Korean J Physiol Pharmacol.  2019 May;23(3):171-179. 10.4196/kjpp.2019.23.3.171.

MicroRNA-186 targets SKP2 to induce p27(Kip1)-mediated pituitary tumor cell cycle deregulation and modulate cell proliferation

• He Z
• Chen L
• Wang Q
• Yin C
• Hu J
• Hu X
• Fei F
• Tang J

• KMID: 2443605
• DOI: http://doi.org/10.4196/kjpp.2019.23.3.171

Abstract

Pituitary tumors are usually benign but can occasionally exhibit hormonal and proliferative behaviors. Dysregulation of the G1/S restriction point largely contributes to the over-proliferation of pituitary tumor cells. F-box protein S-phase kinase-interacting protein-2 (SKP2) reportedly targets and inhibits the expression of p27(Kip1), a well-known negative regulator of G1 cell cycle progression. In this study, SKP2 expression was found to be upregulated while p27(Kip1) expression was determined to be downregulated in rat and human pituitary tumor cells. Furthermore, SKP2 knockdown induced upregulation of p27(Kip1) and cell growth inhibition in rat and human pituitary tumor cells, while SKP2overexpression elicited opposite effects on p27(Kip1) expression and cell growth. The expression of microRNA-186 (miR-186) was reported to be reduced in pituitary tumors. Online tools predicted SKP2 to be a direct downstream target of miR-186, which was further confirmed by luciferase reporter gene assays. Moreover, miR-186 could modulate the cell proliferation and p27(Kip1)-mediated cell cycle alternation of rat and human pituitary tumor cells through SKP2. As further confirmation of these findings, miR-186 and p27(Kip1) expression were downregulated, while SKP2 expression was upregulated in human pituitary tumor tissue samples; thus, SKP2 expression negatively correlated with miR-186 and p27(Kip1) expression. In contrast, miR-186 expression positively associated with p27(Kip1) expression. Taken together, we discovered a novel mechanism by which miR-186/SKP2 axis modulates pituitary tumor cell proliferation through p27(Kip1)-mediated cell cycle alternation.

Keyword

Cell proliferation; Cyclin-dependent kinase inhibitor p27; microRNA-186; Pituitary neoplasms; S-phase kinase-interacting protein-2

MeSH Terms

Animals
Cell Cycle*
Cell Proliferation*
Cyclin-Dependent Kinase Inhibitor p27
Genes, Reporter
Humans
Luciferases
Pituitary Neoplasms*
Rats
Up-Regulation
Cyclin-Dependent Kinase Inhibitor p27
Luciferases

Figure

• 

  Fig. 1 The expression and combined function of SKP2 and p27Kip1 in pituitary tumor cell lines. (A, B) SKP2 expression in rat GH3 and human GH-secreting PA cells was regulated by transfection with si-SKP2 or SKP2 overexpressing vectors, as confirmed by immunoblot assays. Negative control small RNA interference (si-NC) or an empty vector (NC) served as a control. (C, D) Rat GH3 and human GH-secreting PA cells were transfected with si-SKP2 or SKP2 overexpressing vectors, and then DNA synthesis was examined using BrdU assays. (E, F) Cell proliferation was determined using CCK-8 assays. (G) Rat GH3 and human GH-secreting PA cells were cotransfected with si-SKP2 and si-p27Kip1, and then cell cycle analysis was performed using flow cytometer assays. The data are presented as the mean ± standard deviation of three independent experiments. *p < 0.05 and **p < 0.01, compared to the si-NC or SKP2 overexpression vector group. si, small interfering; NC, negative control; GH, growth hormone; PA, pituitary adenoma; BrdU, 5-bromo-2′-deoxyuridine; OD, optical density.

• 

  Fig. 2 SKP2 is a direct target of miR-186, which negatively regulates SKP2 expression. (A) Rat GH3 and human GH-secreting PA cells were transfected with miR-186 mimics or inhibitors to regulate miR-186 expression, as confirmed using real-time PCR. (B) SKP2 mRNA expression in response to miR-186 overexpression or inhibition in rat GH3 and human GH-secreting PA cells was examined using real-time PCR. (C) SKP2 protein levels under the same conditions were examined using immunoblot assays. (D) Luciferase reporter gene assays were performed using wt-SKP2 3′-UTR or mut-SKP2 3′-UTR vectors to verify the direct binding between miR-186 and SKP2 3′-UTR. The data are presented as mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01. miR, microRNA; mRNA, messenger RNA; GH, growth hormone; PA, pituitary adenoma; PCR, polymerase chain reaction; UTR, untranslated region; NC, negative control.

• 

  Fig. 3 The miR-186 modulates pituitary tumor cell proliferation and the cell cycle through SKP2. (A) Rat GH3 and human GH-secreting PA cells were transfected with miR-186 mimics or inhibitors, and then, DNA synthesis was examined using BrdU assays. (B) Cell proliferation using CCK-8 assays. (C) Cell cycle analysis using flow cytometer assays. (D, E) Rat GH3 and human GH-secreting PA cells were cotransfected with miR-186 mimics and SKP2 overexpressing vectors, and then examined for the protein levels of SKP2 and p27Kip1 were examined using Immunoblot assays. (F) DNA synthesis was examined using BrdU assays. (G) Cell proliferation was examined using CCK-8 assays. The data are presented as the mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, compared to the control group; ##p < 0.01, compared to the SKP2 group. miR, microRNA; GH, growth hormone; PA, pituitary adenoma; BrdU, 5-bromo-2′-deoxyuridine; UTR, untranslated region; NC, negative control; OD, optical density.

• 

  Fig. 4 The expression of and correlations among miR-186, SKP2 and p27Kip1 in human tissue samples. (A–C) The mRNA expression of miR-186, SKP2 and p27kip1 in 5 normal human pituitary tissues and 20 human GH-producing pituitary tumor tissues was examined using real-time PCR. The data are presented as the mean ± standard deviation of three independent experiments. **p < 0.01. The protein levels of p27kip1 in normal pituitary tissues and pituitary tumor tissues were determined using immunoblot assays (D) and immunohistochemistry staining (E), respectively. The red arrows indicated p27kip1 positive cells. (F–H) The correlations among miR-186, SKP2 and p27Kip1 in tissue samples were analyzed using Spearman's rank correlation. mRNA, messenger RNA; GH, growth hormone; PCR, polymerase chain reaction.

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