Expression and clinical significance of hsa-miRNA-200c in patients with liver metastasis of epithelial ovarian cancer / 中国实用妇科与产科杂志

OBJECTIVE: To investigate the expressions of hsa-miRNA-200 c and its relationship with metastasis of EOC.METHODS: The expression of hsa-miRNA-200 c was detected by Stem-loop Real-time Quantitative PCR(TaqMan probe method)in 73 cases of EOC,30 cases of benign ovarian epithelial tumors and 30 cases of normal ovarian tissues,which were collected in gynecological operations from Guangdong General Hospital from October 2010 to May 2011.Meantime,the clinical pathologic features data were analyzed.The assessment of the correlation between hsa-miRNA-200 c and clinicopathological features,and the hierarchical analysis of hsa-miRNA-200 c level in 73 cases of ovarian epithelial cancer was further undertaken(Among 73 cases,13 patients suffered liver metastasis and 60 patients had non-liver metastasis).Overexpression or knockdown of hsa-miRNA-200 c,ovarian cancer cell invasion and migration abilitywas detected.RESULTS: The expression of miRNA-200 c in the EOC tissues was 382.18±15.22,which was significantly higher than that in the benign ovarian epithelial tumors(35.61 ± 1.42)and normal ovarian tissues(4.43 ±2.23)(P0.05).The expressions of miRNA-200 c were low in EOC with late clin-ical FIGO stage(670.91±16.88 vs. 129.52±33.3,P0.05).The transwell cabinet invasion experiment showed the expression of miRNA-200 c was negatively correlated with the invasion capability of ovarian cancer cells.CONCLUSION: MiRNA-200 c is likely to play a double regulation role in the development of EOC,whose low-expression has been associated with late EOC,lymph node metastasis,liver metastasis,and poor prognosis.

The mechanism of tenuigenin for eliminating waste product accumulation in cerebral neurons of Alzheimer's disease rats via ubiquitin-proteasome pathway / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the scavenging action of tenuigenin (TEN) on intracerebral amyloid β protein (Aβ) aggregation and the abnormal phosphorylated tau protein and its mechanism in Alzheimer's disease (AD) rats' brain.</p><p><b>METHODS</b>Aβ1-40 was injected into the right CA1 region hippocampus to establish the AD model. Successfully modeled rats were divided into the model group, the low, middle, high TEN group. Rats were administered with TEN (18.5, 37.0, 74.0 mg/kg) by gastrogavage. Besides, a sham-operation group was set up. Expression levels of Aβ1-40 and Tau p-Ser262 were detected by immunohistochemistry. Expression levels of ubiquitin (Ub) and Ub-protein ligase E3 were measured by Western blotting.The content of 26S proteasome was detected by ELISA.</p><p><b>RESULTS</b>Immunohistochemical results showed that the number of Aβ and Tau p-Ser262 positively reacted neurons significantly increased in model group, when compared with the sham-operation group (P < 0.01). Results of Western blot showed expression levels of ubiquitinated protein were up-regulated and those of Ub-protein ligase E3 were down-regulated in the model group (P < 0.01). ELISA results showed that the content of 26S proteasome significantly decreased in AD rats' brain (P < 0.01). Compared with the model group, expression levels of Aβ1-40, Tau p-Ser262, and Ub significantly decreased; expression levels of Ub-protein ligase E3 apparently increased; the content of 26S proteasome significantly increased in each TEN treatment group (P < 0.05, P < 0.01). Best effect was shown in 37.0 mg/kg and 74.0 mg/kg TEN groups.</p><p><b>CONCLUSIONS</b>Ub proteasome pathway (UPP) participated in the occurrence of AD. TEN could obviously reduce intracere- bral Aβ1-40 accumulation and abnormal tau phosphorylation.</p>

Ultrasound-mediated microbubble cavitation enhances gene transduction in rat pulmonary endothelial cells partially by affecting membrane fluidity and cytoskeleton structure / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the effect of therapeutic ultrasound-induced microbubble's cavitation on plasmid gene transduction in rat pulmonary endothelial cells in relation to the changes of membrane fluidity and cytoskeleton structure.</p><p><b>METHODS</b>Rat endothelial cells cultured in vitro were transfected with EGFP plasmid in the presence of protein microbubbles. During the transfection process, the cells were exposed to continuous 2 MHz ultrasonic irradiation for 30, 60, 90, 120 and 180 s (groups A, B, C, D and E, respectively) with the constant mechanical index (MI) of 1.0, or for 60 s with different mechanical index (MI) of 0.5, 0.75, 1.0, 1.5, and 1.8 (groups B1, B2, B3, B4 and B5, respectively). The changes of endothelial cytoskeletal structure and membrane fluidity were evaluated by immunofluorescence staining after the exposure.</p><p><b>RESULTS</b>EGFP gene transduction increase obviously with prolonged echo irradiation and increased MI. The intensity of immunofluorescence staining, which represented endothelial membrane fluidity, was 0.173±0.013, 0.250±0.037, 0.364±0.022, 0.381±0.019, and 0.395±0.009 in groups A-E, as compared with 0.171±0.017, 0.255±0.026, 0.378±0.007, 0.382±0.009 and 0.397±0.008 in groups B1-B5, respectively. The recovery intensity of the immunofluorescence staining representing the changes in microtubulin of the cytoskeleton structure was 159.15±4.79, 188.23±6.20, 205.80±4.48, 208.99±8.34, and 213.70±5.09 in groups A-E, and was 176.84±3.10, 187.57±14.52, 206.41±11.66, 220.12±13.39 and 221.16±12.78 in groups B1-B5, respectively. The endothelial membrane fluidity and microtubule fluorescence recovery intensity increased remarkably compared with the baseline (P<0.01) within the MI range of 0.50-1.0 and the exposure time of 30-90 s, but underwent no further changes in response to prolonged exposure time (180 s) at the MI of 1.5 (P>0.05). No changes in microfilament fluorescence intensity were observed after exposure to different MI or irradiation time.</p><p><b>CONCLUSION</b>Therapeutic ultrasound-mediated albumin microbubble cavitation allows enhances plasmid gene transduction without causing cytoskeleton damages. Increased endothelial membrane fluidity and changes in cytoskeleton structure, especially microtubulin, partially contribute to this enhancement.</p>

Construction of an eukaryotic expression vector for PRL-2 and its effect on human hepatocellular carcinoma cell invasiveness and migration in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct an eukaryotic expression vector for PRL-2 and evaluate its effect on the invasiveness and migration of a human hepatocellular carcinoma cell line.</p><p><b>METHODS</b>RT-PCR was performed to amplify the complete PRL-2 open reading frame using the total mRNA of hepatocellular carcinoma HepG2 cells as the template. PRL-2 gene was inserted into the pGEM T easy vector and sequenced, and the correct PRL-2 sequence was subcloned into the mammalian expression vector pcDNA3.1+. The constructed PRL-2 vector was transfected into CL1 cells via lipofectamine, and the stable expression of PRL-2 mRNA was detected by RT-PCR, the expressed protein identified by immunohistochemistry and Western blotting, and the effect of PRL-2 on the adhesion ability of CL-1 cell evaluated with MTT assay 20 and 120 min after transfection. The effect of PRL-2 on the invasive migration of CL-2 cells was evaluated according to the number of cells penetrating the Matrigel layer of polycarbonate membrane of Boyden chamber.</p><p><b>RESULTS</b>RT-PCR yielded a fragment of 504 bp and the inserted PRL-2 sequence was verified by sequence analysis. The subclones were identified by restriction endonuclease digestion, and a G418-resistant clone, PRL-2-CL1, was obtained after 8 weeks of selection. RT-PCR showed stable expression of PRL-2 mRNA, and Western blotting confirmed overexpression of PRL-2 protein in the transfected cells. PRL-2 increased the adhesion rate of CL-1 cells to fibronectin at 20 min and 120 min after transfection (P<0.05), and also the number of CL-1 cells penetrating the polycarbonate membrane from 10.0+/-3.7 to 44.8+/-2.6 (P<0.05).</p><p><b>CONCLUSION</b>An eukaryotic expression vector of PRL-2 has been successfully constructed, which allows stable and efficient expression in CL-1 cell line. PRL-2 can promote cell adhesion and invasion activity of human hepatocellular carcinoma cells.</p>