Abstract
OBJECTIVE To inhibit the expression of the vascular endothelial growth factor (VEGF) by RNA interference, and to observe the effect in different cells line.
METHODS Using the services of E-RNAi, we designed and constructed two kinds of shRNAs expression vectors which were aimed at the VEGF gene. These vectors were then transfected into HEK293, colon cancer HT29, Hela and HepG2 cells by LIpofectamineTM 2000. The level of VEGF mRNA was determined by RT-PCR and Northern blotting and the VEGF expression was examined by immunofluoresence staining.
RESULTS The two kinds of VEGF specific shRNAs expression vectors were found to efficiently Inhibit the expression of VEGF in HEK293 and HT29 cells by RT-PCR analysis, with inhibition rates of 72% and 42%, respectively; but the inhibition rates were reduced to 28% in Hela cells and 13% in HepG2 cells. Northern blotting showed that the inhibition rates of VEGF mRNA expression were 88% and 89% in HEK293 and HT29 cells, respectively. The inhibition rate of VEGF protein expression in HT29 cells was 73% based on immunofluoresence staining.
CONCLUSION The expression of VEGF was inhibited by RNA interference, but differed with various cells lines, showing that RNA interference was cell-line dependent.
keywords
Angiogenesis is a well-known process that is essential for tumor growth beyond 2 mm.[1] Although numerous growth factors are involved, vascular endothelial growth factor (VEGF), in particular VEGF-A, has been shown to play an important role in tumor angio-genesis.[2] VEGF is a 45 kDa heparin-binding growth factor and is induced by the hypoxia-inducible factorla. Binding of VEGF-A to its receptors, the tyrosine kinase receptors, especially VEGFR-2, mediates many key components of angiogenesis, including endothelial cell proliferation, invasion, migration, and survival, as well as vessel permeability. VEGF is secreted by most tumors, including those of the lung, gastrointestinal tract, kidney, thyroid, bladder, ovary, and cervix, and levels of VEGF have been correlated with tumor progression and invasion.[3]
RNAi is a sequence-specific, posttranscriptional gene-silencing method initiated by double-stranded RNAs, which is homologous to the gene being suppressed. Double-strand RNAs are processed by Dicer, a cellular RNase III, to generate duplexes of about 21nt with 3’-overhanging small interfering RNA (siRNA), which mediate sequence-specific mRNA degradation. RNAi technology is evaluated not only as an extremely powerful instrument for functional genomic analyses, but also as a potentially useful method to develop highly specific gene silencing therapeutics.[4-6] In this study, we constructed vector-based expression systems in which sense and antisense strands of short VEGF sequences were transcribed into the hairpin structure under the control of the U6 promoter.
Several researchers have applied specific anti-VEGF therapy for tumors of the colon, prostate and liver.[7-10]However, due to the “off-target effects” of RNAi,[11, 12] many experiments should be conducted to establish effective sequences to inhibit VEGF before they can be used in the clinic. Furthermore, few studies have employed different cells lines to examine the effect of the same RNAi. So in our study we hoped to find a more specific RNAi for VEGF in several cell lines.
MATERIALS AND METHODS
Construction of RNAi vectors
RNAi vectors pShRNA-Vl and pShRNA-V2 were constructed as previously described.[13] Briefly, 21-nucleotide-long inverted repeats (separated by a 4-nu-cleotide linker, TTCG) were inserted downstream of the U6 promoter. The transcribed RNA thus comprised a 21-base pair of double-stranded RNAs. Five thymidines were inserted downstream on the antisense strand to provide a stop signal for the polymerase III RNA polymerase. The sense strand of the hairpin was homologous to a 21-nucleotide region in the target mRNA. The choice of target sequences was based on the advice of E-RNAi services [14] (http://e-rnai.dkfz. de/). VI and V2 were 5’-TGA AGT TCA TGG ATG TCT ATC-3’ and 5’-ACAT CAC CAT GCA GAT TAT GC-3’ respectively. Construction of an irrelevant RNAi control plasmid for a green fluorescent protein (GFP) gene, pShRNA-GFP, was similar to that described above. The sequences, 5’-GCT GAC CCT GAA GTT CAT C-3’ were designed to target the nu-cleotides 126~144 of the GFP coding region.
Cell culture and transfections
HEK293 human embryonic kidney cells, HT29 human colon cancer cells, human cervical carcinoma Hela cells and HepG2 human hepatoma cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf semm (FCS), 100 µ g/ml streptomycin, and 100 ¼g/ml ampicillin. The cells were plated in 24- or 6-well culture plates at 50%~70% confluence at 24 h prior to transfection. Transfection of cells was carried out by Lipofectamine™ 2000 reagent (Invitrogen, Carlsbad, CA).
VEGF real-time polymerse chain reaction
Total RNA was isolated from cultured cells and the real-time polymerase chain reaction was performed using the RNeasy and one-step RT-PCR kit of Qiagen Corp. RT-PCR for hGAPDH, a housekeeping gene was used as a control. The sequences used for primers were as follows: 5’-CTA CCT CCA CCA TGC CAA GT-3’ (sense); 5’-AAA TGC TTT CTC CGC TCT GA-3’- (antisense) for VEGF (411 bp). The sequences 5’-GGC TCT CCA GAA CAT CAT-3’ (sense) and 5’- CAC CTG GTG CTC AGT GTA-3’ (antisense) were used for hGAPDH (240 bp). For RT-PCR, two pair of primers were added into one reaction tube. The program consisted of an initial reverse transcription at 50°Cfor 30 min, dénaturation at 95°Cfor 10 min, then followed by amplification for 24 cycles (95 °C for 30 s for dénaturation, 55°Cfor 1 min to anneal, and 68°Cfor 1 min to extend) with a final extension step at 68 °C for 10 min. Products were then separated by means of electrophoresis on a 1.5% agarose gel and bands were visualized using UV light followed by analysis using Genetools software.
Northern blotting analysis
Total RNA was extracted from transfected cells at 3 days post-transfection. Total RNA was harvested and purified using the RNeasy Mini Kit (Qiagen). Twenty micrograms of total RNA were separated on 1.2% agarose-formaldehyde gels and transferred onto a positively charged nylon membrane(Amersham). The presence of VEGF mRNA was probed with 32P-labeled VEGF DNA, which was generated with a random-primed labeling kit (Amersham).
Immunofluorescence staining
Cells were harvested for analysis at 2 days post-transfection. They were washed once with PBS and fixed with 4% paraformaldehyde in PBS for 20 min at 4 °C. After blocking with goat serum, the cells were incubated with the appropriate primary monoclonal mouse anti-VEGF antibodies for 2 h at 37 °C. After three washes, the cells were incubated with the Cy3-conjugated rabbit anti-mouse secondary antibodies for lh at 37 °C and washed three times with PBS. The stained cells were mounted and analyzed by fluorescence microscopy.
Results
Identification of recombinant plasmid pShRNA and sequencing
Fig.l shows that the recombinant plasmid pShRNAs could not be digested by Sal I due to the lost of a Sal I site, however, the blank plasmid pTZU6+l could be digested by Sal I. When digested by Hind III and EcoR I, pShRNAs could be separated into two parts (2,800 and 395 bp), while pTZU6+l formed 2,800 and 352 bp. The correct recombinat plasmids were identified by gel electrophresis, then verified by DNA sequencing.
Restriction map of recombinant plasmid pShRNA.
Gel electrophoresis of VEGF RT-PCR
The size of the VEGF RT-PCR product was 41 lbp, as confirmed by gel electrophoresis (Fig.2). After cloning it into a T vector, its sequence was verified by DNA sequencing.
Gel electrophoresis of VEGF products from RT-PCR.
RT-PCR showed the inhibition rate of VEGF in different kinds of cells lines
Fig.3s show the rates of pShRNA-Vl and pShRNA-V2 inhibition were 82% in 293 cells compared with the pTZU6+l control plasmid. However, in HT29 cells the rates of inhibition by pShRNA-Vl and pShRNA-V2 were decreased to 42% and 40%. In contrast, the rates of inhibition by pShRNA-Vl were reduced to 28% in Hela cells, and to 13% in HepG2 cells.
RT-PCR shows the inhibition rate of VEGF in different kinds of cells.
Inhibition rates of VEGF in various cells lines by Northern blotting
As shown in Fig.4, the rates of inhibition by pShRNA-VI and pShRNA-V2 were 77% and 88% in 293 cells, and 87 and 89% in HT29 cells, respectively.
Inhibition rates of VEGF in various cells lines by Northern blotting.
Immunofluorescence staining of VEGF
Fig.5 indicates that the rates of inhibition by pShRNA-VI and pShRNA-V2 were 63% and 73% in HT29 cells. VEGF was stained red and was located in the cytoplasm of the cells.
Immunofluoresence staining for VEGF in HT29 cells.
Discussion
Angiogenesis is the process of generating new capillary blood vessels from pre-existing blood vessels, a process which involves multiple gene products expressed by various cell types. This uncontrolled process of new blood vessel growth from the preexisting circulation network is an important pathogenic cause of tumor growth.[15] Although several proteins such as hepatocyte growth factor, tumor necrosis factor-a, and fibroblast growth factor 2 (FGF2) have been identified as stimulators of angiogenesis in various settings, one of the most important angiogenic growth factors is VEGF, which is overexpressed in many human cancers. VEGF expression in tumors can be induced by more than one mechanism. Hypoxia, which occurs in most tumors, has long been known to be a potent inducer of VEGFJ[16]
In this study, we demonstrated that shRNAs, which were targeted against VEGF, efficiently reduced the transcript levels of VEGF mRNAs, and ultimately resulted in the reduction of levels of the VEGF protein. Furthermore, this inhibition was shown to be highly selective and sequence-specific, since control siRNAs had little inhibitory effect on expression and transcription of VEGF.
There has been considerable interest in the use of RNAi therapeutics to treat a wide range of diseases. In this report, we have further demonstrated that, in addition to the above-reported target sites in the VEGF genome, the specific 21-bp siRNAs targeting VEGF exerted efficient and specific inhibition on VEGF expression, suggesting a good method to inhibit the expression of VEGF.
Taken together, our findings demonstrated that the constructed shRNA is an efficient approach to reducing the level of VEGF transcripts and expression. These findings suggest that shRNA-expressing vectors may provide a practical means for the potential use of RNAi-based anti-VEGF therapeutics. Future studies will be centered on the evaluation of the anti-VEGF efficacy of RNAi vectors in valid animal models, as well as on preclinical research using RNAi technology.
Acknowledgments
We wish to thank Dr. David Engelke of University of Michigan for generously providing the pTZU6+l plasmid.
Footnotes
This work was supported in part by research grants from the National Natural Science Foundation of China (No. 30300298), and the National Natural Science Foundation of China's Joint Research Fund for Overseas Chinese Young Scholars Grant (No. 30228026).
- Received November 27, 2006.
- Accepted December 8, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.