ABSTRACT
RNA interference (RNAi) libraries screens have become widely used for small RNA (sRNA) therapeutic targets development. However, conventional enzymatically libraries, typically prepared using the type 2 restriction enzyme MmeI, produce sRNAs between 18 and 20 bp, much shorter than the usual lengths of 19-23 bp. Here we develop a size unbiased representative enzymatically generated RNAi (SURER) library, which employs type 3 restriction modification enzyme EcoP15I to produce sRNAs ranging from 19 to 23 bp using a group of rationally designed linkers, which can completely mimic the length of sRNAs naturally generated by Dicer enzyme in living cells, and the screening results of SURER libraries showed high recombination rate and knockdown efficiency. SURER library provides a useful tool for RNAi therapeutics screening in a fast and simple way.
Subject(s)
Gene Knockdown Techniques , RNA Interference , Base Sequence , Biocatalysis , DNA-Directed DNA Polymerase/genetics , Gene Expression , Gene Library , Genetic Therapy , Hep G2 Cells , Hepatitis B virus/enzymology , Humans , Inhibitor of Apoptosis Proteins/biosynthesis , Inhibitor of Apoptosis Proteins/genetics , Inverted Repeat Sequences , RNA, Small Interfering/genetics , SurvivinABSTRACT
Purpose. This study was to determine the effect of CTGF-small interfering RNA (siRNA) on TGF- ß 2-induced proliferation in human Tenon capsule fibroblasts (HTFs). Methods. HTFs were transfected with four of CTGF-siRNAs separately for screening of gene silencing efficacy that was determined by transcript level measured by quantitative real-time PCR (qRT-PCR). Recombinant TGF- ß 2 was added into the culture to stimulate the proliferation of HTFs. The gene silencing efficacy of the siRNAs was evaluated by qRT-PCR and immunofluorescence of CTGF transcript and protein levels. The viability of HTFs was determined by cell counting kit-8 (CCK-8). FCM was used to assess cell cycle after CTGF-siRNA transfection. Results. The expression of CTGF and proliferation of HTFs were increased significantly by TGF- ß 2 stimulation. The transfection of CTGF-siRNA abolished the upregulation of CTGF and cell proliferation induced by TGF- ß 2. The analysis of cell cycle indicated that CTGF-siRNA treatment stimulated cells from S phase to G0/G1 phase in comparison with the inverse physiologic function of TGF- ß 2. Conclusion. CTGF targeting siRNA could effectively suppress the expression of CTGF and attenuate the proliferation of HTFs. The siRNA approach may provide a therapeutic option for eliminating filtration bleb scarring after glaucoma filtration surgery (GFS).
ABSTRACT
Epithelial-msenchymal transition (EMT) contributes to posterior capsule opacification (PCO) type of cataract. Transcription factors Snail is a key trigger of EMT activated by transforming growth factor ß (TGF ß ). This study was done to investigate the effect of Snail targeting siRNA on TGF ß 2-induced EMT in human lens epithelial cells. TGF ß 2 treatment of cultured human epithelial cell line (HLEB3) upregulated the expression of Snail and the EMT relevant molecules such as vimentin and α -SMA but downregulated the expression of keratin and E-cadherin. After the stimulation of TGF ß 2, the HLEB3 cells became fibroblast-like in morphology, and the junctions of cell-cell disappeared. TGF ß 2 treatment also enhanced migration ability of HLEB3 cells. TGF ß 2-induced Snail expression and EMT were significantly inhibited by Snail siRNA. By analyzing the response characteristics of HLEB3 in TGF ß 2-induced EMT model with/without Snail-specific siRNA, we concluded that Snail is an element in the EMT of HLEB3 cells induced by TGF ß 2. Snail siRNA targeting can block the induced EMT and therefore has the potential to suppress the development of PCO.
