ABSTRACT
This study aimed to investigate the effect of Wenyang Zhenshuai Granules(WYZSG) on autophagy and apoptosis of myocardial cells in rats with sepsis via regulating the expression of microRNA-132-3p(miR-132-3p)/uncoupling protein 2(UCP2). Sixty SD rats were randomly divided into modeling group(n=50) and sham operation group(n=10). The sepsis rat model was constructed by cecal ligation and perforation in the modeling group. The successfully modeled rats were randomly divided into WYZSG low-, medium-and high-dose groups, model group and positive control group. Rats in the sham operation group underwent opening and cecum division but without perforation and ligation. Hematoxylin-eosin(HE) staining was used to observe the pathological changes of rat myocardial tissue. Myocardial cell apoptosis was detected by TdT-mediated dUTP nick end labeling(TUNEL) assay. Real-time quantitative polymerase chain reaction(RT-qPCR) was performed to detect the expression of miR-132-3p and the mRNA expressions of UCP2, microtubule-associated protein light chain 3(LC3-Ⅱ/LC3-Ⅰ), Beclin-1 and caspase-3 in rat myocardial tissue. The protein expressions of UCP2, LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and caspase-3 in myocardial tissue were detected by Western blot. Dual luciferase reporter assay was used to verify the regulatory relationship between miR-132-3p and UCP2. The myocardial fibers of sepsis model rats were disordered, and there were obvious inflammatory cell infiltration as well as myocardial cell edema and necrosis. With the increase of the WYZSG dose, the histopathological changes of myocardium were improved to varying degrees. Compared with the conditions in the sham operation group, the survival rate and left ventricular ejection fraction(LVEF) of rats in the model group, positive control group and WYZSG low-, medium-and high-dose groups were decreased, and the myocardial injury score and apoptosis rate were increased. Compared with the model group, the positive control group and WYZSG low-, medium-and high-dose groups had elevated survival rate and LVEF, and lowered myocardial injury score and apoptosis rate. The expression of miR-132-3p and the mRNA and protein expressions of UCP2 in myocardial tissue in the model group, positive control group and WYZSG low-, medium-and high-dose groups were lower, while the mRNA and protein expressions of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and caspase-3 were higher than those in the sham operation group. Compared with model group, the positive control group and the WYZSG low-, medium-and high-dose groups had an up-regulation in the expression of miR-132-3p and the mRNA and protein expressions of UCP2, while a down-regulation in the mRNA and protein expressions of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and caspase-3. WYZSG inhibited excessive autophagy and apoptosis of myocardial cells in septic rats and improved myocardial injury, possibly by regulating the expression of miR-132-3p/UCP2.
Subject(s)
Animals , Rats , Rats, Sprague-Dawley , Caspase 3 , Beclin-1/genetics , Stroke Volume , Ventricular Function, Left , Apoptosis/genetics , Autophagy/genetics , Heart Injuries , MicroRNAs/geneticsABSTRACT
Abstract Objective: Mycoplasma pneumoniae pneumonia (MPP) is a common respiratory infection in children. Tumor necrosis factor-cx (TNF-α), interleukin-17 (IL-17), and IL-6 have correlation with Mycoplasma pneumoniae lung infection and MPP pathogenesis. Method: miRNAs participate in the pathogenesis of various diseases by regulating the development and differentiation of the immune cell. Blood was collected and total RNA was isolated. miRNA microarrays were performed to identify differentially expressed miRNAs in MPP patients. The levels of relative miRNAs and mRNAs were evaluated by qRT-PCR. Results: There are 23 differentially expressed miRNAs in MPP children's plasma, 15 miRNAs had enhanced expression and 8 had depressed expression. MPP patients showed lower mir-1323 level in blood samples than healthy controls. MPP patients with pleural effusion had much higher Il6 and Il17a mRNA levels than those without pleural effusion. The expression level of Il6 had a negative correlation with miR-1323 level. In the human THP-1 cell line, the level of miR-1323 was significantly reduced through lipopolysaccharides treatment. In THP-1 cells, overexpression or silencing of miR-1323 significantly reduced or promoted Il6 expression. Conclusion: In conclusion, miR-1323 targets the mRNA of Il6 and inhibits the expression of Il6. The pathogenesis of MPP inhibits the expression of miR-1323 in macrophages, triggers the overexpression of Il6, and enhances inflammation response.
Subject(s)
Humans , Child , Pneumonia, Mycoplasma , MicroRNAs/genetics , Tumor Necrosis Factor-alpha , Leukocyte Count , Mycoplasma pneumoniae/geneticsABSTRACT
BACKGROUND: Mechanical stress can influence the proliferation and differentiation of MC3T3-E1 cells and trigger differential expression of miR-132-3p. However, further research is warranted concerning whether tensile stress can influence the proliferation and differentiation of osteoblasts by regulating miR-132-3p. OBJECTIVE: To determine the expression of osteogenic differentiation markers and miR-132-3p in MC3T3-E1 cells under 12% cyclic stretch and to explore the effect of miR-132-3p on cell proliferation and differentiation. METHODS: MC3T3-E1 cells were loaded with 0% and 12% tensile stress, and alkaline phosphatase activity, osteocalcin mRNA and miR-132-3p expression levels were detected. MC3T3-E1 cells were transiently transfected with miR-132-3p mimics and a negative control transfection group was set up. The expression of alkaline phosphatase, osteocalcin and Runx2 mRNA in transfected cells were detected by qRT-PCR, and the effect of miR-132-3p on cell proliferation were detected by cell counting kit-8 assay. RESULTS AND CONCLUSION: The alkaline phosphatase activity and osteocalcin mRNA expression were down-regulated in MC3T3-E1 cells under 12% stretch stress (P < 0.01), and the expression of miR-132-3p was significantly increased (P < 0.05). QRT-PCR results showed the expression levels of osteogenic differentiation markers alkaline phosphatase activity, osteocalcin, and Runx2 mRNA in miR-132-3p mimics group were significantly decreased after intracellular transfection of miR-132-3p (P < 0.05). Compared with the negative control transfection group, the cell proliferation in the miR-132-3p mimic group was decreased at 24, 48, and 72 hours after transfection (P < 0.001), and the most obvious reduction was observed after 48-hour transfection. These findings indicate that 12% cyclic tensile stress can negatively regulate the proliferation and differentiation ability of MC3T3-E1 cells by overexpressing miR-132-3p.
ABSTRACT
Objective To study the effect of exosomes ( EXs) released from high expression of miR-132-3p mesenchymal stem cells (MSCs) on hypoxia/reoxygenation (H/R) injured endothelial cell function. Methods MSCs extracted from bone marrow of C57BL/6 mice were cultured primarily. MSCmiR-132-3p was obtained from MSCs infected with lentivirus loaded with miR-132-3p vector. At the same time,MSCNC was obtained by infecting MSCs with control lentivirus loaded with scramble sequence. EXs released from MSCNCand MSCmiR-132-3pwas isolated,and MSC-EXs and MSC-EXsmiR-132-3pwere obtained respectively. The obtained EXs and H/R damaged mouse brain microvascular endothelial cells (bend3) were co-cultured. According to culture conditions,the cells were divided into normal culture group (normal cell culture),H/R group (making a H/R model),MSC-EXs group (MSC-EXs co-culture),MSC-EXsmiR-132-3p group (MSC-EXsmiR-132-3pco-culture), and MSC-EXsmiR-132-3p+ LY294002 group ( before the cells and MSC-EXsmiR-132-3pwere co-cultured,treated by adding phosphatidyl alcohol 3 kinase [ PI3K] signaling pathway blocker LY294002 [20 μmol/L]). Quantitative real-time quantitative polymerase chain reaction was used to detect the expression of miR-132-3p in MSCs,MSC-EXs,and bend3 cells. Angiogenesis kit was used to detect angiogenic ability of bend3 cells,and 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferative capacity of bend3 cells. Scratch test was used to detect the migration ability of bend3 cells. hochest33258 staining showed cell apoptosis. Western blot was used to detect the phosphorylation level of protein kinase B ( Akt) . Results Compared with the H/R group, the MSC-EXs treatment group significantly improved the angiogenesis,proliferation,migration abilities, and Akt phosphorylation level of bend 3 cell damage induced by H/R (The H/R group were 3 ± 1,0. 275 ± 0. 020,147 ± 8 μm,and 0. 89 ± 0. 12,respectively;the MSC-EXs treatment group were 8 ± 3,0. 358 ± 0. 030,218 ± 10 μm, and 1. 37 ± 0. 25 μm,respectively;all P<0. 01). Apoptosis was significantly reduced (47 ± 2% vs. 63 ± 2%,all P<0. 01). Compared with the MSC-EXs treatment group,the angiogenesis,proliferation,migration abilities,and Akt phosphorylation level of bend 3 cells in the MSC-EXsmiR-132-3ptreatment group were increased (14 ±3,0. 444 ± 0.050,357±10μm,and1.67±0.23,respectively,all P<0.01).Apoptosis was significantly reduced (34±1%,all P<0. 01) . Compared with the MSC-EXsmiR-132-3ptreatment group, cell proliferation, migration, angiogenesis abilities,and Akt phosphorylation level in the MSC-EXsmiR-132-3p+LY294002 group were significantly reduced (5 ± 2,0. 304 ± 0. 050,175 ± 8 μm and 0. 95 ± 0. 11,respectively,all P<0. 01). Conclusion MSC-EXs with high expression of miR-132-3p may improve many physiological functions of H/R-induced damaged cerebrovascular endothelial cells by activating PI3K/Akt signaling pathway.
ABSTRACT
PURPOSE: Long non-coding RNA taurine upregulated gene 1 (TUG1) is reported to be a vital regulator of the progression of various cancers. This study aimed to explore the exact roles and molecular mechanisms of TUG1 in osteosarcoma (OS) development. MATERIALS AND METHODS: Real-time quantitative PCR was applied to detect the expressions of TUG1 and microRNA-132-3p (miR-132-3p) in OS tissues and cells. Western blot was performed to measure protein levels of sex determining region Y-box 4 (SOX4). Cell viability was assessed using XTT assay. Cell apoptosis was evaluated using flow cytometry and caspase-3 activity detection assays. Bioinformatics analysis and luciferase reporter experiments were employed to confirm relationships among TUG1, miR-132-3p, and SOX4. RESULTS: TUG1 was highly expressed in human OS tissues, OS cell lines, and primary OS cells. TUG1 knockdown hindered proliferation and induced apoptosis in human OS cell lines and primary OS cells. Moreover, TUG1 inhibited miR-132-3p expression by direct interaction, and introduction of miR-132-3p inhibitor partly abrogated the effect of TUG1 knockdown on the proliferation and apoptosis of OS cells. Furthermore, SOX4 was validated as a target of miR-132-3p. Further functional analyses revealed that miR-132-3p inhibited proliferation and induced apoptosis of OS cells, while this effect was greatly abated following SOX4 overexpression. Moreover, TUG1 knockdown suppressed proliferation and promoted apoptosis by upregulating miR-132-3p and downregulating SOX4 in primary OS cells. CONCLUSION: TUG1 facilitated proliferation and suppressed apoptosis by regulating the miR-132-3p/SOX4 axis in human OS cell lines and primary OS cells. This finding provides a potential target for OS therapy.