| BackgroundChronic intermittent hypoxia（CIH）is the main pathologic feature of obstructive sleep apnea（OSA）.Studies have found that OSA and its related CIH promotes tumor progression.We previously found that CIH promotes subcutaneous tumorgenesis of Lewis Lung Carcinoma（LLC）cells in mice,but the specific mechanism remains to be explored.CIH has been found to activate renin-angiotensin system（RAS）.RAS is an important neurohumoral regulatory system,angiotensin Ⅱ（Ang Ⅱ）is the most important active peptide of RAS and plays an important role by binding its specific receptors AT1 R and/or AT2 R.However,weather Ang Ⅱ-AT1 R involved in CIH promote the tumor progression is still unclear.Accordingly,this study will explore the role and mechanism of Ang Ⅱ-AT1 R in CIH promoting LLC cell subcutaneous tumorigenesis in mice through cell and animal experiments.ObjectivesThis study aims to explore the role and mechanism of Ang Ⅱ-AT1 R in the exacerbating effects of CIH on murine lung cancer.It will provide a new diagnosis and treatment idea for patients with obstructive sleep apnea（OSA）combined with lung cancer.Methods1.In vitro,LLC cells and SVEC4-10 cells were directly treated with Ang Ⅱ,it was aimed to investigate the effects of Ang Ⅱ-AT1 R on LLC cells proliferation through MTT,FCM,and examine the effects of Ang Ⅱ-AT1 R on the proliferation,migration,and angiogenesis of SVEC4-10 cells through MTT,scratch test and tube formation.Western blot was used to examine the changes of proteins in both LLC cells and SVEC4-10 cells after above treatments to explore the molecular mechanisms.2.And then,LLC cells and SVEC4-10 cells were treated with intermittent hypoxia（IH）to simulate CIH in mice,ELISA was used to detect the change of Ang Ⅱ in the cell culture medium supernatant of LLC cells or SVEC4-10 cells,and MTT detected the change of LLC cells proliferation to explore weather IH promote LLC cells proliferation by promoting the release of Ang Ⅱ in LLC cells,and MTT、scratch test were used to explore weather IH promote SVEC4-10 cells proliferation and migration by promoting the release of Ang Ⅱ in SVEC4-10 cells;at last,SVEC4-10 cells were treated with the culture medium supernatant of LLC cells after exposing to IH;MTT,scratch test and tube formation were performed to determine whether IH promoted the proliferation and migration of SVEC4-10 cells to promote angiogenesis by promoting the release of Ang Ⅱ from LLC cell,and then,LLC cells were treated with the cell culture medium supernatant of SVEC4-10 cells after IH exposure,MTT was devoted to explore whether IH promoted LLC cells proliferation by promoting the release of Ang Ⅱ from SVEC4-10 cells.Western blot was used to examine the changes of proteins in both LLC cells and SVEC4-10 cells after above treatments to explore the molecular mechanisms.3.In vivo,murine lung cancer model was exposed to normoxia or CIH environment,the mice were treated with NS,Losartan and Enalapril and divided into 6 groups:（1）Con+NS,（2）Con+Ena,（3）Con+Los,（4）CIH+NS,（5）CIH+Ena,（6）CIH+Los;tumor volume and mice body weight were measured every 3 days,ELISA examined the changes of Ang Ⅱ in serum,IHC examined the changes of Ki67 and CD31 in tumor tissues to investigate whether CIH promoted murine lung cancer progression through Ang Ⅱ-AT1 R.Results1.Ang Ⅱ upregulated the expression of Cyclin D1,downregulated p21,Cleaved caspase 9 to promote cell cycle and inhibit apoptosis by activating PI3K/Akt-EGR1 signaling pathway through acting on AT1 R to promote the proliferation of LLC cells.2.Ang Ⅱ promoted the proliferation and migration of SVEC4-10 cells by activating PI3K/Akt-EGR1 signaling pathway through acting on AT1 R to promote the ability of forming tubes of SVEC4-10 cells.3.IH promoted the release of Ang Ⅱ from LLC cells and upregulated the expression of Cyclin D1 and downregulated the expression of p21,Cleaved caspase 9 by activating PI3K/Akt-EGR1 signaling pathway through acting on AT1 R on LLC cells to promote the proliferation of LLC cells.4.IH promoted the release of Ang Ⅱ from LLC cells to activate the PI3K/Akt-EGR1 signaling pathway in SVEC4-10 cells through acting on AT1 R to promote the proliferation and migration of SVEC4-10 cells to induce angiogenesis.5.IH promoted the proliferation and migration of SVEC4-10 cells by promoting the release of Ang Ⅱ from SVEC4-10 cells and activating the PI3K/Akt-EGR1 signaling pathway through acting on AT1 R.6.IH promoted LLC cell proliferation by promoting the release of Ang Ⅱ from SVEC4-10 cells and activating PI3K/Akt-EGR1 through acting on AT1 R in LLC cells.7.CIH upregulated the expression of Cyclin D1,downregulated the expression of p21 and Cleaved caspase 9 to promote tumor cell proliferation,and CIH upregulated the expression of VEGFR2 to promote angiogenesis to promote LLC cells subcutaneous tumor-formation in mice.ConclusionCIH not only increases circulatory Ang Ⅱ but also stimulates the production of local Ang Ⅱ from both tumor cells and endothelia cells.Circulating Ang Ⅱ as well as autocrine or paracrine local Ang Ⅱ,acting on AT1 R in tumor cells and epithelia cells,results in the activation of PI3K/Akt-EGR1 signaling pathway and in turn promotes tumor cell proliferation and angiogenesis,thereby exacerbates subcutaneous lung cancer growth... |
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