Inducing CRFR1Expressions By Different Concentrations Of CRF In Human Glioma Cell Line U87

Corticotropin releasing factor(CRF)is an important neuroendocrinepeptide, and the main physiological function is to stimulate the pituitary glandsynthesis and secretion of adrenocorticotropic hormon(ACTH)and relatedpeptide. CRF and its peptide family common regulate the function ofhypothalamus-pituitary-adrenal (HPA) axis and coordinate the endocrinesystem, the autonomic nervous system, immune system, physiology andbehavior function when the body under stress. And, it has a moderating effecton cardiovascular, gastrointestinal, reproductive, skin, etc. CRF and its relatedpeptide play a role through its receptor(Corticotropin releasing factorreceptor,CRFR). CRF receptors play a role through activation of adenylatecyclase, which belong to the G-protein coupling receptors. As the mainreceptor in the HPA axis, corticotropin releasing hormone receptor1(CRFR1)can promote the release of the pituitary ACTH to mediated CRF by the classicway under stress response. Overexpression of CRFR1recepor has also beenfound in so many human malignant tumors in the central nervous system.However, previous studies have not investigated the relationship of CRF andgliomas.Objective:To explored the expression of CRFR1and the effcet of CRFinducing CRFR1in glioma cell line U87.Methods:1The total RNA were extracted from cultured U87cells. Primer of CRFR1were designed for the RT-PCR amplification. The product was analysis by gelelectrophoresis.2Glioma cell lines U87were treated by different concentrations of CRF. U87cells was divided into four groups, which be dealed with by differentconcentrations of CRF (10-7,10-9,10-12mol/L) and control groups. Then U87 cells were cultured continuely in filled with coverslip of tin plates. Each twoholes were taken out to further immunostainned after12hours. The cells ofsurplus holes continue to culture. Another batch of cells were treated in thesame way after24hours. The last ones were treated in the same way after48hours. The experiment was repeated for three times. Finally, expression ofCRFR1positive rate were carried out by statistical analysis.Results:1CRFR1mRNA was expressed in human glioma cells U87by RT-PCR.2.CRFR1positive expression can be shown in every group byimmunocytochemistry. Positive protein located in the cytoplasm, andpresented a clear yellow or brown stain in the periphery of the nucleus.CRFR1-positive expression rates were carried out by statistically analysis.2.1The single factor analysis were used for different concentration effect onthe expression the CRFR1. Compared with the control groups,differentconcentrations showed significant difference, which including12hours(F=8.099, P<0.05);24hours(F=51.665,P<0.05);48hours (F=14.531,P<0.05). Compare the CRF10-9mol/L group with other concentration ones,there were difference at24hours and48hours. While compare with otherconcentration groups, there is no difference. At24hours, the control groupand CRF10-9mol/L group is significant difference. However, there is nodifference among the control group,CRF10-7mol/L group and CRF10-12mol/Lones. There is significant difference between the CRF10-7mol/L groups andCRF10-12mol/L ones.2.2The single factor analysis were used for different time effect on theexpression the CRFR1. The statistics results are as follows:there is noinfluence to CRFR1expression positive rate in different times byCRF10-7mol/L treated(F=1.839,P=0.238);and there is no influence to CRFR1expression positive rate in different times by CRF10-12mol/L treated(F=3.884,P=0.083)ï¼›there have influence to CRFR1expression positive ratein different times byCRF10-9mol/Ltreated(F=90.66,P=0.0001), multiplecomparisons of the different time points, any two groups of comparisons are differences.2.3Factorial analysis were carried out for different concentrations of CRFand different time to inducing CRFR1positive-expression rate. CRFR1expression rate were inducing by different concentration of CRF(F=21.185,P=0.0001). CRFR1expression rate were influenced by different time(F=27.954, P=0.0001); And, there are interaction between differentconcentration of CRF and different time(F=27.458, P=0.0001). CRFR1expression rate were influenced by different concentration and time of CRF,which about31percent to93percent. And expression intensity was alsodifferences in conclusion. In CRF10-9mol/L and24hours, and the resultsshowed that CRFR1expression was strongest (the brown color), andexpression of the highest positive rate,87%on average. In CRF10-9mol/L and48hours,expression of CRFR1-positive rate were68%on average.Conclusion:The expression of CRFR1mRNA was found by RT-PCR inhuman glioma cells U87. Expression of CRFR1receptor-positive cell ratehave been increased after corticotropin releasing factor(CRF)acting on thelines U87. There was a significant statistically difference. The expression ofCRFR1receptor-positive cell rate were influenced attributed to concentrationof CRF and action time. Concentration and time of CRF may equally to theresults during the process. In CRF10-9mol/L for24hours, expression ofCRFR1-positive cells rate is the highest and the color is more obvious. InCRF10-9mol/L acting on U87cells, from24to48hours, raise the expressionof CRFR1receptors. But this phenomenon was not found in otherconcentration and time point,when CRF10-9mol/L acting on U87cells after12hours showing down-regulation the expression of CRFR1receptors.According to our research results, CRF influence on glioma cells byCRFR1-receptors in vitro experiments.