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LABORATORIO CLÍNICO 509 The association between miRNA SNPs and HCC - W. Li et al Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with high fatality. It is estimated that the incidence of new HCC cases is approximately over half a million per year with 500,000 deaths occurring every year worldwide1,2. This is mainly due to HCC usually being diagnosed at an advanced stage due to a difficult early diagnosis. Although extensive efforts have been done in research related to both diagnosis and treatment, mortality remains third for cancer-related death in the world. Therefore, new diagnostic and prognostic biomarkers for HCC are of paramount significance. MicroRNAs (miRNAs) are a class of small non-coding RNA (19-24 nucleotides) regulating target genes at a post-transcriptional level, which inhibits the mRNA translation or cause the destruction of mRNA. Furthermore, accumulating evidence indicated that single nucleotide polymorphisms (SNPs) in miRNAs may affect the miRNA binding to target gene and, consequently, may result in the alteration of protein expression3,4. Therefore, SNPs within miRNA were related to the risk of various diseases, such as diabetes5 or Parkinson Disease6, in which cancers should be specifically mentioned7,8. Some researches revealed that miR-3152 and miR-449 are involved in cancer17-20. MiR-3152 is up regulated in PDAC (pancreatic ductal adenocarcinoma), and miR-449 is down-regulated or lost in gastric cells, lung adenocarcinoma cells and testicular cancer cells. So, it is possible to connecting these SNPs in miRNAs and HCC to figure out whether relation exists or not. Therefore, this large-scale sample study (498 vs 520) aims at identifying whether rs13299349 in miR-3152 and rs2620381 in miR-449 are associated with susceptibility to HCC. Material and Methods Study subjects In this study, HCC patients diagnosed either by histopathologic or imaging evidence, were enrolled from Shanghai Huashan Hospital and Eastern Hepatobiliary Surgery Hospital. Healthy individuals without any history of neoplasms or other severe diseases were ramdomly recruited as controls from the Taizhou Longitudinal Study. Rev Med Chile 2016; 144: 508-515 Basic characteristics of all enrolled subjects, including age, gender,family history, medication, smoking status, and alcohol intake were collected with questionnaires conducted by well-trained interviewers. For HCC patients, clinicopathologic characteristics and indexes were also collected, such as the number, size, pathologic type/grade and metastasis of the tumor, as well as the serum levels of alpha-fetoprotein (AFP), hepatitis B surface antigen (HBsAg), HBV-DNA, alanine aminotransferase (ALT), aspartate transaminase (AST) and total bilirubin (TB). Informed consent for sample collection and subsequent analysis was obtained from each subject at recruitment. The whole procedure of our study was approved by local ethics review board and fulfilled the international ethical standards. DNA extraction and genotyping assay of SNPs within miRNA Genomic DNA was extracted from peripheral blood with AxyPrepTM Blood Genomic DNA Miniprep Kit (Axygen Biosciences). The technology of nucleic acid electrophoresis and concentration determination were used to ensure the quality and quantity of DNA samples. Genotyping of SNPs in miRNAs was performed by Sequenom- MassARRAY technology. The amplification and extension primers of SNPs, listed in Table 1, were designed by MassARRAY Assay Design Software and synthetized by Invitrogen Corporation Shanghai Representative Office. Sequencing was performed on MassARRAY Compact System (SEQUENOM Corporation) and the data were analyzed by TYPER Analyzer software 4.0. Statistical analysis All statistical analyses were performed using Statistical Package for Social Sciences (SPSS, version 19.0) and Microsoft Excel. Genotype frequencies study and and control groups were assessed for Hardy-Weinberg equilibrium by Pearson chi-square test of goodness-of-fit. Binary logistic regression analysis was conducted to investigate the association between the genotypes/allele types of the miRNA SNPs and the susceptibility to HCC after adjusting for confounders. In subsequent assessment of the relationship between the genotypic/ allelic frequencies of miRNA SNPs and qualitative clinicopathologic characteristics in HCC


Abril 2016
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