Screening And Application Of The Signal Genes In Oral Leukoplakia

In order to screen the marker genes related to OLK, carcinogenesis of OLK and OSCC, laying foundation for the research in the genetic mechanism of oral carcinogenesis, differential gene expression between normal tissues, precancerous oral tissues and oral cancer tissues were examined by Oligo Cancer Microarray (SuperArray, U.S.A).Then we used conventional RT-PCR, Real Time PCR to Validate the positive genes in the results of genechips, searching for the related genes which participated in malignant transformation of OLK. According to the ratio of standard values between two kinds of tissues in gene expression, if the ratio>2 or <0.5, the gene was defined to be positive genes. From normal oral tissue to OLK, then OSCC, if the ratio of a certain gene always exceed 2, or lower than 0.5, then this gene was defined to be a marker for carcinogenesis of OLK .Total RNAs were isolated from 3 tissues, Chemiluminescent Detection Kit (SuperArray Bioscience, Catalog Number D-01), X-ray film and GEArray Expression Analysis Suite(supplied on Internet) were used to analyze the Genechips. In order to quantitate the expression level of the chosen genes in RT-PCR, Kodak Gel image analysis system was used. Lastly, we made a correlation analysis on the results of these two technologies. The gene expression level of ACP-2,BCL-2,SOCS-3,CLK-3,CTNNB-1,FKBP-8,GDF-15,NF-1,XRCC-1 genes has a satisfactory linear correlation with degree of tissue carcinogenesis,R2>0.8, suggesting they are driving genes in OLK occurrence and carcinogenesis. As a result these genes can be markers for the diagnosis of OLK, carcinogenesis of OLK and OSCC.The present study has the following contents: (1) Characteristic of genetic expression on tumor-related genes of OLK; (2) Screening of signal genes related to OLK carcinogenesis; (3) Application of signal genes in diagnosis of oral lesions.ChapterⅠ: Characteristic of genetic expression on tumor-related genes of OLKTo screen the marker genes related to OLK, carcinogenesis of OLK and OSCC,laying foundation for the research in the genetic mechanism of oral carcinogenesis. According to the ratio of standard values between two kinds of tissues in gene expression, if the ratio>2 or <0.5, the gene was defined to be positive genes. Results exhibit that there are 49 genes down-regulated and 157 genes up-regulated at the gene expression level, that is to say the total number of the positive genes is 206 in 440 tumor-related genes. These positive genes distributed in various ring-joints, suggesting they are driving genes in OLK occurrence and carcinogenesis. The malignant transformation of OLK and the occurrence of OSCC have close relations with cell cycle regulation genes and cell growth and differentiation genes. There are 53 genes up or down regulated by more than twice in the expression of cell cycle regulation genes, and 65 genes up or down regulated by more than twice in the expression of cell growth and differentiation genes.After quantization (Kodak Gel image analysis system),it was demonstrated that the RT-PCR results of the positive genes , CTNNB1, GDF15, FKBP8 and NF1 whose expression levels differed significantly between the precancerous tissues and the normal tissues (P<0.05).The genechips test confirmed these results(P<0.05);RT-PCR showed the levels CLK-3, CTNNB-1, GDF-15, FKBP-8, SOCS-3, NF-1, BCL-2, XRCC-1 and ACP-2 genes differed significantly between the normal tissues and the OSCC tissues (P<0.05).The genetic mechanisms of oral leukoplakia were very complicated, particularly associate with cell cycle regulation genes and cell growth and differentiation genes. This research formed the basis for further studies of the genetic mechanisms of oral leukoplakias and marker genes screening.ChapterⅡ: Screening of signal genes related to OLK carcinogenesisThe results showed to us, there were only 3 genes( ACP-2,BCL-2,SOCS-3) down regulated successively by more than twice,6 genes(CLK-3, CTNNB-1, FKBP-8, GDF-15, NF-1 and XRCC-1) up regulated successively by more than twice. The gene expression level of these 9 genes has a satisfactory linear correlation with degree of tissue carcinogenesis, R2>0.8, suggesting they are driving genes in OLK occurrence and carcinogenesis. As a result these genes can be markers for the diagnosis of OLK, carcinogenesis of OLK and OSCC.The genetic mechanisms of OLK malignant transformation were very complicated, particularly associate with signal transduction, DNA damage and repair, oncogene and anti-oncogene et al. This research formed the basis for further studies of the genetic mechanisms of OLK malignant transformation and marker genes screening.ChapterⅢ: Application of signal genes in diagnosis of oral lesionsTo date, the main diagnosis method is still histopathology, which is invasive toward the patients, and has the character of inaccurate in diagnosising. In order to make use of the signal genes in gene diagnosis of OLK, carcinogenesis of OLK, and OSCC, we carry out RT-PCR with CLK-3, CTNNB-1, GDF-15, FKBP-8, SOCS-3, NF-1, BCL-2, XRCC-1 and ACP-2 genes. In order to quantitate the expression level of the chosen genes in RT-PCR, Kodak Gel image analysis system was used.Two kinds of Fisher discrimination methods were used to construct several discrimination formulas. The discrimination formula can used to distinguish OLK is: Y=-27.503 +0.094XGDF-15 -0.122XNF-1+0.368XSOCS-3, the distinguishing point-value Yc=-1.186.That is to say, if the value of a certain oral tissue exceed -1.186,then you can determine it OLK, if the value is lower than -1.186, it's not OLK. Cross-validated (a) method was used to validate the above-mentioned method. The total discrimination coincidence rate and interactive discrimination coincidence rate are 100%, and the sensitivity and specificity are 100%. The discrimination formula can used to distinguish OLK is: Y1=-16.811+0.477XNF-1+0.540XACP-2-0.543XCTNNB-1-0.089XSOCS-3;Y2=-35.832+0.073XNF-1-0.074XACP-2+0.306XCTNNB-1+0.191XSOCS-3, the distinguishing point-value Yc1=8.6513,Yc2=0.1117. If the value of a certain oral tissue exceed 8.6513, then you can determine it haven't taken carcinomatous change, if the value is between 0.1117 and 8.6513, it's a mild cancerous tissue and if the value is lower than 0.1117, it's a cancerous tissue. Cross-validated (a) method was used to validate the above-mentioned method. The total discrimination coincidence rate and interactive discrimination coincidence rate are 100%, and the sensitivity and specificity are 100%. The discrimination formula can used to distinguish OSCC is: Y=-16.099+0.044XGDF-15-0.082XBCL-2+0.234XCTNNB-1, the distinguishing point-value Yc=0. That is to say, if the value of a certain oral tissue exceeds 0, then you can determine it not OSCC, if the value is lower than 0, it's OSCC. Cross-validated(a) method was used to Validate the above-mentioned method, the total discrimination coincidence rate is 100%, the interactive discrimination coincidence rate is 91.67%, the sensitivity is 85.71% and the specificity is 100%.The present study formed a convenient and simple method that can be used to gene diagnosis of OLK, carcinogenesis of OLK, and OSCC.