Study On Mechanism Of Inhibiting Human Glioblastoma U251 Cells’ Proliferation By Depleting STAT5b Expression

Background and Objective Glioma is a most common primary malignant neurological tumor, of which more than 82% for glioblastoma (glioblastoma multiforme, GBM), and glioblastoma multiforme belongs to a highly malignant glioma. The median survival period of glioblastoma multiforme is only 15 months. Glioblastoma multiforme placed a severe mental pressure and huge economic burden on the individuals and families. Studying the glioblastoma multiforme pathogenesis and targeting the proper gene activity to search for effective drugs and theraputic methods are the hotspots of clinical glioblastoma research. Signal transducers and activators of transcription (STATs) are an important signal pathway involved in oncogenesis. STATs are a family of transcription factors and are involved in a wide varietyof cellular physiological processes, including differentiation, survival, or cell growth. Currently, more and more studies focused on STAT5 in mammals seven STAT family members in recent years. STAT5 has been identified as an oncogene, is over-expressed in a variety of tumors, including cervical cancer and other malignancies. STAT5 has been shown to regulate proliferation and inhibition of apoptosis in several cancer cells. For instance, STATS activation has been shown primarily in hematopoietic malignancies. Blocking STAT5 signaling pathway can inhibit cell proliferation of pancreatic cancer cells. STAT5 is comprised two highly homologous isoforms, STAT5a and STAT5b. Although STAT5a and STAT5b have different carboxyl terminal, these are functionally not redundant.Signal transducer and activator of transcriptionSb has important implications for the tumorgenesis and development of human glioblastoma multiforme. Over-expression of STAT5 plays an important role on the promotion of glioblastoma multiforme development. Each character of STAT5a and STAT5b gene in tumors has not been distinguished. Liang, et al investigated the detail role of STAT5a and STAT5b in human gliomas. The results of the study showed that STAT5b have a key role in cell formation, cell cycle and transfer of pleomorphic gliomas by adjusting the expression of genes, such as Bcl-2、p21(waf1/cipl)、p27(kip1)、FAK and VEGF. Immunohistochemistry staining results showed the dyeing positive rate of STAT5b(57.1%) in cytoplasmic of glioblastoma multiforme significantly increased compared with normal cortex (22.2%) and diffuse astrocytoma (27.3%).In human glioma cells, study on expression of STAT5 and mechanism of down-regulated STAT5 is relative less. This study focused on glioma and detected the expressions of JAK1 and STAT5b in mRNA levels and protein levels, investigated STAT5b over-activation and abnormal expression in glioma tissues, explored JAK1/STAT5 signaling pathway in the role of the development of glioma, and explored VEGF, Bcl-2, survivin, bax expression and human glioblastoma U251 cells proliferation ability by depleting STAT5b expression. This study may provide theoretical basis for glioma gene therapy.Methods The glioma tissue samples, adjacent tissue and different glioma cells (human glioma cell lines U-373MG, U251, TJ905, TJ899 and A172, mouse cell line G422) were collected. The mRNA expression levels of JAK1 and STAT5b were detected by RT-PCR method. The protein expression levels of JAK1, STAT5b, phosphorylated JAK1 and phosphorylated STAT5b were measured by Western blot assay. The protein expression levels of VEGF, Bcl-2, survivin and bax were also detected by Western blot assay. Furthermore, STAT5b expression was depleted by transfecting Pegenesil-l-ShSTAT5b plasmids. The mRNA and protein expression levels of STAT5b, JAK1, VEGF, Bcl-2, survivin and bax were measured by RT-PCR and Western blot assay respectively. The proliferatioal ability of glioma cells U251 treated with or without silencing STAT5b was measured by MTT assay.Results STAT5b was high expressed in glioblastoma cells, and it was also found that the JAK1 was over expressed in glioblastoma cells. The statistics results showed that STAT5b expression in glioblastoma was positively correlated with JAK1 expression. Phosphorylaed JAK1 and phosphorylaed STAT5b were significantly activated in glioma cell lines U-373MG, U251, A172, and mouse glioma cell line G422. It indicated that JAKl/STAT5b might be an important signaling pathway in glioma. We also found the expreesion of VEGF, anti-apoptosic protein bcl-2 and survivin were significantly increased in four glioma cell lines, while bax expression was decreased in four glioma cell lines. STAT5b small RNA interference plasmid was constructed by RNA assay. The known targeting sequence of STAT5b was indexed in Gene Bank (NCBI Pumed). The single chain of STAT5b siRNA was synthesized in vitro, and the double-stranded chain was formed after annealing. The RNA was no pollution, high purity, integrity and no degradation shown in 1.5% agarose gel electrophoresis. The sequencing results showed that the STAT5b interference plasmid (Pgenesil-1-ShSTAT5b) was constructed successfully. The scamble siRNA sequence was constructed with Pgenesil-1 vector labled EGFP as negative control plasmid (Pegenesil-1-HK). The transfection efficiency was determined by green fluorescence using laser fluorescence microscopy. There was strong green fluorescence in U251 cells transfected with Pegenesil-1-HK and Pgenesil-1-ShSTAT5b plasmids. otherwise, there was no fluorescence signal in U251 cells untransfected with plasmid. The silece effect was tested in mRNA level and protein level respectively. STAT5b expression levels were decreased in U251 cells transfected with Pgenesil-1-ShSTAT5b plasmids than cells transfected with Pegenesil-1-HK plasmids and blank vector plasmids. There were no differences of JAK1 expression in U251 cells transfected with Pgenesil-1-ShSTAT5b, Pegenesil-1-HK and vector plasmids either in mRNA level or protein level. The results showed that STAT5b could be specificly depleted by the small interference RNA (ShSTAT5b). Ferthermore. the expressions of VEGF, Survivin, Bcl- 2 in U251 Cells transfected with Pgenesil-1-ShSTAT5b plasmids were significantly decreased than cells transfected with Pegenesil-1-HK and blank vector plasmids. But the bax expression was increased in glioma cells silenced STAT5b expression. The proliferation ability of U251 cells silenced STAT5b were dramatically slower than the blank and negative control. The apoptotic U251 cells silenced STAT5b significantly increased than the blank and negative control. These results indicated that STAT5b was a hub in JAK1/STAT5 signaling pathways.Conclusion STAT5b was activated and abnormal expressed in glioma tissues and glioma cells. JAK1/STAT5 signaling pathway played an important role in the development of in the pathogenesis of glioma. STAT5b was a hub in JAK1/STAT5 signaling pathways. The expressions of VEGF, Survivin and Bcl-2 in U251 Cells depleting STAT5b were dramatically decreased than the blank and negative control. But the bax expression was increased in glioma cells silenced STAT5b. The apoptotic U251 cells were significantly increased when they were silenced STAT5b. This study may provide theoretical basis for glioma gene therapy.