| Objective: Laryngeal carcinoma is one of the common malignant tumors in otolaryngology-head and neck surgery,accounting for about 26%-30% of global head and neck cancer.In recent years,with the smoking population increasing and the living environment gradually deteriorating,the incidence of laryngeal carcinoma has been increased than before,and it's more threaten to human's health and life.Recently,with the development of modern medicine and experimental techniques,some scholars have proposed mitochondrial DNA changes which is one of the important factors in tumorigenesis,and it is related to tumor occurrence and development.In higher animals,mitochondrial DNA is the only space,where exist extranuclear genetic material.Human mt DNA is a closed,double-stranded molecular structure,consisting of 16,569 bp,which contained 13 polypeptide-coding genes,2ribosomal RNA genes and 22 transfer RNAs,they play an indispensable role in the process of the synthesis of mitochondrial proteins.The D-loop region is a non-coding region of human mitochondrial DNA,which located at mt DNA16024 bp to 576 bp and only accounting for 6% of the entire mtDNA sequence,but with high mutation and polymorphism.Because of this region contains important structures such as replication origin,transcription promoter and so on.,it controls the replication and transcription of mtDNA.In this study,PCR and DNA sequencing techniques were used,and the D-Loop region of mt DNA was sequenced in 71 laryngeal carcinoma patients and 159 healthy controls,the relationship between the laryngeal carcinoma risk-associated SNPs and clinical features was evaluated.We hope the result could provide a new way for the early diagnosis,effective treatment and prognosis of laryngeal cancer.1 Object of study and sample collection: In this study,blood samples were collected from 71 patients with laryngeal carcinoma and treated by surgery in the Fourth Hospital of Hebei Medical University from 2013 to 2016,aged among 39 to 73 years.All cases are males.After the informed consent was agreed with from patients and their relatives,we collected peripheral venous blood from the 71 patients and stored in the refrigerator at-4 ° C.In addition,159 normal subjects were selected from the fourth hospital of Hebei Medical University from 2010 to 2013 as control group,we make detailed records of their medical information and exclude laryngeal carcinoma-related diseases,we took peripheral venous blood 3ml each person as our samples.Informed consents were obtained from all participants before the enrollment and the samples were collected according to the Human Tissue Research Committee of the hospital.2 Experimental and analytical methods: The genomic DNA was immediately extracted with the TIANGEN? Genomic DNA extraction kit.Use a PCR Master Mix Kit(Promega)to run PCR,so that the mtDNA D-Loop region of the purpose of this fragment can be amplified.The amplified products were bi-directional repeated sequenced.The results were compared with the currently accepted Cambridge standard sequence,and the connection between mtDNA D-loop mutation and laryngeal carcinoma was obtained.3 Statistical analysis: All the statistical analysis was done by the SPSS21.0 software.X2test was used to analyze enumeration data to calculate the P value,odds ratio(OR)and 95% confidence intervals(CI).P value <0.05 was premediated statistically significant.Results:1 The clinical data of laryngeal carcinoma group and control group were compared and found that: gender,smoking and drinking history of the two group have statistically different(P<0.05).There was no obvious difference between laryngeal carcinoma group and control group compared with the age under 40 years and the age over 40 years(P>0.05).2 71 laryngeal carcinoma patients and 159 healthy controls were enrolled Methods:in this study.SNPs were detected in 146 sites within the 982 bp mitochondrial D-Loop region from the blood samples of the laryngeal carcinoma patients and healthy controls,referring to the standard RA Cambridge sequence.The frequency of the mtDNA alteration was 14.9%.Among these,two SNPs without reporting in the Mitomap database were classified as new polymorphisms.The SNPs with sample size beyond four in either laryngeal carcinoma group or healthy control group were used for cancer risk analysis,a total of 25 SNPs were selected.3 When individual SNPs were analyzed comparing laryngeal carcinoma patients with the healthy controls,a statistically significant difference of distribution frequency was observed for 73G/A ? 309C/C insert ? 315 C/C insert ?524C/del?556A/del?16288C/T?16291T/C?16311 C/T and 16319A/G alleles.The frequency of genotype 73G/A was 100% in laryngeal carcinoma patients and 56% in healthy controls.The alleles frequency of 73G/A in laryngeal carcinoma group was higher than that in control group(P<0.01).One C insertion at 309 site were detected in 24 of 71 laryngeal carcinoma patients(33.8%)and 116 of 159 healthy controls(73.0%).The alleles frequency of 309 C insert in laryngeal carcinoma group was lower than that in healthy controls(P<0.01).One C insertion at 315 site was detected in 30 of 71 laryngeal carcinoma patients(42.3%)and 149 of 159 healthy controls(93.7%).The alleles frequency of 315 C was lower in laryngeal carcinoma group than that in control group(P<0.01).One C deletion at 524 site was detected in 20 of 71 patients(28.2%)and 1 of 159 controls(0.6%).The alleles frequency of 524 C Del in laryngeal carcinoma group was higher than that in healthy controls(P<0.01).One A deletion at 556 site was detected in 6 of 71 patients(8.5%)and none of 159 controls(0%).The alleles frequency of 556 A Del was higher in patients than that in controls(P<0.01).The frequency of genotype 16288C/T was 5.6% in laryngeal carcinoma patients and 0% in healthy controls.The alleles frequency of 16288C/T in laryngeal carcinoma group was higher than that in control group(P=0.013).In addition,the frequency of genotype 16291 T was 11.3% in laryngeal carcinoma patients and1.3% in controls.The alleles frequency of 16291T/C in laryngeal carcinoma group was higher than that in control group(P=0.020).The frequency of genotype 16311 C was 19.7% in laryngeal carcinoma patients and 9.4% in control group.The alleles frequency of 16311C/T in laryngeal carcinoma group was higher than that in control group(P=0.030).The frequency of genotype 16319 A was 5.6% in laryngeal carcinoma patients and 15.7% in controls.The alleles frequency of 16319A/G in laryngeal carcinoma group was lower than that in control group(P=0.033).4 We then analyzed the relationship between the laryngeal carcinoma risk-associated SNPs and clinical features of laryngeal carcinoma patients.There was no association of the laryngeal carcinoma risk-associated SNPs with clinical features,including tumor size,extend of classification and UICC stage(2002 vision)(P>0.05).Conclusions:1 People who addicted in smoking and drinking for so many years prefers to suffer from laryngeal carcinoma.2 The D-Loop region of laryngeal carcinoma patients has a high degree of polymorphism,the high frequency polymorphic sites were located in the hypervariable segment one(HV1),hypervariable segment two(HV2)and hypervariable segment three(HV3).Most of the mtDNA variants were single base transition,part of the variants are insertion and deletion.3 The change in73G/A was found in all laryngeal carcinoma samples,it demand to have a further study.4 The mtDNA D-Loop region polymorphisms may be associated with the tumorigenesis and progression of laryngeal carcinoma.The 73G?524del?556del?16288C?16291T and 16311 C were significantly associated with an increased risk for laryngeal carcinoma,whereas the 309 C?315 C insert and16319 A genotype might be associated with resistance to laryngeal carcinoma.5 There was no relations between D-Loop region polymorphisms and clinical features of laryngeal carcinoma patients,which meant that the D-Loop region polymorphisms may potentially play an important role in early stage oftumor progression and be useful molecular marker for early diagnosis of laryngeal carcinoma.It may supply a new theoretical basis for tumor diagnosis. |
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