| Glimoas are the most common primary brain tumors with significant morbidity and mortality in central nervous system. Surgery operation is the primary therapy to glioma. Because of its infiltrating growth characterization without boundary between the normal brain and glioma, it is diffcult to be eradicated by surgery operation.More than seventy-five percent of glioma patiets will die one year after the surgery operation because of the recurrence of glioma. As one of the major therapeutic methods of the malignant tumor, chemotherapy is the important supplement to treat glioma and plays an irreplaceable role. Because of the considerable side-effect on the central nervous system even whole body and existence of the blood brain barrier, chemotherapeutical medicines that can be used in clinic are too few.It has a significance to seek for a kind of drug that has high effect and low side-effect.Hepatoma-derived growth factor (HDGF) is a heparin-binding growth factor originally purified from media conditioned with the human hepatoma cell line HuH-7 and can stimulate proliferation of Swiss 3T3 cells . Its precise function is unclear, but HDGF is known to be highly expressed during the early development of many tissues, including cardiovascular , kidney , and liver . In our early stage of study, we found that a high level of HDGF expression in tumors correlates with a high level of glioma. However, the mechanisms of the HDGF associated aggressive biological behavior are unknown. In this study, we knocked down HDGF expression in U87 cells to determine the biological consequences. Transfection with HDGF-specific small interfering RNA (siRNA) resulted in down-regulation of HDGF expression in U87 cell lines.AIM: (1) To confirm the relationship between glioma tumor and HDGF.(2) Design siRNA to knock down the expression of HDGF in a glioma cell line with high HDGF expression.(3) Use mouse model in vivo to confirm the effect of HDGF in tumor growth and choose DNA chip to study the correlated gene changes of the HDGF associated aggressive biological behavior.METHODS:(1) Use immunohistochemistry to test the expression of HDGF in glioma tumor .(2) Use RT-PCR and Western-Blot to choose one cell line with high HDGF expression from five glioma cell lines , then use siRNA to knock down HDGF expression in U87 cells.(3) Construct mouse model in vivo with U87 cells with or without HDGF-siRNA transfection. And compare the speed of tumor growth and the tumor volume at the same time spot between two mice groups treated with different U87 cells.(4) Perform global gene expression analysis using Agilent chip to elucidate potential mechanism of HDGF-siRNA-induced growth inhibition in vivo model. And compare the gene expression profiles between tumor tissues with wild U87 cells and those with U87 cells treated with HDGF-siRNA.RESULTS:(1) We find that the high expression level of HDGF is correlated with the high grade of glioma tumors.(2) The expression of HDGF in U87 cell line is the highest in five glioma cell lines(U87,U118,DBTRG,SW1783,U373).(3) RT-PCR and Western-blot both confirmed that the siRNA-1 can knock down the expression of HDGF in U87 cell line effectively.(4) In the vivo model,we can see visable differnence between siRNA-1 treated group and wild group from both tumor volume and tumor weight in the early stage,the specific statistics is : the first group which was killed 15 day after inoculation,the average tumor volume of siRNA-1 treated group is 100.840±48.027 mm~3,wild group is222.912±85.614mm~3,P is 0.030.the average tumor weight is 0.062±0.050g compared to 0.130±0.036g,P is 0.040.The second group was killed 20 days after inoculation , the average tumor volume of siRNA-1 treated group is 139.104±39.082 mm~3,wild group is280.628±74.224 mm~3,P is 0.008.the average tumor weight is 0.113±0.024g compared to0.194±0.053g,P is 0.020.The third group was killed 28 days after inoculation, the average tumor volume of siRNA-1 treated group is 454.231±297.767 mm~3,wild group is 608.361±224.303 mm~3,P is 0.384.the average tumor weight is 0.325±0.170g compared to 0.473±0.057g,P is 0.126.(5) We can concluded from the gene chip statistics that after the knock down of HDGF,a lot of gene got changed in vivo tumor .For example,HGF was impressed obviously.CONCLUSION:(1) The siRNA-1 can knock down the expression of HDGF effectively.(2) Knocking down HDGF by SiRNA can obviously inhibit the early tumor growth of U87 cells.(3) It can be concluded from the DNA-chip result that HDGF might regulate the biological behavior of glioma cells through HGF and NF-kB pathway. |
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