (MDR) analysis of the interaction between DACH1 gene SNPs and general clinical features. A, Age, BMI, Smoking, Alcohol, T2DM, rs9285274, rs9529895, rs17088351, rs59352399. The data at the apex represent the influence of this factor on the risk for EC disease, and the larger the value represents the greater the influence of this factor on the risk for EC disease. The magnitude of the value on the line between the 2 factors represents the magnitude of the interaction between the 2 factors. B, Age, BMI, Smoking, Alcohol, T2DM, rs9285274, rs9529895, rs17088351, rs59352399 factors. The strength of the interaction depends on the proximity of these 2 factors.
Fig 5: DACH1 expression in tumor tissue from EC patients. A. Comparison of DACH1 expression levels in cancer tissues and normal tissues adjacent from EC patients. B. Correlation between DACH1 mRNA expression level and TNM stage in EC patients. C, Comparison of DACH1 mRNA expression levels in tumor tissues of EC patients with different genotypes of rs9285274 at DACH1. D, Comparison of DACH1 mRNA expression levels in tumor tissues of EC patients with different genotypes at rs9529895 locus of DACH1. E, DAHC1 mRNA expression levels in tumor tissues of patients with different genotypes with EC at DACH1 rs17088351 loci. F, DACH1 mRNA expression levels in tumor tissues of patients with different genotype with EC at the DACH1 rs59352399 locus.
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