LIM-domain binding protein 2 was down-regulated by miRNA-96-5p inhibited the proliferation, invasion and metastasis of lung cancer H1299 cells

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.

Emodin reduces the injury of glomerular mesangial cells in lupus nephritis by targeting forkhead protein K2 through miR-96-5p / 中国临床药理学与治疗学

AIM: To investigate the injury of emodin (EMO) in reduce of glomerular mesangial cells (MCs) in lupus nephritis by targeting forkhead protein K2 (FOXK2) through miR-96-5p. METHODS: The contents of 24 h urine protein, serum urea nitrogen (BUN) and serum creatinine (Scr) in MRL / faslpr mice (lupus nephritis group) and MRL / MPJ mice (control group) were detected. MCs were separated, purified and divided into: MCs group (MCs without any treatment), L-EMO group (MCs treated with 10 μmol/L Emodin), M-EMO group (MCs treated with 25 μmol / L Emodin), H-EMO group (MCs treated with 50 μmol / L Emodin), H-EMO + miR-96-5p-NC group (MCs treated with 50 μmol / L Emodin and transfected with miR-96-5p-NC), and H-EMO + miR-96-5p-minic group (MCs treated with 50 μmol/ L Emodin and transfected with miR-96-5p-minic). Double luciferase report experiment was used to verify the targeting relationship between miR-96-5p and FOXK2. The real-time quantitative fluorescent polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-96-5p. Western blot was used to detect the expression of FOXK2 and apoptosis related proteins. The enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors in MCs. cell count kit 8 (CCK-8) was used to determine the activity of MCs. Annexin-V FITC/PI double staining was used to detect apoptosis of MCs. RESULTS: Compared with the control group, 24 h urinary protein content, serum BUN and Scr levels in the lupus nephritis group were significantly increased (P< 0.05). Compared with the MCs group, the miR-96-5p expression, interleukin1β (IL-1β), interleukin6 (IL-6), tumor necrosis factor-α (TNF-α), A450 value and B-lymphoblastoma-2 (Bcl-2) protein in the L-EMO group, M-EMO group and H-EMO group were significantly decreased (P<0.05), the FOXK2 level, cell apoptosis rate, Bcl-2 related X gene (Bax), aspartate specific cysteine proteinase-3 (cleaved Caspase-3) protein levels were significantly increased, respectively (P<0.05), the effect of Emodin was dose-dependent. Compared with the H-EMO group and H-EMO+miR-96-5p-NC group, H-EMO+miR-96-5p-minic group obviously increased the miR-96-5p expression, inflammatory factor levels, A450 value and Bcl-2 protein level (P<0.05), and obviously decreased FOXK2 level and cell apoptosis rate (P< 0.05). CONCLUSION: EMO can reduce the injury of lupus nephritis MCs by down-regulating miR-96-5p and then up-regulating FOXK2.

Effect of miR-96-5p targeting IRS1 on apoptosis of PC12 cells induced by aluminum maltol / 中华劳动卫生职业病杂志

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.

Effect of miR-96-5p targeting FOXO4 on proliferation and apoptosis of rat retinal vascular endothelial cells induced by high glucose / 国际眼科杂志(Guoji Yanke Zazhi)

@#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.

Expression and clinical significance of miR-96-5p in primary hepatocellular carcinoma at early recurrence after radical surgery / 实用医学杂志

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.