Design Of STAT3 Antisense Oligonucleotide And Variation Of Radiosensitivity Of A549 Cells Transfected By STAT3 Antisense Oligodeoxynucleotide

Radiation therapy and chemotherapy in conjunction with surgical operation have been commonly used for the treatment of many tumors. However a significant number of tumors fail respond to radiation therapy and/or chemotherapy because many forms of tumors appear less sensitive to radiation and anticancer drugs. Over the last few years, studies manipulating the expression of certain genes have led to a better understanding of the genetic basis of sensitivity to ionizing radiation. Such studies are important not only to gain fundamental insights into the response of cells to radiation, but also to provide clues as to how and where one could intervene to make tumor cells more radiosensitive. Examples of genetic manipulations affecting radiosensitivity have proliferated in the literature over the last few years, probing the many processes and pathways that influence whether a cell lives or dies after radiation. There are many manipulation strategies and also lots of genes to be selected or targeted to intervene tumor radiosensitivity. This study was to design effective antisense olgonuccleotide targeting STAT3mRNA and explore its effect on the proliferation and apoptosis of human non-small cell lung cancer cell line A549; and then we use the effective STAT3 antisense oligodeoxynucleotides to prohibit the expression and activity of STAT3 protein so as to enhance the radiosensitivity of A549 cells.Signal transducers and activators of transcription (STATs) were identified originally as key components of cytokine signaling pathways, involved in mediating responses to IFNs, IL-6 and other cytokines. On phosphorylation of STAT monomers by tyrosine kinases, the monomers dimerized, translocate to the nucleus, and bind to specific promoter sequences, thereby inducing expression of multiple genes associated with cellular proliferation and survival. As a point of convergence for many tyrosine kinase signaling pathways, such as EGFR, IL-6/JAK and Src, STAT3, one of the most important members of the seven STATs family, has been found to be constitutively activated in a wide variety of human tumor specimens and tumor cell lines, including breast cancer, head and neck aquamous cell carcinoma, prostate cancer, various leukemias, multiple myeloma and melanoma cells. Furthermore, accumulating evidence is defining a critical role for STAT3 in oncogenesis. In particular, STAT3 signaling has been shown to prevent apoptosis and enhance cell proliferation through regulating genes involved in cell growth and programmed cell death, including Bcl-xL, Mcl-1, c-Myc, CyclinD1, and p53. In addition to its role in promoting tumor cell growth and survival, constitutive STAT3 activation has also been shown to up-regulate VEGF expression and to promote tumor angiogenesis. Numerous studies have also demonstrated that abrogation of STAT3 signaling blocks oncogenesis in vitro and in vivo. Interrupting STAT3 signaling has been shown to induce tumor apoptosis and /or cell cycle arrest in a wide range of cancer cell lines which provided a rationale for designing new drugs for therapy of these cancers.Antisense nucleotide technique bears a promising prospect in tumor therapy due to its functional specificity and relatively low toxicity. With more than 20 years study, the antisense technique has become relatively mature and used extensively in gene silence or knockout experiments. A variety of target-specific antisense anti-tumor drugs are on phase II or III clinical trials, and an antisense drug for topical use has been approved for clinical use. It is reported that antisense drugs for systemic use can also reduce expression of a target protein in a target tissue, indicating that topical use is no longer the only route of antisense drug intake. The third generation peptide nucleic acid (PNA) and phosphodiamidate Morpholino oligomers (PMO) being used for research have overcome almost all instability of oligonucleotides, have few non-specific actions and low toxicity, and are not devalued by ribozyme. Although in vivo absorption efficiency and target-specificity of antisense nucleotides need to be perfected by further study, they are common concerns in nucleotide drug research. Breakthrough of these problems is sure to bring about a new heat of research on antisense drugs. This study was to design effective antisense olgonucleotide targeting STAT3mRNA and explore its effect on the proliferation and apoptosis of human non-small cell lung cancer cell line A549; and then to investigate the influence of effective STAT3 antisense oligonucleotide(AS10) on radiosensitivity of A549 cells, observe the variation of the radiosensitivity of A549 cells after transfected with AS10-STAT3 and probe into the mechanism of the observed phenomena.Ten antisense sequences targeting STAT3 were designed with RNAStructure4.2 software and STAT3 mRNA total sequence, and synthesized using phosphorothioate chemistry according to reference articles to improve its stabilization in cells, and transfected them at 200 nM respectively into A549. Forty-eight hours later after transfected by Oligofectamine method, Cell proliferation inhibition measured by Cell Count Kit (CCK-8) assay. The designed sequences inhibited proliferation of A549 cells, seven of them have a significant effect on proliferation inhibition, compared with positive control group(P<0.01), the proliferation inhibition of AS10 reach 75.46%. Then transfected AS10 into A549 cell at different concentration, Forty-eight hours later after transfected, CCK-8 was practiced, and the result showed that the higher the concentration of the AS10-STAT3 was, the heavier the inhibition was displayed in a certain scope varied from 0 to 300 nM (P<0.01), while no statistic inhibitive effects were found in blank or nonsense oligodeoxynucleotide-STAT3 (NS- STAT3) control. When we tried to transfect more than 400nM of oligodeoxynucleotides, proliferation inhibition was found in both groups of the cells transfected by antisense and nonsense oligodeoxynucleotide STAT3 (P<0.05). We suggest it as a nonspecific toxic reaction of oligodeoxynucleotides causes by too high relative concentration of the short DNA sequences.Cells were transfected with 75 nM AS10-STAT3 or control NS-STAT3 in 96 wells cultures (or 300 nM in 6 wells), exposed to different dose of irradiation 24 hours later, then compared for cell viability and apoptotic response. Effects on cells activity were confirmed using the CCK-8 assay. Apoptotic assays were performed using Hoechst33258 and Annexin V/PI with FACS analysis. Obvious enhancement of inhibition were observed when AS10-STAT3 transfected combining withγirradiation within low dose scope characterized from 4Gy to 12Gy, yet no obvious changes in radiosensitivity could be detected when the radiation dose increased to 12Gy higher, which could be interpreted as that high dose of radiation itself could kill so much that little damage effect of ASO-STAT3 could be detected.Similar results were obtained when refered to levels of apoptosis of different experimental groups. In the visible light, apoptotic cells were characterized as shrinkage of cell membrane, loss cell volume, condensation of cytoplasm, some cells falling off from the wall of the culture, et al. After Hoechst 33258 staining was performed, cells were viewed with a fluorescence microscope. Chromatin condensation, nuclear shrinkage or nucleosomal fragmentations were considered morphologic markers of apoptosis. We found that part of the cells underwent apoptosis 48 hours after transfected with AS10-STAT3, more apoptotic cells could be detected following AS10-STAT3 transfected combining withγirradiation, but no statistic difference could be found between the NS-STAT3 and blank control group. No typical apoptotic bodies were found in NS-STAT3 and blank control group. In the groups of AS10-STAT3 transfection with subsequent exposure to 16Gy irradiation, increasing number of apoptotic cells and apoptotic bodies were detected, but not found in NS-STAT3 transfected combining withγirradiation and cells treated with irradiation only.Annexin V/PI assay was then performed using FACs for detecting early apoptosis of A549 cells after different treatment of each group in 6 wells. Statistic difference was found between the blank control and groups of AS10-STAT3 and NS-STAT3 transfected at 400nM (P<0.01), and no obvious difference could be found in groups between blank control and 400nM NS-STAT3 groups, the AS10-STAT3 group's early-apoptotic cells rate is 11.51%, while the blank control groulp's early-apoptotic rate is only 5.18%. When treated with irradiation combined, statistic contrast could be obtained among different groups (P<0.01). In groups irradiated with different dose after transfected with 300nM ASO-STAT3, ratio of early apoptosis was found increasing in different extent contrast to blank control, irradiated only, NS-STAT3 transfection with subsequent irradiation. All these data support that AS10-STAT3 itself can induce apoptosis of A549 cells, and it could produce more apoptosis cells together withγirradiation than NS-STAT3 could, which supports a role for STAT3 as a potential molecular target for tumor therapies as has been testified by many researchers.Cells were transfected with 400 nM AS10-STAT3 or control NS-STAT3 in 6 wells cultures, after 48h, the cells were collected, protein in the cytoplasm and the nucleus was extracted. Western blot was used to observe the change of total STAT3 protein and Tyr-705 phosphorylated STAT3 protein and Bcl-xL,CyclinD1 protein in different groups. We found that the expression of STAT3,P-STAT3,Bcl-xL and CyclinD1 were downregulated after transfected with AS10-STAT3. Flow cytometry was applied to analyze the cell cycle, we found that the ratio of G1 phase A549 cells is increased after tranfected with antisense oligonucleotide(P<0.01), the antisens group's G1 phase cells rate is 63.96%, while the control groulp's G1 phase cells rate is 44.47%.In summary, the computer aided design was useful method to obtain the effective antisense oligonucleotide, and the effective antisense oligonucleotide(AS10-STAT3) had a very potent effect of proliferation inhibition and induction of apoptosis on A549 cells. When practiced together withγirradiation, AS10-STAT3 can not only kill the tumor cells itself but also partly enhance the cells radiosensitivity which improves the therapeutic effects of radiotherapy to radioresistant tumors or cancers. These results support roles for STAT3 as a potential molecular target for tumor therapies and also as a potential molecular radioenhancer for those cancers that are not so sensitive to radiotherapies. Further preclinical studies of STAT3 targeted therapies, as potential radiosensitizers will be presented in the late work.