Single Nucleotide Polymorphisms Of MicroRNA Processing Machinery Genes And Risk Of Colorectal Cancer

Objective: Colorectal cancer(CRC) is one of the most common malignant tumor. The CRC incidence displays a trend of rapid rise in Asian area including China. Micro RNAs(mi RNAs) are RNA molecules with 22 nucleotides that implicated in various biological processes such as embryonic development, cell differentiation, proliferation, apoptosis, cancer development and insulin secretion. Single nucleotide polymorphisms(SNP), a variation that occurs during DNA duplication, is widely distributed in the entire human genome. Mi RNA related single nucleotide polymorphisms(mi R-SNPs), defined as single nucleotide polymorphisms(SNPs) in mi RNA genes, mi RNA binding site and mi RNA processing machinery, are able to modulate mi RNA and target gene expressions so as to influence cancer risk, treatment efficacy and patient prognosis. SNPs of micro RNA processing machinery genes were identified for their association with cancer risk of the tumors in recent studies, but fews had focused on the relationship for SNPs of micro RNA processing machinery genes and CRC. We genotyped 6 mi R-SNPs of mi RNA processing machinery genes including XPO5(rs11077), RAN(rs14035), Dicer(rs3742330), TNRC6B(rs9623117), GEMIN3(rs197412), GEMIN4(rs2740348) in a case-control study to evaluated the impact of these mi R-SNPs on CRC risk, in order to screen the high risk population for CRC susceptibility.Methods:1 Samples: Blood samples for experimental group were collected from 137 CRC patients who received CRC 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 134 healthy controls without cancer history in the Fourth Hospital of Hebei Medical University between October of 2013 and October of 2014.2 DNA extraction: Genomic DNA was extracted immediately with a Wizard Genomic DNA extraction kit(Promega, Madison, WI, USA) from blood samples. Then the DNA was frozen immediately under 4℃ until used.3 DNA amplification and mi R-SNP screening: Polymerase chain reaction(PCR) was performed using a PCR Master Mix Kit according to manufactor’s instructions. The products were confirmed by agarose gel electrophoresis. Sequencing was done by Generay Biotech(Shanghai) Company. The mi R-SNP of the mi RNA processing genes including XPO5(rs11077), RAN(rs14035), Dicer(rs3742330), TNRC6B(rs9623117), GEMIN3(rs197412), GEMIN4(rs2740348) were genotyped using the Polymerase Chain Reaction- Ligase Detection Reaction(PCR-LDR) assay based on the NCBI SNP database(http://www.ncbi.nlm.nih.gov/snp/).4 Statistical analysis: Student’s t-test was used to analysis quantitative data. The χ2 test was used to analyse dichotomous values, such as the presence or absence of smoking, alcohol consumption, obesity and any individual SNP in CRC patients and healthy controls. Statistical analyses were performed using the SPSS 17.0 software(SPSS Inc., Chicago, IL, USA). For all the statistical tests, P<0.05 was considered to indicate a statistically significant difference.Results:1 No statistical difference exist referring to clinical characteristics such as age, gender between CRC patients and heathy controls(P>0.05).2 No statistical difference exist referring to smoking, alcohol consumption, and obesity between CRC patients and healthy controls(P>0.05).3 The genotype distribution frequency for each SNP(rs11077, rs14035, rs9623117, rs2740348) showed no statistical difference between CRC patients and matched controls. Two SNPs of these mi R-SNP were identified for their association with CRC cancer risk by χ2 test. For the rs3742330 located in Dicer genes, the frequencies of genotype AA and AG+GG were 55.5% and 45.5% in patients whereas 37.3% and 62.7% in the controls. The AA genotype carrier of rs3742330 was associated with a 2.09-fold increased risk when compared with that of AG+GG genotype carrier(odds ratio, 2.09; 95% CI, 1.29-3.40; P=0.003). As for the rs197412 located in GEMIN3, the frequencies of genotype TT and CT+CC were 54.7% and 45.3% in the patients, 40.3% and 59.7% in controls. The TT genotype carrier show a significantly increased risk for CRC compared with CT+CC carrier(odds ratio, 1.79; 95% CI, 1.11-2.90; P=0.017).Conclusions:1 For the SNPs rs3742330 targeting in Dicer and rs197412 targeting in GEMIN3, the frequency distribution for each SNP was significant difference between CRC patients and healthy controls. The SNPs for rs3742330 and rs197412 were associated with the cancer risk for CRC development.2 SNPs of mi RNA processing machinery genes can be identified as a prediction of molecular markers of CRC risk.