Downregulation of VEGF and KSP Gene Expression Inhibits Proliferation of A549 Cells

To investigate the proliferation and invasion of A549 cell by down-regulating KSP and VEGF gene. Designing and screening the siRNA of targeting VEGF and KSP promoter, transfected into A549 cells with transfetion reagent. According to the difference of transfection mixture the cells were divided into five groups. Western Blotting was used to detect the expression of VEGF and KSP. MTT assay was used to detect the cell proliferation, qPCR was used to measure the expression of VEGF and KSP, the MTT results indicated that the proliferation of the cells was significantly decreased; The results of qPCR showed that the siRNA obviously decreased the expression of VEGF and KSP gene;. The Western Blotting showed that the expression of VEGF and KSP were significantly reduced; The proliferation of the co-transfected cell were inhibited more than the single group transfected cell. These results indicate that co-regulation of VEGF and KSP gene expression more effectively inhibit A549 cell proliferation, better than only regulating VEGF or KSP gene expression.


Intruduction
Non-small cell lung cancer is a common primary Lung cancer. Laboratory and clinical studies have demonstrated that cancer is a gradual process of long-term accumulation, involving multiple genetic and epigenetic variation, involving decrease of anti-oncogene gene expression and increase of oncogene expression [1]. VEGF is vascular endothelial growth factor gene, cancer cell growth and metastasis depend on angiogenesis, vascular endothelial growth factor is effective angiogenic growth factor [2]. VEGF and VEGFR has become targets for the treatment of cancer.Therefore, VEGF is expected to become a new molecular marker of cancer diagnosis and treatment [3]. KSP is a member of the kinesin family, and plays an important role in early mitosis, formation of the spindle, centrosome separation and chromosome division during mitosis [4]. In addition, KSP is also closely related to tumor development and progression, and highly expressed in many tumor cell lines [5]. It has become an important new target for cancer chemotherapy.
Most research focused on siRNA or targeting a single gene, which is an effective way to control single-gene diseases [6]. There are various compensatory mechanisms and complex signal transduction pathway in cancer cell, therefore the effect becomes poor of siRNA controlling cancer [7]. We think that downregulate KSP and VEGF will have better effect in cancer therapy. In this study, we first designed and screened siRNA of cancer gene VEGF and KSP.
Then, transfected siRNA into A549 cell respectively, detected its impact on proliferation, invasion, expression of the target gene. Finally,

siRNA sequences design
The sequences of the RNAs oligonucleotides were sequences did not share any homology with any of the known mRNA databases.

Transfection of RNAs oligonucleotides
The siRNAs were transfected into cultured cells at At 5-6 h post-transfect, the medium was replaced with 10% serum-supplemented RPMI-1640, and the cells were incubated for additional 24-96 h. Next, the cells were harvested by centrifugation, rinsed with phosphate buffered saline, and subjected to total RNA or protein extraction.

Cell proliferation assay
Cell viability was examined by MTT assay (Roche Diagnostics

24
Each RNA sample was analyzed in triplicate.

Western blot analysis
The western blot was used to detected the expression of VEGF, KSP and bcl2.

Statistical analysis
All of the data were used statistical software SPSS17.0 do statistical analysis , and the results expressed inxs, using the single factor ANOVA method do multiple comparison and the LSD methodhe do comparison between two groups.

Cell morphology was observed in all groups after transfection 48h
cells in NC and CT groups has a good shape, fusiform. The difference was not significant. KSP and VEGF groups cells increased apoptosis, cell rounding, and the two groups has no significant difference of apoptosis number. the number of apoptotic cells and round cells of KSP+VEGF groups was significant increased. Comparing VEGF+KSP group with KSP or VEGF group, the difference of was also significant . Figure 1. The results showed in Table 2

the protein and miRNA expression levels after transfection
The results showed that the expressions of KSP, VEGF, bcl2 protein and mRNA in KSP, VEGF and KSP+VEGF groups were significantly lower than the CT and NC groups (p<0.05 ); The expressions of mRNA and protein in VEGF+KSP groups were significantly lower than KSP or VEGF groups (p<0.05). Table 3.

Table 3. The protein and miRNA expression levels of each groups 3 Discussion
Nearly half a century, with the development of industrialization, lung cancer incidence and mortality has been on a clear upward trend [8] , but there is no fully effective drug to treat lung cancer. More and more studies show that siRNA technology can effectively inhibit the proliferation of cancer cells [9] .
Therefore, finding an effective siRNAs target site may be one of the most popular cancer treatment.
In recent years studies have shown that lung cancer development, metastasis and prognosis had closely related to angiogenesis [10] . Some studies suggest that immortalized tumor cell formation and angiogenesis are closely related. In recent years, we found that many of angiogenesis factors, including VEGF is a new discovery of the role of the current strong and specific vascular regulatory factors [11] . VEGF, also known as vascular permeability factor, vascular endothelial cell proliferation, vascular basement membrane hydrolysis and build stronger role, and high specificity, tumor angiogenesis is induced strong and specific role of regulator [12] . Transfection of targeting VEGF siRNAs, which can effectively lower mRNA and Protein levels. With the reduced VEGF expression, Bcl2 expression was significantly reduced, the degree of apoptosis was significantly increased.
KSP has conserved ATP enzyme domain and dynamic domains, it play an important role in formation of a bipolar spindle and separation processes in mitotic metaphase [13] . Studies have shown, the KSP siRNA can lead to the generation of a single star spindle promote mitotic arrest in M phase and induce apoptosis of tumor cells [14] . protein involved in cell mitosis [15] , the body's normal proliferation of cells, there are also the expression of KSP, but its expression level was significantly lower than the malignant tissue; the body's normal differentiation and maturation of cells and tissues, such as nerve cells were not detected in the KSP expression [16] .