Intron-specific shRNA-mediated downregulation of survivin and promotion of apoptosis in HeLa cells.

Overexpression of the survivin gene contributes to tumorigenesis; it has been recognized as an important target for cancer therapy. In the present study, survivin expression was suppressed using recombinant plasmid mediated short hairpin RNAs (shRNAs) that were constructed to target exonic or intronic sequences of the survivin gene. In addition, a negative control shRNA was constructed. HeLa cells were transfected with specific shRNA constructs and the blocking efficiency of each shRNA was assessed at the mRNA and protein levels; and the five shRNA constructs with higher blocking efficiency were selected. Cell apoptosis was assessed by flow cytometry (FCM) following Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Hoechst staining was used to detect the morphological diversity of the nuclei in apoptotic cells. The results demonstrated that survivin expression was effectively reduced by the transfection of shRNAs in HeLa cells. In addition, the apoptotic rates of the shRNA-treated groups were significantly increased compared with the negative control group according to the FCM results. The nuclei of HeLa cells exhibited apoptotic characteristics in the shRNA-treated groups as identified by Hoechst staining. Survivin-targeting shRNAs effectively downregulated the expression of the gene and markedly increased the apoptotic rate of HeLa cells. Data from the present study also indicated that the intron-specific shRNA demonstrate a high efficiency of inhibition of survivin expression and were able to induce cell apoptosis of HeLa cells through RNAi, potentially providing novel target sites for tumor therapy. In conclusion, the present study suggests that intron-specific blocking of survivin by RNAi may provide a tool for anticancer therapy.

Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α.

The aim of the present study was to construct a fast-acting, eukaryotic expression vector in eukaryotic cells based on transmembrane-tumor necrosis factor­α (TM­TNF­α) structure. Two types of recombinant eukaryotic expression vectors were constructed, pcDNA3.1-TM-enterokinase-TNF­α and pcDNA3.1­TM­Factor Xa­TNF­α, according to the TNF­α transmembrane segments. Following the generation of these vectors, mouse embryonic 3T3 fibroblasts were transfected and reverse transcription­polymerase chain reaction and western blotting analyses were used to analyze mTNF­α mRNA and protein expression levels, respectively, in total cellular protein extracts and extracellular fluid. The biological activity of TNF-α in the extracellular fluid was then measured using an MTT assay. The vectors were successfully constructed, and mRNA and fusion proteins were detected in the 3T3 cells. Among the fusion proteins, the one observed in pcDNA3.1-TM-FactorXa-TNF-α-transfected 3T3 cells remained as a transmembrane protein. In addition, treatment of L929 cells with TNF­α derived extracellular fluid samples from pcDNA3.1­TM­FactorXa­TNF­α­transfected 3T3 cells was associated with a dose­dependent reduction in in cell­specific activity. The results indicate that proteins expressed using pcDNA3.1­TM­FactorXa­TNF­α vectors form transmembrane proteins. In addition, the results indicate that, only when coupled with FactorXa activity, the extracellular region of TM­TNF­α forms s­TNF­α, and the controlled expression of the fusion protein is initiated.

Construction of the POT1 promoter report gene vector, and the effect and underlying mechanism of the POT1 promoter in regulating telomerase and telomere length.

By using human genomic DNA as a template to clone protection of telomere 1 (POT1) promoter gene segments and construct the POT1 promoter luciferase report gene vector (pGL3-Control-POT1-promoter), the association between POT1, and the regulation of telomerase and telomere length was investigated. In the present study, two recombinant luciferase report gene vectors were constructed, which included different regions of the POT1 promoter. The plasmids were transformed into DH5α and the positive clones were obtained. The two plasmids termed as pGL3-Control-POT1-promoter-1 and pGL3-Control-POT1-promoter-2, were confirmed using restriction enzyme analysis and sequencing. They were separately and transiently transfected into four types of human tumor cells (A549, H460, HepG2 and HeLa). The transcriptional activities of the POT1 promoter were verified using the dual-luciferase assay. The relative expression of POT1 and human telomerase reverse transcriptase (hTERT), and telomere length were analyzed using quantitative polymerase chain reaction in the four types of non-transfected tumor cells. Using SPSS software, correlations between POT1 promoter activity, and POT1 expression, hTERT expression and telomere length were analyzed. Two POT1 promoter fragments (POT1-promoter-1 and -2) were successfully constructed into the pGL3-Control luciferase report gene vector. POT1-promoter-1 exhibited significantly stronger transcription activity compared with POT1-promoter-2. The results of the partial correlation and linear regression analyses were similar: POT1 promoter activity was identified to be significantly and positively correlated with POT1 expression and telomere length (partial correlation coefficients, both P<0.05; linear regression, both P<0.01). However, POT1 promoter activity and hTERT expression were significantly negatively correlated (both P<0.05). The results obtained in the present study suggest that the POT1 promoter influences telomere length. Furthermore, these data indicated that POT1 promoter activity and POT1, as well as telomere length, may be a useful biomarker for tumor detection and future patient prognosis.

[Preliminary study on the relationship between sow maternal behaviour during early lactation and polymorphism of PRLR gene].

PCR-RFLP technique was applied to analyze single nucleotide polymorphisms of PRLR gene in Minpig and Landrace to investigate the possible effect of PRLR gene on sow maternal behaviour. A Nae I-RFLP site was detected in the PRLP gene. The single nucleotide polymorphism, a T-->C transition at nucleotide 1,620 of the cDNA sequence, was a silent mutation. Least square analysis between the genotypes and the maternal behavioural traits showed that sows with genotype AB had a significantly higher frequency of lateral-lying-to- other-posture trait and percentage of sow-terminated nursing trait than sows with the AA and BB genotypes, although no significant differences were found in other behavioural traits. It is possible that allele A is the unfavorable allele for sow maternal behaviour.