Upregulation of MiR-196a promotes cell proliferation by downregulating p27kip1 in laryngeal cancer

Abstract

Background: Accumulating evidence has confirmed that miR-196a plays a critical role in tumorigenesis and tumor progression in a variety of cancers. It has been demonstrated that miR-196a is highly up-regulated in laryngeal cancer by miRNA profiling analysis. However, the functional mechanism of miR-196a in laryngeal cancer remains unclear. This study aims to explore the mechanism of miR-196a in laryngeal cancer.

Methods: In the present study, we conducted qPCR analysis of miR-196a expression in human laryngeal cancer and showed that miR-196a was overexpressed in tumor-derived samples and laryngeal cancer cell lines compared with matched normal controls. Further functional analysis of miR-196a demonstrated that the inhibition of miR-196a could inhibit laryngeal cell-cycle progression and proliferation in vitro. Luciferase reporter assay and western blot confirmed that miR-196a directly targeted p27kip1. Moreover, in order to investigate whether miR-196a regulated cell growth in laryngeal cancer cells by targeting p27kip1, rescue studies were performed in laryngeal cancer cells.

Results: Results showed that overexpression of p27kip1 rescue decreased cell proliferation caused by miR-196a inhibitors. A negative relation between miR-196a and p27kip1 expression in laryngeal cancer tissues were also noted by further analyses.

Conclusions: The present study showed that miR-196a was upregulated in laryngeal cancer and promoted cell proliferation by downregulating p27kip1 in laryngeal cancer. However, further studies are needed to verify this finding.

Keywords: Laryngeal cancer; miR-196a; p27kip1.

Fig. 1

Expression levels of miR-196a in laryngeal tissues and cell lines by real-time PCR analysis. a MiR-196a expression was examined by qRT-PCR in 20 laryngeal tissues and in 20 adjacent noncancerous counterparts (NCs) MiR-196a was significantly higher in laryngeal tissues than their counterparts. b MiR-196a was also up-regulated in Hep-2, TUU212, and SNU899 laryngeal cell lines compared with normal cell line (b). *p < 0.05; **p < 0.01

Fig. 2

Effect of miR-196a on cell proliferation and apoptosis. a The relative expression level of miR-196a in Hep-2 and TU212 cells, transfected with miR-196a negative control (miR-NC) or inhibitors, was tested by qPCR. b and c WST assay was conducted to detect the proliferation of Hep-2 cells and TU212 cells, respectively. d The apoptotic rates of cells were detected by flow cytometry. e The bar chart represents the percentage of cells in G0/G1, S, or G2/M phase, as indicated. *p < 0.05; **p < 0.01

Fig. 3

miR-196a directly targets the p27^kip1 gene. a The luciferase reporter plasmid containing wild-type or mutant p27^kip1 3′-UTR was cotransfected into HEK-293T cells with miR-196a mimic or mimic NC. Luciferase activity was determined by the dual luciferase assay and shown as the relative firefly activity normalized to Renilla activity. b The level of p27^kip1 mRNA was determined by qPCR. c the expression of p27^kip1 protein was analyzed by Western blotting. β-actin was used as control. *p < 0.05; **p < 0.01

Fig. 4

miR-196a regulated cell proliferation through targeting p27^kip1. a, b siRNA of p27^kip1 rescue decreased cell profileration caused by miR-196a inhibitors in Hep-2 (a) and TU212 (b) cell lines, respectively. c An inverse relationship between the expression of p27kip1 and miR-196a was detected