miR-149抑制结直肠癌细胞迁移的分子机制研究

The molecular mechanism of inhibiting effect of miR-149 on the metastasis of human colonic carcinoma cells

目的探讨miR-149对结直肠癌(CRC)细胞增殖、侵袭、迁移及凋亡的影响及其作用机制。方法实时荧光定量PCR(q-PCR)方法检测miR-149在CRC细胞SW620、LS174T和人结肠上皮细胞FHC中的表达水平;荧光素酶分析法验证STAT3与miR-149之间的靶向结合关系;q-PCR和Western blot检测miR-149过表达对CRC细胞中STAT3、p-STAT3 mRNA和蛋白表达的影响。将miR-149 mimics与STAT3过表达质粒单独或者共转染至CRC细胞中,并分为miR-NC组、miR-149 mimics组、miR-149 mimics+pEGFP/STAT3组。采用CCK-8法、Transwell法、细胞划痕法、流式细胞术分别检测各分组CRC细胞的增殖、侵袭、迁移和凋亡情况。结果与正常结肠上皮细胞FHC相比,CRC细胞SW620、LS174T中miR-149表达水平受到抑制(P<0.01)。荧光素酶实验证实,在CRC细胞中STAT3是miR-149的一个直接靶基因。过量表达miR-149抑制STAT3、p-STAT3 mRNA和蛋白的表达,降低CRC细胞增殖能力、侵袭能力以及迁移能力(P<0.01)。同时,过量表达miR-149也可促进CRC细胞的凋亡(P<0.01)。但过表达STAT3可解除miR-149对CRC细胞增殖、侵袭以及迁移能力的抑制作用。结论 miR-149通过靶向STAT3抑制结直肠癌细胞的增殖、侵袭与迁移,同时促进细胞的凋亡。

Objective To investigate the regulation and function of miR-149 in human colorectal cancer cell lines.Methods miR-149 expression patterns were detected in SW620 and LS174 T cell lines using quantitative real-time fluorescence PCR(q-PCR) method.And then,the target gene of miR-149 was explored via luciferase reporter assay.The expressions of STAT3 and p-STAT3 mRNA and proteins in CRC cells were detected by q-PCR and Western blot assay,respectively.miR-149 mimics and pEGFP/STAT3 vector were transfected or co-transformation into CRC cells.CRC cells were divided into miR-NC group,miR-149 mimics group and miR-149 mimics+pEGFP/STAT3 group.Proliferation,migration,invasion and apoptosis were determined by CCK-8 assay,wound-healing assay,transwell assay and flow cytometry,respectively.Results miR-149 expression was down-regulated in SW620 and LS174 T cell lines compared to that of the normal colon epithelial cell FHC detected using q-PCR methods(P<0.01).Then,STAT3 was identified as a direct target gene of miR-149 in CRC cells by luciferase reporter assay.Further studies indicated that the introduction of miR-149 was able to down-regulate the mRNA and proteins expression of STAT3 and p-STAT3,suppress cell proliferation,migration and invasion,and promote apoptosis of CRC cells(P<0.01).However,the over-expression of STAT3 could decrease the inhibiting effect of miR-149 on the proliferation,migration and invasion of CRC cells.Conclusion miR-149 can inhibit proliferation,invasion and migration and promote apoptosis of CRC cells via targeting STAT3.