Specificity of miR-378a-5p targeting rodent fibronectin.
One criterion for microRNA identification is based on their conservation across species, and prediction of miRNA targets by empirical approaches using computational analysis relies on the presence of conservative mRNA 3'UTR. Because most miRNA target sites identified are highly conserved across different species, it is not clear whether miRNA targeting is species-specific. To predict miRNA targeting, we aligned all available fibronectin 3'UTRs and observed significant conservation of all 20 species. Twelve miRNAs were predicted to target most fibronectin 3'UTRs, but rodent fibronectin showed potential binding sites specific for five different miRNAs. One of them, the miR-378a-5p, contained a complete matching seed-region for all rodent fibronectin, which could not be found in any other species. We designed experiments to test whether the species-specific targeting possessed biological function and found that expression of miR-378a-5p decreased cancer cell proliferation, migration, and invasion, resulting in inhibition of tumor growth. Silencing fibronectin expression produced similar effects as miR-378a-5p, while transfection with a construct targeting miR-378-5p produced opposite results. Tumor formation assay showed that enhanced expression of fibronectin in the stromal tissues as a background environment suppressed tumor growth, while increased fibronectin expression inside the tumor cells promoted tumor growth. This was likely due to the different signaling direction, either inside-out or outside-in signal. Our results demonstrated that species-specific targeting by miRNA could also exert functional effects. Thus, one layer of regulation has been added to the complex network of miRNA signaling.
Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Canada; School of Public Health, Sun Yat-sen University, Guangzhou, PR China; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
Fengqiong
F
Liu
Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China.
Qing
Q
Lv
Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
William W
WW
Du
Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
Haoran
H
Li
Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
Xiangling
X
Yang
Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
Danyang
D
Liu
Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
Zhaoqun
Z
Deng
School of Public Health, Sun Yat-sen University, Guangzhou, PR China.
Wenhua
W
Ling
Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China. Electronic address: zhangyo@sz.tsinghua.edu.cn.
Yaou
Y
Zhang
Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada. Electronic address: byang@sri.utoronto.ca.
Burton B
BB
Yang
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D005353:Fibronectins / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009024:Morphogenesis / Q000235:genetics; D009361:Neoplasm Invasiveness; D013045:Species Specificity
D005353:Fibronectins; C546119:MIRN378 microRNA, mouse; D035683:MicroRNAs
3′-UTR; 3′-untranslated region; 3′UTR; DMEM; Dulbecco's modified Eagle's medium; FBS; Fibronectin; GFP; Invasion; MicroRNA; Species specificity; fetal bovine serum; green fluorescent protein; miRNA; microRNA; siRNA; small interfering RNA