| BackgroundMitochondria are important cellular organelles which locate in eukaryotic cells and generate the vast majority of ATP to the cell and regulate several signaling cascades.Human mitochondrial DNA(mtDNA)is a double-stranded molecule with a total length of 16569 bp.The mitochondrial genome contains a non-coding displacement-loop(D-loop)region and a coding region.The D-loop region locates in np1-576 and np16024-16569 which is about 1124 bp in length,accounting for 6% of the whole mtDNA.It serves as the main site for transcription and replication.D-loop region is the most variable portion of the human mitochondrial genome and mutations within this region are present in a variety of human cancers.Thyroid carcinoma represents only 2% of all malignant diseases,it is the most common endocrine system malignancy.Thyroid papillary carcinoma(PTC),thyroid follicular carcinoma(FTC),undifferentiated thyroid carcinoma(ATC)and thyroid medullary carcinoma(MTC)are the main pathologic types of primary thyroid carcinoma.Thyroid papillary carcinoma is the most common pathological type,accounting for about 80% of thyroid cancer.Although the pathogenesis of thyroid cancer in recent years has made great progress,but the molecular mechanism of the disease is still unclear.Some researchers suggest that oxidative damage to macromolecules and alterations in cell energy metabolism may result in thyroid dysfunction and affect tumor progression.Because mitochondria are the main places in energy metabolism in the normal thyroid gland and thyroid tumors,the effect of mitochondrial alterations in tumorigenesis in the thyroid gland require to be investigated.ObjectiveThe aim of the present study was to analyze the genetic alterations in D-loop region in thyroid carcinoma and benign lesions and investigate the correlation between mitochondrial mutations and clinic pathological and biological characteristics.Methods(1)Sample collectionParaffin-embedded samples of 196 papillary thyroid carcinoma(155 females and41 males with a mean age of 46 years)and 205 thyroid benign lesions(146 females and 59 males with a mean age of 48 years)which including 106 thyroid adenoma,62non-tumor nodular goiters,17 Hashimoto's thyroiditis and 20 sub-acute thyroiditis were obtained from the pathology units of Jiangsu University Affiliated People's Hospital from Jan 2010 to Jan 2016.The age and gender of patients,tumor size,cell differentiation and status of lymph node metastasis were obtained from histopathology records.All patients and volunteers were consent informed.(2)DNA ExtractionDNA was extracted with the RecoverAll? Total Nucleic Acid Isolation Kit(Life Technologies,CA,USA)according to the manufacturer's protocols.The extracted nucleic acid was store at-20?.(3)Design primer and Polymerase Chain ReactionNested PCR was used to multiply mtDNA D-loop region.PCR products were mixed with loading buffer and electrophoresed through 2% agarose gels.(4)Direct sequence determination and analysisThe remaining PCR products were sent to Beijing Liuhe Huada Gene Technology Co.,Ltd.for sequencing.The mtDNA D-loop sequence was compared to the reference sequence(human mitochondrial DNA revised Cambridge reference sequence,rCRS)using the Mitomaster software.When the mutation site was identified,the sequencing peak was observed with Chromas software and the mutation site was compared with mtDB database.If there were no reports,the mutations were considered as new mutations.At the same time,these mutations were compared with the Polymorphic Sites subdatabase.If the mutation frequency was more than 1%,it was considered as a single nucleotide polymorphism(SNP).If the mutation frequency was less than 1% in all the tested populations,it was considered as a mutation site.(5)Statistical AnalysisSPSS 17.0 statistical analysis software(SPSS,Inc.,Chicago,USA)was used for analysis.Chi-square tests were conducted to identify mtDNA D-loop alterations between papillary thyroid carcinoma and thyroid benign lesions.The relationship between the number of mutations and the clinical data was determined by measuring the Pearson's correlation coefficient.A two tailed Student t test was employed to compare the difference in the number of mutations between genders.P value 0.05 was considered to be statistically significant.Results(1)The 389 samples(97%)were successfully amplified and clearly visible bands were observed.(2)MtDNA alterations in D-loop region were detected in 171 of 196(87.2%)of papillary thyroid carcinomas and 175 of 205(85.4%)of the benign lesions and the statistically difference was not significant(P>0.05).In addition,151 of 196(77%)papillary thyroid carcinomas and 156 of 205(76%)thyroid benign lesions harbored somatic insertions at the polymorphic homopolymeric C stretch(D310);56 of 196(29%)papillary thyroid carcinomas and 71 of 205(35%)thyroid benign lesions harbored somatic deletions at the dinucleotide(CA)repeat region(D514).No significant difference was observed between the two groups(P>0.05).(3)There was no significant correlation between age,sex,tumor size,TNM staging and mtDNA D-loop mutation numbers.ConclusionsThese data indicate that mtDNA D-Loop region mutations are frequent in benign and malignant thyroid lesions and it may be involved in the early stage of thyroid tumor development.MtDNA D-Loop region mutations cannot be considered a marker of malignancy... |
PDF Full Text Request