A total of 20 human pituitary tumor tissues were collected from patients who had undergone surgery. In additon, 5 normal pituitary tissues were obtained from autopsies in which the deceased had no history of any endocrine abnormalities. Tissues were collected under research protocol approved by Sichuan Provincial People's Hospital of University of Electronic of Science and Technology of China. Informed consent was obtained from all patients or their families. Tissue samples were fixed in 10% formalin or immediately frozen in liquid nitrogen and stored at −70℃ until further use.

The fixed tissue samples were embedded in paraffin and then cut into 4-µm-thick tissue sections. After removing the wax, the sections were incubated in 10% normal goat serum (Beyotime, Haimen, China) for 30 min at 37℃ and then with the anti-p27^Kip1 antibody (ab32034; Abcam, Cambridge, MA, USA), overnight at 4℃. The sections were incubated with a secondary antibody linked with biotin and then with HRP-conjugated anti-biotin (Beyotime) for 30 min at 37℃. Subsequently, the sections were incubated in freshly prepared DAB reagent and then counterstained with hematoxylin (Beyotime). The sections were visualized by light microscopy (Olympus, Tokyo, Japan).

The rat pituitary tumor cell line GH3 and the human embryonic kidney epithelial cell line HEK293 were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). HEK293 cells were cultured in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). GH3 cells were cultured in Ham's F12K medium enriched with 15% horse serum and 2.5% FBS, as previously described [25]. All cells were cultured at 37℃ with 5% CO₂.

Human primary GH-secreting PA cells were isolated from adenoma tissues as previously described [26]. Adenoma tissues were minced with a scalpel suspended in MEM and treated with collagenase I at a final concentration of 1 mg/ml for 2 h at 37℃. Undissociated tissue was removed using a 200-µm filter. Cells were collected by centrifugation, washed twice and then resuspended in MEM supplemented with 10% FBS.

The expression of p27^Kip1 and SKP2 was regulated by transfection with either small interfering RNA for p27 or SKP2 (si-p27/si-SKP2) or p27/SKP2 overexpressing vectors (GeneCopoecia, Guangzhou, China). The expression of miR-186 was controlled by transfection with miR-186 mimics or inhibitors (GenePharma, Shanghai, China). Cell transfection was performed using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA).

Total RNA was extracted using the TRIzol reagent (Invitrogen) following the manufacturer's protocol. SYBR green PCR Master Mix (Qiagen, Hilden, Germany) was used to detect messenger RNA (mRNA) expression following the manufacturer's protocol. GAPDH expression was used as an endogenous control. miRNA expression was determined using a Hairpin-it TM miRNAs qPCR kit (Genepharma) with RNU6B serving as an endogenous control. The 2^−ΔΔCT method was used to analyze the relative fold changes.

Target cells were lysed using RIPA buffer supplemented with 1% phenylmethylsulfonyl fluoride. The proteins were extracted and analyzed to determine their concentration using the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, China). Proteins were then loaded onto a SDS-PAGE minigel and transferred onto a PVDF membrane. Afterward, the membrane was probed with the following antibodies at 4℃ overnight: anti-p27^Kip1 (ab32034), anti-SKP2 (ab68455, Abcam) and anti-tubulin (ab6160, Abcam). Next, the blots were incubated with the horseradish peroxidase-conjugated secondary antibody immunoglobulin G (cat. P0448; Dako, Milano, Italy). Signal visualization was conducted using ECL Substrates (Millipore, Billerica, MA, USA) with tubulin serving as an endogenous protein for normalization. Gray intensity analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

A Cell Counting Kit-8 (CCK-8; Beyotime) was used to determine the cell proliferation rate. Transfected cells were seeded in 96-well culture plates at a density of 0.5 × 10⁴ cells/well, and the plates were incubated for 24, 48, 72 h. 1 h before the endpoint, 10 µl CCK-8 reagents was added into each well. The valus of optical density at 490 nm were measured using a microplate reader (Bio-Rad, Hercules, CA, USA).

The DNA synthesis in proliferating cells was determined by measuring 5-bromo-2′-deoxyuridine (BrdU) incorporation. BrdU assays were conducted at 24 and 48 h after transfection. Cells were seeded in 96-well culture plates at a density of 2 × 10³ cells/well, cultured for 24 or 48 h, and then incubated with a 10 µM solution of BrdU (BD Pharmingen; San Diego, CA, USA) for 2 h. After the incubation, the medium was removed, and the cells were fixed for 30 min at room temperature, incubated with peroxidase-coupled anti-BrdU antibody (Sigma-Aldrich, St. Louis, MO, USA) for 60 min at room temperature, washed three times with phosphate-buffered saline (PBS), incubated with peroxidase substrate (tetramethylbenzidine) for 30 min, and then the 450 nm absorbance values were measured for each well. Background BrdU immunofluorescence was determined in cells not exposed to BrdU but stained with the BrdU antibody.

For the cell cycle analysis, cells were washed two times with ice-cold PBS, resuspended in 1 ml of ice-cold 70% ethanol with gentle vortexing and incubated at 4℃ overnight. The following day, the cells were washed with 1 × PBS and resuspended in buffer containing 10 µg/ml of propidium iodide, 10 µg/ml of RNase A and 0.1% Triton X-100. After incubating for 30 min at room temperature in the dark, the DNA content in the cells was measured using a flow cytometer (Guava easyCyte 8HT), and the data were analyzed using InCyte software.

Data were processed using SPSS ver. 17.0 statistical software (SPSS Inc., Chicago, IL, USA) and presented as the mean ± standard deviation of results from at least three independent experiments. Student's t-test was used for statistical comparison between means where applicable. Differences among more than two groups in the above assays were estimated using one-way ANOVA. A p-value less than 0.05 was considered statistically significant.

To assess the role of SKP2 and p27^Kip1, we achieved SKP2 expressionby transfection with either si-SKP2 or SKP2 overexpressing vectors, as confirmed by immunoblotting (Fig. 1A, B). In rat GH3 and human GH-secreting PA cells, si-SKP2 increased p27^Kip1 protein levels, while p27^Kip1 protein levels were reduced by SKP2 overexpression (Fig. 1A, B).

The two cell lines transfected with either si-SKP2 or SKP2-overexpressing vectors were then examined for their DNA synthesis capacity and cell proliferation. As shown in Fig. 1C–F, SKP2 overexpression significantly promoted DNA synthesis and cell proliferation in both cell lines, while SKP2 knockdown resulted in opposite effects. Cell cycle alternation was examined. As shown in Fig. 1E, SKP knockdown induced cell cycle arrest in the G0/G1 phase, while SKP2 overexpression resulted in opposite effects. Importantly, the effects of SKP2 knockdown could be partially attenuated by the p27^Kip1 knockdown. The above findings indicate that SKP2 modulates the proliferation and cell cycle of rat and human pituitary tumor cells via through p27^Kip1.

Rat GH3 and human GH-secreting PA cells were transfected with miR-186 mimics or inhibitors to achieve miR-186 expression, as confirmed using real-time PCR (Fig. 2A). The mRNA expression and protein levels of SKP2 were significantly upregulated by miR-186 inhibition and downregulated by miR-186 overexpression in both cell lines (Fig. 2B, C).

To confirm direct binding of miR-186 to the 3′-untranslated region (UTR) of SKP2, two different SKP2 3′-UTR luciferase reporter gene vectors were constructed: wt-SKP2 3′-UTR containing the wild-type SKP2 3′-UTR with the predicted miR-186 binding site and mut-SKP2 3′-UTR containing a mutation in the predicted miR-186 binding site within the SKP2 3′-UTR (Fig. 2D). HEK293 cells were cotransfected with these two vectors and either miR-186 mimics or miR-186 inhibitors and then examined for luciferase activity. As shown in Fig. 2D, the luciferase activity of the wt-SKP2 3′-UTR was significantly suppressed by miR-186 mimics and while enhanced by miR-186 inhibitors. Importantly, mutation of the putative miR-186 binding site eliminated these effects on the luciferase activity.

After confirming that SKP2 is a direct downstream target of miR-186, functional experiments were performed. Rat GH3 and human GH-secreting PA cells were transfected with either miR-186 mimics or inhibitors, and then DNA synthesis capacity, cell proliferation, and cell cycle analyses were performed. The DNA synthesis capacity and proliferation of the two pituitary tumor cell lines were significantly suppressed by miR-186 overexpression and promoted by miR-186 inhibition (Fig. 3A, B). Overexpression miR-186 consistently induced cell cycle arrest of both two cell lines at G0/G1 phase, while miR-186 inhibition resulted in opposite effect (Fig. 3C).

To gain insight into the molecular mechanism behind these effects, we investigated the role of SKP2 in miR-186 function in pituitary tumor cell lines. Rat GH3 and human GH-secreting PA cells were cotransfected with SKP2 vector and miR-186 mimics, and then, the protein levels of SKP2 and p27^Kip1 and the DNA synthesis capacity were examined. MiR-186 overexpression remarkably reduced the SKP2 protein level and increased the p27^Kip1 protein level, while SKP2 overexpression resulted in opposite effects. Importantly, the effect of miR-186 overexpression could be partially attenuated by SKP2 overexpression (Fig. 3D). Consistently, the DNA synthesis capacity and proliferation of rat GH3 and human GH-secreting PA cells was suppressed by miR-186 overexpression and enhanced by SKP2 overexpression; the effects of miR-186 overexpression could be partially reversed by SKP2 overexpression (Fig. 3F, G), suggesting that miR-186 modulates the proliferation of pituitary tumor cell lines via SKP2 and downstream p27^Kip1.

To further confirm the above findings, the expression of miR-186, SKP2 and p27^Kip1 were examined in 5 normal pituitary tissues and 20 GH-producing pituitary tumor tissues. As shown in Fig. 4A–C, miR-186 and p27^Kip1 mRNA expression were significantly downregulated while SKP2 expression was upregulated in tumor tissues. Moreover, immunoblotting and IHC results showed that the p27^Kip1 protein levels were also lower in pituitary tumor tissues (Fig. 4D–E). Spearman's rank correlation analysis showed that SKP2 expression was negatively correlated with miR-186 and p27^Kip1 expression and that miR-186 expression was positively correlated with p27^Kip1 expression (Fig. 4F–H).