Zhoushan Putuo District People's Hospital, Zhoushan, 316100, China.
Sci Rep. 2021 Nov 30;11(1):23186. doi: 10.1038/s41598-021-02613-4.
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.
结直肠癌（CRC）发病率和死亡率均较高。目前的研究已经证实，多种 microRNA 多态性与肿瘤易感性相关，但其机制尚不清楚。本研究旨在阐明 rs2682818 多态性如何参与 CRC 的进展。首先，通过定量实时聚合酶链反应评估 CRC 患者不同 rs2682818 多态性基因型（TT 基因型、GG 基因型和 TG 基因型）中 miR-618 的差异表达。其次，将携带 rs2682818（SNP 型）或不携带 rs2682818（野生型）的 miR-168 前体序列的质粒转染至 293T 细胞，以验证 rs2682818 多态性是否影响 miR-618 的表达。再次，通过 CCK-8 测定、流式细胞术测定、Transwell 测定和小鼠异种移植实验来测定 miR-618 在 CRC 中的生物学功能。然后，通过荧光素酶报告实验验证生物信息学工具预测的 miR-618 的候选靶基因。最后，为了解释潜在的分子机制，进行 Western blot 实验以验证通路相关蛋白的差异表达和磷酸化。结果显示，miR-618 在结肠癌中下调，特别是在 rs2682818 GG 纯合基因型的 CRC 患者中。TT 纯合基因型患者中成熟 miR-618 的表达水平较高，这些患者通常具有更长的生存时间。此外，miR-618 模拟物明显损害 CRC 细胞的生长和侵袭能力，并且 miR-618 模拟物也明显促进 CRC 细胞凋亡。我们的荧光素酶实验证实，TIMP1 是 CRC 细胞中 miR-618 的靶标。TIMP1 的敲低也显著抑制了 CRC 的恶性细胞特征，包括恶性生长和侵袭以及抗凋亡。总之，rs2682818 多态性通过影响 CRC 细胞中成熟 miR-618 的表达参与 CRC 的进展，而 miR-618 通过靶向 TIMP1 表达抑制 CRC 的进展。
