PkgⅡ Inhibits Osteosarcoma Progression By Regulating The JAK2/STAT3 Signaling Pathway Induced By EGFR

Objective:Osteosarcoma(OS)is the most frequently observed primary malignant bone tumor.It is known that c GMP-dependent protein kinase II(PKG II)encodes a membraneanchored enzyme with specific serine/threonine protein kinase activity,which could inhibit the growth of various malignant tumor cells.This study aims to elucidate the role of PKG II in osteosarcoma and explore its potential mechanism through both in vitro and in vivo experiments.Method:(1)Quantitative Real-time PCR(q RT-PCR)and Western Blotting were used to detect m RNA and protein expression levels of PKG II in three human osteosarcoma cell lines and human osteoblast cell line.(2)c GMP was used to activate endogenous PKG II in MNNG/HOS and U2 OS cell lines.Cell Counting Kit-8 and clone formation assay were employed to detect the effect of endogenous PKG II on the proliferation activity of human osteosarcoma cells,wound-healing and Transwell invasion assays were used to judge the activities of migration and invasion.(3)Recombinant adenovirus was used as the carrier of PKG II gene to transfect human osteosarcoma cell lines.Western Blotting and q RT-PCR were used to verify the overexpression effect of PKG II.Western Blotting was used to detect Proliferating Cell Nuclear Antigen(PCNA),Matrix Metalloproteinase-2(MMP-2),Matrix Metalloproteinase-13(MMP-13)expression.To observe the activities of proliferation,migration and invasion induced by epidermal growth factor(EGF),functional experiments were employed as before.(4)Osteosarcoma cells transfected with Ad-PKG II,were injected into the right back of nude mice subcutaneously to construct a nude mouse subcutaneous xenograft osteosarcoma model.Peripheral blood was collected to measure the level of hemoglobin by Enzyme-linked Immunosorbent Assay(ELISA).(5)Osteosarcoma cells transfected with Ad-PKG II,were used to inject into the bone marrow cavity of the left tibia to construct a nude mouse orthotopic transplantation osteosarcoma model.The weight the mice,the size and weight of the tumor were measured,and peripheral blood were collected to study the level of alkaline phosphatase by ELISA.Micro CT and Hematoxylin-eosin(HE)staining were used to observe the bone destruction and tumor infiltration of orthotopic transplanted tumors.Immunohistochemistry(IHC)was employed to detect the protein expression level of PKG II in tumor tissues.(6)HE staining was used to detect the angiogenesis in the orthotopic xenograft tumor model in nude mice,and the IHC method was used to detect the protein expression level of CD105 in tumor tissues.(7)Osteosarcoma cells were transfected with Ad-PKG II and stimulated by EGF.Western Blotting was used to detect the protein expressions of VEGFR2,EGFR,pEGFR,JAK2,p-JAK2,STAT and p-STAT3.IHC was used to detect the expression of EFGR protein in tumor tissues in orthotopic xenograft tumor models.Results:(1)Compared with the normal human osteoblast cell line,the m RNA and protein expressions of PKG II line were suppressed in the human osteosarcoma cells.(2)Activated endogenous PKG II significantly reduced the proliferation,migration and invasion of human osteosarcoma cells.(3)Overexpression effect of adenovirus transfected PKG II was verified.After the stimulation of EGF,the expression levels of PCNA,MMP-2 and MMP-13 were increased significantly in osteosarcoma cells.Overexpression and activation of PKG Ⅱcan reduce the increased expression of PCNA,MMP-2,and MMP-13 caused by EGF stimulation.The proliferation,migration and invasion activities of osteosarcoma cells were significantly enhanced after EGF stimulation.Besides,overexpression and activation of PKG II significantly inhibited the increased proliferation,migration and invasion activities stimulated by EGF in osteosarcoma.(4)Overexpression of PKG II could significantly inhibit the growth of subcutaneous xenograft tumors and orthotopic xenograft tumors in nude mice.Besides,it also significantly inhibited the bone destruction and neovascularization in vivo.(5)The protein expression of p-EGFR,p-JAK2 and p-STAT3 in the PKG II group was significantly much lower.Compared with NC group,the expression of EFGR was decreased in PKG II group.Conclusion:(1)The expression of PKG II was suppressed in OS.Overexpression and activation of PKG II markedly suppressed the increased OS biological behaviors such as proliferation,migration,invasion and angiogenesis stimulated by EGF.(2)Moreover,in vivo,PKG II significantly inhibited tumor growth and progression and delayed bone destruction and angiogenesis.(3)Mechanistically,the negative effect of PKG II was related to the blockade of EGFR activation and abrogation of the downstream signaling as JAK2/STAT3 pathway.These findings may bring new insights into understanding the OS tumorigenesis and providing a potential therapeutic target for its molecular therapy.