Expression of TUBB4B in mouse primary spermatocyte GC-2 cells and its regulatory effect on NF-κB and MAPK signaling pathway / 南方医科大学学报

OBJECTIVE@#To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells) and the role of TUBB4B in regulating the development of GC-2 cells.@*METHODS@#Lentiviral vectors were used to infect GC-2 cells to construct TUBB4B knockdown and negative control (NC-KD) cells. The stable cell lines with TUBB4B overexpression (Tubb4b-OE) and the negative control (NC-OE) cells were screened using purinomycin. RT-qPCR and Western blotting were used to verify successful cell modeling and explore the relationship between TUBB4B and CCP1 expressions in GC-2 cells. The effects of TUBB4B silencing and overexpression on the proliferation and cell cycle of GC-2 cells were evaluated using CCK8 assay and flow cytometry. The signaling pathway proteins showing significant changes in response to TUBB4B silencing or overexpression were identified using Western blotting and immunofluorescence assay and then labeled for verification at the cellular level.@*RESULTS@#Both TUBB4B silencing and overexpression in GC-2 cells caused consistent changes in the mRNA and protein expressions of CCP1 (P < 0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration (P < 0.05). TUBB4B knockdown and overexpression had no significant effect on proliferation rate or cell cycle of GC-2 cells, but caused significant changes in the key proteins of the nuclear factor kappa-B (NF-κB) signaling pathway (p65 and p-p65) and the mitogen-activated protein kinase (MAPK) signaling pathway (ErK1/2 and p-Erk1/2) (P < 0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells (P < 0.05).@*CONCLUSIONS@#TUBB4B and CCP1 interact via a mutual positive regulation mechanism in GC-2 cells. CCP-1 can deglutamize TUBB4B, and the latter is involved in the regulation of NF-κB and MAPK signaling pathways in primary spermatocytes.

Protective effect of miR-9-5p regulating transient receptor potential melastatin 7 on myocardial ischemia-reperfusion in rats / 解剖学报

Objective To investigate the effect of microRNA-9-5p (miR-9-5p) regulating transient receptor potential melastatin 7 (TRPM7) on myocardial ischemia-reperfusion (MIR) in rats. Methods Thirty-two SD rats were divided into sham operation group, model group, miR-9-5p overexpression group and empty vector control group. The MIR model was established by ligation of left coronary artery. The sham operation group was not ligated. miR-9-5p agomir and agomir NC were injected into tail vein 24 hours before model establishment in miR-9-5p overexpression group and empty vector control group. The myocardial injury was observed by HE staining. The expression of miR-9-5p was detected by Real-time PCR. The serum levels of interleukin(IL)-6, tumor necrosis factor alpha(TNF-α), IL-1β, creatine kinase isoenzyme MB (CK-MB), cardiac troponin Ⅰ (cTnI), lactate dehydrogenase (LDH) and the contents of malondialdehyde (MDA) and superoxide dismutase (SOD) in myocardium were measured were measured by ELISA. Cardiomyocyte apoptosis was detected by TUNEL. Double luciferase assay verified the relationship between miR-9-5p and TRPM7. The protein expressions of TRPM7, Bcl-2, Bcl-2 associated X (Bax), phosphorylated nuclear factor kappa-B 65 (p-NF-κB p65) and toll like receptor 4 (TLR4) were detected by Western blotting. Results The expression of miR-9-5p was low in myocardial tissue of rats (P<0.05). Overexpression of miR-9-5p could reduce the expression levels of CK-MB, cTnI and LDH, and improve the degree of myocardial injury. Compared with the model group, the apoptosis rate, Bax protein expression, MDA, IL-6, TNF-α and IL-1β contents in myocardial cells of miR-9-5p overexpression group decreased, while Bcl-2 protein expression and SOD content increased (P<0.05). The result of dual luciferase assay showed that TRPM7 was the target gene of miR-9-5p, and the protein expressions of TRPM7, p-NF-κB p65 and TLR4 in miR-9-5p overexpression group were lower than those in model group (P<0.05). Conclusion MiR-9-5p can inhibit myocardial cell apoptosis, oxidative stress and inflammation induced by myocardial ischemia-reperfusion, and inhibit TLR4/NF-κB pathway by regulating TRPM7.

Effects of CENP-A on invasion and migration of ovarian cancer cells by regulating PI3K/AKT/NF-κB signaling pathway / 中华内分泌外科杂志

Objective:To investigate the effects of centromere protein-A (CENP-A) on the invasion and migration of ovarian cancer (OC) cells and explore the related mechanism.Methods:OC cell line A2780 was cultured in vitro, and they were divided into Ng Group (Blank Control Group) , pcDNA group (negative transfection group:PCDNA vector plasmid) , pcDNA-CENP-A group (over-expression Group: pcDNA-CENP-A Vector Plasmid) and pathway inhibitor group (TRANSFECTION-CENP-A+ PI3K pathway inhibitor LY294002) . The cell proliferation was detected by CCK-8 method; the cell migration and invasion was detected by Scratch test and Transwell test; the expression of CENP-A, E-cadherin, N-cadherin and phosphatidylinositol 3-kinase/protein kinase B/nuclear factor-kappa B (PI3K/AKT/NF-κB) pathway related proteins was detected by Western blot.Results:A2780 cells were successfully transfected. After 24 hours, with the extension of culture time, compared with that in NG group [ (0.50±0.07) , (0.72±0.11) , (0.99±0.14) ] and pcDNA group [ (0.55±0.08) , (0.78±0.12) , (1.02±0.15) ], the viability of A2780 cells in pcDNA-CENP-A group [ (0.78±0.12) , (1.03±0.15) , (1.67±0.25) ] and pathway inhibitor group [ (0.63±0.09) , (0.87±0.13) , (1.39±0.20) ] increased significantly ( P<0.05) , compared with that in the pcDNA-CENP-A group, the viability of A2780 cells in the pathway inhibitor group was significantly decreased ( P<0.05) , in a time-dependent manner. Compared with those in NG group [ (15.83±1.46) %, (105.32±15.78) individual] and pcDNA group [ (16.79±1.46) %, (108.98±16.35) individual], the migration rate [ (37.96±5.80) %, (25.15± 2.19) %] and invasion number [ (327.87±49.18) individual, 206.53±30.97) individual] of A2780 cells, protein expression of CENP-A, N-cadherin, Vimentin, p-PI3K/PI3K, p-AKT/AKT, NF-κB, interleukin (IL-1β) , tumor necrosis factor-α (TNF-α) in pcDNA-CENP-A group and pathway inhibitor group were significantly higher ( P<0.05) , the expression of E-cadherin was significantly lower ( P<0.05) ; compared with those in the pcDNA-CENP-A group, the migration rate and invasion number of A2780 cells, protein expression of CENP-A, N-cadherin, Vimentin, p-PI3K/PI3K, p-AKT/AKT, NF-κB, interleukin (IL-1β) , tumor necrosis factor-α (TNF-α) in pathway inhibitor group were significantly lower ( P<0.05) , and the expression of E-cadherin was significantly higher ( P<0.05) . Conclusion:Overexpression of CENP-A can promote the proliferation, invasion and migration of ovarian cancer cells, which may be achieved by activating PI3K/AKT/NF-κB signaling pathway.