miR-451 inhibits inflammatory responses in glomerular mesangial cells by targeting Psmb8 in diabetic nephropathy mice / 中国病理生理杂志

AIM:To investigate the effect of microRNA(miR)-451 by targeting proteasome subunit βtype 8 (Psmb8)on the inflammatory responses in mouse glomerular mesangial cells(MCs)under high-and low-glucose condi-tions.METHODS:The expression levels of miR-451,IL-18 mRNA and TNF-αmRNA were detected by qPCR.The pro-tein expression levels of IL-18,TNF-αand Psmb8 were determined by Western blot when miR-451 was over-expressed and down-expressed in the MCs.Moreover,the expression of IL-18 and TNF-αwas detected when Psmb8 was silenced by si-Psmb8 in MCs.RESULTS:The expression of miR-451 was significantly decreased in the MCs treated with high glucose compared with low glucose group(P<0.01).However,the expression of Psmb8 was increased in high glucose group as compared with low glucose group(P<0.01).Moreover, the expression levels of Psmb8, IL-18 and TNF-αwere signifi-cantly decreased when miR-451 was over-expressed in high glucose group(P<0.01).Additionally,the expression levels of IL-18 and TNF-αwere significantly reduced when Psmb8 was silenced in the MCs under high glucose condition.CON-CLUSION:miR-451 reduces the expression of inflammatory factors via targeting Psmb8 in the MCs under high glucose condition.Therefore,miR-451 may play a role in inflammation of diabetic nephropathy.

Construction of let-7a expression plasmid and its inhibitory effect on k-Ras protein in A549 lung cancer cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To elucidate the role of let-7a-mediated gene regulation in the pathogenesis of lung cancer.</p><p><b>METHODS</b>Two template DNA sequences were designed based on hsa-let-7a sequence in miRBase database. The let-7a expression construct and a control plasmid, namely pGenesil-let-7a and pGenesil-control, respectively, were generated by cloning the annealed oligonucleotides into pGenesil-1 and then transfected into A549 cells, which were selected by G418 to establish the lung cancer cell lines stably expressing let-7a-GFP and control-GFP. The living cells were counted by MTT assay and cell growth curves were drawn to analyze the cell proliferation. The k-Ras mRNA level was assessed by semi-quantitative RT-PCR, and the expression of k-Ras protein was determined by Western blotting and immunocytochemistry.</p><p><b>RESULTS</b>The recombinant vectors were verified by sequencing. The cell growth curves indicated that the proliferation of the cells transfected with pGenesil- let-7a were inhibited significantly compared with that of cells transfected with pGenesil-control and A549 cells. Semi- quantitative RT-PCR analysis showed that the levels of k-Ras mRNA almost remained unchanged in cells with or without the treatments. Western blotting and immunocytochemistry demonstrated a significant decrease of k-Ras protein levels in cells transfected with pGenesil-let-7a, but not in cells transfected with pGenesil-control, when compared to A549 cells.</p><p><b>CONCLUSION</b>let-7a over-expression represses the expression of k-Ras protein and significantly inhibits the growth of lung cancer cells.</p>

Inducement effect of ginsenoside Rg3 on apoptosis of human bladder transitional cell carcinoma cell line EJ / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the effect of Rg3 on inhibiting and inducing apoptosis of bladder cancer cells.</p><p><b>METHOD</b>The bladder cancer cell line EJ was treated with Rg3 of various concentrations. Cell proliferation was measured by MTT assay. Morphological changes of cells were observed by fluorescent staining of Hoechst 33258. Cell cycle and apoptosis rate were analyzed by flow cytometry (FCM). The expression of caspase-3 in cells was detected by immunocytochemistry. DNA ladder was showed by agarose gel electrophoresis.</p><p><b>RESULT</b>Rg3 inhibited proliferation of EJ cells in a manner of concentration-dependent relationship, IC50 of Rg3 in 48 h treatment was 125.5 mg x L(-1) to EJ cells. When treated with 150 mg x L(-1) of Rg3 for 24 h and 48 h, the cells showed apoptotic morphological characteristics including the condensed chromatin, the nuclear fragmentation, the apoptotic body and bright fluorescent granules as well as a higher caspase-3 expression. FCM assay indicated that Rg3 regulated cell cycle and induced apoptosis of EJ cells. When treated for 24 h and 48 h with 75 mg x L(-1) of Rg3 as well as for 48 h with 150 mg x L(-1) of Rg3, the percentages of cells in S phase and G2/M phase were increased, whereas the percentage of cells in G0-G1 was decreased. The apoptosis rates were increased from (1.05 +/- 0.17)% in control group cells to (8.41 +/- 0.98)%, (18.57 +/- 2.20)% and (33.98 +/- 1.64)%, respectively. Remarkable DNA ladders were revealed. The effects showed a manner in dose and time dependent of Rg3.</p><p><b>CONCLUSION</b>The results suggest that ginsenoside Rg3 exerts an inhibiting effect on proliferation of EJ cells by inducing apoptosis.</p>