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. 2018 Sep 17;234(2):1522–1533. doi: 10.1002/jcp.27017

Figure 5.

Figure 5

MiR‐24‐3p and MEN1 act in a negative feedback model. (a–d) Mammary epithelial cells were transfected with miR‐24‐3p mimics/mimics NC (mimics/mimics NC; a and c) and/or miR‐24‐3p inhibitor/inhibitor NC (inhibitor/ inhibitor NC; b,d), and cells were harvested for RNA and protein extraction at 24, 48, and 72 hr after transfection. The expression of bovine MEN1 mRNA at 24 hr (a,c) and protein menin in a time course (b,d) were assessed using qRT‐PCR and western blot technology, respectively. Representative WB images of the expression of protein menin at 24, 28, and 72 hr after transfection of indicated miR‐24‐3p are shown. The data are shown as the relative expression levels normalized to the internal control, β‐actin. * p < 0.05. (e,f) Mammary epithelial cells were transfected with pEGFP‐C2‐bMEN1 (bMEN1)/pEGFP‐C2‐Vector (vector) for MEN1 overexpression system (e) and/or bovine MEN1 specific siRNA/nonspecific negative control siRNA (control) for MEN1 low‐expression system (f). Cells were harvested for RNA extraction and then the expression detection of miR‐24‐3p at 24 hr after transfection. The data are shown as the relative expression levels normalized to the internal control, small nuclear (sn) RNA U6. (g,h) The expression of miR‐24‐3p (g) and MEN1 (h) were measured in mammary tissues of dairy cows at dry period stage (n = 3) and peak lactation stage (n = 3). The data are shown as the relative expression levels normalized to the internal controls, small nuclear RNA U6 for miR‐24‐3p expression (g) and β‐actin for MEN1 expression (h). *p < 0.05. (i) MiR‐24‐3p can negatively modulate the expression level of menin in mammary epithelial cells, whereas menin is a positive regulator of miR‐24‐3p expression. MiR‐24‐3p and menin were suggestive of forming a “negative feedback loop” in mammary epithelial cells, dynamically maintaining the metabolic balance in mammary glands. MEN1: multiple endocrine tumor type 1; mRNA: messenger RNA; NC: negative control; qRT‐PCR: quantitative real‐time polymerase chain reaction; siRNA: small interfering RNA