Role Of Zinc Finger Protein ZFX In Glioma And Non - Small Cell Lung Cancer And The Function Of PLCH1 In Mouse Anxiety And Fear Memory

ZFX (zinc finger protein, X-linked) is a member of ZFX/ZF Y (zinc finger protein, Y-linked) protein family. Zfx and Zfy gene normally exists in mammal genomes, locating respectively at the end of X and Y chromesome but not at the homologous region. It has been reported that ZFX and ZFY are related to sex choice, sperm formation and Turner syndrome. The full length ZFX protein consists of a transcription activation domain, a nuclear localization sequence and a DNA binding region, which is a thirteen C2H2[Cys (2) His (2)] type zinc finger structure. C2H2 zinc finger structure is one of the common structures in proteins with important physiological functions in mammalian cells. ZFX has been found to be related to self-renewal of stem cells in embryonic and adult mammals. ZFX together with MYC and cofactors Cnot3 and Trim28 forms a network that regulates the transcription of genes involved in cell cycle, cell death and cancer in mouse embryonic stem cells. Therefore the function of ZFX in cancer becomes the research hotspot in recent years.Glioma occurs in the brain or spinal cord, but most in the brain. It is called glioma because the similar cell morphology as glial cell. Accounting for 30% of brain and central nervous system tumors, and 80% of malignant brain tumors, glioma is a serious threat to human health. Glioma is hard to cure and easy to recur. It is still a problem in medical field. Investigations into the molecular pathology of gliomas are therefore urgently needed, in order to develop new therapeutic strategies and improve patient outcomes. In the present study, lentiviral vector mediated RNA interference was used to knock down ZFX to study its role in glioma. MTT assay and colony formation assay showed that ZFX knockdown can effectively inhibit the growth and clone formation ability of human glioma cell line U87 and U373. Further study revealed that this growth inhibition upon ZFX knockdown was due to the cell cycle arrest in G0/G1, senescence and apoptosis induction. Western blots showed that ZFX knockdown changed the expression level of several proteins involved in cell cycle, apoptosis and classical signal pathway such as Akt and MAPK/ERK signaling. We also show for the first time that ZFX knockdown decreases the in vivo growth potential of U87 glioma xenografts in both subcutaneous and intracranial models in nude mice. Our results indicate that ZFX plays an oncogene role in glioma.Since ZFX has been reported to regulate lymph node metastasis in lung cancer, we proposed that ZFX may also play an important role in lung cancer. RNA interference technology was also used to knockdown ZFX to study its function in non-small-cell lung cancer. It’s revealed that ZFX Knockdown inhibits the proliferation rate and colony formation ablity of H1299 cell line. Further studies showed that ZFX knockdown mainly caused the cell migration defect, increased senescene and apoptosis, and cell cycle arrested at G0/G1 to inhibit the growth of H1299. Western blots showed that knockdown of ZFX changed expression level of proteins involved in cell cycle, apoptosis and Akt, MAPK/ERK signaling pathway, which is in accordance with the growth inhibition phenomenon. In addition, subcutaneous nude mice tumor model of H1299 cell line were also established. ZFX knockdown led to decreased proportion of tumor bearing mice and reduced mean tumor volume in the subcutaneous tumor model.Taken together, our studies revealed that ZFX promotes the growth of glioma and lung cancer cells, which indicated that ZFX may be a potential therapy target for glioma and non-small-cell lung cancer.Phospholipase C (PLC) can catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which are important second messengers in cell. So PLC is considered to play a central role in many important signaling pathways, integrating different signal inputs to regulate PIP2, DAG and IP3 levels.13 members were identified in PLC family. PLCH1 is a newly identified protein in this family, also known as PLCηl. Although it’s highly expressed in the brain, its function in the brain is remained to be explored. Since other members in PLC family have shown to be involved in advanced brain function, such as learning, memory and mood regulation. Therefore we use PLCH1 knockout mice to study its role in memory and anxiety.We first examined the basic moving ablility of PLCH1 knockout mice. Total distance moved in open field test showed that the basic moving ability was not impaired by PLCH1 konckout. Less time on rota rod indicated that PLCH1 knockout impaired the motor coordination of mice. In the open field test, PLCH1 knockout mice showed anxiety phenotype. Time spent in the center, distance moved in the center, center zone crossing number of PLCH1 knockout mice were all less than those of wild type mice. In order to further examine the anxiety phenotype, the elevated plus maze test was used. Elevated plus maze test showed that PLCH1 knockout mice spent less time in the open arm, moved less distance in the open arm than the wild type mice. Although there were no significant differences, the trend was in accordance with the anxiety phenotype from open field test. In addition, PLCH1 knockout mice showed a stronger memory of fear at 2.4 h and 10 day after the contextual fear training. Water maze showed that the latency to platform of PLCH1 knockout mouse was higher than the wild type mice, suggestting that spatial memory may be impaired. But the one way ANOVA analysis results showed no significant difference. Taken together, PLCH1 knockout mice showed normal moving ability, impaired motor coordination, stronger fear memory and anxiety-like phenotype.Taken together, our preliminary data supported that the deletion of PLCH1 can increase the anxiety level of mice, which is contrast to the phenotype of PICβ1 knockout mice.