Effect Of Silencing CD40 Gene Expression By RNAi On Characteristics Of Brain Glioma Cells U87MG

CD40, a transmembrane glycoprotein I, is one member of tumor necrosis factor receptor (TNFR) super family. CD40 is expressed on dendritic cells, but also on B cells, macrophages, hematopoietic cells, epithelial cells, vascular endothelial cells as well as carcinoma cells.Increasing evidence suggests that CD40 fosters tumor growth, progression and tumor angiogenesis. CD40 not only participates in the process of tumor onset, but also promotes tumor growth and metastasis. Studies have shown that blocking CD40 signals can inhibit tumor growth and early onset, and inhibit tumor angiogenesis and metastasis. Many studies have shown that the expression of CD40 is closely related to glioma. The high expression of CD40 has a relationship with glioma neovascularization, suggesting that CD40 enhances the glioma angiogenesis and tumor progression.Objective:Glioma is the most frequent primary brain tumor and is life-threatening, invading surrounding normal brain tissue, and usually growing rapidly. The complete surgical resection is very difficult, and the glioma is so easy to relapse after surgery. Moreover, the radiotherapy and chemotherapy are lack of treatment specificity, which may cause great damage to the normal brain function and overall immune system of patients. And thus, current research focuses on inhibiting the invasive growth of glioma and finding the best effective treatment. While increasing evidence showed that the expression of CD40 had a very close relationship with glioma angiogenesis, invasive growth and other biological characteristics. Therefore, our study investigated the proliferation and migration of glioma cells U87MG through gene silencing of CD40, which provided a theoretical basis for the clinical treatment of gliomas.Methods: ①According to genes Blast and references, we designed a length of 19 bp specific oligonucleotide sequence:5'GCG AA UUC CUAGACACC UG-3'.We have systhesised siRNA oligonucleotide chain with hairpin structure ends with a pair, then systhesised its complementary strand. At 5'and 3'end of this template, HindⅢand BamHⅠrestriction sites were introduced. CD40 plasmid pSilencer3.1-H1 neo was constructed, then enzyme digestion and gene sequencing.②RT-PCR and FCM were used to detect the CD40mRNA levels and protein expressions before and after transfected by pSilencer3.1-CD40 siRNA in U87MG cells.③To observe the proliferation rates, U87MG cells in three groups were placed in 37℃,5% CO2 incubator, with 10% FBS and 2% FBS in DMEM, respectively. After 24h, 28h,72h, the number of the cells were counted and the growth curves were described.④24 hours after adding sCD40L, cell migration of U87MG, U87MG-siRNA-control and U87MG -siRNA-CD40 cell groups was detected with transwell by counting the number of cells below upper booth.⑤U87MG cell lines were cultivation expandly, pretreated with sCD40L, adjusted to a concentration of 1×106/50μl, then injected into the armpit of nude mice at 50ul. The left armpit of nude mice was control group, the right was interference group. After inoculation of tumor, the time of tumor growth, tumor size, tumor vascular hyperplasia of the nude mice were recorded.Conclusions:①The mRNA expression of CD40 in 87MG-siRNA-control group was no significant lower compared with parental U87MG cells. However, U87MG-siRNA-CD40 group was significantly decreased.②Compared with parental U87MG cells group, there were no difference between U87MG-siRNA-control cells group and U87MG-siRNA-CD40 cell group in proliferation rate. When the concentration of FBS was lowered to 2% FBS, adding 50ng/ml sCD40L, the tumor proliferation rate in U87MG-siRNA-CD40 group was significantly lower than the parental U87MG cells group (P<0.01), but there was no difference between U87MG-siRNA-control group and the parental U87MG cells group.③The number of cells were (70±8)/HP, (74±9)/HP, (36±5)/HP in U87MG group, U87MG-siRNA-control cell group, U87MG-siRNA-CD40 cell group respectively.④7-10 days after inoculation, the time of tumor formation in control group was earlier than interference group. Moreover, the volume and vascular hyperplasia of tumor were reduced in interference group.Summary:Decreasing the expression of CD40 in brain glioma cells U87MG by RNAi could effectively inhibit the growth of glioma invasion and metastasis, which provided a new theoretical basis for the clinical treatment of glioma.