Retinoic Acid, Rapamycin On Glioma Cells RNA Binding Protein Expression

Recent advances in RNA biology reveal unexpected diversity and complexity of cellular RNA metabolism. RNAs in cells are associated with RNA-binding proteins (RBPs) to form ribonucleoprotein (RNP) complexes, which influence the structure and interactions of the RNAs and play critical roles in their biogenesis, splicing, stability, transport, translation and cellular localization. Aberrant expression of RBPs affects many steps of RNA metabolism, significantly altering expression of RNA. Thus, altered expression and dysfuncting of RBPs are implicated in the development of various diseases including cancer. Emerging roles of RBPs as a global coordinator of post-transcriptional steps and altered RBP as a generator of cancer related RNA alternative splicing have been well described. Previous work of our lab showed highly expressed PTB (Polypyrimidine-Tract Binding protein) and PCBP2(Poly(rC) Binding Protein2) in glioblastoma cells.ATRA (All-Trans Retinoic Acid) and rapamycin suppress the proliferation of tumor cells, inducing differentiation and apoptosis through RARE(Retinoic Acid Responding Element)and mTOR(mammalian Target Of Rapamycin) pathway, respectively.In our research, we firstly confirmed the drug effect by MTT assay, IMF(immunofluorescence) test and TUNEL(TdT-mediated dUTP nick end labeling) assay. The result was consistent with what was reported in published papers and gave us the proper methods of treating T98G cells with the two drugs. Later, we discovered UNR (Upstream of N-Ras), UNRIP (UNR-Interacting Protein), PCBP2were down regulated in ATRA and rapamycin treated T98G cells, which was related to the dose and duration of drug application. This implied the RBPs may play a role as the downstream in the pathway of anti-cancer drugs.Then, in order to over express the RBPs in T98G cells to see wether they can reverse the drug effect, we tried identification and transfection of UNR, UNRIP, PCBP2expression vectors into eucaryotic cells. By this method we can verify the relationship between the RBPs and the biological effect caused by ATRA and rapamycin. Also, the mechanism were researched on. We cloned5’UTR sequence which may include IRES (Internal Ribosome Entry Sites) of UNR UNRIP PCBP2and ligate them to dicistronic fluorescent vectors. If ATRA and rapamycin down regulate the RBPs by the way of suppressing the function of IRES, there can be certain changes in the expression of the Renilla and Firefly fluorescent lights.We can conclude from the research that ATRA and rapamycin could suppress the proliferation of tumor cells, inducing differentiation and apoptosis, and meanwhile down regulate UNR, UNRIP and PCBP2. The UNR, UNRIP, PCBP2expression vectors may be used to prove that over-expressed RBP might rescue the differentiation or apoptosis induced by ATRA and rapmycin. Dicistronic fluorescent vectors with IRES sequence in them may help to value wether the down-regulation of the RBPs are mediated by IRES. The findings imply a new pathway of tumor metabolism thus help find a new target or resistance mechanism of anti-tumor drugs.