VARIANTS IN MITOCHONDRIAL tRNA GENE MAY NOT BE ASSOCIATED WITH THYROID CARCINOMA
Lv F1,a, Qian G2,a, You W1,a, Lin H3, Wang XF3, Qiu GS2, Jiang YS2, Pang LX3, Kang YM4, Jia BF4, Xu JZ5,*, Yu Y1,*
*Corresponding Author: Dr. Jinzhong Xu, Department of Clinical Pharmacy, the Affiliated Wenling Hospital of Wenzhou Medial University, Taiping Nan Road 190, Wenling 317500, People’s Republic of China. Tel./Fax: +86-(0)576-8620-6288. E-mail: xujzwl@163.com and Dr. Yang Yu, Department of Breast Surgery, Henan Provincial People’s Hospital, Weiwu Road 7, Zhengzhou 450003, People’s Republic of China. Tel./Fax: +86-(0)371- 6558-0014. E-mail: 510790135@qq.com
page: 59

MATERIALS AND METHODS

Database Searches. We systematically searched for unrestricted language articles included in PubMed, Embase, Google Scholar, Cochrane Library, China National Knowledge Infrastructure (CNKI), Chinese VIP, and Chinese Wan-fang databases from inception to December 2015. The literature search in PubMed was carried out using the following keywords “mitochondrial tRNA mutations, thyroid carcinoma,” or “mt-tRNA mutations, thyroid cancer,” or “mt-tRNA variants, thyroid cancer.” We excluded studies if the crucial data were not reported in original papers, or if they had a very high probability of inaccurate reporting. Data Extraction. Data were independently extracted by two authors (F. Lv and G. Qian), and checked by another author (W. You). Any disagreement was resolved by discussions until consensus was reached. Determining the Conservation Index of These mt-tRNA Variants. With the purpose of understanding the possible role of mt-tRNA variants in thyroid cancer, we performed a phylogentic approach to see the conservation index (CI) of each mt-tRNA variant. In brief, the mtDNA sequences of 15 vertebrates were used in the inter-specific analyses; these species included Mus musculus, Gorilla gorilla, Hylobates lar, Lemur catta, Pan paniscus, Homo sapiens, Thylamys elegans, Procavia capensis, Cavia porcellus, Orycteropus afer, Bos taurus, Sus scrofa, Felis catus, Platanista minor and Herpestes javanicus. The CI was then calculated by comparing the human nucleotide (nt) variants with 14 other vertebrates. Notably, the CI of >70.0% was regarded to have a functional potential [10]. Calculation of the ⊿G of mt-tRNA With and Without These Variants. To see whether these variants affected the ⊿G of mt-tRNAs, we used the RNA Fold Web server program to measure the ⊿G of each mt-tRNA with and without these variants (http://rna. tbi.univie.ac.at/cgi-bin/ RNAfold. cgi) [11]. Screening for the Frequency of the A12308G Variant in Thyroid Carcinoma. We then performed the polymerase chain reaction (PCR)-Sanger sequence to detect the occurrence of the “well-known” A12308G variant in patients with thyroid cancer. Briefly, a total of 300 blood samples of thyroid cancer (150 male and 150 females; average age 52 years) and 200 healthy subjects (100 males and 100 females; average age 49 years) were recruited from the Henan Provincial People’s Hospital, Zhengzhou, People’s Republic of China (PRC). Informed consent and clinical evaluation were obtained from all these subjects. The study protocol was approved by the Ethics Committee of Henan Provincial People’s Hospital. We first extracted the genomic DNA from each sample, then we used the PCR to amplify the gene using the following primer sequences: forward (5’-TGC TAG TAA CCA CGT TCT CC-3’); reverse (5’-TTT GTT AGG GTT AAC GAG GG-3’). The PCR product was subsequently examined for specificity using 1.5% agarose gel electrophoresis. Double-stranded automated sequencing was performed using an ABI PRISM™ 3700 sequencing machine (Applied Biosystems Inc., Foster City, CA, USA). The sequence was then compared with the human mitochondrial reference sequence (NC_012920) [6].



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