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微小RNA-506通过靶向层粘连蛋白γ1抑制结肠癌细胞的恶性程度。

MicroRNA-506 Inhibits Malignancy of Colorectal Carcinoma Cells by Targeting LAMC1.

作者信息

Zu Caihua, Liu Tao, Zhang Guoliang

机构信息

First Central Clinical College, Tianjin Medical University, Tianjin, China.

Key Laboratory for Critical Care Medicine of the Ministry of Health, Tianjin, China

出版信息

Ann Clin Lab Sci. 2016 Dec;46(6):666-674.

Abstract

OBJECTIVE

To investigate the effects of microRNA-506 (miR-506) on malignancy of colorectal carcinoma (CRC) cells and to elucidate the underlying mechanism.

METHODS

Human colorectal carcinoma cell lines SW480, SW620, HCT116, and HT29 were served as model. Five experimental groups are established in this study, including cell control, pcDNA3 blank vector control, miR-506 over-expression, pSIH1 blank vector control, and miR-506 suppression groups. Quantitative reverse transcription PCR (qRT-PCR) assay was performed to measure miR-506 level. Transwell, Cell counting kit8 (CCK-8), and colony formation assays were performed to detect migration and invasion, viability, and colony formation abilities of CRC cell lines, respectively. Furthermore, bioinformatics method was applied to predict potential target genes of miR-506. Green fluorescent protein (GFP) reporter assays were used to verify the direct regulation of miR-506 on target mRNA in CRC cell lines. The LAMC1 mRNA and protein levels were detected by qRT-PCR and Western blot, respectively.

RESULTS

In the CRC cell lines, miR-506 level increased in the miR-506 over-expression group (P<0.05), compared with the blank vector control group. In the miR-506 over-expression group, cellular viability was significantly reduced (P<0.05). Migrated and invasive cell numbers and cell colony numbers were decreased (P<0.05). LAMC1 mRNA and protein levels in the miR-506 over-expression groups were lower than those in the control groups (P<0.05). However, there were no difference on the above indexes between pSIH1 blank vector control and miR-506 suppression groups.

CONCLUSION

miR-506 acts as a tumor suppressor and inhibits malignancy of colorectal cancer cells through directly targeting LAMC1.

摘要

目的

探讨微小RNA-506(miR-506)对大肠癌细胞恶性生物学行为的影响并阐明其潜在机制。

方法

以人结肠癌细胞系SW480、SW620、HCT116和HT29为模型。本研究设立5个实验组,包括细胞对照组、pcDNA3空载体对照组、miR-506过表达组、pSIH1空载体对照组和miR-506抑制组。采用定量逆转录聚合酶链反应(qRT-PCR)检测miR-506水平。分别采用Transwell实验、细胞计数试剂盒8(CCK-8)法和集落形成实验检测大肠癌细胞系的迁移侵袭能力、活力及集落形成能力。此外,应用生物信息学方法预测miR-506的潜在靶基因。采用绿色荧光蛋白(GFP)报告基因实验验证miR-506对大肠癌细胞系靶mRNA的直接调控作用。分别采用qRT-PCR和蛋白质免疫印迹法检测层粘连蛋白γ1(LAMC1)mRNA和蛋白水平。

结果

与空载体对照组相比,miR-506过表达组大肠癌细胞系中miR-506水平升高(P<0.05)。miR-506过表达组细胞活力显著降低(P<0.05),迁移侵袭细胞数和细胞集落数减少(P<0.05)。miR-506过表达组LAMC1 mRNA和蛋白水平低于对照组(P<0.05)。然而,pSIH1空载体对照组和miR-506抑制组上述指标无差异。

结论

miR-506作为一种肿瘤抑制因子,通过直接靶向LAMC1抑制大肠癌细胞的恶性生物学行为。

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