Three dysregulated miRNAs control kallikrein 10 expression and cell proliferation in ovarian cancer - PubMed Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Apr 13;102(8):1244-53.
doi: 10.1038/sj.bjc.6605634. Epub 2010 Mar 30.

Three dysregulated miRNAs control kallikrein 10 expression and cell proliferation in ovarian cancer

N M A White  1 , T-F F Chow, S Mejia-Guerrero, M Diamandis, Y Rofael, H Faragalla, M Mankaruous, M Gabril, A Girgis, G M Yousef
Affiliations

Three dysregulated miRNAs control kallikrein 10 expression and cell proliferation in ovarian cancer

N M A White et al. Br J Cancer. .

Abstract

Background: Kallikrein-related peptidases (KLKs) are a family of serine proteases that have been shown to be dysregulated in several malignancies including ovarian cancer. The control of kallikrein genes and their physiological function in cancer is not well understood. We hypothesized that microRNAs (miRNAs) represent a novel mechanism for post-transcriptional control of KLK expression in cancer.

Methods: We first analysed miRNA expression in ovarian cancer in silico. A total of 98 miRNAs were reported to have altered expression in ovarian cancer. Three of these miRNAs were predicted to target KLK10. We experimentally verified the predicted miR-KLK10 interaction using two independent techniques, a luciferase assay with a construct containing the KLK10 3' untranslated region (UTR), pMIR-KLK10, and measuring KLK10 protein levels after transfection with miRNA.

Results: When we co-transfected cells with pMIR-KLK10 and either let-7f, miR-224, or mR-516a, we saw decreased luciferase signal, suggesting that these miRNAs can target KLK10. We then examined the effect of these three miRNAs on KLK10 protein expression and cell growth. Transfection of all miRNAs, let-7f, miR-224, and miR-516a led to a decrease in protein expression and cellular growth. This effect was shown to be dose dependent. The KLK10 protein levels were partially restored by co-transfecting let-7f and its inhibitor. In addition, there was a slight decrease in KLK10 mRNA expression after transfection with let-7f.

Conclusion: Our results confirm that KLKs can be targeted by more than one miRNA. Increased expression of certain miRNAs in ovarian cancer can lead to decreased KLK protein expression and subsequently have a negative effect on cell proliferation. This dose-dependent effect suggests that a 'tweaking' or 'fine-tuning' mechanism exists in which the expression of one KLK can be controlled by multiple miRNAs. These data together suggest that miRNA may be used as potential therapeutic options and further studies are required.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Partial sequence of the 3′UTR region for KLK10 (transcript variant 1). Nucleotide positions refer to GenBank accession # NM_002776. The position of the stop codon is circled. The miRNA predicted target sites are highlighted in grey with the targeting miRNA indicated above each site.
Figure 2
Figure 2
Effect of transfection of hsa-let-7f with pMIR–KLK10 on luciferase activity. Cells were transfected with either pMIR–KLK10, pMIR–KLK10 and has-let-7f precursor molecule, empty vector and has-let-7f precursor molecule, pMIR–KLK10 and a random miR sequence, or the pMIR–KLK10 with has-let-7f and the let-7f inhibitor. There was a decrease in luciferase signal when cells were transfected with pMIR–KLK10 and let-7f. The signal was partially restored when cells were co-transfected with pMIR–KLK10, let-7f, and anti-let-7f. Luciferase signals were normalised with β-gal.
Figure 3
Figure 3
Effect of transfection of miR-224 with pMIR–KLK10 on luciferase activity. Luciferase activity was significantly decreased in OVCAR-3 cells when miR-224 was co-transfected with pMIR–KLK10 (P<0.001). Luciferase signal was partially restored when cells were co-transfected with pMIR–KLK10 and miR-224 and its inhibitor anti-miR-224. *P<0.001.
Figure 4
Figure 4
Effect of transfection of miR-224 and miR-516a on luciferase activity as measured with the pMIR–KLK10. Cells were co-transfected with the empty vector, pMIR and hsa-miR-224 precursor molecule, the empty vector, pMIR and hsa-miR-516a precursor molecule, the cloned vector, pMIR–KLK10 and hsa-miR-224 precursor molecule, pMIR–KLK10 and hsa-miR-516a precursor molecule, or pMIR–KLK10 only. There was a decrease in luciferase activity when cells were co-transfected with pMIR–KLK10 and either miR-224 or miR-516a.
Figure 5
Figure 5
A bar graph showing the effect of miRNA transfection on KLK10 protein level (dashed bars) and cell proliferation (grey bars) in ovarian cancer cells. The transfection of any of the three miRNAs, let-7f, miR-224, and miR-516a decreased both KLK10 protein levels and cellular proliferation in the OVCAR-5 ovarian cancer cell line when compared with untransfected controls. The transfection of miR-18 was used as positive control, as it is known to have an oncogenic effect on cellular proliferation.
Figure 6
Figure 6
A dose curve analysis of the effect of transfected let-7f concentration on KLK10 protein level. Transfection of let-7f was found to inhibit KLK10 protein production in a dose-dependent manner.
Figure 7
Figure 7
The effect of let-7f on KLK10 protein and mRNA expression in the OVCAR-3 cell line. (A) KLK10 protein expression was significantly decreased in cells transfected with let-7f (P<0.01). Protein expression was partially restored when cells were co-transfected with let-7f and its inhibitor anti-let-7f. There was a slight decrease in KLK10 protein expression when cells were transfected with a random sequence miR and miR-17. (B) There was a slight decrease in OVCAR-3 KLK10 mRNA expression when compared with cells transfected with either let-7f, let-7f and anti-let-7f, random sequence miR, or miR-17. *, P<0.01.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC, Chen X, Dreyfuss G, Eddy SR, Griffiths-Jones S, Marshall M, Matzke M, Ruvkun G, Tuschl T (2003) A uniform system for microRNA annotation. RNA 9: 277–279 - PMC - PubMed
    1. Arora A, Guduric-Fuchs J, Harwood L, Dellett M, Cogliati T, Simpson DA (2010) Prediction of microRNAs affecting mRNA expression during retinal development. BMC Dev Biol 10: 1. - PMC - PubMed
    1. Bando Y, Ito S, Nagai Y, Terayama R, Kishibe M, Jiang YP, Mitrovic B, Takahashi T, Yoshida S (2006) Implications of protease M/neurosin in myelination during experimental demyelination and remyelination. Neurosci Lett 405: 175–180 - PubMed
    1. Baudis M, Cleary ML (2001) Progenetix.net: an online repository for molecular cytogenetic aberration data. Bioinformatics 17: 1228–1229 - PubMed
    1. Bayani J, Paliouras M, Planque C, Shan SJ, Graham C, Squire JA, Diamandis EP (2008) Impact of cytogenetic and genomic aberrations of the kallikrein locus in ovarian cancer. Mol Oncol 2: 250–260 - PMC - PubMed

Publication types