Identification Of Subtle Differences In D-loop Of Mitochondrial DNA Between Alcohol Related HCC And Hepatitis B Virus Related HCC Pations

Objective: Human mitochondrial genome is a 16 kb length, closed-circular, duplex molecule that containing 37 genes, two ribosome RNAs, and a complete sets of 22 tRNA . mtDNA (mitochondrial DNA) is believed to be more susceptible to DNA damage and acuquired mutations at a higher rate than nuclear DNA (nDNA) because of exposure to high levels of reactive oxygen species (ROS) generated in mitochondria, lack of protective histones, and limited capacity for DNA repair . Thus somatic mtDNA mutation occur in a wide variety of degenerative diseases and cancer, and mutation becomes homoplasmic by clonal expansion in tumor tissues . In many cancers including hepatitis virus caused HCC, most somatic mutations and polymorphisms accurate in a hotspot in mtDNA noncoding region called displacement loop (D-loop) , which is important for both replication and expression of the mitochondrial genome because it contains the leading-strand origin of replication and the promoter of transcription.Through ethanol consumption increases ROS generation in hepatic mitochondria and cause multiple hepatic mitochondrial DNA deletions, somatic mutations and polymorphisms in the D-loop were rarely studied in alcohol related HCC. Other than oxidative stress caused by viral infection, the hepatitis virus also integrated into target genes involved in cell growth or apoptosis for further oncogenic effect , so subtle differences for carcinogenesis mechanism would exist between alcoholic cirrhosis related HCC and virus related HCC. We therefore sequenced cancerous tissues, noncancerous tissues and blood mitochondrial D-loop of hepatitis B virus related HCC and alcohol related HCC patients to identify these differences.Methods: 1 Samples collection and patients follow up: We obtained histologically confirmed cancerous and noncancerous liver tissues from 60 HCC patients including 11 alcoholic cirrhosis related HCC (average alcohol consumption > 40g per day for at least five years) and 49 HBV related HCC patients, and 38 normal livers tissue from hepatic hemangioma patients at the Fourth Hospital of Hebei Medical University between 2007~2008. All the patients were not given any treatment before surgery .2 mtDNA extraction: All the tissues were kept in liquid nitrogen immediately after surgical resection according to guideline by the human tissue research committee at the hospital. mtDNA of the liver was extracted with Mitochondrial DNA Extraction Kit. The blood was obtained from corresponding patients and mtDNA was obtained with Blood Mitochondrial DNA Extraction Kit. All mtDNA was stored at -20°C until use.3 PCR amplification and sequence analysis:The forward primer 5'-CCCCATGCTTACAAGCAAGT-3'and reverse primer 5'-GCTTTGAGGAGGTAAGCTAC-3'were used for amplified a 982 bp DNA fragment from mtDNA D-loop region as described elsewhere. PCR was performed with PCR Master Mix Kit (Promega, Madison, WI) and purified with Ultra Clean DNA purification Kit (Bio101, Solana Beach, CA) prior to sequencing. Cycle sequencing was run with Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Bio system, Foster City CA) and then separated on the ABIPRISM Genetic Analyzer 3100 (Applied Biosystem). Mutation and polymorphism were confirmed by repeated analysis.4 The measurement of liver function and AFP: venous blood specimens were drawn on fasting before operation in 49 hepatitis B virus related HCC and 11 alcohol related HCC (all of them were newly diagnosed patients).AFP was detected with the electrochemiluminescence immunoassay ( ECL ) , whose principle was specific Chemiluminescence Reaction on electrode surface caused by electrochemistry, including two courses of electrochemistry and chemiluminescence. (Roche, BASLE,SWITZERLAND), ALT,AST,GGT were detected with the enzyme rate method to test the enzyme activity, whose principle was the determination of enzyme activity according to the rate caused by enzyme. TBIL,DBIL,TP,Album were detected with end-point method, using SYNCHRON LX20 biochemistry analysor. (BECKMAN COULTER INC. Somerset, NJ ). 5 Statistical analysis: The student's t-test was used to analyze differences in age and sex,Fisher's extact test or x2 test was used to compare categorical data between groups.Resluts: 1 There were no statistical difference of Age, TP,ALB,TBIL,DBIL,GGT between alcohol cirrhosis related HCC and HBV related HCC. The ALT (Z=-2.341, P=0.019), the AST (Z=-3.097, P=0.002), AFP (Z=-3.582, P=0.000) and the positive rate of AFP(χ2=20.093,P=0.000) were statistical different between the two groups2 Compared the sequences of mtDNA with the Cambridge sequence, we found 91 polymorphic loci (blood, cancerous and noncancerous tissues were all unanimously, but inconsistent with the Cambridge sequence, called polymorphism),and the multi-state rate was 9.27%.3 At nucleotide 152,there were 18 samples contained C in Hepatitis B virus-related HCC group,while no one contained C in alcoholic cirrhosis related HCC group (P=0.012) ; at nucleotide 249,there were 15 samples without containing A in Hepatitis B virus-related HCC group,while A were contained in all samples from alcoholic cirrhosis related HCC group(P=0.030);at nucleotide 16293,there were 2 samples contained G in Hepatitis B virus-related HCC group,while no one in control group (P=0.047);4 By analyzing,of 60patients, 29 DNA mutations were found on 26 patients, and the mutation rate was 43.3%; 10 loci was nucleotide mismatch, and the mutation frequency of another loci was up to 18 (18/60 ), and the form of mutation contained single nucleotide deletion and insertion, long nucleotides deletion.No mutation was found in the control group of Hepatic hemangioma. We perform further follow up for survival rate of these HCC patients to search the mutations or polymorphism associated with outcome of HCC.Conclusions: 1 The ALT,AST,AFP and the positive rate of AFP were statistical different between the two groups.The values of ALT and AST increased significantly in hepatitis B virus related HCC group. Compared with alcohol cirrhosis related HCC group, the liver dysfunction of hepatitis B virus related HCC was severe.The AFP values have no increased in the most of alcohol cirrhosis related HCC ,so we should find a new clinical markers to replace AFP.2 At nucleotide 152 and 249, there were statistical difference between HBV related HCC,alcoholic cirrhosis related HCC and control samples; At nucleotide 16293,there were statistical difference between alcoholic cirrhosis related HCC and control samples, and the positive rate of AFP were statistical different between the two groups.It's suggested that there would be Subtle differences between hepatitis B virus related HCC and alcohol related HCC.3 The D-loop of Mitochondrial DNA is a highly polymorphic and mutation region. The frequency of polymorphic was 9.27% (91/982),and the rate of mutation was 43.3%. The main way of mutation was replacement of nucleotide,all of mutations were homogeneity. In the group of hepatic hemangioma, there were no mutations in the corresponding loci.