Effects Of OSAS Model IH On The Expression Of Tumor Metastasis-associated Proteins

Sleep apnea syndrome(SAS)was first reported in the 50 s of last century.With the quickly increasing incidence of sleep apnea syndrome,more and more attention has been paid to it.According to the results of polysomnography,SAS can be divided into three different types: obstructive,central and mixed.The pathogenesis and clinical manifestations of the three groups are different.Epidemiological studies have shown that obstructive sleep apnea syndrome(OSAS)is the most prevalent in the adult population.The pathogenic process of OSAS is complex,mainly related to anatomic factors,neurohumoral factors.Anatomical factors mainly refer to the anatomical structure of upper airway in patients with varying degrees of stenosis.The stenosis of patients in the stationary state,and in the state of sleep increased.The degree of stenosis of nerve humoral factors including patients with various endocrine disorders such as hypothyroidism etc..The main pathophysiological features of OSAS are nocturnal intermittent hypoxia(IH)and persistent hypercapnia.OSAS is a great harm,and the harm is not only limited to the disease itself,but also in the other complications.The common complications of OSAS include diseases of cardiovascular system,respiratory system and endocrine system.In recent years,it has been found that the probability of OSAS patients with tumors and the mortality of the patients are increased with different degrees,and the disease progresses rapidly.In this regard,some scholars speculate that OSAS induced by IH may be one of the important risk factors for tumor development and tumor cell proliferation.In the development and progression of tumors with different degrees of epithelial mesenchymal transition(EMT),oxidative stress,inflammatory reaction and other events.If OSAS mode IH has any effect on these events in the occurrence and development of tumors,the detailed mechanism is not very clear.In this study,A549 human lung cancer cells and mouse melanoma B16F10 cells as the research objects,simulating the intermittent hypoxia environment,to establish tumor cells-the intermittent hypoxia model,and to study the effects of IH on protein expression and biological behavior of tumor cells,and to explore the mechanism of that,and to provide new angles and methods for the prevention and treatment of tumors.Objectives1.To explore the effect of OSAS IH on the expression level of HIF-1?,YAP and P-YAP protein in human lung cancer cell line A549.2.To explore the effect of OSAS IH on the expression of NF-kappa B,NRF2 and MMP-9 protein in mouse melanoma B16F10 cells.MethodHuman lung cancer A549 cells and mouse melanoma B16F10 cells were cultured for normal cell culture,and then exposed to IH environment.The environment of the IH culture tank was set up to be IH 5mins and re-oxygenation 5mins,and the hypoxia re-oxygenation cycle was set up depends on the need.The minimum oxygen concentration in the tank is controlled at about 5% when oxygen is low,and the oxygen concentration in the tank is about 21%.The time of exposure was set up as1 H,3h,6h,which were divided into IH1 group(IH1),IH3 group(IH3),IH6 group(IH6),and the cells were cultured in normal cell culture medium as control group(N).Western-blot method was used to detect the expression of hypoxia inducible factor-1alpha(HIF-1),YAP,P-YAP protein in A549 cells of human lung cancer cell line,and the expression leve of HIF-1 and YAP m RNA was measured by q RT-PCR method.Western-blot method was used to detect the expression level of nuclear factor-2(NRF2),an inflammatory related nuclear factor(NF-?b),and the expression level of matrix metalloproteinase-9(MMP-9).Results1.The expression of HIF-1? and YAP The results of WB showed that the expression of HIF-1? protein in H1,IH3 and IH6 groups were obviously higher than that in the control group,and the expression of HIF-1? protein increased gradually with the increasing of treating time.The expression of YAP protein in IH1,IH3 and IH6 groups were higher than that in coontrol group,and the trend of YAP protein expression was time dependent.The expression of P-YAP protein in IH1,IH3,IH6 groups were obviously lower than that in control group,and the expression level of P-YAP protein and time of IH treatment were also connected.q RT-PCR results showed that the IH1 group(2.91 + 0.04),IH3 group(5.69 + 0.17)relative expression of HIF-1? m RNA compared with N group(1.01 + 0.05)was higher(P<0.05),IH6 group(9.18 + 0.59)relative expression of HIF-1 m RNA was significantly higher than that in group N(P<0.01),and it was found that the relative expression of HIF-1 ? m RNA in cells was dependent on the time of IH treatment.Group IH1(2.50 + 0.18),IH3 group(4.07 + 0.25)YAP relative expression amount of m RNA compared with group N(1.00 + 0.01)was significantly higher(P<0.05),IH6 group(9.18 + 0.58)relative expression of YAP m RNA was significantly higher than that in group N(P<0.01),and found that the relative expression of cells in YAP the amount of m RNA and IH with time-dependent(P<0.05).2.The expression of NRF2,NF-kappa B P65,I kappa B alpha,MMP-9 protein Compared with N group,the expression of NRF2 protein in IH1 group,IH3 group and IH6 group increased significantly.Compared with N group,the expression of NF kappa B protein in IH1 group,IH3 group and IH6 group was significantly higher than that in P65 group.Compared with N group,the expression of I kappa B protein in CH group and IH group were significantly decreased.Moreover,the degradation degree of I kappa B alpha protein was higher than that of CH when exposed to IH.Compared with N group,the expression of MMP-9 protein in IH group was significantly increased.Conclusion1.OSAS IH could promote the expression of HIF-1? and YAP in human lung cancer A549 cells,and reduce the expression of P-YAP protein.And OSAS IH promoted the expression of HIF-1? m RNA and YAP m RNA which point out that the dysregulation of Hippo-YAP signaling pathway may be one of the reasons for the increased risk and progression of patients with OSAS and tumors.2.OSAS model IH can promote the high expression of NRF2 in mice melanoma B16F10 cells,which indicates us the rapid progress of OSAS patients with a tumor may be associated with oxidative stress events caused by OSAS.3.OSAS IH can promote NF-kappa B p65 high expression in mice melanoma B16F10 cells,and can lead to low expression of I kappa B protein in lung cancer cells of Lewis mice,which indicate us that inflammatory response plays an important role in the increased risk of OSAS patients with tumors.4.Tumor cell-hypoxia model showed that the expression of MMP-9 protein increased in Lewis mice,which suggests that intermittent hypoxia can promote tumor invasion and metastasis.