| Backgrounds Glioma is the most common intracranial tumor, with a high degree of malignancy, a powerful capacity for invasiveness and a poor prognosis. Its high rate of postoperative recurrence makes it one of the most refractory tumors. To find out the pathogenesis of glioma and seeking for new therapeutic targets are basic problems to be solved. Neuropilin-1which belongs to the family of auxiliary non-tyrosine kinase receptors is a single-pass transmembrane glycoprotein, and mainly expressed in vascular endothelial cells, tumour cells and neurons. It has been found that NRP-1was overexpressed in many kinds of human tumors, playing an important role in the malignant progress of these tumors. According to previous researches, analysis of human glioma specimens of different malignancy degrees, discovered that glioma showed a high NRP-1protein level, and the content of NRP-1protein was associated closely with the malignancy degree and patients’prognosis. Currently, the patho mechanisms of NRP-1protein remains unclear, which has interfered seriously the advance of glioma treatment. Mitogen activated protein kinase (MAPK) pathway is a typical cellular signal pathway. Apart from regulating cell proliferation, differentiation, survival, transformation and apoptosis, activiated MAPK path also promotes the invasiveness and metastasis in glioma. Whether the impact exerted on glioma by NRP-1is related with MAPK pathway remains to be determined.Part one The expression of NRP-1in different glioma cell lines and the design, synthesis and screening of efficient targeting sequences of NRP-1siRNAObjectiveTo observe the expression of NRP-1in different glioblastoma cell lines, and then to design and select effective siRNA targeting sequences of NRP-1gene for subsequent research. Methods1. Western blot was utilized to detect the expression of NRP-1in malignant glioma cell line LN319,U373MG, U87MG, U118MG and U251MG.2. U373MG showed a high expression of NRP-1and was chosed as the glioma cell line in this research. Two siRNA(NRP-1siRNA1and NRP-1siRNA2) of the targeting human NRP-1mRNA were designed and synthesized by Ambion company. There were four groups:blank control group, negative control group (control siRNA), NRP-1siRNA1group and NRP-1siRNA2group. Using Lipofectamine2000as transfection reagent, U373MG cells were transfected respectively with NRP-1siRNA1. NRP-1siRNA2and control siRNA. Then RT-PCR (reverse transcription-polymerase chain reaction) and Western blot are adopted to detect the difference of the expression of NRP-1mRNA and NRP-1protein between these groups.Results1. NRP-1is positively expressed in malignant glioma cell lines of U373MG, U87MG, LN319and U118MG. LN319and U373MG cell lines show a relatively higher expression of NRP-1protein. However. NRP-1was not significantly expressed in U251MG.2. The expression of NRP-1mRNA was suppressed by approximately50%and85%, in NRP-1siRNAl and NRP-1siRNA2group respectively, compared to the blank control group. Also, The expression of NRP-1protein was suppressed by approximately47%and77%, in NRP-1siRNA1and NRP-1siRNA2group respectively, compared to the blank control group.ConclusionNRP-1is highly expressed in LN319and U373MG cell lines. Because LN319isnot glioblastoma multiforme cell line, so we choose U373MG cell line to carry out our experiments. Both targeting sequences of NRP-1siRNA1and NRP-1siRNA2can obviously inhibit the expression of NRP-1in U373MG, but NRP-1siRNA2shows a more powerful inhibition effect. So, siRNA2was chosed as the NRP-1gene silencing tool in the following experiments. Part two The effects of NRP-1gene silence by siRNA interference on U373MG glioma cell growthObjectiveTo observe the effects of siRNA interference targeting NRP-1gene expression on the cell proliferation, apoptosis and cell cycle of U373MG cell line. MethodsAfter transfecting U373cell line with NRP-1gene targeted siRNA, we examined the cell proliferation using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay, detected the cell cycle using FACScan flow cytometer, and analyzed cell apoptosis after staining the cells with Annexin V-FITC and propidium iodide.Results1. After transfection for48hours and72hours, the cell proliferation rate of NRP-1siRNA2group decreased by32%(P<0.05) and43%(P<0.01) respectively, compared to that of blank control group.2. Compared to blank control group, the apoptotic cells number of NRP-1siRNA2group increased nearly two times (P<0.01)3. Compared to blank control group, the phase G1cell number of NRP-1siRNA2group increased obviousely (P<0.01), however, the phase S cell number decreased significantly (P<0.01)ConclusionAfter NRP-1gene was silenced by siRNA, U373cell proliferation was markedly inhibited, cell apoptosis was dramatically accelerated and cell mitosis was delayed. The impeded effects of NRP-1siRNA on U373MG glioma cell proliferation may be associated with its induction of a cell cycle arrest at G1phase, which delayed the cell cycle progress to mitosis. Part three The effects of NRP-1gene silence by siRNA interference on MAPK signal pathways in U373MG glioma cellObjectiveTo observe the impact of siRNA interference targeting NRP-1gene expression on MAPK signal pathways in U373MG glioma cell aiming at studying wether the MAPK signal pathways was related to the pathomechanisms of NRP-1protein in gliomas.Methods1. After transfected U373MG cells with NRP-1siRNA or control siRNA48hours, we used Western blot method to examine the expressions of pro-apoptotic proteins BAD and antiapoptotic protein BCL-2, which proteins both belonged to BCL-2family.2. After transfected U373MG cells with NRP-1siRNA or control siRNA48hours, we used Western blot method to examine the expression levels of p-ERK1/2, ERK1/2, p-JNK and JNK proteins.which proteins all belonged to MAPK pathway.Results1. Compared to blank control group, the expression of BCL-2and p-BAD-Serl12of NRP-1siRNA2group decreased by approximately55%and45%respectively, however, the level of total BAD protein has no obvious changes.2. Compared to blank control group, the expression of p-ERK1/2and p-JNK of NRP-1siRNA2group decreased by approximately87%and72%respectively, yet the level of total ERK1/2and JNK protein have no marked differences.Conclusion1. The inactivation of ERK, JNK/MAPK signal pathways might be one of the molecular mechnisms that NRP-1siRNA affects U373MG cells growth. The MAPK signal pathways’inactivation might be due to that NRP-1siRNA blocks the phosphorylation of ERK1/2and JNK protein, but not the process of signal proteins expression.2. After transfected with NRP-1siRNA2, the expression of BCL-2family proteins were changed in U373MG cells, resulting in its pro-apoptotic effects increased. This change may be associated with the inactivation of JNK pathway, followed by the decreased activation of Bcl-2and BAD protein. |
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