Pcbp2 In Human Glioma Cells And Its Molecular Mechanism

Poly(C)-binding proteins (PCBPs) are known as RNA-binding proteins that interact in a sequence-specific fashion with single-stranded poly(C). They can be divided into two groups:hnRNP K and PCBP1-4. These proteins are involved mainly in various posttranscriptional regulations (e.g., mRNA stabilization or translational activation/silencing). The PCBPs contain three KH domains, two consecutive KH domains at the amino terminus and a third KH domain at the carboxyl terminus, separated by an intervening sequence of variable length. The ability of PCBPs to recognize and bind poly (C) RNA sequences via their KH domains is critical for their function in mammalian cells. Several studies have reported that members of the family hnRNP K, PCBP1and PCBP2play important roles through a combination of their target mRNAs in various human tumor cells. However, there were no reports in the literature about the expression or function of the family members in human glioma cells. This PhD dissertation is mainly focused on the preliminary studies of the expression, function and downstream molecular mechanisms of PCBP2in human gliomas.We applied Real-time PCR, Western blot and immunohistochemistry to detect the expression of PCBP2in both glioma cell lines and primary glioma tissues. The result indicated that PCBP2was up-regulated in glioma cell lines and primary glioma tissues. For further study of the function of PCBP2, we synthesized a small interfering RNA(siRNA) which can specifically target PCBP2in T98G, U87MG and U251glioma cell lines. Then MTT assay and Cell count assay were employed to find that knockdown of PCBP2would inhibit glioma cells proliferation. We constructed recombinant adenovirus to interfer PCBP2in xenograft model in nude mice and it was also suggested that knockdown of PCBP2could suppress tumor growth of glioma cells in nude mice. BrdU incorporation assay and flow cytometry were employed to find that knockdown of PCBP2in glioma cells would inhibit DNA synthesis and cell cycle process. Hoechst staining, TUNEL and detection of cleaved caspase-3and cleaved PARP were employed to find that knockdown of PCBP2in glioma cells would induce caspase-3pathway mediated cell apoptosis. Therefore, we concluded that PCBP2may work as a potential molecular target for anti-glioma therapy. Meanwhile, we also discovered that PCBP2may be involved in TGF-β1/Smads and P53pathways, more studies should be provided for detail molecular mechanism.For studying the mechanism of PCBP2regulating glioma cell proliferation and apoptosis, RIP-Chip was applied to screen target mRNAs binding to PCBP2in glioma cell line. We compared the results from the three sets of experiments and identified35mRNAs that met threshold in all experiments. Combining bioinformatics prediction, we find9genes encoded by the PCBP2associated mRNAs involved in cell cycle or apoptosis from35candidate mRNAs. Real-time PCR was used to preliminarily validate the microarray results. Subsequently, we confirmed PCBP2binding sites in the candidate target mRNAs by biotin pull-down. Eventually we confirmed8fragments distributed among7mRNAs can be combined with PCBP2. We also found that FHL3mRNA3'UTR-A can highly specifically bind to PCBP2, and its core recognition sequence was further identified.FHL3,as one of targets binding to PCBP2we newly identified, belongs to the four and a half LIM (FHL) family, composed of the five members, FHL1～FHL5. They are expressed in a cell and tissue specific nlanner. It has been proved that FHL is an important regulatory factor in cell growth and differentiation by participating in transcriptional regulation, signal transduction, apoptosis and so on. We applied Western blot and immunohistochemistry to detect the expression of FHL3. The result indicated that FHL3was down-regulated in glioma cell lines and primary glioma tissues. Knockdown of PCBP2enhanced the level of FHL3protein, suggesting that PCBP2may negatively regulate FHL3protein expression. Through dual luciferase and mRNA half-life assay, we verified that knockdown of PCBP2up-regulated FHL3protein expression by binding FHL3mRNA3'UTR and enhancing its mRNA stability in glioma cells. In order to understand the role of FHL3in glioma cells, we overexpressed FHL3in T98G glioma cells, and found the glioma cells proliferation ability was abated, the occurrence of apoptosis increased. Co-knockdown of PCBP2and FHL3can rescue most of the proliferative capacity, inhibit apoptosis of glioma cells, which illustrated knockdown of PCBP2can inhibit glioma cell proliferation and induce glioma cells apoptosis through up-regulation of FHL3.In order to further study the molecular mechanism of FHL3in glioma cells, by biotin pull-down combined with mass spectrometry identification, we found22proteins can directly or indirectly associate with FHL3mRNA3'UTR besides PCBP2. In addition, we also found FHL2protein levels may be affected by the negative regulation of FHL3, but its mRNA level is not affected. DNA gene biochip was applied to screen the different gene expression pattern of T98G overexpression of FHL3.708differently expressed genes were found, including420down-regulated and288up-regulated. According to bioinformatic prediction and references report, we found28genes involved in glioma cell proliferation, apoptosis or invasion. We compared Real-time PCR and chip results, finally from the28genes we screened11genes for next step verifying. The mechanism of FHL3regulating the genes needs more experimental data for support.Our findings suggest for the first time that PCBP2is involved in gliomagenesis. Through small interfering RNA technology, PCBP2reveals a tumor oncogene like potential in glioma growth and apoptosis. Our identification of FHL3as a novel target mRNA binding to PCBP2is stabilized by knockdown of PCBP2. Overexpression of FHL3inhibits glioma growth and induces glioma apoptosis by regulating downstream genes expression. The axis of PCBP2-FHL3-downstream genes may be highly significant as new pathways in controlling glioma progression.