RNA Interference-mediated ADAM9 And Stat3 Silencing In Nonsmall-cell Lung Cancer

Background:The incidence rate of lung cancer, a therioma with the highest morbidity and mortality, shows a sustained growth trend, seriously threatening human health. Non-small cell lung cancer(NSCLC) is the most popular and accounts for ~80% of all type of lung cancer. Metastasis is a major cause of morbidity and mortality in NSCLC cancer. However,the mechanism of such metastasis is unclear, which worsens the prevention and treatment of NSCLC. Surgery, radiotherapy and chemotherapy are the primary treatment of NSCLC. However, most patients have been diagnosed at the advanced stage, a stage is not the best time for treatment, and treated by chemotherapy. Although chemotherapy would effectively improves the life quality and symptoms of patients with advanced NSCLC, it also causes serious damage to normal cells because of the lack of selectivity, impairs the patient’s immune system and induces serious complications. Thus, the effective targets and therapeutic strategies for the treatment of NSCLC are required for development.In recent years, targeted molecular therapy gradually applied to the clinical treatment of cancer. This approach has received good curative effect and provided a new strategy for NSCLC.A disintegrin and metalloproteinases(ADAMs) comprise a family of zinc dependent type I transmembrane protein with proteolysis and cell adhesion activity, which hydrolysize a variety of protein sextracellular domain and induce cell signal transduction. Further, ADAMs have a variety of functions including the degradation of extracellular matrix,cell-cell or cell-matrix adhesion, cell fusion, sperm-egg fusion, wound healing and cancer metastasis, formation and progression.ADAM9, an important member of the ADAM protein family, is frequently upregulated in various types of cancer which triggering tumorigenesis and metastasis of tumor cell. Consistent with these results, a previous study demonstrated that ADAM9 is highly expressed in NSCLC and the increasing levels of ADAM9 correlates with a shortened survival time.Additionally, ADAM9 overexpression increases cell adhesion and invasion of NSCLC cells; RNA interference(RNAi)-mediated downregulation or knockout of endogenous ADAM9 inhibit adenoid cystic carcinoma cell growth, metastasis, proliferation, migration and invasion of A549 cells. The above studies indicating that ADAM9 is an ideal target for the treatment of NSCLC.Signal transducer and activator of transcription(Stat3) is one of the most important members of the STAT family and could be activated by many cytokines and growth factors, which is involved in signal transduction. It is has been demonstrated that activated Stat3 is capable of regulating cell growth, cell apoptosis and cell cycle. Increased expression of Stat3 is observed in the cancer including prostate cancer, breast cancer,NSCLC and ovarian cancer, which inhibit cell apoptosis and facilitate the development of cancer. Inhibition of the expression of Stat3 by RNAi attenuates tumor cell proliferation and invasion and induce cell apoptosis.Therefore, Stat3 can represent an attractive target for therapeutic intervention against NSCLC.Epidermal growth factor receptor(EGFR) is a multifunctional membrane glycoprotein found in a variety of tissue types. The overexpression of EGFR leads to excessive cell growth and malignancy.Constitutively activated Stats proteins are found in many tumor cells and cancer tissues, which show that after treatment with anti-EGFR monoclonal antibodies lung cancer cells are inhibited Stat3 phesphorylation and result in slowed proliferation, accelerates apoptosis,weakened invasiveness, and arrested cell cycles. These results suggest a key role for EGFR-STAT3 signaling in the proliferation of lung cancer cells.By now, the research indicate that ADAM9-EGFR and JAK-STAT3 signaling pathway promote the development of non-small cell lung cancer,but the relationship in tumorigenesis development between the two signal pathway is no clear.Objective:To investigate the relation between JAK-STAT3 and ADAM9-EGFR signaling pathway in NSCLCMethods:To recombinant lentiviral small hairpin RNA expression vector carrying ADAM9 and Stat3, then transfect into 293 T cells for ADAM9 sh RNA-expressing lentivirus(Lv/sh ADAM9), Stat3 sh RNA-expressing lentivirus(Lv/sh-Stat3) or non-targeting sh RNA-expressing lentivirus(Lv/sh NC). The m RNA and protein levels expression in A549 cells were determined by RT-q PCR and western blot after the cells were infected with the lentivirus carrying the ADAM9 si RNA, Stat3 si RNA, negative control si RNA or Combined ADAN9. The viability level of A549 cells was determined by CCK-8. The cell apoptosis was determined by TUNEL assay and caspase(caspase-, caspase-8 and caspase-9) activity assay. The cell migration and invasion determined by wound healing assay and transwell system assay. The MMP-2 and MMP-9 level were determined by western blot. A male BALB/c mice model bearing A549 tumor cell following treated with sh RNA-expressing lentivirus(Lv/sh-ADAM9, Lv/sh-Stat3 and Lv/sh NC) was constructed, and determined by TUNEL assay.Results:A recombinant lentiviral small hairpin RNA expression vector carrying ADAM9, Stat3 and control was constructed and then transfected into 293 T cells to generate sh RNA-expressing lentivirus(Lv/sh ADAM9,Lv/sh-Stat3 and Lv/sh NC). The expression level of ADAM9 or Stat3 m RNA in Lv/sh-ADAM9-infected or Lv/sh- Stat3-infected A549 cells were decreased compared with that in untreated cells or Ad/sh-NC-infected cells. The results from CCK-8 assays further reveal that the control had no cytotoxic effect on A549 cells, while the viability of A549 cells was markedly inhibited by ADAM9 and Stat3 silencing.RNAi-mediated ADAM9 and Stat3 silencing significantly induce cell apoptosis. The level of cell migration and cell invasion were inhibited in the ADAM9 and Stat3 silencing group. The tumor growth in male BALB/c mice bearing A549 tumor cells following treated ADAM9 and Stat3 silencing was significantly inhibited.Conclusions:Silencing of ADAM9 and Stat3 reduces proliferation,migration and invasion of NSCLC,and increases apoptosis in vitro and in vivo.Therefore, silencing of ADAM9 and STAT3 inhibits the growth of NSCLC. Our results further demonstrates JAK-STAT3 and ADAM9-EGFR signaling pathways have synergistic effect on the growth of NSCLC. Moreover,Stat3 is not the key downstream mediator in the two signaling pathways.