miR-93­mediated collagen expression in stress urinary incontinence via calpain-2.

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.

Cholesterol metabolism and expression of its relevant genes in cultured steatotic hepatocytes.

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.