Corticotropin-releasing Factor Receptors 1 To Activate CAMP/PKA Signaling Pathways In Apoptosis Mechanisms U87MG

Backgroud: Glioma is the most common primary malignant tumor in the central nervous system, and accounts for approximately 40-50%of total intracraniai tumor. These tumors are highly invasive, and have no obvious dividing line with normal brain tissue,often infiltrate critical neurological areas within the brain.It has the characteristics of high incidence rate and high fatality rate. With traditional treatment of surgical treatment, radiotherapy and chemotherapy, treatment effect is still not ideal. Growth, the exsistance of BBB(Blood-brain barrier), glioma is hard to be removed completely by the currently therapy. To elucidate its pathogenesis and to find new treatments is a research hotspot in neurosurgery.Corticotropin-releasing factor is a 41-amino acid peptide, which is extracted and depurated from hypothalamus of goats by Vale and his colleagues. It mainly distributes in the human central nervous system,especially in hypothalamus, and peripheral systems are widely distributed. Corticotropin-releasing factor has a major role in hypothalamus- pituitary- adrenal axis, regulates the body of endocrine, autonomic nerve, immune response in the stress state.Such as Grazia found that CRF inhibited cell growth of human endometrial adenocarcinoma cells and breast cancer cells by CRFR1 activation. Erini found that CRF facilitate apoptosis of activated rat adrenal phaeochromocytoma cell line(PC12) cells by increasing the expression of Fas L. Minas found that CRH, its receptors CRHR1, and the proapoptotic molecule Fas L were produced by human ovarian tumour cells in situ. However, the function and mechanism of CRF on glioma is rarely reported in the domestic and abroad.Objective: Compare the change of content c AMP and PKA after processing U87 cells with CRF and receptor inhibitor Antalarmin. Explore the regulation effects of CRF in c AMP/PKA singnal pathway and on human glioblastoma U87 MG cell line apoptosis.Methods:1 c AMP analysis by ELISAU87 cells in the logarithmic phase of growth were seeded into 6-well plates at a density of 10-7 cells per well. After culture for 24 h, they were random Ly assigned to the following treatment groups: control, 10-7mol/L CRF, 10-6 mol/L antalarmin, 10-7 mol/L CRF + 10-6 mol/L antalarmin(by adding to CRF after antalarmin half-hour later). After treatment for 24 h, cells were harvested and washed with PBS, followed by exposure for 30 min to IBMX(1 mg/m L) to prevent the enzymatic dissociation of intracellular c AMP. Cells were disrupted by ultrasound lysed cell lysis method. Quantitative analyses of c AMP were conducted using a c AMP ELISA kit.2 Detecting PKA m RNA expression by Realtime fluorescence quantitative PCRThe total RNA was extracted from cells with Trizol reagents and detected its integrity by 1.5% agarose gel electrophoresis(AGE). Briefly, the RNA was converted to c DNA with c DNA synthesis kit. Designing PKA primer sequences by DNA man software and synthesizing from SBS Genetech CO.Ltd. PCR was proceeded in the Rotor-Gene Q Real-Time PCR system. The amplication ramp included an initial hold step of 2min, at 95 degree centigrade followed by a three-step cycle consisting of 30 sec at 95 degree centigrade, and 30 sec at 60 degree centigrade,repeated 40 times. In the end, the product was extended 5min at 72 degree centigrade. The results were analyzed using the 2-△△CT method and counted as the percentage of β-actin m RNA. That each of the other samples relative to the control sample of target gene m RNA transcription level, recording the results of the data.Results:1 c AMP analysis by ELISA: In the same time,cell samples of different treatments are variant, with statistical significance(F12h=8.18,P=0.008<0.05, F24h=7.821,P=0.009<0.05,F36h=5.820, P=0.021<0.05). c AMP concentration in the presence of different time is decreasing trend.2 Analysis of RTFQ-PCR results, In the same time, U87 cells PKA m RNA of different treatments Cells of different treatments are variant, with statistical significance(F12h=175.926, P<0.05, F24h=137.956, P<0.05, F36h=129.280, P<0.05).It show that the expression of CRF m PNA PKA treatment group was higher than the same time the other treatment groups.Conclusions:1 Elisa results showed:CRF acting on the U87 cells, increasing the concentration of intracellular c AMP, and may promote apoptosis through the second messenger c AMP, and over time, the concentration of intracellular c AMP reduction.2 RT-PCR showed consistent trends m RNA of PKA expression and intracellular c AMP. Combined with previous studies, the CRF are consistent with the trend of apoptosis.In summary, we deduced that CRF may play a role for the apoptosis of U87 MG cell line on some degree and may play a role via c AMP-PKA signal pathway in the apoptosis promotes of cell U87.