Zhang Donglei, Yang Ning
Department of Gastroenterology, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China.
J BUON. 2019 May-Jun;24(3):1128-1136.
Colorectal cancer (CRC) is a common malignancy which has a high mortality rate around the world. The advancement of new therapeutic strategies is crucial for the efficient treatment of CRC. Many miRNAs play a central role in the progression of cancer cells. There are still few studies on miR-335-5p and CRC. In this study, the potential of miR-335-5p in CRC development and progression was explored.
The expression level of miR-335-5p in CRC cell lines and tissues was detected by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Cell counting kit-8 (CCK8) assay and colony formation assay were applied for evaluating the ability of cell proliferation. Wound healing assay and Transwell assay were applied for detecting cell migration and invasion ability. Moreover, dual luciferase reporter assay was performed to validate if lactic dehydrogenase B (LDHB) is a downstream target of miR-335-5p. Western blotting was used to estimate the expression of protein.
The expression of miR-335-5p was significantly low in CRC tissues and cells. To investigate the function of miR-335-5p in CRC, two CRC cell lines (HCT-116 and SW620) were selected for further experiments. After transfection with mimics and inhibitor to up-regulate or down-regulate miR-335-5p, it was found that overexpression of miR-335-5p obviously decreased cell proliferation and inhibited migration ability and invasion, while the knockdown of miR-335-5p could obtain the opposite results. Then it was validated in dual luciferase reporter assay that LDHB could be a potential directive target gene of miR-335-5p. Moreover, the rescue assay confirmed that miR-335-5p executed its function as a tumor suppressor gene through targeting LDHB in CRC.
To sum up, the present study demonstrated that miR-335-5p regulates cell proliferation, migration as well as invasion of CRC cells through down-regulating LDHB, and demonstrated that miR-335-5p/LDHB axis may be an underlying therapeutic strategy in CRC.
结直肠癌（CRC）是一种常见的恶性肿瘤，在全球范围内具有较高的死亡率。新治疗策略的进展对于CRC的有效治疗至关重要。许多微小RNA（miRNA）在癌细胞的进展中起着核心作用。关于miR-335-5p与CRC的研究仍然很少。在本研究中，探讨了miR-335-5p在CRC发生发展中的潜力。
通过定量实时聚合酶链反应（qRT-PCR）检测CRC细胞系和组织中miR-335-5p的表达水平。应用细胞计数试剂盒-8（CCK8）检测和集落形成检测评估细胞增殖能力。采用伤口愈合检测和Transwell检测检测细胞迁移和侵袭能力。此外，进行双荧光素酶报告基因检测以验证乳酸脱氢酶B（LDHB）是否为miR-335-5p的下游靶标。使用蛋白质印迹法估计蛋白质的表达。
miR-335-5p在CRC组织和细胞中的表达显著降低。为了研究miR-335-5p在CRC中的功能，选择了两种CRC细胞系（HCT-116和SW620）进行进一步实验。在用模拟物和抑制剂转染以上调或下调miR-335-5p后，发现miR-335-5p的过表达明显降低细胞增殖并抑制迁移能力和侵袭，而敲低miR-335-5p可得到相反的结果。然后在双荧光素酶报告基因检测中验证LDHB可能是miR-335-5p的潜在直接靶基因。此外，挽救实验证实miR-335-5p通过靶向CRC中的LDHB发挥其作为肿瘤抑制基因的功能。
综上所述，本研究表明miR-335-5p通过下调LDHB调节CRC细胞的增殖、迁移和侵袭，并表明miR-335-5p/LDHB轴可能是CRC的一种潜在治疗策略。
