Effect Of CRF On U87MG Proliferation And P53Expression

Backgroud: Corticotropin-releasing factor （CRF）, containing41aminoacids, is an important neuroendocrine peptide and a member of CRF family.CRF can coordinate functions of endocrine system, autonomic nerve system,immunological system and behavior under stress viahypothalamic-pituitary-adrenal axis （HPA）. Also, CRF can modulate thefunctions of cardiovascular system, gastrointestinal system, skin andproductive system. Recent studies suggested that CRF may have an influenceon tumor proliferation. Possibly because CRF involved in growth of tumorvessels, development of tumors and their metastasis. Besides, CRF may play arole in anti-proliferation and neuron-protection. Some studies showed thatCRF also had an effect on central nervous system neoplasms. CRF receptorsexpressed in some central and peripheral nervous system neoplasms, likeCRFR1expressed in medatloblastoma, ganglioneuroma, neuroblastoma andmeningioma. Recently, the existence of CRFR in glioblastoma had beenproved. However, it is not clear that how CRF influences glioblastoma and itspathway so far.Objective： Find out the effect of CRF on human glioblastoma U87MGcell line proliferation and its influence on U87MG p53gene expressionunder different CRF concentrations.Methods：1MTT tested for U87MG cell line proliferationThawed and passaged U87MG cellsand divided cells into test groups andcontrol group with96-well plates（6sample sizes in each group）. Add DMEMcontaining different concentrations of CRF in test groups (0,10^-7,10^-8,10^-9mol/L). Tested OD at24-hour,48-hour and72-hour cultured, and evaluatethe influence of CRF on U87MG proliferation. Analyzed the data withstatistical analysis. 2FCM tested for U87MG cell cycle and apoptosisDivided logarithmic phase cells randomly into4groups（3sample sizes ineach group） and added DMEM containing10^-7mol/L,10^-8mol/L,10^-9mol/Land0mol/L CRF respectively. After48hours cultured, fixed the cells with70%ice ethanol overnight. Tested the cell cycle and apoptosis with FCM.Analyzed the data with statistical analysis.3The effect of CRF on p53gene expression tested by realtimefluores-cence quantitative PCR.Passaged the U87MG cells four times and divided them into4groups （3sample sizes in each group） randomly. Added DMEM containing10^-7mol/L,10^-8mol/L,10^-9mol/L and0mol/L CRF in50ml culture flasks respectively.Extracted RNA at6h,12h,24h and48h cultured and tested p53geneexpression with realtime fluores-cence quantitative PCR. Analyzed the datawith statistical analysis.Results:1MTT data suggested that CRF inhibited human glioblastoma U87MGcell line proliferation. In the test groups, CRF showed no statistical differenceson U87MG proliferation among each timepoint（P>0.05）. Differences existedstatistical significance among test groups （P<0.01）. Compared with controlgroup, cell apoptosis rate in test groups increased with CRF concentration, andpresented a positive correlation.2FCM data revealed that, contrasted with control group, cell cycles weremostly arrested before phase G2. The inhibiting effect correlated positivelywith the CRF concentrations. CRF induced U87MG apoptosis. There werestatistical significances for each test group compared with control group（P<0.01）. Our data suggested the dose-depended relationship between CRFconcentration and cell apoptosis.3Our PCR data suggested that different concentrations had differenteffects on expression of p53gene at the same timepoint （F=7.878, P=0.002）and different timepoints had different effects on p53gene expression at thesame concentrations,（F=9.268, P=0.001）between test group with control groups. But in multiple comparisons, There was no significance among eachtest group（P>0.05）. Analysis revealed that different times and differentconcentrations all could affect expression of p53gene on U87MG cell line,but this effect was not related with concentration gradient.Conclusions:1MTT results exhibited that CRF could inhibit proliferation of U87MGcell line,and the inhibition increased with CRF concentration. Presented linearrelation.2FCM revealed that cell cycle arrested before phase G2. the cell cycleinhibition increased with CRF concentration. Apoptosis: CRF could inducedapoptosis of U87MG cell line. Presented dose-effect relationship betweenCRF concentration and cell apoptosis.3Realtime fluores-cence quantitative PCR results suggested the effect ofCRF on p53gene expression of U87MG had statistical significance.Concentration and time both could affect the expression of p53gene. CRFcould up-regulate expression of p53gene, but the p53gene expression leveldid not change much with different concentrations CRF.In summary,we deduced that CRF could inhibit proliferation of U87MGcell line on some degree via up-regulating p53gene expression whichmediated apoptosis of neoplasm cells.