Association Between Mitochondrial DNA Mutations And MiR-1 And Hepatocellular Carcinoma

Objective Reprogrammed metabolism is a key feature of cancer cells, which is characterized by an enhanced uptake and utilization of glucose. It has repeatedly been reported that the metabolic hallmark of glucose catabolism was enhanced aerobic glycolysis and PPP (pentose phosphate pathway) in HCC (hepatocellular carcinoma), regardless of oxidative phosphorylation functioning. Mitochondrion, the site of oxidative phosphorylation, provides energy for cells, and mitochondrial genome involves coding structural components of oxidative phosphorylation chain. Whether mitochondrial dysfunction or genome alterations play a role in disordered oxidative phosphorylation of tumor cells is still obscure. Although hundreds of miRNAs (microRNAs) have now been identified in humans and an overwhelming body of evidence implicated miRNA in the process of gene expression, the direct relevance of such regulation in PPP is unclear. SnoRNAs (small nucleolar RNAs) direct chemical modification of rRNA in ribosome, influencing protein synthesis and thus the expression profile and pathological role of snoRNAs in HCC needs to be explored. The study aims to characterize the reprogrammed glucose catabolism in HCC with the following objectives:1. comprehensively profiling somatic mitochonrial mutations and its relevance with oxidative phosphorylation in HCC at the gene level;2. verifying the regulator role of miR-1 (microRNA-1) in TKT (transketolase) and G6PD (glucose-6-phosphate dehydrogenase) gene and its association with PPP at the post-transcription level;3. depicting SNORD78 (small nucleolar RNA 78) expression and its pathological significance in HCC at the translation level and preliminarily establishing the modified stem-loop reversed transcipted method of SNORD78.Methods1. Moitochondrial somatic mutations were screened by amplifying mitochondrial genome, followed by Sanger sequencing and cloning sequencing. We have applied the long-range gene synthesis technique to convert COX3 (cytochrome c oxidase 3) gene directly into nuclear code and subcloned the gene into the pcDNA3.1-Mito vector containing mitochondrial target sequence. We transfected wild-type and mutant COX3 vectors into HepG2, and detected the levels of ATP, lactate, apoptosis ratio and ROS.2. The expression of essential enzyme in PPP, G6PD and TKT was predicted using bioinformatics software to be regulated by miR-1. miR-1 expression profile in HCC was quantified by real-time PCR (RT-PCR). We transfected miR-1-3p and 3’UTR of TKT or G6PD vectors into HepG2 simultaneously; fluorescence intensity was performed to detect by dual-luciferase reporter assay and expression of target genes by Western blot assay to verify the regulator role of miR-1-3p. Apoptosis ratio, ROS production, cell viability, NADPH and lactate levels were analysed in HepG2 with miR-1-3p overexpression, respectively.3. The expression signature of SNORD78 in HCC tissues and plasma was quantified by RT-PCR. We constructed SK-Hep-1 cell line that downregulated SNORD78 with siRNA. RT-PCR analysis was peformed to detect the SNORD78 and its host gene GAS5 (growth arrest-specific transcript 5) expression following treatment with two siRNAs. And the pathological significance of SNORD78 with loss-of-function analyses were to be explored. Moreover, SNORD78 in plasma was reversed transcipted by stem-loop method and quantified by Taqman PCR and digital PCR, respectively.Results1. Somatic mutations of mitochondrial genome in HCC(1) We sequenced the entire mitochondrial genome of 56 HCC tumor tissue and matching peripheral leukocyte samples. Sequencing results of mitochondrial genome in HCC tumor were compared with those from leukocytes and 51 somatic mutations were identified in (27/56) 48.2%HCC patients,11 nonsynonymous mutations were found among (6/56) 10.7%HCC patients, with a nonrandom distribution in code genes of mitochondrial genome; heterplasmies were identified in somatic cells and germline cells, with the ferquency of 21.4%(12/56) and 12.5%(7/56), respectively.(2) COX3 is a highly conserved gene in human mitochondrial genome and we chose G9267A as a model to explore somatic mutation function in HCC.(3) ATP production was decreased markedly in cells transfected with G9267A and declined progressively with increasing mutant COX3 mtDNA (P for trend= 0.0152). Lactate production was increased in mutant cells and increased with increasing mutant COX3 mtDNA (P for trend= 0.0461). Compared with wild-type cells, more ROS were detected in mutant cells and increased ROS with increasing mutant content (P for trend<0.0001). The mutant-types are more apoptotic than wild-type, and more apoptotic in the less mutant cells (P for trend= 0.0005), suggesting a switch from mitochondrial oxidative phophorylation to glycolysis for ATP production in HepG2 cell line.2. miR-1 expression and the impact on PPP in HCC(1) The expression of miR-1-3p was downregulated in HCC tissues than adjacent tissues, and the expression was negatively associated with the TNM stage of HCC.(2) The relative luciferase activity and gene expression of TKT and G6PD decreased in HepG2 transfected with miR-1 (P<0.001), which suggested the impact of miR-1 on PPP activity through regulation on G6PD and TKT.(3) miR-1-3p promoted cell cycle arrest in S phase, inhibiting cell proliferation, moreover, miR-1-3p promoted ROS production, cell apoptosis and the synthesize of lactic acid, yet decreased NAPDH production, which indicated that miR-1 not only play a role in the growth, proliferation and apoptosis of HepG2, but also in regulating the activity of PPP.3. SNORD78 expression in HCC and the biological function in HCC cell line(1) SNORD78 was upregulated in HCC tissues than adjacent tissues (P=0.004) and SNORD78 was positively associated with tumor foci, stage and distant metastasis (P= 0.02,0.014,0.01); Kaplan-Meier survival curve analysis showed that high-level expression of SNORD78 was associated with short overall survival and recurrence free survival of patients with HCC (P=0.023,0.014), which suggests SNORD78 may provide a potential biomarker for the progression and prognosis of HCC.(2) RT-PCR analysis of SNORD78 following treatment of SK-Hep-1 cells with siRNAs showed the markable interference effect on SNORD78 (P all<0.05) and yet no significant chage of GAS5, the host gene of SNORD78. Knockdown of SNORD78 promoted cell apotosis, altered cell cycle distribution, inhibited cell proliferation, restrained migration and invasion of SK-Hep-1 cell line (P all<0.05).(3) The expression of SNORD78 in plasma was downregulated in HCC patients than healthy controls (P=0.0138), yet no difference compared with liver cirrhosis patients (P=0.1281) and moreover SNORD78 expression was negatively associated with tumor number (P=0.016) and stage (P=0.039).(4) After performed reverse transcription by A-tailing, specific primer and stem-loop respectively, the expression of SNORD78 was quantified, which showed the correlation between stem-loop and A-tailing was (R2=0.7626,P=0.0015), between stem-loop and specific RT was (R2=0.8198, P=0.00103). The copy number of SNORD78 in plasma was decreased in HCC patients than controls using Taqman PCR (P<0.05), correspondent with analysis by digital PCR. Therefore, the modified stem-loop reversed transcription of SNORD78 proved to be reliable.Conclusions1. The somatic mutation COX3-G9267A in mitochondrial genome damaged oxidative phosphorylation activity and enhanced glycolysis ability.2. miR-1 was considered to be associated with the enhanced PPP by targeting to 3’UTR of TKT and G6PD in HCC.3. SNORD78 was considered as a potental biomarker of HCC. And modified stem-loop reversed transcription of SNORD78 proved to be reliable.