Dihydrofolate Reductase Interacts With SuFu And Regulates Hedgehog Signaling Pathway

Background：Malignant cancer a leading cause of death in the world, ann also in China.According to the epidemiological study shows that approximately3.12millioncancers were diagnosed and2.7million people died of cancer erery year.nearly. Theincidence and mortality of cancer have been increasing, become the first cause ofdeath in city and rural residents in recent20years. The five most common cancers arelung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, as amajor health threat to our lives. The most commonly used cytotoxic anti-cancer drugshave some disadvantages: low-specificity and high side-effect. Targeted cancertherapies are expected to be more effective than current anti-cancer treatments andless harmful to normal cells, therefore, looking for an effective targeted drug hasbecome a new topic in cancer research.The hedgehog (Hh) signaling pathway plays a critical role in embryogenesisacross multiple species and its activity is reduced or absent in adult organisms. Recentstudies that uncontrolled activation of the Hh signalling pathway results in distinctcancers of the brain, muscle and skin have received significant attention.Dihydrofolate reductase (DHFR), a interacting protein with Suppressor of fused(SuFu) that one such negative regulator of Hh signaling. DHFR is an enzyme thatreduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as electron donor,thereby regulating the regeneration of tetrahydrofolic acid. Knocking-down oroverexpression of DHFR can affect tumor cells proliferation and apoptosis, so, toexplore the biological function of DHFR is very important to deeply understand theHh signaling pathway during cancer incidence and progression.Objective：1. To study DHFR and negative regulator SuFu interaction in the Hh signalingpathway, using different protein-protein interaction methods;2. Study on the influence of DHFR on Hh signaling pathway key factors SuFu, Gli1, Gli2and cell cycle regulatory protein Cyclin D1, as well as the effects on tumorcell apoptosis, explore the biological function of DHFR in tumorigenesis anddevelopment.Methods：1. Plasmid DNAs construction1.1Using gene cloning method, to construct different DHFR expression plasmids,pGADT7-DHFR, DHFR-pcDNA3.1/myc-his, pEGFP-DHFR,pcDNA3-FLAG-DHFR;1.2Designment and construction of DHFR RNAi vector, and confirmation of itsknock-down efficiency.2. DHFR and SuFu protein-protein interactionUsing Yeast-2-hybrid and GST pull-down assay, to test whether DHFR caninteract with SuFu.3. Study on the influence of DHFR on Hh signaling pathway3.1Detection of DHFR、SuFu、Gli and Cyclin D1protein levels under thefollowing conditions:a. before and after methotrexate (MTX) treatmentb. ovexpression of DHFR or knocking-down DHFRc.in GnRH-expressing neuronal cell line NLT and human glioma cell lines U87,U251, H4.3.2Using real-time PCR method, to detection of some key regulators mRNAregulation in the Hh signaling pathway follow by a. before and after methotrexate(MTX) treatment, b. ovexpression of DHFR or knocking-down DHFR.3.3Establishment of Gli-luciferase stable transfected cell line, observation of theeffect on the transcriptional activity of Gli, with different concentrations of MTXtreatment.3.4MTT assay measures cell survival rate by different concentrations of MTXtreatment.Results：1. The successful construction of DHFR ovexpression and RNAi vectors.2. Confirmation of DHFR and SuFu protein-protein interaction by Y-2-H and GST pull-down methods.3. Compared with the mouse GnRH neural cell line NLT, Gli1and Gli2higher,expressed in human glioma cell lines U87, U251, H4, DHFR, suggesting highexpression level of DHFR and elevated activity of Hh pathway.4. Gli1and Gli2mRNA and protein decreased after MTX treatment orknocking-down DHFR, but SuFu was increased, indicates DHFR may positivelyregulate Hh signaling pathway.5. Luciferase reporter assay shows Gli transcription activity decreased with theincrease of MTX concentration in Gli-8GBS stable expression293T cell line,suggesting that DHFR may positively regulate Hh signaling pathway.6. H4cells growth inhibition rate gradually increased, in correlation with theincreasing concentration of MTX treatment for24h or48h respectively, suggestingthat DHFR can positively regulate Hh signalling activity.Conclusion：DHFR may positively regulate Hh signaling pathway by interacting withnegative regulator SuFu.