Experimental Study Of Loss Of Heterozygosity And Imprinting Of CDKN1C On Laryngeal Squamous Cell Carcinoma

PrefaceLaryngeal carcinoma is one of the most common malignant tumors occurred in head and neck,which accounts for about 1-5%in human malignant tumors.In recent years,its prevalence increased greatly and it has destroyed many people's health.At present,the primary treatments for laryngeal carcinoma include operation,radiotherapy, biotherapy and chemotherapy according to the biological behavior of the tumor.With the development of diagnostic and therapeutic technology,some important progresses have been made in retention of laryngeal function,prolonging patients life after different treatment.However,the effect of all these measures is not as good as expected in advanced and re-occurred cancer patients.Therefore,the issue need badly to be solved is to find and treat the laryngeal carcinoma early with new medicines and techniques to reduce the damage to normal tissues.Recently,it has been found that except for the well known abnormality in DNA sequence,epigenetics changes in gene regulation plays an important role in the pathogenesis of tumor.Epigenetics is a new subject studying the situations of hereditable expression changes without DNA sequence change.Genomic imprinting is one of the most important contents of it.It means that the variable phenotypic expression of a gene depending on whether it is of paternal or maternal origin,which is a function of the DNA methylation pattern.Imprinted regions are observed to be more methylated and less transcriptionally active.If one allele was imprinted,the ante-oncogene will be dysfunctioned,and causes the cancer-predisposing.Many important ante-oncogenes located on Human chromosome 11p15.5,where contains lots of imprinted genes.CDKN1C(p57kip2)maps to there.CDKN1C gene binds to CDKs(cyclin-dependent kinases)and inhibits the function of it.Through this way,it can stop the cell cycle in G1 phase.It plays an important role in cell growth. There's CDKN1C gene is closely related to tumorigenesis.Normally,CDKN1C gene shows maternal expression.Manr studys indicate CDKN1C gene mutation is seldom seen in human tumors.It mainly appears loss of heterozygosity(LOH),loss of imprinting(LOI)or mistake of imprinting and aberrant methylation of CpG island, which thereby induce low level of gene expression or even gene deletion.In order to investigate the heterozygosity and imprinting state of CDKN1C gene in laryngeal carcinoma,we selected two micro satellite sites TH01(in CDKN1C), D11S2359(on a backward position of CDKN1C)and an enzyme- resecting site rs3852522(in CDKN1C)to study.The methods of PCR,RT-PCR,polyacrylamide gel electrophoresis-silver staining and enzyme shearing were used to reveal the molecular and biological mechanism of the tumor.Materials and methods1.Subjects40 samples of laryngeal lquamous cell carcinoma were collected from the department of otorhinolaryngology of Shengjing Hospital affiliated to China Medical University during May to December in 2007.The patients included 35 men and 5 women,mean age was 57.84±9.84.All cases were typed and staged according to the TNM standard in UICC 2002.Normal laryngeal mucosa were collected from 11 patinents,more than 2 cms away from laryngeal carcinoma areas.All the patients did not accept systemic radiotherapy or chemotherapy before operation.Samples abstraction was ratified by the ethics committee of Shengjing Hospital affiliated to China Medical University,and all the patients signed informed consents. 2.Methods(1)PCR primer sequenceTH01(146-190 bp):Forward:5'-GTGGGCTGAAAAGCTCCCGATTAT-3' Reverse:5'- GTGATTCCCATTGGCCTGTTCCTC-3'D11S2359(197-221 bp):Forward:5'-TCCTCTCACAAAAATTGATGG-3' Reverse:5'- CTAACCTGACATTGTGCACA-3'rs3852522(391bp):Forward:5'-GCGAACCCGACGCAGAAGAG-3' Reverse:5'- GCATGTCCTGCTGGAAGTCGTAATC-3'(2)Detection of CDKN1C gene heterozygosityTotal genomic DNA was extracted by the phenol-chloroform method.TH01and D11S2359 were amplified.PCR products were analyzed on polyacrylamide gel after stained with silver nitrate.(3)RT-PCRTotal genomic RNA was extracted from all the heterozygotes and then cDNA was synthesized by reverse transcription PCR method.Amplified rs3852522 fragment by PCR.(4)Detection of CDKN1C imprinted stateThe amplified products of heterozygote cDNA were resected by FspBI enzyme, and then were analyzed on agarose gel.(5)Statistical analysisThe data were analyzed by the x~2 test with Fisher's exact test where appropriate (SPSS 12.0).Statistical significance was defined at P＜0.05.Results1.TH01 and D11S2359 amplificationThe PCR products of TH01 and D11S2359 should be 146-190bp and 197-221bp,respectively.On the basis of semi logarithmic table,the two alleles of TH01 should be 158bp and 170bp,and the two allele of D11S2359 should be 200bp and 218bp,respectively.2.LOH of CDKN1C gene at TH01 and D11S2359Two micro satellite sites of CDKN1C gene TH01 and D11S2359 were selected to detect the heterozygosity of it.LOH was detected in 18 laryngeal lquamous cell carcinoma samples(3 of stageⅡ,3 of stageⅢand 12 of stageⅣ).9 was detected at TH01 site and 14 was detected at D11S2359 site.In 5 samples,LOH was found at the two sites.But in the control group,LOH was detected only in 1 sample at D11S2359 site.There was significant difference between the two groups(P= 0.037＜0.05)and among different stage samples(P=0.034＜0.05).LOH was seen in 12 samples of supra glottis laryngeal carcinoma and 6 samples of glottis laryngeal carcinoma.It was not seen in the sanples of infra glottis laryngeal carcinoma.There's no significant difference among the three different stages(P=0.108＞0.05).3.Imprinting state of CDKN1C geneThe cDNA amplification product of CDKN1C gene is 391bp and was resected by FspBI.The undigested fragement was defined as a(391bp),and the digested fragment(253bp and 138bp)was defined as b.The products had both a and b fragement were diallele expressed(imprinting deletion),otherwise were monoallelic expressed.Among 22 heterogous tumor samples,13 were diallele expressed.2 normal samples were diallele expressed,too.The prevalence of diallele expression in was much higher than that in cinreol group There was no significant difference between laryngeal carcinoma samples and the control group(P = 0.060＞0.05).Conclusions1.In the development of LSCC,LOH at TH01 and D11S2359 sites of CDKN1C gene may be an important reason.2.In some LSCC patients,LOI of CDKN1C gene was be observed,but there was no significant difference between the two groups.3.Except for LOH,there's other mechanisms that can change the imprint state of CDKN1C gene.