LIT1 Gene Imprinting Status Is Associated With The Biology Behavior Of Human Colorectal Cancer

Colorectal cancer is the most common malignant tumor,the incidence of colorectal cancer in our country are relatively low,but the trend is upward in recent years.Reports have shown that the incidence of colorectal cancer ranks third in malignant tumors,the mortality rate ranks fifth in cancer deaths.The research found the occurrence and development of colorectal cancer have much relation with genetic changes,such as APC gene mutation,k2ras,p53 and so on.In recent years,epigenetic changes in the tumor received people's attention gradually,the so-called epigenetic gene sequence has not changed,but its expression has had the inheritability change.Genomic imprinting is an epigenetic modification,it is one kind which we discovered in recent years does not comply with the Mendel's law,but is a phenomenon depended upon the single parenthood to transmit certain genetics character.That is to say,certain gene were dependent single allele to express,its another allele does not express or the express is extremely weak.As if these genes of different parental origin on a pair of alleles for identification with some western and named,which such a genetic phemomenon to be called the imprinted gene.Research suggests that disruption of imprinting mechanisms may play a critical role in the development of cancer.LIT1 (long QT intronic transcriptl) is an imprinted gene located on human chromosome 11p 15.5,because the intron of KvLQT1 gene has antisense strand transcription. KvLQT1 was one kind of gene which was responsible for encoding the cardiac potassium ion channel protein. Gene deletion and mutation changed the ammo acid sequence of proteins,thereby affecting potassium ion channel protein function,resulting in myocardial cell membrane disorder inside and outside the ion flow,so that cell repolarization time extended,showing a series of symptoms of LQTS.In many organizations,LIT1 maternal gene expression ,the paternal gene imprinting and the expression was inhibited.Disruption of imprinting mechanisms may play a critical role in the development of cancer. This study team formerly studied loss of imprinting of LIT1 has relation with BWS(giant tongue-omphalocele syndrome).The so-called loss of imprinting(LOI) refers to a specific parental allele of the normal source of the loss of expression.This experiment used reverse transcription-polymerase chain reaction-restriction fragment length polymorphism(RT-PCR-RFLP) method, studied LIT1 gene loss of heterozygosity and imprinting ststus of the changes in colorectal cancer and the relationship between them,analysised the imprinted gene LIT1 and colorectal cancer's relation,explored LIT1 at the occurrence and development of colorectal cancer in imprint control mechanism and its relationship with the biological behavior of colorectal cancer.Material and methods1 .Experimental meterials1.1 Experimental specimens:Resected cancerous,and non-cancerous colon tissue of 57 primary colorectal cancer patients were collected.DNA and RNA were extracted from tissue specimens.1.2 Major pharmaceutical reagents and equipmentAgarose (USA Amresco); DNA restriction endonuclease (Japan TAKARA); TRIZOL (KGI); RT-PCR Kit, Genefmder nucleic acid dyes purchased from TAKARA JAPAN companies. Primers, P-actin provided by the Shanghai Public Health. The autoclave LS3020(SanYo), enzyme immunity instrumentation SUNRISE (TACAN), low speed centrifuge LM-2A (Beijing Medical Centrifuge Factory), deep frozen refrigerator FORMA SCIENTIFIC (the US), electrophoresis box BCK-181A(HAIRE), PCR increases meter PTC-100TM continually (the US), low temperature table model centrifuge ST-21 (SORVALL SURPER), ultraviolet spectrophotometer UV-260 (island Tianjin), electrophoretic apparatus DYY-â…¢5 (US), electrophoresis gelatin image formation automated analysis system OLYMPUS. Electric heating constant temperature water bath box HETW21600 (Shanghai leaps forward medical instrument factory).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 specimenWe used polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP) analysis of the genotype in all specimens.Only when the state of specimens is heterozygous genotype,can distinguish between two alleles of the expression product to provide information. LIT1-F, 5'-CAgCACAAAgAggTTTTTgA-CAg-3', LIT1-R, 5'-gAgTTTAAAACACgTgTgTgCATT-3', annealing temperature 54℃.The expected size of the PCR fragment of the LIT1 gene is 410 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, ddH₂O 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,enzyme cuts 37℃the water bath overnight,polyacrylamide gel electrophoresis, genefinder nucleic acid dyes for heterozygote screening.2.3 Gene imprinting status detectedChose heterozygous specimens,cut their RT-PCR product with the same restriction endonuclease,after that 12% non-denaturing polyacrylamide gel electrophoresis. If the LIT1 gene expression showed biallelic expression of type (AB type) is 410bp, 222 bp and 188bp simultaneously exist that have taken place in the loss of imprinting. If monoallelic expression showed that only 410bp (A type) or only 222bp and 188bp (B type), indicating that transcription from one allele, namely, the maintenance of normal imprinting status (probably because of the difference between 222bp and 188bp fragmentssmaller overlapping and can not clearly show, therefore as enzyme cuts judgment standard take 222 bp).2.4Statistical analysisUsing Fisher exact test analysis of loss of imprinting of LIT1 and the clinical patholgy material relevance. All data were used SPSS 16.0 software for analysis. Results1 .PCR product DetectedIn our experiment,we confirm that the LIT1 gene is 410bp, through after PCR response fluid agarose gel electrophoresis the image analysis, by succeeds the synthetic product.2. Heterozygous specimen screeningOf the 57 patients enrolled for analysis,9 were heterozygous and thus informative for LIT1, and the positive rate of LIT1 of colorectal cancer were 15.79%(9/57). 9 of heterozygosity of tumor tissue specimens for DNA-wide heterozygosity, and no loss of heterozygosity.3.Imprinting status detected9 heterozygous normal tissue specimens, reverse transcription of the cDNA, by PCR and then digested by Rsa I after polyacrylamide gel electrophoresis separation, all homozygous samples. And loss of imprinting for the 4 specimens (4/9 = 44.44%).Conclusion1.LOI of LIT1 was observed in tumor samples2. LIT1 gene and clinicopathological features (age, sex, location, etc.) without statistical study related to each other, but with statistical study related to lymph node metastasis. We can only surmise that in colorectal, the LOI of LIT1 has relation with lymph node metastasis.