A Significant Increase of RNAi Efficiency in Human Cells by the CMV Enhancer with a tRNAlys Promoter

Abstract

RNA interference (RNAi) is the process of mRNA degradation induced by double-stranded RNA in a sequence-specific manner. Different types of promoters, such as U6, H1, tRNA, and CMV, have been used to control the inhibitory effect of RNAi expression vectors. In the present study, we constructed two shRNA expression vectors, respectively, controlled by tRNAlys and CMV enhancer-tRNAlys promoters. Compared to the vectors with tRNAlys or U6 promoter, the vector with a CMV enhancer-tRNAlys promoter silenced pokemon more efficiently on both the mRNA and the protein levels. Meanwhile, the silencing of pokemon inhibited the proliferation of MCF7 cells, but the induction of apoptosis of MCF7 cells was not observed. We conclude that the CMV enhancer-tRNAlys promoter may be a powerful tool in driving intracellular expression of shRNA which can efficiently silence targeted gene.

Antitumor potential of SLPI promoter controlled recombinant caspase-3 expression in laryngeal carcinoma

“…Combination of revCasp3 and target genes including other apoptosisinducing factors involved in caspase-dependent or caspaseindependent pathways, 25 --27 such as Granzyme B, AIF, Bid and so on, will increase the apoptosis of target cells. Another way to improve the therapeutic effect is to introduce some transcriptional regulatory elements, such as the CMV enhancer, 28 which may significantly increase the transcriptional activity of SLPI promoter while maintaining its tissue specificity. For gene therapy of head and neck squamous cell carcinoma specifically, application of inducible promoters such as Egr-1, which may increase the transcriptional efficiency in response to the radiotherapy, 29 is also a promising approach.…”

Short Hairpin RNA-Mediated Gene Silencing

“…Transcription from these promoters leads ( 87 ) to the production of long RNAs that can also be engineered to encode downstream shRNA precursor sequences that are processed to form active molecules for RNAi ( 79 ) . Numerous studies have successfully employed this approach using a number of different tRNA promoters resulting in gene knockdown at comparable levels to U6 promoters ( 37, 51,60,[88][89][90] .…”

RNAi and miRNA in Viral Infections and Cancers

“…The miRNA for hCMV is miR-UL112-1 that can target the viral gene IE1 which is an immediate-early gene product requiring for activating transcription of hCMV early genes. When miRUL112-1 expressed early in hCMV infection, a decrease in viral replication was observed (Weiwei et al, 2009).Herpes Simplex Virus Type 1 (HSV-1) causes long term latent infections in neurons. HSV-1 express two miRNAs in latently infected neurons which caballing of ICP0 and ICP4 down-regulating, mmediate early proteins (Hill et al, , 2009).…”

“…Suppression of PUMA by miR-BART5 may protect EBV infected cells from virus-induced apoptosis. BCLAF1, another apoptotic factor is a cellular target of the KSHV miRNAs miR-K5, miR-K9, and miR-K10 which raises the levels of BCLAF1 and decreased virus production after induction of viral replication (Tian et al, 2009;Weiwei et al, 2009). THBS1 is blocks by multiple KSHV miRNAs, including miR-K1, miRK3-3p, miR-K6-3p, and miR-K11 may help KSHV-infected cells in avoiding detection by the host immune system (Malterer et al, 2011;Wu et al, 2011).…”

“…MICB is a stress-induced cell surface ligand, and the hCMV miRNA miR-UL112-1 was reported by Stern-Ginossar et.al to inhibit the expression of MICB by binding sites within the 39UTR of this mRNA. So, down-regulation of MICB by miR-UL112-1 may allow hCMV to escape NK cell immune surveillance (Malterer et al, 2011;Qiu et al, 2011;Weiwei et al, 2009). …”