Zhou Jianda, Liu Rui, Luo Chengqun, Zhou Xiao, Xia Kun, Chen Xiang, Zhou Ming, Zou Qiong, Cao Peiguo, Cao Ke
Department of Plastic and Reconstructive Surgery; Third Xiangya Hospital; Central South University; Changsha City, Hunan, PR China.
Department of Oncoplastic and Reconstructive Surgery; The Affiliated Tumor Hospital of Xiangya Medical School; Changsha City, Hunan, PR China.
Cancer Biol Ther. 2014 Oct;15(10):1340-9. doi: 10.4161/cbt.29821. Epub 2014 Jul 14.
MicroRNA-20a (miR-20a) plays a key role in tumorigenesis and progression. But its function is reverse in different kinds of malignant tumor, and its role and mechanism in cutaneous squamous cell carcinoma (CSCC) remains unclear.
To determine the miR-20a's roles in CSCC and confirm whether LIMK1 is a direct target gene of miR-20a.
First miR-20a and LIMK1 expression levels were detected in six pairs of CSCC tissues and corresponding normal skin by qRT-PCR. Then MTT assays and colony formation assays were performed to evaluate the impact of miR-20a on cell proliferation. In addition, scratch migration assays and transwell invasion assays were performed to check miR-20a's effect on cell metastasis. Since LIMK1 (LIM kinase-1) was predicted as a target gene of miR-20a, the changes of LIMK1 protein and mRNA were measured by western blot and qRT-RCR methods after miR-20a overexpression. Moreover the dual reporter gene assay was performed to confirm whether LIMK1 is a direct target gene of miR-20a. Finally LIMK1 mRNA and miR-20a in other 30 cases of CSCC pathological specimens were determined and a correlation analysis was evaluated.
The miR-20a significantly low-expressed in CSCC tissues compared with that in matched normal tissues while LIMK1 has a relative higher expression. MiR-20a inhibited A431 and SCL-1 proliferation and metastasis. Both of LIMK1 protein and mRNA levels were downregulated after miR-20a overexpression. The dual reporter gene assays revealed that LIMK1 is a direct target gene of miR-20a. Furthermore, qRT-PCR results of LIMK1 mRNA and miR-20a in 30 cases of CSCC pathological specimens showed miR-20a is inversely correlated with LIMK1 expression.
Our study demonstrated that miR-20a is involved in the tumor inhibition of CSCC by directly targeting LIMK1 gene. This finding provides potential novel strategies for therapeutic interventions of CSCC.
微小RNA-20a（miR-20a）在肿瘤发生和进展中起关键作用。但其在不同类型恶性肿瘤中的功能相反，其在皮肤鳞状细胞癌（CSCC）中的作用及机制仍不清楚。
确定miR-20a在CSCC中的作用，并证实LIMK1是否为miR-20a的直接靶基因。
首先通过qRT-PCR检测6对CSCC组织及相应正常皮肤中miR-20a和LIMK1的表达水平。然后进行MTT试验和集落形成试验以评估miR-20a对细胞增殖的影响。此外，进行划痕迁移试验和Transwell侵袭试验以检测miR-20a对细胞转移的作用。由于LIMK1（LIM激酶-1）被预测为miR-20a的靶基因，在miR-20a过表达后，通过蛋白质印迹法和qRT-RCR方法检测LIMK1蛋白和mRNA的变化。此外，进行双荧光素酶报告基因试验以证实LIMK1是否为miR-20a的直接靶基因。最后，测定另外30例CSCC病理标本中的LIMK1 mRNA和miR-20a，并进行相关性分析。
与配对的正常组织相比，CSCC组织中miR-20a显著低表达，而LIMK1表达相对较高。miR-20a抑制A431和SCL-1的增殖和转移。miR-20a过表达后，LIMK1蛋白和mRNA水平均下调。双荧光素酶报告基因试验显示LIMK1是miR-20a的直接靶基因。此外，30例CSCC病理标本中LIMK1 mRNA和miR-20a的qRT-PCR结果显示miR-20a与LIMK1表达呈负相关。
我们的研究表明，miR-20a通过直接靶向LIMK1基因参与CSCC的肿瘤抑制作用。这一发现为CSCC的治疗干预提供了潜在的新策略。
