Preliminary Study On The Bone Metastasis Genes Of Nasopharyngeal Carcinoma By Tumor Cells Co-cultured With Tissue Model

As a common complication of malignant tumor at advanced stage,incidence rate of bone metastasis is 15%-20%in whole body,the third after lung metastasis and liver metastasis.In some malignant tumor,bone metastasis's incidence is far more higher than other tumor,such as multiple myeloma(95%-100%),breast caner and prostatic carcinoma(65%-75%),lung carcinoma.Clinically,bone metastasis often lead to severe pain regionly,pathologic fracture,hyper calcinemia,spinal compression, and have a serious influence on animation of the patients and could not be cured usually.The mechanism of bone metastasis often has a relation with the characteristics of the tumor cells,the interaction between the tumor cells and the microenvironment of the bone tissue.In the process of the bone metastasis,because of the interaction between the microenvironmet and the tumor cells,the tumor cells have a specified genotype,this kind signature contributes to the diagnose,evaluation of the therapeutic effect and the therapeutics of the malignant tumor's bone metastasis.According to the research of the multiple myeloma,breast caner,prostatic carcinoma and lung carcinoma and the analysis of the MILANO software,we select CXCR4,CTGF,MMP-1,IL-11,MST1R,ETV-1,Cox2,OPN,ITGB3(CD61),EZH2,Tiaml,PTHrP,SALP,PLAU,CD44,OPG,Pleiotrophin,ADAMTS1,Erizin,TGF-βas the gene of nasopharyngeal carcinoma related with bone metastasis, they can improve bone metastasis of malignant tumor by chemistrical chemistry, strengthening the adhesiveness between tumor cells and vascular endothelial cells, degradation of regional stroma,promoting angiogenesis.Nasopharyngeal carcinoma is a malignant epithelial tumor which is common in south China.The incidence of NPC can reach 14.68-18.53 per 100 thousand people, and the mortality rate is 12.46 per 100 thousand people in some area where nasopharyngeal carcinoma is high incidence.The most common location of the NPC's distant metastasis is bone,and the meso-life span of the patients is 5-9months.The severe pain regionly,pathologic fracture,hypercalcinemia,spinal compression due to bone metastasis have a serious influence on animation of the patients and could not be cured by the routine chemotherapy,radiotherapy and operation,thus to study the mechanism of the NPC's bone metastasis profoundly can reduce the bone metastasis and play a significant role in the diagnose of bone metastasis and the method of control the bone ache.Quantitative Real time PCR(QRT-PCR) is a new technique which developed in recent years,it has proven to be a powerful method to detect the gene expression level Here we use SYBR GreenⅠas fluorescent dye.SYBR GreenⅠis a dye that binds the minor groove of double strand DNA.As more double strand amplicons are produced,SYBR GreenⅠdye signal will increase.Therefore,fluorescence signal intensity of the SYBR GreenⅠis related to the quantity of double strand DNA.So based on the intensity of fluorescence signal we can know the quantity of double strand DNA in the PCR system.And through use the analysis of dissociation curve, we could exclude the interference of nonspecific augmentation of the double strand DNA such as double body of primer.In quantitative real time PCR,the target gene can be relatively quantitatively analyzed,whose accuracy and sensitivity is higher than the conventional RT-PCR.Compared with using Taqman probe,SYBR GreenⅠtechnique has more widely application,and is also cheaper.Through 2^{-ΔΔCT} method, the expression difference of relative gene can be well known.To analyze the relative quantity of gene expression using the 2^{-ΔΔCT} method is reported in many papers,and because this method does not need standard curve,it is more convenient and feasible than absolute quantity method.At present,the reseach of NPC's bone metastasis is not common,and the establishment of the animal model of NPC's bone metastasis is hard to achieve,so we use the model of the tumor cells co-cultured with tissue to simulate the the microenvironment of the tumor,tissue releases secrete factors to culture,then those factors can play an immediate action on tumor cells.And tumor cells also release some facors that can effect tissue directly,the interaction between tissue and tumor cells can improve tumor metastasis.So,firstly,we compare the expression of the target genes in NPC cell line 5-8F before and after co-cultured with bone tissue by QRT-PCR and we can select the genes that relate with the bone metastasis of 5-8F; secondly,we compare the expression of the genes that we have selected in 5-8F before and after co-cultured with other tissue(liver,lung,brain) to determine the tissue specificity;thirdly,we use fluorescence activated cell sorter(FACS),Western Blot and other NPC cell line to validate the genes selected after two step above;all of these are to aproach the mechanism of NPC's bone metastasis and to provide potential target of the treatment of NPC's bone metastasis. We apply 2^{-ΔΔCT}method and t test to analyse the result of the QRT-PCR,the data show that the expressions of CXCR4,CTGF,MST1R,ETV-1,COX2 in the 5-8F after co-cultured with bone tissue are higher than before co-culture with significance(P＜0.05);the expression of the TGF-βwas lower(P＜0.05);the expressions of MMP-1,IL-11,OPN,ITGB3,EZH2,Tiaml,PTHrP,SALP,PLAU,CD44,OPG,Pleiotrophin,ADAMTS1,Erizin have no significance (P＞0.05).So,we can conclude that CXCR4,CTGF,MST1R,ETV-land COX2 may promote the bone metastasis of 5-8F.To determine the tissue specificity of the expression of CXCR4,CTGF,MST1R,ETV-1and COX2,we compare the expression of these five genes in 5-8F before and after co-cultured with other tissue(liver,lung,brain).The data shows that:1) CXCR4:higher in 5-8F co-cultured with bone tissue,lower in 5-8F co-cultured with liver tissue,no significant diffrencein 5-8F co-cultured with lung tissue,lower in 5-8F co-cultured with brain tissue.2)CTGF:higher in 5-8F co-cultured with bone tissue,higher in 5-8F co-cultured with liver tissue,higher in 5-8F co-cultured with lung tissue,higher in 5-8F co-cultured with brain tissue.3)MST1R:higher in 5-8F co-cultured with bone tissue,no significant diffrence in 5-8F co-cultured with liver tissue,lower in 5-8F co-cultured with lung tissue,lower in 5-8F co-cultured with lung tissue.4)ETV-1:higher in 5-8F co-cultured with bone tissue,no significant diffrence in 5-8F co-cultured with liver tissue,higher in 5-8F co-cultured with lung tissue,higher in 5-8F co-cultured with brain tissue. 5)COX2:higher in 5-8F co-cultured with bone tissue,no significant diffrence in 5-8F co-cultured with liver tissue,no significant diffrence in 5-8F co-cultured with lung tissue,lower in 5-8F co-cultured with brain tissue.So the expressions of CXCR4,MST1R,COX2 have the specificity of bone tissue,CTGF may be a general factor of NP C's metastasis,CXCR4,MST1R,COX2 can improve the bone metastasis of NPC patient.Then we use fluorescence activated cell sorter(FACS),Western Blot and other NPC cell lines to validate CXCR4,MST1R and COX2 selected above;all of these are to aproach the mechanism of NPC's bone metastasis and to provide potential target of the treatment of NPC's bone metastasis,but we need more clinical validation.