摘要
目的:探讨miR-31对皮肤鳞状细胞癌(鳞癌)生长的影响及作用机制。方法:在鳞癌细胞中转染miR-31的2'-羟基甲基化的反义寡核苷酸(antisense oligonueleotide,ASO),运用平板克隆形成和体外成瘤实验检测miR-31对鳞癌生长的影响。运用Western blot印迹及GFP报告基因实验验证miR-31的靶基因。在鳞癌细胞中转染靶基因的siRNA,检测其对细胞生长的影响。最后,运用实时定量PCR以及免疫组织化学检测miR-31和靶基因在鳞癌组织中的水平。结果:转染miR-31反义寡核苷酸(miR-31 ASO)可以抑制克隆数目及体内裸鼠肿瘤体积(P<0.05)。LATS2(large tumor suppressor homolog 2)是miR-31的直接靶基因。抑制LATS2表达后,鳞癌细胞克隆数目增加(P<0.05)。miR-31在鳞癌中高表达,与LATS2的表达呈负相关。免疫组织化学结果也显示LATS2在鳞癌组织中低表达。结论:miR-31通过负调控LATS2抑制鳞癌的生长。因此,miR-31具有作为鳞癌分子治疗靶标的潜能。
Objective：To investigate the roles and regulation mechanism of miR-31 in human cutaneous squamous cell carcino-ma （cSCC） growth. Methods：cSCC cells were transfected with the antisense oligonucleotide （ASO） of miR-31, and the cSCC growth was tested by colony formation and in vivo tumor formation assays. The target gene of miR-31 was validated by Western blot and green fluorescent protein （GFP） reporter assay. The cells were then transfected with the siRNA of the target gene, and the effect of the target gene on cell growth was preformed by colony formation assay. Finally, real-time PCR and immunohistochemistry were used for analy-sis of the expression of miR-31 and its target gene. Results：miR-31 ASO resulted in a low number of cell colonies and small tumor vol-ume （P〈0.05）. Western blot showed that the cells with miR-31 ASO had a higher protein level of large tumor suppressor homolog 2 （LATS2） than the control. The 3＆#39; UTR of LATS2 had a binding site with miR-31, and miR-31 ASO increased the GFP intensity con-trolled by LATS2 3＆#39; UTR, whereas no effect was observed on the mutant LATS2 3＆#39; UTR. Western blot showed that LATS2 siRNA inhib-ited the expression of LATS2 protein by about 80%. Knocking down of LATS2 increased the colony number by about 70%or 1.3-fold in cSCC cells. Real-time PCR showed that miR-31 was overexpressed in most cSCC tissues, compared with normal tissues. An inverse relationship existed between miR-31 and LATS2 expression levels. Immunohistochemistry validated that LATS2 was downregulated in cSCC tissues. Conclusion：miR-31, which functions as an oncogene, promotes cSCC growth by suppressing LATS2 expression. Our da-ta suggest that miR-31 is a potential miRNA-based therapeutic target for cSCC growth.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2014年第9期555-559,共5页
Chinese Journal of Clinical Oncology
关键词
鳞状细胞癌
microRNA
squamous cell carcinoma
fluorescence-based quantitative PCR
