MiR-145 is involved in the proliferation of bovine mammary epithelial cells and regulates bovine insulin receptor substrate 1

Figures & data

Table 1. Sequences of bta-miR-145 mimic, mimic NC, inhibitor and inhibitor NC.

Products for bta-miR-145	Sequence
mimic	GUCCAGUUUUCCCAGGAAUCCCU
	GGAUUCCUGGGAAAACUGGAC uu
mimic NC	UUCUCCGAACGUGUCACGUTT
	ACGUGACACGUUCGGAGAATT
inhibitor	AGGGAUUCCUGGGAAAACUGGAC
inhibitor NC	CAGUACUUUUGUGUAGUACAA

NC: negative control.

Table 2. Primers for microRNA reverse transcription and real-time qPCR analysis.

Gene^a	Primers (5′–3′)^b	Amplification length, bp
RT-5SrRNA	5′GTCGTATCCAGTGCAGTGCAGGGTTCCGAGGTATTCGCACTGCACTGGATACGACTCCAA3′
RT-bta-miR-145	5′GTCGTATCCAGTGCAGTGCAGGGTTCCGAGGTATTCGCACTGCACTGGATACGACAGGGA3′
5SrRNA	F：GCCGCGTCGGGCCTGGTTAGTAC	67
	R：GTGCAGGGTTCCGAGGT
U6	F：GCTTCGGCAGCACATATACTAAAAT	89
	R：CGCTTCACGAATTTGCGTGTCAT
bta-miR-145	F：GCCGCGTCCAGTTTTCCCAGGAA	68
	R：GTGCAGGGTTCCGAGGT
IRS1	F：TGTTGACTGAACTGCACGTTCT	112
	R：CATGTGGCCAGCTAAGTCCTT
GAPDH	F：GGCAAGTTCAACGGCACA	128
	R：ACCACATACTCAGCACCAGCA

^a RT: reverse transcription primer; IRS1: insulin receptor substrate 1.

^b F: forward; R: reverse.

Figure 1. Cells images without or with bta-miR-145 transfection under fluorescence microscopy. (A) Bovine mammary epithelial cells without transfection (10 × 10). (B) Bovine mammary epithelial cells with bta-miR-145 mimic-FAM transfection(10 × 10). (C) Bovine mammary epithelial cells with bta-miR-145 inhibitor-FAM transfection (10 × 10).

Figure 2. The bta-miR-145 overexpression and inhibition. Overexpression effect is caused by transfecting 150 pmol bta-miR-145 mimic. Inhibition effect is caused by transfecting 300 pmol bta-miR-145 inhibitor. The same as in Figures 3, 7 and 8.

Figure 3. The effect of bta-miR-145 overexpression and inhibition on the cells proliferation. Optical density value indicates the number of live cells. NC means negative control. (A) Overexpression of bta-miR-145 significantly decreases the proliferation of mammary epithelial cells (p < .05). (B) Inhibition of bta-miR-145 significantly increases the proliferation of mammary epithelial cells (p < .05). * indicate significant differences at levels of p < .05.

Figure 4. Phylogenetic tree of bta-mir-145. The bovine, human, mouse, rabbit and swine miR-145 precursor were selected. Bta-mir-145 and bta-mir-143 are in the same cluster.

Figure 5. Two major signalling pathways related to IRS1.

Table 3. Signalling pathway of bta-miR-145.

Category	Term	Count	%
KEGG_PATHWAY	Axon quidance	18	3.0
KEGG_PATHWAY	Endocytosis	21	3.5
KEGG_PATHWAY	MAPK signalling pathway	25	4.1
KEGG_PATHWAY	TGF-beta signalling pathway	12	2.0
KEGG_PATHWAY	Chronic myeloid leukaemia	11	1.8
KEGG_PATHWAY	Colorectal cancer	11	1.8
KEGG_PATHWAY	Adherens junction	10	1.7
KEGG_PATHWAY	Wnt signalling pathway	13	2.2
KEGG_PATHWAY	Pathway in cancer	21	3.5
KEGG_PATHWAY	Pancreatic cancer	8	1.3
KEGG_PATHWAY	Focal adhesion	14	2.3
KEGG_PATHWAY	ErbB signalling pathway	8	1.3
KEGG_PATHWAY	Regulation of actin cytoskeleton	14	2.3
KEGG_PATHWAY	Neurotrophin signalling pathway	9	1.5
KEGG_PATHWAY	Cell cycle	9	1.5
KEGG_PATHWAY	Hypertrophic cardiomyopathy (HCM)	7	1.2
KEGG_PATHWAY	Calcium signalling pathway	11	1.8
KEGG_PATHWAY	Oocyte meiosis	8	1.2
KEGG_PATHWAY	Long-term potentiation	6	1.0
KEGG_PATHWAY	Renal cell carcinoma	6	1.0
KEGG_PATHWAY	Dilated cardiomyopathy	7	1.2

KEGG PATHWAY analysis was based on the predicted target of bta-miR-145.

Figure 6. The two probable binding sites on the bovine IRS1 3’UTR for bta-miR-145. Matching bases were displayed with shadow.

Table 4. The minimum free energy and the secondary structure of the binding sites.

IRS1: insulin receptor substrate 1.

Figure 7. The effect of bta-miR-145 overexpression or inhibition on IRS1 mRNA level. No significant change for IRS1 mRNA expression appeared after bta-miR-145 mimic or inhibitor transfection (p > .05).

Figure 8. Protein greyscale scanning and statistical results of IRS1 by Western blotting. A and C respectively show IRS1 immunoblot in pBMECs after 72 h of transfect with the miR-145 mimic or inhibitor. B and D, respectively, show statistical result of protein relative expression levels. Protein expression was normalised to β-actin. * indicate significant differences at levels of p < .05.