| Objective: To evaluate the relationship between hepatoma derived growth factor (HDGF) and glioma. siRNA targeting human HDGF gene was designed and transiently transfected into glioma cells expressing the high level of HDGF by lipofectamine. The effect and mechanism of HDGF-siRNA on the proliferation, migration, invasion and non-anchor dependent growth in glioma cells was studied primarily.Methods:1. The expression of HDGF in glioma tissues of different pathologic grading was detected by Imunohistochmistry(IHC).2. The protein and mRNA level of HDGF in five glioma cell lines was detected by Western blot and RT-PCR respectively. IHC in cell chip was used to screen the one with the highest expression level of HDGF from the five glioma cell lines for the further research.3. HDGF specific siRNA was synthesized and transiently transfected into U87 cells by Lipofectamine 2000. The HDGF expression was detected by RT-PCR and Western-blot to compare the its expression level before and after transfection. The optimal time was chosen,when the effect of RNA interference was best. Negative control(cells were treated by Lipofectamine only without siRNA) and blank control(cells were untreated ) were set up in the experiment. 4. To study the effect of HDGF-siRNA on U87 cell proliferation, migration and invasion, MTS proliferation assay, wound healing assay, migration assay, invasion assay and soft agar clony formation assay was performed.5. To investigate the mechanism of HDGF on glioma U87 cells, gene expression profile was analyzed by microarray, and some results of microarray were further confirmed by RT-PCR and Western blot after HDGF down-regulation in U87.Results:1. The IHC results of glioma tissue showed that HDGF expressed differently in glioma tissues with different pathologic grading. Compared with its expression in grade one and two, HDGF expression in high grade glioma showed stronger staining in nucleus and more positive cell numbers.2. The results of WB, RT-PCR and cell chip IHC showed that HDGF protein was expressed at the highest level in U87 cells, at the moderate level in U118, U373 and SW1873, while expressed at the lowest level in DBTRG cells among all five cell lines.3. HDGF gene expression was obviously down-regulated after transient transfection of siRNA targeting HDGF into U87 cells, especially during 48 to 72 hours .after transfection.4. Compared with the blank and the negative control groups, the proliferation potent of RNAi group cells was greatly attenuated in vitro (p<0.01), especially in serum free condition. The proliferation inhibition ratio is 18% in serum free condition, 9% in the condition with 10% FBS and 28% in serum free matrigel pre-treated group.5. Compared with the blank and the negative control groups. The colony number in soft agar assay was 7±1.4 in RNAi group(p<0.01), 42.5±3.5 in negative control group, and 45±5.6 in blank group.The migrated cell numbers after 6 hours were 23.4±2.9 in RNAi group(p<0.01), 65.8±3.0 in blank group,and 59.4±7.4 in negative control group. The invasion numbers was 6.4±1.1 in RNAi group, 22.6±2.8 in blank group and 16.6±1.8 in negative control group(p<0.01).6. Compared with those in the blank and the negative control groups, not only the migration distance but also the migrated cell numbers were less in RNAi group in the wound experiment.7. The results of microarray showed that HGF had markedly down-regulated after RNAi in the nude mice xenograft, which was confirmed by RT-PCR and Western-blot.Conclusion:1. HDGF was highly expressed in glioma , with a positive correlation with pathologic grading.2. HDGF expression was variable among five types of glioma cell lines with U87 having the highest expressing level.3. HDGF-siRNA we designed and synthesized chemically can specifically down-regulate the expression of HDGF.4. U87 cell proliferation, migration, invasion, non-anchor dependent growth were obviously inhibited after RNA interference.5. The result of microarray was further confirmed by Western-blot, indicating HDGF regulate glioma biological behavior through HGF signal pathway.
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