Lei K, Xie W, Sun T, Liu Y, Wang X
Department of Urology, First Affiliated Hospital of Nanchang University, Nanchang 330006, China.
Department of Pathology, First Affiliated Hospital of Nanchang University, Nanchang 330006, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2022 May 20;42(5):712-717. doi: 10.12122/j.issn.1673-4254.2022.05.12.
To explore the role of miR-744-5p/CCND1 axis in clear-cell renal cell carcinoma (ccRCC).
We examined the expression levels of miR-744-5p in 65 pairs of ccRCC and adjacent tissue specimens and in 5 ccRCC cell lines and human renal tubular epithelial (HK2) cells using qRT-PCR. The ccRCC cell lines 786-O and OSRC2 were transfected with miR-744-5p mimic, CCND1 mimic, or their negative control mimics, and the changes in cell proliferation, migration, and invasion were evaluated with CCK-8, wound healing, and Transwell assays. The downstream target molecules of miR-744-5p were predicted by bioinformatics analysis, and the expression level of CCND1 in ccRCC cells was verified by qRT-PCR and Western blotting. The relationship between miR-744-5p and CCND1 was further validated by dual luciferase reporter assay, and the role of the miR-744-5p/CCND1 axis in ccRCC was explored by rescue experiments.
MiR-744-5p was significantly downregulated in ccRCC tissues and cell lines (all < 0.05), and its overexpression inhibited the proliferation, migration, and invasion of ccRCC cells (all < 0.05). Bioinformatics analysis and dual luciferase reporter assay showed that CCND1 was a downstream target of miR-744-5p. The results of rescue experiments showed that upregulation of CCND1 could partially reverse the inhibitory effect of miR-744-5p overexpression on ccRCC cell proliferation, migration, and invasion (all < 0.05).
MiR-744-5p inhibits the malignant phenotype of ccRCC cells by targeting CCND1, and the miR-744-5p/CCND1 axis may be a novel target for diagnosis and treatment of ccRCC.
探讨miR-744-5p/CCND1轴在透明细胞肾细胞癌(ccRCC)中的作用。
采用qRT-PCR检测65对ccRCC组织及癌旁组织标本、5种ccRCC细胞系和人肾小管上皮(HK2)细胞中miR-744-5p的表达水平。将ccRCC细胞系786-O和OSRC2分别转染miR-744-5p模拟物、CCND1模拟物或其阴性对照模拟物,采用CCK-8、伤口愈合和Transwell实验评估细胞增殖、迁移和侵袭的变化。通过生物信息学分析预测miR-744-5p的下游靶分子,采用qRT-PCR和蛋白质免疫印迹法验证ccRCC细胞中CCND1的表达水平。通过双荧光素酶报告基因实验进一步验证miR-744-5p与CCND1的关系,并通过挽救实验探讨miR-744-5p/CCND1轴在ccRCC中的作用。
miR-744-5p在ccRCC组织和细胞系中显著下调(均P<0.05),其过表达抑制ccRCC细胞的增殖、迁移和侵袭(均P<0.05)。生物信息学分析和双荧光素酶报告基因实验表明CCND1是miR-744-5p的下游靶标。挽救实验结果显示,上调CCND1可部分逆转miR-744-5p过表达对ccRCC细胞增殖、迁移和侵袭的抑制作用(均P<0.05)。
miR-744-5p通过靶向CCND1抑制ccRCC细胞的恶性表型,miR-744-5p/CCND1轴可能是ccRCC诊断和治疗的新靶点。