ABSTRACT
The aim of the present study was to investigate the expression and mechanism of microRNA (miR)93 in collagen expression in stress urinary incontinence (SUI). Vaginal tissue, primary fibroblasts and SUI primary fibroblasts were obtained to detect the expression of miR93, interstitial collagenase (MMP1), collagen I and calpain2. Reverse transcriptionquantitative polymerase chain reaction analysis was performed to detect the levels of miR93 and MMP1. Western blotting was used to evaluate the protein levels of calpain2, MMP1 and collagen I. MMP1 and hydroxyproline levels in the supernatant were measured by ELISA. The association between miR93 and calpain2 was investigated by luciferase reporter assays. The expression of miR93 and collagen I was significantly downregulated in the SUI group, while the expression of calpain2 and MMP1 was significantly upregulated. ELISA analysis demonstrated that the MMP1 level increased and the hydroxyproline level decreased in the SUI group. Additionally, calpain2 was identified to be a target of miR93, and miR93 was able to negatively regulate the expression of calpain2. Restoration of calpain2 in miR93overexpresseing SUI primary fibroblasts reversed the alteration in hydroxyproline expression, indicating that calpain2 was negatively associated with collagen expression. The results of the present study suggested that miR93 regulated MMP1 and collagen I expression in fibroblasts via calpain2. miR93 mediated collagen expression in stress urinary incontinence via calpain2.
Subject(s)
Calpain/genetics , Collagen Type I/genetics , MicroRNAs/genetics , Urinary Incontinence, Stress/genetics , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Expression Regulation/genetics , Humans , Matrix Metalloproteinase 1/genetics , Middle Aged , Urinary Incontinence, Stress/pathology , Vagina/metabolism , Vagina/pathologyABSTRACT
OBJECTIVE: To investigate the cholesterol metabolism and mRNA expression of the relevant genes in cholesterol synthesis of the cultured steatotic hepatocytes model. METHODS: A steatotic model of hepatocytes was constructed by adding palmitic acid to the growing L-02 cells. These cells were collected at day 3 and 6, respectively. Cells with the culture solution without palmitic acid added served as the control. The contents of intracellular triglyceride (TG) and total cholesterol (TC) were detected by the analysis kit. The expression of sterol-regulatory element binding protein-2 (SREBP-2) and its target gene hydroxymethylglutaryl CoA reductase (HMGCR) and low-density lipoprotein receptor (LDLR) were measured by RT-PCR. RESULTS: Hepatocyte steatosis was observed at day 3 and became more intense at day 6. The contents of intracellular TG and TC were increased and the expression of the SREBP-2, HMGCR and LDLR mRNA were upregulated in a time-dependent manner in the model group. Compared with the control group, the content of intracellular TG was higher at both day 3 and 6 (P < 0.05), while the content of intracellular TC was significantly increased only at day 6; The expression of HMGCR and LDLR mRNA was upregulated in steatotic hepatocytes at both day 3 and 6 (P < 0.05), whereas the SREBP-2 mRNA was increased only at day 6 (P < 0.05). CONCLUSION: Cholesterol accumulation is probably due to the upregulated expression of the relevant genes in the cholesterol synthesis of the steatotic hepatocytes.