Effects of silencing cyclooxygenase-2 expression via RNA interference on the tumorigenicity of the SMMC-7721 human hepatocarcinoma cell line.

We constructed a vector carrying a shRNA sequence against cyclooxygenase-2 (COX-2) that was subsequently transfected into the human hepatocarcinoma cell line SMMC­7721. Furthermore, we established a COX-2-deficient stable cell line and a model of tumor-shRNA transplantation in nude mice. Negative shRNA was used as the control. The tumor volume in the experimental group was smaller compared to that in the control group. Hematoxylin and eosin staining indicated that the cells in the experimental group differentiated better than those in the control group. The COX-2 mRNA level in the tumor tissues injected with SMMC-7721/COX-2i was markedly downregulated compared to that in the tumor tissues injected with SMMC-7721/negative shRNA. The inhibition rate reached 68.6%. Immunohistological study showed a significantly strong COX-2 expression in the control group tumor cells, whereas the experimental group exhibited moderate expression, indicating the inhibition of COX-2 expression after transfection of cells with shRNA against COX-2. Western blot analysis further proved the inhibition of COX-2 expression. In conclusion, RNAi-mediated regulation of COX-2 expression could efficiently inhibit liver-transplanted tumor growth in BALB/c nude mice.

Optimization of conditions for transfection with the Sofast gene vector.

We previously reported the synthesis and characterization of a novel cationic polymer gene vector. The present article further explored and optimized the working conditions of the Sofast gene vector both in vitro and in vivo, and improved its performance. The transfection conditions of Sofast, such as cell type, cell density, transfection time, N/P values and analysis time after transfection, were further explored. Moreover, the effects of the fusion peptide diINF-7 on transfection efficiency were examined. Sofast was successfully applied for the transfection of exogenous genes into more than 40 types of cell lines derived from humans, mice, monkeys and other species. When the cells were 50-80% confluent, Sofast possessed a better transfection efficiency. In most cases, Sofast also had a higher transfection efficiency when it was used to transfect cells that were seeded for several hours and had adhered to the substrate. The results from in vitro experiments indicate that the recommended Sofast to DNA mass ratio is 16:1, and the optimum analysis time after transfection is 48 h. The salt concentration in the Sofast working solution markedly affected the transfection efficiency. When conducting in vivo transfection, the working solution should be salt-free, whereas for in vitro transfection, it is more appropriate for the working solution to include certain salt concentrations. Finally, the results confirm that diINF-7 significantly promotes the transfection efficiency of Sofast. In conclusion, the present research not only established the optimal conditions for Sofast in the transfection of commonly used cells, but also built the foundations for in vivo and in vitro applications of Sofast, as well as its use in clinical practice.

Further evaluation of a novel nano-scale gene vector for in vivo transfection of siRNA.

In this research, a lipid-cationic polymer (LCP) containing the side-chain branching of brassidic acid was synthesized using chemical methods. As a gene vector for small interfering ribonucleic acid (siRNA) transfection, the efficiency and biosafety of LCP were preliminarily evaluated to investigate its possible application on tumor gene therapy. The toxicity, side-effects, and biosafety of LCP were investigated in animals based on the results of in vitro experiments. The siRNA against cyclooxygenase-2 (COX-2) was transfected by LCP to interfere with the COX-2 expression in nude-transplanted tumors. Hematoxylin and eosin stains, immunohistochemistry, reverse transcription-polymerase chain reaction, and Western blot were performed to evaluate the efficiency of LCP for siRNA transfection. The animal toxicity experiment showed that a high concentration of LCP had a low toxic effect on animals and did not induce allergic or pyrogenic reactions. The results from the in vivo transfection indicated that LCP could efficiently transfect siRNA and silence the target gene expression. The LCP gene vector for siRNA transfection is highly efficient during in vivo transfection and had low toxicity. From all aspects of tumor gene therapy and basic research, LCP is valuable for scientific research and medical applications.