Abstract
Colorectal carcinoma (CRC) has a high morbidity and mortality. Current studies have confirmed a variety of microRNA polymorphisms were associated with tumor susceptibility, however, the mechanisms are still unknown. In this study, we were aimed to clarify how polymorphism rs2682818 participated in the progression of CRC. First of all, the differential expression of miR-618 was assessed by quantitative real-time polymerase chain reaction in CRC patients with different genotypes of polymorphism rs2682818, including homozygous (TT) genotype, homozygous (GG) genotype and heterozygous (TG) genotype. Secondly, plasmids carried miR-168 precursor sequences harboring rs2682818 (SNP type) or without rs2682818 (wild type) were transfected into 293T cells to verify that polymorphism rs2682818 affected miR-618 expression. Thirdly, CCK-8 assay, flow cytometry assay, transwell assay and mouse xenograft assay were performed to measure the biological functions of miR-618 in CRC. Fourthly, the candidate target genes of miR-618 which were predicted by bioinformatics tools were verified by luciferase reporter assay. Finally, in order to explain the potential molecular mechanisms, western blotting was performed to demonstrate the differential expression and phosphorylation of pathway related proteins. The results showed that miR-618 was down-regulated in colon cancer, especially in CRC patients with rs2682818 GG homozygous genotype. Higher expression of mature miR-618 occurred in patients with TT homozygous genotype, and these patients usually had a longer survival time. Moreover, miR-618 mimic obviously impaired the growth and invasion ability of CRC cells, and miR-618 mimic also remarkably promoted CRC cell apoptosis. Our luciferase experiments confirmed that TIMP1 was a target of miR-618 in CRC cells. Knockdown of TIMP1 also significantly inhibited the malignant cytological features of CRC, including malignant growth and invasion as well as apoptosis resistance. In summary, polymorphism rs2682818 participated in the progression of CRC via affecting the expression of mature miR-618 in CRC cells, and miR-618 inhibited the progression of CRC via targeting TIMP1expression.
Highlights
Colorectal carcinoma (CRC) has a high morbidity and mortality
We found that polymorphism rs2682818 was associated with CRC risk, and the survival of CRC patients with rs2682818 TT homozygous allele were significantly better than those patients with TG heterozygous genotype or GG homozygous genotype
Survival analysis were performed to assess the impact of polymorphism rs2682818 in CRC patients, our result revealed that patients with GG homozygous or TG heterozygous genotype had a worse overall survival compared with those patients with TT homozygous genotype (Fig. 1a)
Summary
Colorectal carcinoma (CRC) has a high morbidity and mortality. Current studies have confirmed a variety of microRNA polymorphisms were associated with tumor susceptibility, the mechanisms are still unknown. Studies have confirmed that many risk factors are related to the occurrence and progression of CRC, including germline genetic mutations[2], associated diseases[3], environmental exposures[4], lifestyle and dietary factors[5]. Numerous studies have confirmed that microRNAs participate in cancer development and progression, functioning as oncogenes and tumor suppressor genes via interfering with expression of downstream key g enes[10,11,12]. Rs2682818 polymorphism is located on the precursor’s stem-loop of the miR-618 sequence and several studies have demonstrated that rs2682818 is associated with diseases susceptibility, including ischemic stroke and hirschsprung disease[22,23]. It is a very attractive and significant scientific problem to confirm the relationship between rs2682818 polymorphism of miR-618 and colorectal carcinoma susceptibility and explain how is rs2682818 polymorphism involved in the occurrence and progression of colorectal carcinoma
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