Exploration On The Pathogenesis And Metabasis Related Genes Of Osteosarcoma With SBC Gene Microarray

Background:Osteosarcoma is a malignant bone tumor of the most common,its distinguishing feature is the high incidence of youth,high degree of malignancy,and the high rate of early metastasis and poor prognosis.Therefore to seek occurrence,development and transfer mechanisms for the effective treatment of osteosarcoma of the original lesion,prevention, early detection and effective treatment for pulmonary metastasis is essential for improving the prognosis of patients with sarcoma.At present study,The occurrence of tumor is related to tumor oncogene activation,anti-cancer gene inactivation and out control of the expression of other genes,which is a multi-stage and multi-channel synergy results.Gene chip is known as DNA chips or DNA microarray.The technology has the outstanding advantages of high efficiency and high-information as a result of the micro-parallel processing and high-density integrated concept.In this study,we use osteosarcoma cell line as a model to study the important significance of gene expression level in the incidence and the evolution of osteosarcoma.In this experiment,osteosarcoma cell lines(MG-63, U-2OS) and osteoblast cell lines hFOB1.19 are our research objects.The application of SBC were 16 K cDNA microarray gene expression profiles(including known genes 12,581),through repeated,the difference in the expression of the known genes are identified in osteosarcoma cells and normal osteoblasts and the removal of osteosarcoma fresh specimens and two osteosarcoma cell line are targeted to verify the significantly different expression genes in the mRNA level.Objective:1,screen the pathogenesis-related and metastasis-associated gene cluster Of osteosarcoma in the genomic level.2,explore initially the incidence and transfer mechanisms,Provide target genes for osteosarcoma genetic diagnosis,gene therapy.3,lay the initial foundation for further knockout and transfection of pathogenesis-related and metastasis-associated genes for research.Methods:1,cell culture and osteosarcoma samples collected:two osteosarcoma cell lines (MG-63,U-2OS) and an osteoblast cell lines(hFOB1.19) purchased from the Chinese Academy of Sciences Shanghai cells.All chips for cell culture experiments on cells in the logarithmic growth phase.Experimental use of tissue osteosarcoma from June 2007 to August of the Second Military Medical University,Changhai Hospital orthopedic surgical resction speciment.The speciments are implanted and cryopreserved in liquid nitrogen tank standby Immediately after resecting.2,the experimental cells's total RNA extraction and probe synthesis:using TRIzol total RNA extraction,three experimental cells's total RNA are extracted and the total RNA are Purified by QIAGEN's RNAeasy mini kit(refer to the QIAGEN's instruction manual). The concentration and quality of the total RNA are determined by the spectrophotometer and agarose gel electrophoresis.Double-stranded cDNA synthesis,synthesis of fluorescent-labeled cRNA and cRNA fragmentation of the process,strictly Comply with SBC chip Expression Analysis of experimental methods.3,chip hybridization:Preparation will be good in front of the biotin-labeled cRNA expression profile in the four SBC chip hybridization overnigh(the chip to do a repeat,a total of four).Washing,dyeing process are in accordance with the standard expression of the SBC chip experiment guide following chip hybridization.4,data analysis:we obtain images with the Aligent Scanner in the chips,get the different value multiples through transformating from Imagene into numerical applying Genespring numerical analysis software standardization.We filter out the genes whose average multiples of the different expression(chip repeat) is=2.0 or=0.5 times between the two osteosarcoma cell lines and osteoblast cell lines,then get the different expression of common.The analysis of differentially expressed genes are recoursed to GenBank database, Genespring and so on.5,real-time quantitative RT-PCR test:the design of primers,Sign GENEBANK site to design the upstream and downstream primers with the four differentially expressed genes as a template.Chimeric fluorescence(SYBR~R Greenâ… ) real-time RT-PCR:Using ABI SYBR~R Green fluorescence quantitative PCR(see specific reaction system of ABI SYBR~R Green RT-PCR).Results:1,the different gene expression of osteosarcoma cell lines and osteoblast cell lines:to express difference=2.0 or=0.5 times among the people's SBC 16 K CDNA microarray gene expression profiles,which containing about 12,581 known genes,the two osteosarcoma cell lines and hFOB1.19 osteoblast cell lines were differentially expressed as follows:MG-63 up 842 genes,down 1255 genes;U-2OS up 1,040 genes,down 1,430 genes.Taken with the osteoblast cell lines there were commonly 131 genes increased and 63 genes declined.In the 131 up-regulated genes in common,we selected MUC1(an average of 5.513 and 3.297 times increased) and ASS gene(average increase of 16.18,3.234 times) in the tissues and cell lines to express their quantitative PCR test. Among the 63 downturn common genes,we chosen the IL-1B(an average of 0.019 and 0.01 times lower),GDF15(an average of 0.0232 and 0.0473 times lower) gene in tissue specimens and cell lines to express their quantitative PCR test.2,differentially expressed genes in real-time quantitative PCR test:we used real-time quantitative PCR method to verify the different expression of the four genes in the four clinical osteosarcoma samples and the two osteosarcoma cell lines compared with osteoblast cell lines.In the mRNA level compared with osteoblast cell hFOB 1.19,the original osteosarcoma tissues and osteosarcoma cell lines showed the same results with SBC expression differences.This further validates the reliability of chips.Conclusion:The incidence of osteosarcoma are highly related to oncogenes,tumor suppressor genes,tumor-associated genes,and other genes.Some genes,which had been determined related to the incidence of other sources of tumor,such as TPD5,CLU,LSP1,MUC1, CXCL6,SERPINB2 and so on,were proved to be closely related to the pathogenesis and metabasisis of osteosarcoma.To study the function of differentially expressed genes helps to uncover the the incidence and transfer mechanism of osteosarcoma.Gene chip technology can be successfully Screening tumor related genes,which is a powerful tool to explore tumor incidence and transfer mechanisms.