SnoVectors for nuclear expression of RNA.

Many long noncoding RNAs (lncRNAs) are constrained to the nucleus to exert their functions. However, commonly used vectors that were designed to express mRNAs have not been optimized for the study of nuclear RNAs. We reported recently that sno-lncRNAs are not capped or polyadenylated but rather are terminated on each end by snoRNAs and their associated proteins. These RNAs are processed from introns and are strictly confined to the nucleus. Here we have used these features to design expression vectors that can stably express virtually any sequence of interest and constrain its accumulation to the nucleus. Further, these RNAs appear to retain normal nuclear associations and function. SnoVectors should be useful in conditions where nuclear RNA function is studied or where export to the cytoplasm needs to be avoided.

A quantitative method for measuring the antitumor potency of recombinant human endostatin in vivo.

Recombinant human endostatin (rhEndostatin) has been shown to inhibit tumor growth, but the variable antitumor activity of different rhEndostatin preparations has necessitated the development of an accurate, reproducible in vivo bioassay for evaluating the rhEndostatin activity. To assess the in vivo antitumor efficacy of rhEndostatin, H22 tumor-bearing mice received three doses of rhEndostatin and the potency of rhEndostatin preparations in inhibiting tumor growth was determined by ED(50)-potency assay and validated by dose-response parallel-line assay. There was a consistent and highly reproducible linear regression relationship between rhEndostatin dosage and tumor growth inhibition rate. The ED(50) values were determined from dose-response regression lines for seven rhEndostatin preparations with high reproducibility. On the basis of the current study, the potency of rhEndostatin preparations was assigned a value of 6.09 x 10(5) U/ampoule and a 95% confidence limit of 5.96 x 10(5)-6.22 x 10(5). We consider that this procedure can be served as a potential candidate pharmacopoeial method for potency measurement of different rhEndostatin preparations.

RNA interference remarkably suppresses bcl-2 gene expression in cancer cells in vitro and in vivo.

Bcl-2 is an anti-apoptotic protein. If the level of Bcl-2 protein can be reduced sufficiently in tumors using RNA interference (RNAi) to target the gene message, the apoptosis of tumor cells may be promoted. In this study, we synthesized 19 nucleotides (nts) small interference RNA (siRNA) constructs suppressing bcl-2 gene expression in human tumor cells (HeLaB2 and BGC-823 cell lines) in vitro. The bcl-2 gene expression levels were significantly reduced when these siRNA were transfected into experimental two tumor cells for 72 hours. The apoptosis process was also examined in the tumor cells. Here we synthesized siRNA from a DNA template under the control of the RNA polymerase III promoter in transfected tumor cells. Using this DNA vector-based approach, we found that the siRNA efficiently and specifically inhibited the synthesis of protein encoded by the bcl-2 gene in HeLaB2 and BGC-823 tumor cells. Tumor growth was inhibited by 66.5% with 2mg/kg pSilencer 3.1H1-bcl-2 in mouse liver tumor-bearing BALB/c mice. This approach may prove to be a valuable clinical technique for the analysis of specific gene functions and gene therapy of malignant tumors that utilize the bcl-2 gene via RNA interference.

The methods for determining the purity and in vitro or in vivo activity of recombinant human endostatin.

In order to establish the methods of high-performance liquid chromatography (HPLC) for determining the purity of recombinant human endostatin (rhEndostatin) and in vitro or in vivo activity of rhEndostatin, two columns were firstly used in HPLC analysis for determining the purity of rhEndostatin, including Waters Symmetry 300C4 (4.6 mm x 250 mm, 5 microm) and the Superdex75 HR 10/30. Cell lines, bovine capillary endothelial cells (BCEs) or human umbilical vein endothelial cells (HUVECs) expression human vascular endothelial growth factor (hVEGF) were used in method MTT or LDH as substrate, respectively. The bioactivity in vivo was assayed by the anti-tumor proliferation rate in H22 liver tumor-bearing mice. The results showed that the retention time of rhEndostatin sample was stable at 19.066 min or 11.506 min in reverse phase HPLC (RP-HPLC) or gel filtering HPLC (GF-HPLC). The stableness, repeat and recovery rates were over 99% in both methods and there was no statistical difference between these two methods (p > 0.05). In nonserum culture medium, rhEndostatin can sensitively and stably inhibit the proliferation of the HUVEC cells that were transfected with plasmid encoding hVEGF. LDH substrate methods is the most sensitive and stable method. The anti-tumor activity in H22 tumor-bearing mice was also highly repeatable and had an inhibition rate over 50% at 20 mg kg(-1) weight. As a conclusion, the RP-HPLC and GF-HPLC set up in this paper are highly repeatable, accurate and sensitive for detecting the purity of rhEndostatin. The bioactivity of rhEndostatin can be measured through detection the proliferation-inhibition on HUVECs transfectants with hVEGF in vitro or on H22 liver tumor in vivo.