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. 2021 Aug 17;11(8):2417.
doi: 10.3390/ani11082417.

MicroRNA-200b Regulates the Proliferation and Differentiation of Ovine Preadipocytes by Targeting p27 and KLF9

Xiayang Jin  1 , Jiqing Wang  1 , Jiang Hu  1 , Xiu Liu  1 , Shaobin Li  1 , Yujie Lu  1 , Huimin Zhen  1 , Mingna Li  1 , Zhidong Zhao  1 , Yuzhu Luo  1
Affiliations

MicroRNA-200b Regulates the Proliferation and Differentiation of Ovine Preadipocytes by Targeting p27 and KLF9

Xiayang Jin et al. Animals (Basel). .

Abstract

MicroRNAs (miRNAs) are crucial regulatory molecules in lipid deposition and metabolism. However, the effect of miR-200b on the regulation of proliferation and adipogenesis of ovine preadipocytes is unknown in the sheep (Ovis aries). In this study, the expression profiles of miR-200b were investigated in the seven tissues of Tibetan ewes and differentiated preadipocytes. The effect of miR-200b, as well as its target genes p27 and KLF9, on the proliferation of ovine preadipocytes and adipogenesis was also investigated, using cell viability analysis, EdU staining, Oil Red O staining and reverse transcription-quantitative PCR (RT-qRCR). The miR-200b was expressed in all the tissues investigated, and it was highly expressed in lung, liver, subcutaneous adipose and spleen tissues. The expression of miR-200b continuously decreased when the differentiation of ovine preadipocytes initiated. The miR-200b mimic dramatically accelerated the proliferation but inhibited differentiation of ovine preadipocytes. The miR-200b inhibitor resulted in an opposite effect on the proliferation and differentiation of ovine preadipocytes. The dual luciferase reporter assay results showed that miR-200b mimic significantly decreased the luciferase activity of p27 and KLF9 in HEK293 cells transfected with wild-type dual luciferase reporter vectors. This suggests that p27 and KLF9 are the target genes of miR-200b. In over-expressed-p27 preadipocytes, the number of EdU-labeled preadipocytes and the expression levels of proliferation marker genes CDK2, CDK4, CCND1 and PCNA significantly decreased. In addition, the transfection of over-expressed-KLF9 vector into adipocytes remarkably increased the accumulation of lipid droplets and the expression levels of differentiation marker genes aP2, PPARγ, LPL and GLUT4. These results suggest that miR-200b accelerated the proliferation but inhibited the adipogenic differentiation of ovine preadipocytes by targeting p27 and KLF9, respectively.

Keywords: KLF9; differentiation; miR-200b; ovine preadipocytes; p27; proliferation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The expression profiles of miR-200b and PPARγ. (A) The expression level of miR-200b in the ovine seven different tissues. The expression levels of PPARγ (B) and miR-200b (C) during ovine preadipocyte differentiation. The values are shown as mean ± SD (n = 4). Values with different lowercase letters are different at p < 0.05.
Figure 2
Figure 2
The effects of miR-200b on the proliferation of ovine preadipocytes transfected with miR-200b mimic, miR-200b inhibitor and negative control (NC). (A) The transfection efficiency of miR-200b in ovine preadipocytes on day 2 after transfection. (B) The proliferation activity of ovine preadipocytes was analyzed using CCK-8. (C,E) The EdU analysis of ovine preadipocytes. The images of DAPI and EdU reflect the total number of preadipocytes and the number of EdU-labeled preadipocytes, respectively. The image of Merged reflects the proportion of EdU-labeled preadipocytes in the total preadipocytes. (D,F) The proportion of EdU-labeled preadipocytes in the total preadipocytes. The values represent mean ± SD (n = 3), ** p < 0.01.
Figure 3
Figure 3
The expression levels of CDK2, CDK4, CCNB1, CCND1, PCNA and p53 in ovine preadipocytes on day 2 after the transfection with miR-200b mimic, miR-200b inhibitor and NC. The values represent mean ± SD (n = 3), ** p < 0.01.
Figure 4
Figure 4
The effect of miR-200b on ovine adipocyte differentiation on day 8 after transfection with miR-200b mimic, miR-200b inhibitor and negative control (NC). (A) The transfection efficiency of miR-200b. (B,C) The Oil Red O staining results of ovine adipocytes. (D,E) The area of Oil Red O staining counted by the ImageJ software. The values represent mean ± SD (n = 3), * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
The expression levels of aP2, PPARγ, FASN, GLUT4, LPL and C/EBPβ in ovine adipocytes on day 8 after transfection with miR-200b mimic, miR-200b inhibitor and NC. The values represent mean ± SD (n = 3), * p < 0.05, ** p < 0.01.
Figure 6
Figure 6
The miR-200b targets p27 and KLF9 in ovine preadipocytes. (A) The structural diagram of wild-type and mutant-type dual luciferase reporter vectors. (B,D) The luciferase activity was analyzed when miR-200b mimic, NC and wild-type or mutant-type dual luciferase reporter vectors were co-transfected into HEK293 cells. (C) The expression level of p27 was detected in preadipocytes on day 2 after the transfection of miR-200b mimic, miR-200b inhibitor, and NC. (E) The expression level of KLF9 in adipocytes on day 8 after transfecting with miR-200b mimic, miR-200b inhibitor, and NC. The values represent mean ± SD (n = 3), * p < 0.05, ** p < 0.01.
Figure 7
Figure 7
The effect of p27 on ovine preadipocyte proliferation when pcDNA3.1-p27, si-p27 and their NC groups were transfected into preadipocytes. (A) The expression level of p27. (B) The expression levels of CDK2, CDK4, CCND1 and PCNA. (C,D) The EdU staining result. (E,F) The percentage of EdU positive preadipocytes counted by the ImageJ software. The values represent mean ± SD (n = 3), ** p < 0.01.
Figure 8
Figure 8
The effect of KLF9 on ovine adipocyte differentiation when pcDNA3.1-KLF9, si-KLF9 and their NC were transfected into differentiated preadipocytes. (A) The expression level of KLF9. (B) The expression levels of aP2, PPARγ, LPL and GLUT4. (C,D) The Oil Red O staining results. (E,F) The relative area of Oil Red O staining was counted using the ImageJ software. The values represent mean ± SD (n = 3), ** p < 0.01.
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References

    1. Wood J.D., Enser M., Fisher A.V., Nute G.R., Sheard P.R., Richardson R.I., Hughes S.I., Whittington F.M. Fat deposition, fatty acid composition and meat quality: A review. Meat Sci. 2008;78:343–358. doi: 10.1016/j.meatsci.2007.07.019. - DOI - PubMed
    1. Fernandez X., Monin G., Talmant A., Mourot J., Lebret B. Influence of intramuscular fat content on the quality of pig meat-1. Composition of the lipid fraction and sensory characteristics of m.Longissimus lumborum. Meat Sci. 1999;53:59–65. doi: 10.1016/S0309-1740(99)00037-6. - DOI - PubMed
    1. Hausman D.B., DiGirolamo M., Bartness T.J., Hausman G.J., Martin R.J. The biology of white adipocyte proliferation. Obes. Rev. 2001;2:239–254. doi: 10.1046/j.1467-789X.2001.00042.x. - DOI - PubMed
    1. Li M.X., Sun X.M., Cai H.F., Sun Y.J., Plath M., Li C.J., Lan X.Y., Lei C.Z., Lin F.P., Bai Y.Y., et al. Long non-coding RNA ADNCR suppresses adipogenic differentiation by targeting miR-204. Biochim. Biophys. Acta. 2016;1859:871–882. doi: 10.1016/j.bbagrm.2016.05.003. - DOI - PubMed
    1. Hilton C., Karpe F., Pinnick K.E. Role of developmental transcription factors in white, brown and beige adipose tissues. Biochim. Biophys. Acta. 2015;1851:686–696. doi: 10.1016/j.bbalip.2015.02.003. - DOI - PubMed