| Objective: Colorectal cancer(CRC)is the third most common cancer type and is responsible about seven hundred thousand deaths each year,which makes it the fourth leading cause of cancer-related mortality in both sexes worldwide.The evidence that the disease is significantly increasing in most developing countries heralds an even more remarkable disease burden in the near future.Environmental,dietary patterns(high fat consumption),genetic,and epigenetic changes are considered to affect CRC risk.Recent studies also show that mitochondrial genome instability is associated with colorectal carcinogenesis.Human mitochondrial DNA(mtDNA)is a 16569 bp closed double strand circular molecule that contains 37 genes,including two ribosomal RNAs and 22 transfer RNAs who participate in the protein synthesis in mitochondria.The D-Loop region is important for regulating both replication and expression of mitochondrial genome because it contains both the leading-strand origin of replication and the main promoter for transcription.The accumulation of single nucleotide polymorphisms(SNPs)in the displacement loop(D-Loop)of mitochondrial DNA has been identified for their association with cancer risk in a number of cancers.In present study,individual SNPs in the D-Loop of mtDNA were analyzed between 150 colon cancer patients and 159 healthy controls to investigate the predictive value of D-loop SNPs on colon cancer.Methods:1 Samples collection: Blood samples for experimental group were collected from 150 colon cancer patients who underwent colon cancer resection at the Deparment ? of Sugery in the Fourth Hospital of Hebei Medical University between January of 2006 and December of 2008.Blood samples were collected from 159 healthy controls without history of anycancer who were checked-up in the Fourth Hospital of Hebei Medical University between October of 2013 and October of 2014.Informed consent was obtained from all participants prior to enrollment and all the samples were collected according to the Human Tissue Research Committee at the hospital.2 DNA extraction: Genomic DNA was extracted immediately with a Wizard Genomic DNA extraction kit(Promega,Madison,WI,USA)from blood samples and stored at 4?.3 DNA amplification and sequence analysis: The forward primer5'-CCCCATGCTTACAAGCAAGT-3' and reverse primer 5'-GCTTTGAG GAGGTAAGCTAC-3' were used for amplification of a 982 bp product from the mtDNA D-loop region based on Cambridge mtDNA sequence.PCR was performed according to the protocol of PCR Master Mix Kit(Promega Corporation,Fitchburg,WI)and purified before sequencing.Cycle sequencing was performed with the Big Dye Terminator v3.1 Cycle Sequencing Kit(Life Technologies,Carlsbad,CA),and the products were then read with the ABI PRISM 3100 Genetic Analyzer(Life Technologies,Carlsbad,CA).Polymorphisms were confirmed by repeated analyses from both strands.4 Statistical analysis: Student's u-test was used to analysis quantitative data.The ?2 test was used to analyze dichotomous values,such as such as the presence or absence of any individual SNP in colon cancer patients and healthy controls.Statistical analyses were performed using the SPSS 17.0software(SPSS Inc.,Chicago,IL,USA).For all the statistical tests,a P-value of <0.05 was considered to statistically significant.Results:1 A total of 150 colon cancer patients and 159 healthy controls were enrolled in this study.No statistical difference exists referring to clinical characteristics such as age and gender between colon cancer patients and healthy controls(P>0.05).2 SNPs were detected in 138 sites within the D-Loop region from the healthy controls and colon cancer patients,and the frequency of the mtDNA alterations was 14.1%.Twenty-four SNPs with a minor allele frequency >5%(high frequency)in either controls or colon cancer patients were used for further cancer risk analysis.3 When individual SNPs were analyzed between colon cancer patients and the healthy controls,the alleles at positions 73,146,195,309,324,16,261,and 16,304 were identified as colon cancer risk-associated SNP sites.The frequency of genotype 73 G was 92.7% in colon cancer patients and 56% in controls,it was higher than that in the healthy controls(P=0.000).The frequency of genotype 146 T was 92.0% in colon cancer patients and 84.3% in controls,it was higher than that in the healthy controls(P=0.037).The frequency of genotype 195 T was 97.3% in colon cancer patients and 91.2% in controls,it was higher than that in the healthy controls(P=0.021).47.3%colon cancer patients were detected the insertions of one or two C at the 309 sites,and in the healthy controls was 63.5%.The allele frequency of 309 C insert in colon cancer group was lower than that in control group(P=0.004).The allele 324 C at 324 sites was detected in all colon cancer patients(100%)and 151 of 159 controls(95%),therefore the allele frequency of 324 C was higher in colon cancer group compared with control group(P=0.005).In addition,the frequency of genotype 16261 C was 99.3% in colon cancer patients and 91.8% in controls(P=0.002).The allele frequency of 16304 T in colon cancer group was higher than that in control group(P=0.004).4 The relationship between the colon cancer risk-associated high frequency SNPs in colon cancer patients and clinical features was then analyzed.There was no association of cancer risk-associated SNPs with clinical features,including gender,age,tumor size and stage.Conclusions:1 The D-Loop region of mtDNA was a hotspot for polymorphisms in colon cancer.Most of the mtDNA variants were single base transition and insertion,and SNPs mainly located in the hypervariable segment one(HV1)and hypervariable segment two(HV2).2 We identified a number of cancer risk-associated SNPs in mitochondrial D-loop region in colon cancer.A statistically significantincrease in the SNP frequency was observed for 73 G,146T,195 T,309C,324 C,16261C and 16304 T in colon cancer patients,therefore the patients who carry these alleles were more susceptible to colon Cancer.3 SNPs in the mtDNA D-loop were found to be valuable markers for colon cancer risk evaluation. |
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