| The morbidity of lung cancer, one of the most common primary malignant carcinomas in the world, has increased annually. Furthermore, to date, the therapeutic efficacy for this malignancy has not improved due to current high mortality and poor long-term survival rates.Stat3, a member of the signal transducers and activators of transcription (STAT) family, is a key signal transduction protein that mediates signaling by numerous cytokines, peptide growth factors, and oncoproteins. Accumulating evidence demonstrates that Stat3 activation plays important roles in cell differentiation, proliferation, development, apoptosis, and inflammation . Elevated activity of Stat3 has been frequently found in a variety of human tumors, including hematologic malignancies, head and neck cancer, breast cancer, and prostate cancer.Upon stimulation, Stat3 can be activated by tyrosine or serine phosphorylation or acetylation. Under physiological conditions, Stat3 activation is transient and lasts from several minutes to several hours because of the transient nature of cytokine and growth factor signaling and the presence of proteins such as suppressor of cytokine signaling (SOCS) and PIAS (STAT blockade) that inhibit Stat3 expression. Aberrantly active Stat3 promotes uncontrolled growth and survival through dysregulation of expression of downstream targeted genes, such as cyclin D1, cyclin D2, c-Myc, p53, Bcl-xL, Bcl-2, Mcl-1, and Survivin; these genes influence cell cycle progression or inhibit apoptosis. In addition, constitutively activated Stat3 induces cellular transformation in vitro and tumor formation in nude mice. Thus, Stat3 may be an important molecular target for tumor therapy.Studies have shown that using a variety of approaches, such as tyrosine kinase inhibitors, antisense oligonucleotides,decoy oligonucleotides, and dominant-negative Stat3 proteins can inhibit Stat3 expression in cancer cells to suppress proliferation and induce apoptosis. In human head and neck squamous carcinoma cells, blocking of Stat3 signaling by decoy oligonucleotides or antisense oligonucleotides inhibits transforming growth factor effects and suppresses oncogenic growth of these cells. Stat3βis a naturally occurring dominant negative Stat3 mutant that is identical to Stat3 except for the absence of the transactivation domain. Blockade of Stat3 signaling by Stat3βin human myeloma cells and breast cancer cells in vivo down-regulates IL-6-induced expression of the Bcl-xL antiapoptotic gene and induces apoptosis, thereby suggesting that targeting Stat3 by Stat3βmay enhance in vivo antitumor responses.The use of RNA interference (RNAi) represents a novel alternative to gene inhibition that may be capable of inhibiting Stat3 expression in cancer cells leading to suppression of proliferation and induction of apoptosis. RNAi is triggered by introducing long double-stranded RNA (dsRNA) molecules into cells where the dsRNAs are cleaved by an endonuclease named dicer into 21- to 23-nt RNAs referred to as short interference RNAs (siRNAs). The siRNA molecules that serve as a guide for sequence-specific degradation of homologous mRNAs have been used for functional analysis of genes in many species. siRNA targeting Stat3 has been successfully used for treating numerous cancers. However, to our knowledge, no reports have been published to date concerning the effect of siRNA against the Stat3 gene in lung cancers cells. In our studies we have used Lewis lung cancer cells, which have shown to be of value for the study of lung cancer.Study objectives:(1) to determine if Stat3 siRNA can inhibit the expression of Stat3 gene in Lewis lung cancer cells(2) to investigate the effect of Stat3 siRNA on the growth and apoptosis in these cells.(3) to investigate the effect of Stat3 siRNA on the growth,apoptosis and metastasis in vivo.Methods:(1) Construction of a recombinant plasmid expressing Stat3 siRNA Using Stat3 gene sequences from GenBank, selected suitable target site, synthesized oligonucleotides as DNA template encoding Stat3 siRNA, annealed and ligated into pSilencerTMneo 3.1-H1 expression vector to construct plasmid pSH1Si-Stat3.(2) In vitro studies:The Lewis lung cancer cell lines were transfected with plasmids pH1Si-Stat3. To determine the expression of the Stat3 at 72h after transfection, semi-quantitative RT-PCR analysis and Western blot analyses with the samples extracted from transfected and control cells were performed. The cells were also analyzed for cell cycle phase distribution and apoptosis rate by flow cytometry, DNA Ladder, and stained by Annexin V-CY3 apoptosis detection kit to detect the early apoptosis.(3) In vivo studies:To study the effects of pH1Si-Stat3 on Lewis lung cancer growth in vivo, we developed a C57BL/6 mouse tumor xenograft model. Mice were transplanted with Lewis lung cancer cells into the right flank.On day 7, palpable tumors had developed, and the pH1Si-Stat3 plasmids and scramble control, mock control buffer were injected. Effects of the different constructs on suppression of tumors were analysed by various procedures. For example, the primary tumor and spontaneous lung metastasis were measured. Hematoxylin- Eosin stain and TUNEL assay were used to detect the apoptosis of tumor cells. RT-PCR and Western blot analysis were used to detect the expression of related genes and proteins, Immunohistochemistries for VEGF and MMP-2 were performed to determine the expression of related proteins.Statistical analysisData were expressed as mean±SE. Comparison between groups was performed with the Student's t test and analysis using a SigmaStat statistical software package (SPSS, Chicago, IL). P<0.05 was taken as showing significance.Results:(1) The plasmids pH1Si-Stat3 which was designed for expression of Stat3 siRNA was constructed, and confirmed by restriction enzyme digest and sequence analysis.(2) Transfection with pH1Si-Stat3 inhibited the growth of Lewis lung cancer cell cells and induced apoptosis. The results from semi-quantitative RT-PCR analysis and Western blot analyses for the samples from Lewis lung cancer cell cells after transfection demonstrated that the plasmids containing Stat3 siRNA could specifically reduce Stat3 expression. MTT assay showed that the growth-inhibition rate of the Stat3 siRNA groups was significantly higher than that of the vehicle group or plasmid group. The results of DNA ladder electrophoresis, FCM and Annexin V-CY3 apoptosis detection assay demonstrated that the pH1Si-Stat3 group showed remarkable apoptosis effects compared to control groups.(3) Examination of subcutaneous tumors in C57BL/6 mouse showed that in the group transfected with pH1Si-Stat3, both the average weights and volumes of the tumors were lower than control groups. The lung metastatic rate and Ioce in the pH1Si-Stat3 group was signicantly lower than those in the control groups. The results from semi-quantitative RT-PCR analysis and Western blot analyses provided strong evidence that Stat3 siRNA can significantly suppress Stat3 expression. Western blot and immunohistochemistry showed that the protein expression of VEGF and MMP-2 in siRNA group were significantly lower than those in control group.Conclusions1.we successfully constructed pH1Si-Stat3 plasmid.2.Stat3 siRNA blocked the constitutively actived Stat3 signaling pathway efficiently and peculialy in vitro, which induced tumor cells apoptosis, arrested cell circle, and inhibited tumor cells proliferation, indicating that activated Stat3 signaling pathway is an ideal molecule target to gene therapy of lung cancer.3.This study demonstrated that in vivo delivery of siRNA into tumor mass results in effective inhibition of expression of gene encoding Stat3 and suppresseed growth and induced apoptosis of the Lewis lung cancer cells. VEGF and MMP-2 are the downstream gene regulated by Stat3 signaling pathway in Lewis lung cancer, and the mechanism of Stat3 signaling pathway contribute to carcinogenenisis at least partly might be due to promoting transcriptional activation of VEGF and MMP-2.Furthermore, RNAi technique may be a promising tool for cancer therapy.
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