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Abstract
Background
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.
Object
To determine the miR-20a’s roles in CSCC and confirm whether LIMK1 is a direct target gene of miR-20a.
Methods
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.
Results
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.
Conclusion
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.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
This work was supported by National Natural Science Foundation of China (81372140, 81301688, 81272192, 81171882, and 81071645); Ph.D. Programs Foundation of Ministry of Education of China (20130162110050 and 20130162120093); Natural Science Foundation of Hunan Province (Grant No.13JJ4028); Technology Project of Hunan Province (2012SK3229); Project of the Department of Science and Technology of Hunan Province (2013FJ6003); Program for New Century Excellent Talents in University (NCET-11-0527) ; Fundamental Research Funds for the Central Universities (2011JQ028); Post-doctoral Foundation of Central South University (131425) ; 125 Talent Project of the Third Xiangya Hospital of Central South University and Hunan Provincial Innovation Foundation for Postgraduate. Thanks for the kindly giving of SCL-1 cell line from the Dermatology Department of China Medical University.