[Inhibition of VEGF expression by plasmid-based RNA interference in the retinoblastoma cells].

OBJECTIVE: To explore the inhibition effect of small hairpin RNA (shRNA) expression plasmid targeting vascular endothelial growth factor (VEGF) on VEGF expression in cultured retinoblastoma (RB) cells. METHODS: VEGF shRNA plasmid p4.1CMV-VEGF was constructed and transfected into retinoblastoma cell lines SO-RB50 and HXO-RB44. Using neomycin G418 in conjunction with gradient dilution, p4.1CMV-VEGF shRNA positive single clone of RB cells was selected and subsequently enriched. Real-time polymerase chain reaction (PCR) was applied to detect VEGF mRNA levels of RB cells. VEGF protein concentration in culture supernatants of RB cells were determined by enzyme-linked immunosorbent assay (ELISA). Plasmid p4.1CMV-Neg shRNA, which expressed shRNA lacking significant sequence identity to human and mouse genome databases, was transfected into RB cells as negative control. Cells without any treatment were used as blank controls. RESULTS: p4.1CMV-VEGF shRNA was constructed successfully and VEGF shRNA construct positive clone of RB cells was developed. VEGF mRNA level of SO-RB50 (HXO-RB44) cells in negative control and blank control was 5.02 (5.70) folds and 6.32 (4.86) folds greater than that in p4.1CMV-VEGF shRNA treated SO-RB50 (HXO-RB44) cells. VEGF protein concentration in culture supernatants of p4.1CMV-VEGF shRNA treated SO-RB50 cells (187.69 +/- 83.89) microg/L was significantly lower than that of negative control (822.98 +/- 187.98) microg/L and blank control (865.76 +/- 170.33) microg/L (P < 0.01). There was also significant difference of VEGF protein concentration between p4.1CMV-VEGF shRNA treated HXO-RB44 cells (162.20 +/- 66.33) microg/L and controls (764.33 +/- 164.79) microg/L in negative control and (828.22 +/- 145.94) microg/L in blank control (P < 0.01). CONCLUSIONS: Stable transfection of VEGF shRNA expression plasmid can potently suppress VEGF expression in RB cells. RNA interference (RNAi) targeting VEGF promises to be a substantial tool for the study of the treatment of RB.

Inhibition of Bcl-2 expression by a novel tumor-specific RNA interference system increases chemosensitivity to 5-fluorouracil in Hela cells.

AIM: RNA interference (RNAi) has been proposed as a potential treatment for cancer, but the lack of cellular targets limits its use in cancer gene therapy. No current technology has achieved direct tumor-specific gene silencing using RNAi. In the present study we attempt to develop a tumor-specific RNAi system using the human telomerase reverse transcriptase (hTERT) promoter; furthermore, we analyzed its inhibitive effect on Bcl-2 expression. METHODS: The vectors containing a small hairpin RNA (shRNA) to target exogenous reporters [firefly luciferase and enhanced green fluorescent protein (EGFP)] and endogenous gene (Bcl-2) were constructed. Luciferase expression was determined by dual luciferase assay. Reverse transcription-polymerase chain reaction (RT-PCR), fluorescence microscopy and fluorescence-activated cell sorting (FACS) were used to measure EGFP expression. Inhibition of Bcl-2 was evaluated by RT-PCR and Western blotting. Cell proliferation and viability were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. FACS was used to analyze the cell cycle distribution profile. RESULTS: We showed that with the hTERT promoter directly driving shRNA transcription, expression of the exogenous reporters (LUC and EGFP) in tumor cells, but not normal cells, was specifically inhibited in vitro. The hTERT promoter-driven shRNA also depressed the expression of Bcl-2. Inhibition of Bcl-2 did not affect cell proliferation, but increased the chemosensitivity of HeLa cells to 5-fluorouracil. CONCLUSION: The present study describes an efficient RNAi system for gene silencing that is specific to tumor cells using the hTERT promoter. Suppression of Bcl-2 by using this system sensitized HeLa cells to 5-fluorouracil. This system may be useful for RNAi therapy.

[Activity analysis of human beta ß2-microglobulin gene promoter in P815 cells].

AIM: To clone the human beta2-microglobulin(beta2m) gene promoter and study its activity in P815 cells. METHODS: PCR amplification was performed using primers based on human beta2m gene sequence from GenBank and human genomic DNA as a template. The PCR product was directedly ligated into pBluescript II vector for sequencing. The promoter fragment was subcloned into a pcDNA3-EGFP plasmid after it had been identified correctly. A mouse mastocytoma cell line P815 was transiently and stably transfected with the plasmid containing human beta2m gene promoter and enhanced green fluorescence protein(EGFP) gene. The mRNA expression of EGFP in transiently transfected cells was quantified by RT-PCR and that in stably transfected cells was detected by fluorescence microscope and flow cytometry(FCM). RESULTS: A 302 bp DNA fragment was amplified and cloned into the pcDNA3-EGFP vector. RT-PCR analysis showed that EGFP mRNA expression was induced by IFN-gamma in a dose-dependent manner. There was no difference in the fluorescence positive cell rate between the IFN-gamma-treated group and the control group. But the fluorescence intensity of the 5 x 10(5) U/L IFN-gamma-treated group increased about 2 folds compared with that of the control group. CONCLUSION: Human beta2m gene promoter is active in mouse mastocytoma P815 cells. It can regulate the expression of reporter gene under the control of IFN-gamma.