Association of polymorphisms at the microRNA binding site of the caprine KITLG 3'-UTR with litter size

Abstract

This study identified three novel single nucleotide polymorphisms (SNPs) (c.1389C > T, c.1457A > C and c.1520G > A) in the caprine KITLG 3'-UTR through DNA sequencing. The three SNP loci were closely linked in Guanzhong dairy (GD) goats. Two alleles of the c.1457A > C SNP introduced two miRNA sites (chi-miR-204-5p and chi-miR-211). Individuals with combined genotype TT-CC-AA had a higher litter size compared with those with combined genotypes CC-AA-GG, TC-CC-GA and CC-AC-GG (P < 0.05). Luciferase assays showed that chi-miR-204-5p and chi-miR-211 suppressed luciferase expression in the presence of allele 1457A compared with negative control (NC) and allele 1457C (P < 0.05). Western blot revealed that KITLG significantly decreased in the granulosa cells (GCs) of genotype AA compared with that in the GCs of genotype CC and NC (P < 0.05). The KITLG mRNA levels of the CC-AA-GG carriers significantly decreased compared with those of the TT-CC-AA, TC-CC-GA and CC-AC-GG carriers. In addition, cell proliferation was reduced in haplotype C-A-G GCs compared with that in haplotype T-C-A GCs. These results suggest that SNPs c.1389C > T, c.1457A > C and c.1520G > A account for differences in the litter size of GD goats because chi-miR-204-5p and chi-miR-211 could change the expression levels of the KITLG gene and reduce GC proliferation.

Figure 1. Sequencing chromatograms of different genotypes at the c.1389C > T, c.1457A > C and c.1520G > A loci.

Figure 2. Characterisation and functional analysis of KITLG 3′-UTR.

(A) Double fluorescein enzyme expression vector linked with KITLG 3′-UTR. (B) Granulosa cells seeded on 24-well plates were transiently co-transfected with psiCHECK-2-KITLG 3′-UTR and miRNA mimics (chi-miR-204-5p and chi-miR-211) or stable negative control (NC). Luciferase activity was measured after 36 h of transfection. Renilla luciferase was normalisedwith firefly luciferase activity, and the mean activities ± standard deviation from three independent experiments are shown. The different small letters represent significant difference at the 5% level.

Figure 3. KITLG expression is suppressed by chi-miR-204-5p in granulosa cells.

(A) Western blot analysis of KITLG expression in CC and AA genotype granulosa cells transfected with either chi-miR-204-5p or negative control (NC). The expression of β-actin was used as a loading control. (B) Densitometric quantification of three Western blot results for KITLG relative to β-actin levels. Data represent the means ± standard deviation of three independent experiments, where an asterisk denotes significance between CC and AA genotype granulosa cells at P < 0.05. Note: The 16 bases inthe 5′region sequence of chi-miR-204-5p are the same as that of chi-miR-211. Thus, chi-miR-204-5p was used in Westernblot analysis.

Figure 4. Comparison of mRNA expression levels of caprine KITLG in granulosa cells among four combined genotypes.

The number of animals of combined genotypes CC-AA-GG, TT-CC-AA, TC-CC-GA and CC-AC-GG were 9, 10, 5 and 6, respectively. The fold change was normalised against the β-actin and GAPDH genes. The different small letters represent significant difference at the 5% level.

Figure 5. Effect of chi-miR-204-5p on granulosa cell viability.

The cells were cultured in chi-miR-204-5p mimics or stable negative control (NC) for 36 h. Granulosa cell viability was measured using the MTT assay (mean ± standard deviation, n = 3 independent culture experiments). Small letters represent significant difference at the 5% level.