The Research Into HIF- 2 Alpha Of Human Lung Adenocarcinoma A549 Stem Cells Characteristics And Antiapoptotic Ability

Background and purpose:Currently, lung cancer has shown a gradually increase in the morbidity and mortality. Although chemotherapy is still one of the most important means in the treatment of lung cancer, the survival and prognosis of the patients are not ideal, so there is an urgent need for our deeper insight of tumor development to solve the problem of lung cancer from the root. The “cancer stem cell hypothesis” holds that the tumor is derived from the cells with atypia who can proliferate, self-renew and differentiation extensively, which play a key role in the invasion, metastasis and recurrence of tumor.Hypoxic microenvironment is a typical feature of solid tumors and is supposed to be an independent risk factor of tumor progression and bad prognosis. As an crucial component of cancer stem cell niche, hypoxic microenvironment plays an important role in the evolution and anti-apoptosis ability of cancer stem cells in tumor. Hypoxia inducible factors are central regulatory factors for the tumor adaption to the hypoxic microenvironment, mediating physiological and pathological responses to hypoxia. HIF-1 is a heterodimeric complex consisting of a hypoxically inducible subunit HIF-1α and a constitutively expressed subunit HIF-1β.HIF-1α also includes three subunits: HIF-1α,HIF-2α and HIF-3α.Although HIF-1α and HIF-2α have a gene sequence homology,they regulate their unique target genes respectively in the tumour progression. As an major regulator of cancer stem cells evolution, HIF-2α plays an important role in the maintenance of properties and the anti-apoptosis ability of the stem cells.Therefore, this study aimed to discuss whether HIF-2α can regulate stem cell marker gene OCT-4 by Wnt/β-catenin pathway to maintain the stem cell properties of A549 cell spheres and enhance their anti-apoptotic ability under hypoxic microenvironment.Objective:1. To study the affect of HIF-2α on maintain A549 stem cells characteristics2. To study the influence of HIF-2α on enhancing the antiapoptotic ability of A549 cell spheres under hypoxia preliminarily.Methods:1. A549 cells were cultured with serum- free medium(SFM)to induce the formation of A549 cell spheres. 2. The interfering lentivirus harboring sh RNA-HIF-2α was transfected into A549 cells and A549 cell spheres. 3. Western Blot was used to measure the protein levels of HIF-2α, OCT-4 and β-catenin of A549 cells and A549 cell spheres under normal and hypoxia 24 h respectively. The levels of protein of HIF-2α, OCT-4 and β-catenin of the A549 cells and A549 cell spheres transfected with the sh RN A-HIF-2α in normal and hypoxia 24 h conditions were measured by Western Blot as well. 4. The m RNA expressions of HIF-2α, OCT-4 and β-catenin were measured by RTPCR. 5. Flow cytometry was used to detect the apoptosis and the ROS concentration of A549 cells and A549 cell spheres with or without being transfected with the sh RNA-HIF-2α in normal and hypoxia 24 h conditions. 6. CCK-8 was used to detect the proliferation of A549 cells and A549 cell spheres with or without being transfected with the sh RN A-HIF-2α in normal and hypoxia conditions.Results:1. A549 cell spheres of different sizes were suspended in the SFM, which were composed of 20 to 100 cells with strong refractivity observed by inverted phase contrast microscope. 2. The second generation of A549 cell spheres was scattered in the hypoxia medium after hypoxia for 24 hours, with a stronger refractivity compared with the normal group. 3.(1) There was a slight protein and m RNA expression of HIF-2α of A549 cells and A549 cell spheres under normal conditions(P>0.05).(2) The protein and m RNA levels of HIF-2α, OCT-4 and β-catenin of A549 cells and A549 cell spheres in the hypoxia group were higher than the normal group(P<0.05).(3) The protein and m RNA levels of HIF-2α, OCT-4 and β-catenin of A549 cellspheres were higher than the A549 cells under hypoxia conditions(P<0.05). 4.(1) There was a slight protein and m RNA expression of HIF-2α of A549 cells and A549 cell spheres under both the normal and hypoxia conditions after sh RNAHIF-2α was transfected into cells(P<0.05).(2) The protein and m RNA levels of OCT-4 and β-catenin of A549 cells and A549 cell spheres in the hypoxia group reduced slightly or remained unchanged compared with the normal group after sh RNA-HIF-2α was transfected into cells(P<0.05). 5. The apoptosis of A549 cells and A549 cell spheres in the hypoxia group reduced compared with the normal group( P<0.05); The apoptosis increased significantly in the hypoxia group after sh RNA-HIF-2α was transfected into cells(P<0.05). 6. The levels of ROS of A549 cells and A549 cell spheres in the hypoxia group reduced compared with the normal group(P<0.05), and the falls were even more serious in A549 cell spheres( P<0.05);The ROS levels increased significantly in the hypoxia group after sh RNA-HIF-2α was transfected into cells, and the rise was more obviously in A549 cell spheres(P<0.05). 7. The proliferation of A549 cells and A549 cell spheres increased unde r the hypoxia condition at the 8h,16 h,24h,36, and the increase was more obviously in A549 cell spheres(P<0.05).Conclusion:1. The expressions of HIF-2α and its target gene OCT-4 of A549 cell spheres under the hypoxia condition, as well as the stem cell properties. 2. The hypoxia can induce the proliferation of A549 cell spheres, maintaining the intracellular low levels of ROS and inhibiting the apoptosis. 3. Silent HIF- 2 alpha can promote the apoptosis of A549 cell spherers.