MicroRNA-128-3p inhibiting epithelial-mesenchymal transition of ovarian cancer cells by regulating zince finger E-bonx binding homeobox 1 / 解剖学报

Objective To investigate the effect of mircoRNA-128-3p (miR-128-3p) on epithelial-mesencfrymal transition (EMT) of ovarian cancer cells and its regulatory mechanism on zinc finger E-box binding homobox 1(ZEB1). Methods Real-time PCR technology was used to detect the expression of miR-128-3p in epithelial ovarian cancer tissue (EOC) and adjacent normal tissue (30 cases each), and to observe whether there was a difference. Two human ovarian cancer cell lines, SK0V3 and A2780, were selected and transfected respectively. MiR-128-3p mimic (miR-128-3p mimic group) and negative control mimic (NC mimic group) were used to detect the expression of miR-128-3p in 4 groups by Real-time PCR to verify the interference effect. Transwell assay was used. The migration and invasion abilities of the four groups of cells were observed. The regulatory relationship between miR-128-3p and ZEBl was verified by dual luciferase assay, and the expression level of ZEBl protein after overexpression of miR-128-3p was detected by Western blotting; pcDNA-ZEBl was transfected into SK0V3 and A2780 cell lines to make it overexpression of ZEBl was divided into NC mimic group, miR-128-3p mimic group, and miR-128-3p mimic+pcDNA-ZEBl group. Western blotting was used to detect the EMT-related protein E-cadherin in 6 groups of cells and the expression level of vimentin. Results Real-time PCR result showed that the expression of miR-128-3p in EOC tissues decreased compared with that in adjacent tissues (P < 0. 01); The relative expression of miR-128-3p in the miR-128-3p mimic group was higher than that in the NC mimic group, while the numbers of migrating cells and invasive cells were lower than those in the NC mimic group (P < 0 . 0 1) . The result of dual luciferase experiments showed that miR-128-3p had a negative regulatory effect on ZEBl. In SK0V3 and A2780 cells, the relative expression of ZEBl protein in the miR-128-3p mimic group was lower than that in the NC mimic group, while the relative protein expression of E-cadhein was higher than that in the NC mimic group (P < 0 . 0 1) . The relative protein expression of E-cadhein in the miR-128-3p mimic+pcDNA-ZEBl group was lower than that in the miR-128-3p mimic group (P < 0 . 0 1) . In SKOV3 and A2780 cells, the relative protein expression of vimentin in the miR-128-3p mimic group was lower than that in the NC mimic group, and the relative protein expression of vimentin in the miR-128-3p mimic+pcDNA-ZEBl group was higher than that in the miR-128-3p mimic group (P < 0 . 0 1) . Conclusion The expression of miR-128-3p decreases in epithelial ovarian cancer tissues, which ma)' be due to the regulation of ZEBl to affect the expression of EMT-related proteins and participate in the EMT process of ovarian cancer cells.

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>

Inhibitory effect of ginsenoside-Rg3 on lung metastasis of mouse melanoma transfected with ribonuclease inhibitor / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effect of ginsenoside-Rg3 on lung metastasis of ribonuclease inhibitor (RI) gene-transfected mouse B16 melanoma.</p><p><b>METHODS</b>C57BL/6 mice were iv injected with parental or RI-transfected B16 melanoma cells. Lung metastasis was assessed by the number of surface tumor nodules. Mice were divided into 6 groups. Group I, II and III of mice were given parental, mock-transfected and RI-transfected B16 melanoma cells, respectively while in group IV, V and VI, Rg3 (1.5 mg/kg, iv q.o.d. x 10) was given to mice bearing parental, mock-transfected and RI-transfected B16 melanoma, respectively. Micovessel density (MVD) of the lung metastatic tumor was assessed by immunohistochemical staining of factor VIII-R expression.</p><p><b>RESULTS</b>The number of tumor nodules was significantly decreased in mice injected with RI-transfected B16 melanoma (Gp III, compared to Gp I and II). Rg3 treatment per se could also decrease the number of lung tumor nodules but to a lesser extent (Gp IV and V compared to Gp III). However, Rg3 synergized with RI transfection resulting in most significant inhibition of lung metastasis (Gp VI). Mice in Gp I and II died within 26 days of the experiment, whereas all the mice in Gp VI were alive during the observation period of one and one half month. MVD was significantly decreased in the lung tumor nodules in mice injected with RI-transfected B16 melanoma. It was further decreased when additional Rg3 was given (Gp VI).</p><p><b>CONCLUSION</b>Transfection of ribonuclease inhibitor gene significantly reduces the metastatic potential of B16 melanoma. Ginsenoside-Rg3 has a synergistic effect.</p>