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Objective: To investigate the effect and mechanism of miR-96-5p on apoptosis of PC12 cells induced by maltol aluminum. Methods: In January 2021, PC12 cells at logarithmic growth phase were divided into blank control group and low, medium and high dose group. Cells in each group were treated with 0, 100, 200 and 400 μmol/L maltol aluminum for 24 hours respectively. Cells were collected and cell apoptosis rates were detected by flow cytometry, miR-96-5p and insulin receptor substrate 1 (IRS1) mRNA expressions were detected by qRT-PCR, and the protein expression levels of cysteine protease 3 (Caspase3) 、activated cysteine protease 3 (Cleaved-caspase3) 、IRS1、phosphorylated protein kinase B (p-AKT) and phosphorylated glucose synthesis kinase 3β (p-GSK3β) were detected by western blotting. The target binding relationship between miR-96-5p and IRS1 was detected by double luciferase reporter gene experiment. The miR-96-5p inhibitor cells and negative control cells were constructed after transfecting PC12 cells with miR-96-5p inhibitor for 24 hours. The cells were divided into blank control group, negative control group, aluminum exposure group, aluminum exposure+negative control group, aluminum exposure+miR-96-5p inhibition group, and miR-96-5p inhibition group. After transfecting PC12 cells with miR-96-5p inhibition and IRS1 siRNA for 24 h, the cells were divided into aluminum exposure+miR-96-5p inhibition+negative control group and aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group. The control group was cultured in complete culture medium, and cells in the aluminum exposure group were treated with 200 μmol/L maltol aluminum for 24 hours. Cells in each group were collected and the apoptosis rate, miR-96-5p and IRS1 mRNA expression levels, as well as protein expression levels of Caspase3, Cleaved-caspase3, IRS1, p-AKT, and p-GSK3β were measured. Results: After 24 hours of exposure, compared with blank control group and low-dose group, the apoptosis rates, relative expressions of Caspase3 and Cleaved-caspase3 proteins, and relative expressions of miR-96-5p in the medium and high-dose groups of PC12 cells were significantly increased, while the relative expression levels of IRS1 mRNA, IRS1, p-AKT and p-GSK3β proteins were significantly decreased (P<0.05). Targetscan prediction and double luciferase report experiment both proved that IRS1 was a direct target gene of miR-96-5p. In the transfection experiment, compared with the aluminum exposure group, the apoptosis rate, the relative expressions of Caspase3 and Cleaved-caspase3 proteins, the relative expression of miR-96-5p in the aluminum exposure+miR-96-5p inhibition group were significantly decreased, while the relative expression levels of IRS1 mRNA and IRS1, p-AKT and p-GSK3β proteins were significantly increased (P<0.05). In the IRS1 low expression experiment, compared with the aluminum exposure+miR-96-5p inhibition+negative control group, the apoptosis rate, the relative expressions of Caspase3 and Cleaved-caspase3 proteins in the aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group were significantly increased, while the relative expression levels of IRS1 mRNA and IRS1, p-AKT and p-GSK3β proteins were significantly decreased (P<0.05) . Conclusion: The increased expression of miR-96-5p and the targeted inhibition of IRS1 may be one of the mechanisms of apoptosis of PC12 cells induced by maltol aluminum exposure.
Subject(s)
Animals , Rats , Aluminum/toxicity , Apoptosis , Cell Proliferation , Glycogen Synthase Kinase 3 beta/metabolism , Insulin Receptor Substrate Proteins/metabolism , MicroRNAs/metabolism , PC12 Cells , Proto-Oncogene Proteins c-akt/metabolism , RNA, MessengerABSTRACT
Abstract Objectives: Lung cancer was one of the most common malignancies around the world. It has great significance in to search for the mechanism of occurrence and development of lung cancer. LIM Domain Binding protein 2 (LDB2) belongs to the LIM-domain binding family, it can be used as a binding protein that combined with other transcription factors to form the transcription complex for regulating the expression of target genes. The expression of microRNA-96-5p (miR-96-5p) has been investigated in various tumors. The aim of this study is to investigate the potential role of LDB2 and miR-96-5p in lung cancer. Methods: Real-time quantitative PCR was applied to detect the expression of LDB2 and miR-96-5p. The proliferation, invasion, and metastasis of H1299 cells were analyzed by CCK8, transwell, and wound healing assay after LDB2 or miR-96-5p transfection. Luciferase activities assay and western blot were used to reveal the targeted regulation between LDB2 and miR-96-5p. Results: Here the authors found LDB2 was down-regulated in lung cancer tissues and negatively correlated with miR-96-5p expression, it could promote or inhibit the proliferation, invasion and metastasis of H1299 cells after LDB2 knockdown or overexpression and regulate the expression of cyclinD1, MMP9, Bcl-2, and Bax via ERK1/2 signaling pathway. Furthermore, miR-96-5p exerted its function by directly binding to 3′-UTR of LDB2 and regulating expression of LDB2. miR-96-5p could promote the proliferation, invasion, and metastasis of H1299 cells. Conclusion: These findings demonstrate that LDB2 can act as a new regulator to inhibit cell proliferation, invasion, and metastasis via the ERK1/2 signaling pathway, and miR-96-5p may be a potential promising molecular by targeting LDB2 in lung cancer.
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@#[Abstract] Objective: To investigate the expression changes of miR-96 in endometrial cancer tissues and cells, and to explore its effect on tumor malignant phenotypes as well as the possible mechanisms. Methods:From April 2016 to December 2018, 76 cases of endometrial cancer tissues from 76 patients who were surgically treated in the Department of Obstetrics and Gynecology of our hospital were selected for this study. qPCR was used to detect the expression of miR-96 in human endometrial cancer tissues and cells, and the correlation between the miR-96 expression and the clinicopathological characteristics of patients was analyzed. miR-96 inhibitor was transfected into human endometrial cancer Ishikawa cells in vitro. After transfection, the expression of miR-96 in Ishikawa cells was detected by qPCR; the tumor biological behaviors of Ishikawa cells were detected by CCK-8 test, Clone formation test, Flow cytometry, Scratch test and Transwell test; and the FOXO1 protein expression in Ishikawa cells was detected by WB. At the same time, Dual luciferase reporter gene assay was used to observe the targeting relationship between miR-96 and FOXO1. Results: The results of qPCR showed that the expression of miR-96 was abnormally high in human endometrial cancer cells (JEC, Ishikawa, HEC-1B) and endometrial cancer tissues (all P<0.01), and the expression of miR-96 was closely related to FIGO stage and lymph node metastasis (all P<0.05). After transfection with miR-96 inhibitor, the expression level of miR-96 in Ishikawa cells decreased significantly (P<0.01), the proliferation activity and clone formation ability decreased significantly (all P<0.01), the apoptotic rate increased significantly (P<0.01), and the scratch healing rate and the number of invasive transmembrane cells decreased significantly (P<0.01). Dual luciferase reporter gene assay showed that miR-96 could directly target FOXO1, and WB showed that miR-96 could negatively regulate FOXO1 protein expression in Ishikawa cells (P<0.01). Conclusion: The expression of miR-96 is abnormally high in endometrial cancer tissues and cells. Inhibiting the expression of miR-96 can inhibit the proliferation, invasion and migration of endometrial cancer cells and promote their apoptosis. The mechanism may be related to the targeted regulation of FOXO1.
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@#AIM: To investigate the effect of microRNA-96-5p(miR-96-5p)on proliferation and apoptosis of rat retinal vascular endothelial cells induced by high glucose and to explore its mechanism. <p>METHODS: SD rat retinal vascular endothelial cells(RRVEC)were cultured and the RRVEC was divided into control group(NG)and high glucose group(HG). The high glucose-induced RRVECs were harvested separately or co-transfected with miR-96-5p mimic, miR-NC, si-FOXO4, si-NC. The expression of miR-96-5p and FOXO4 was detected by qRT-PCR and Western blotting, respectively. MTT assay was used to detect the proliferation activity. Flow cytometry was used to detect the apoptosis rate. The dual luciferase reporter assay validated the target gene of miR-96-5p. Western blotting was used to detect the expression of CyclinD1, p21, p27, Bcl-2, Bax and cleaved-caspased-3. <p>RESULTS:The expression levels of miR-96-5p, CyclinD1 and Bcl-2 in RRVEC were significantly decreased after high glucose treatment, and the expression levels of FOXO4, p21, p27, Bax and cleaved-caspased-3 were significantly increased, inhibiting cell proliferation activity, but promoting apoptosis. Overexpression of miR-96-5p and inhibition of FOXO4 expression increased the expression levels of CyclinD1 and Bcl-2, inhibited the expression of p21, p27, Bax, cleaved-caspased-3, enhanced cell proliferation and inhibited apoptosis. Dual luciferase reporter assay demonstrated that FOXO4 was a target gene for miR-96-5p. Overexpression of FOXO4 reversed the effect of miR-96-5p overexpression on high glucose-induced proliferation and apoptosis of RRVEC. <p>CONCLUSION:miR-96-5p inhibits high glucose-induced apoptosis of rat retinal vascular endothelial cells and promotes cell proliferation by targeting FOXO4.
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Objective To explore the effects of hsa-miR-96 to A549. Method A549 were transfected with miR-96 inhibitor/mimics and shRNA-mTOR. The proliferation were detected by MTT,the expression of p21, cyclin D1,p-4E-BP,S6K were detected via RT-PCR and WB. The luciferase assay were used to anaylse the target of miR-96. Result With or without metformin treated,up-regulated the expression of miR-96 could inhibit A549 proliferation,increase p21 expression and decrease cyclin D1 expression instead. The results may related with G1 arrest which induced by miR-96 up-regulation. The ratio of firefly fluorescence value/Renilla fluorescence value was lower than that in wild type NC or mutant group. Conclusion miR-96 target with mTOR to inhibit the growth of A549.
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Objective To investigate the expression and clinical significance of miR-96-5p in primary hepatocellular carcinoma (HCC) at early recurrence after radical surgery. Methods 61 HCC eryopreservation tissue samples from the liver carcinoma specimens data obtained after radical surgery and banked in our hospital were divided into 2 groups: early recurrence group (33 cases) and non-early recurrence group (28 cases). Aquantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-96-5p. Results Compared with the non-early recurrence group , the expression of miR-96-5p was observably down-regulated [(0.634 ± 0.783) vs (5.182 ± 11.321), P = 0.043]. The expression of miR-96-5p was correlated to tumor diameter, early recurrence and vascular invasion (P<0.05). Conclusions miR-96-5p are significantly related to early liver cancer recurrence and metastasis. miR-96-5p may be a molecular marker of HCC at early recurrence as well as a target for targeted therapy of liver cancer in future.
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10.3969/j.issn.1007-3969.2013.04.008