The Relationship Between The Loss Of Imprinting IGF2 And Lymph Node Metastasis In Human Colorectal Cancer

ObjectiveThis experiment studied the changes of IGF2 gene of imprinting ststus in colorectal cancer,analysised the relation of the imprinted gene IGF2 and colorectal cancer,explored IGF2 at the occurrence and development of colorectal cancer in imprint control mechanism and its relationship with the biological behavior of colorectal cancer,with reverse transcription-polymerase chain reaction-restriction fragment length polymorphism(RT-PCR-RFLP) method.Methods1.Experimental meterials:Experimental specimens:Resected cancerous,and non-cancerous colon tissue of 56 primary colorectal cancer patients were collected.DNA and RNA were extracted from tissue specimens.2.Testing method2.1 Organization of DNA/RNA extraction and identification:Take about 100mg frozen tissue samples,the conventional use of 10%SDS/chloroform/ethanol extraction of DNA.UV Spectrophotometric Determination of A260/A280,DNA samples as the best ratio of 1.8-2.0.TRIzol reagent/chloroform/isopropanol/ethanol extraction of RNA.2.2.Screening heterozygote specimen:Only when the state of specimens is heterozygous genotype,can distinguish between two alleles of the expression product to provide information. IGF2-F,5'-CTTGGACTTTGAGTCAAATTGG-3',IGF2-R, 5'-GGTCGTGCCAATTACATTTCA-3',annealing temperature 54℃.The expected size of the PCR fragment of the IGF2 gene is 292 bp,primer were synthesized by Shanghai Sangon.The PCR reaction was conducted in 10xPCR buffer with 2μl,50mM MgCl2 1μl,10mM dNTP 0.4μl,10μM primer 1.6μl,ddH20 14μl,TaqE 0.2μl,DNA 20μl。Conditions for amplification were 94℃for 2 min followed by 30 cycles at 94℃for 30 sec,54℃for 30 sec,and 72℃for 1 min.A final step was 72℃for 5 min.Taked the PCR product 8μl agarose gel electrophoresis,ApaⅠenzyme cuts 37℃the water bath overnight,polyacrylamide gel electrophoresis,genefinder nucleic acid dyes for heterozygote screening.Because of the 36bp fragments smaller and can not clearly show,therefore as enzyme cuts judgment standard take 256 bp.Heterozygous case exhibt two bands of 292 and 256bp(AB type),homazygous case exhibt band of 292bp(A type) or band of 256bp(B type).2.3.Gene imprinting status detected:Chose heterozygous specimens,cut their RT-PCR product with the same restriction endonuclease,after that 12%non-denaturing polyacrylamide gel electrophoresis.If the IGF2 gene expression showed biallelic expression of type(AB type) is 292bp and 256bp simultaneously exist that have taken place in the loss of imprinting.If monoallelic expression showed that only 292bp(A type) or only 256bp (B type),indicating that transcription from one allele,namely,the maintenance of normal imprinting status.2.4.Statistical analysis:Using Fisher exact test analysis of loss of imprinting of IGF2 and the clinical pathology material relevance.All data were used SPSS 16.0 software for analysis.Results1.PCR product DetectedIn our experiment,we confirm that the IGF2 gene is 292bp,through after PCR response fluid agarose gel electrophoresis the image analysis, by succeeds the synthetic product.2.Heterozygous specimen screeningOf the 56 patients enrolled for analysis,29 were heterozygous and thus informative for IGF2,and the positive rate of IGF2 of colorectal cancer were 51.79%(29/56)and 1 case is loss of heterozygosity.Imprinting status detected:Heterozygous normal tissue specimens,reverse transcription of the cDNA,by PCR and then digested by ApaⅠafter polyacrylamide gel electrophoresis separation,In 28 cases no loss of heterozygosity heterozygote,17cases of tumor organization have had LOI.17 cases of tumor in this organization corresponds to adjacent tissues,14 cases(82.35%) have had LOI.Tumor tissue and adjacent tissues there is no loss of imprinting Example 7(25%).Relationship between the LOI of IGF2 and clinical pathological characteristics:28 informative IGF2 tumout samples,11cases of tumor organization have had LOI in 13 patients with lymph node metastasis and 6case have had LOI in 15 patients without lymph node metastasis.ConclusionLOI of IGF2 was observed in tumor samples and in nontumorus normal mucosa, the prevalence of LOI in nontumorus normal mucosa was significantly higher in cases with LOI-positive cancer than in those with LOI-negative cancer(p＜0.05).IGF2 gene and clinicopathological features(age,sex,location) without statistical study related to each other and with lymph node metastasis positive correlation exit.