Effect Of HIF-1? Dephosphorylation On Apoptosis Of Osteosarcoma Cells Induced By ILKAP Under Hypoxic Condition

Osteosarcoma?OS?is a kind of malignant bone tumor originated from mesenchymal.It is the most common disease spectrum of primary malignant bone tumor in children and adolescents.The age distribution of osteosarcoma patients showed a bimodal pattern,which was divided into adolescence and old age.The peak of puberty is about 15 years old.0-24years old group,the incidence of osteosarcoma is 4.4/million.The second peak was about 75years old and consisted mainly of secondary osteosarcoma associated with Paget's disease or other bony lesions.The ratio of male to female morbidity was 1.22:1.Metastasis is still the most important thing in osteosarcoma.About 80%of osteosarcoma patients had metastases or micrometastases at the time of diagnosis.The 5-year survival rate increased from less than30%to more than 70%due to advances in treatment principles and chemotherapy.Despite the above progress,there is still a long way to go in the study of the biological mechanism of osteosarcoma.How to cure osteosarcoma is one of the difficult problems in the medical field.Integrin-linked kinase-associated phosphatase?ILKAP?is a serine/threonine?S/T?phosphorylase.ILKAP is also a tumor suppressor gene expression product which has the function of regulating tumorigenesis and development.ILKAP is widely expressed in human tissues,mainly in kidney,skeletal muscle,liver and other organs.Mediated apoptosis is the main physiological function of ILKAP.ILKAP and tumor cells occurrence and development are closely related.Hypoxia inducible factor-1?HIF-1?is one of the most important factors in the study of tumor microenvironment and tumor etiology.As a transcription factor,HIF-1 has a series of effects,such as promoting cell proliferation and inhibiting cell apoptosis.Once the content of HIF-1 is out of balance,the cells will have a variety of malignant ability and develop into tumor.Early studies suggested that HIF-1 was mainly regulated by hydroxylase and entered the ubiquitin degradation pathway in normal microenvironment,but HIF-1 could not be degraded and functioned only when the cells were in hypoxia.Recent years,with the further study of HIF-1 regulation,it has been found that hydroxylation is not the only way to regulate HIF-1 transcriptional activity,and phosphorylation is also an important way to regulate HIF-1,especially in non-hypoxic conditions.Objective:It was proved that hypoxia could lead to apoptosis,and the effect of ILKAP on apoptosis of osteosarcoma cells was studied by interaction with HIF-1?.Methods:1?Construction and monitoring of cell hypoxia model:osteosarcoma cells were cultured in a carbon dioxide incubator produced by Billups Rothenberg Corporation.The canisters were continuously filled with mixed O₂?1%?,N₂?94%?and CO₂?5%?from 0h to 96h.The temperature of carbon dioxide incubator was 37°C,humidity was 95%.The culture medium was changed every 24 hours.The dissolved oxygen content was monitored by YSI model 55,thus monitoring the hypoxic environment.2?The viability of osteosarcoma cells was determined by trypan blue staining.Mix single cell suspension with 0.4%trypan blue solution at 9:1.One drop of trypan blue dye was added to 0.1mL single cell suspension and stained at room temperature for 3min to record the number of living and dead cells.The treated bone tumor cell material was observed under high power microscope.The number of living cells and dead cells in 1000 cells were counted and the unstained cells were counted.The survival rate of cells was calculated by formula.3?Apoptosis of osteosarcoma cells was detected by laser confocal microscopy.The co-location of DAPI and TUNEL was observed in osteosarcoma cells.4?HIF-1?and apoptotic marker were detected by Western blots-substrate chemiluminescence ECL assay.5?To study the role of ILKAP in the process of apoptosis:ILKAP shRNA adenovirus transfection and Ad-ILKAP overexpression were used.6?To study the interaction among HIF-1?,ILKAP and p53:the cells were treated with hypoxia for 72hours,and then immunoprecipitation was carried out.Results:1?Cell viability decreased with time,apoptosis increased?P<0.05?.HIF-1?was induced gradually with hypoxia treatment,and phosphorylated HIF-1?decreased gradually.HIF-1?-PBS was treated with 400U ILKAP as a negative control with 400U?phosphatase as positive control.It was found that ILKAP and?phosphatase had similar effects on HIF-1?,and the dephosphorylation of HIF-1?increased gradually.2?Cell survival is maintained by ILKAP shRNA under hypoxia.Apoptosis was associated with ILKAP under hypoxia.At the same hypoxia time,the number of apoptosis in the shRNA group was significantly lower than that in the control group?P<0.05?.Western blot bands showed that HIF-1?did not change to dephosphorylation in the ILKAP shRNA group as compared with the control group.3?The results of immunoprecipitation showed that phosphorylated HIF-1?preferentially precipitated with anti ILKAP antibody.There was no interaction between dephosphorylated HIF-1?and p53 antibody immunoprecipitation.4?Compared with control group,the cell survival rate of Ad-ILKAP group was significantly decreased in a time dependent manner?P<0.05?.Compared with the control group,the apoptotic marker and TUNEL method showed a marked increase in apoptosis in Ad-ILKAP group.Normoxic conditions could not induce HIF-1?expression,but could be detected in Ad-ILKAP group.Conclusion:1?With the prolongation of hypoxic time,apoptosis increased significantly,especially after 48h of hypoxia treatment.2?ILKAP can play a dephosphorylation effect similar to?phosphatase,and can promote HIF-1?from phosphorylation state to dephosphorylation state.It is revealed that ILKAP plays an important role in the process of HIF-1?dephosphorylation.3?Under the condition of long-term hypoxia,if the expression of ILKAP in the cells decreases,the apoptosis behavior of the cells is weakened.Therefore,ILKAP is essential for the long-term hypoxia induced apoptosis of cells.4?ILKAP directly binds to phosphorylated HIF-1?,so that HIF-1?dephosphorylation of ilkap and dephosphorylation of HIF-1?are separated.5?Dephosphorylated HIF-1?directly binds to p53.ILKAP does not directly interact with p53.6?In the case of normoxia,ILKAP can induce apoptosis and dephosphorylation of HIF-1?.In summary,ILKAP directly dephosphorylates HIF-1?and plays an important role in the subsequent p53 dependent apoptosis.Considering its complex role with p53 and p53 itself,our present experimental results provide a basis for further study on the role of different HIF-1?types in many diseases.