Demethylation of TIMP2 and TIMP3 inhibit cell proliferation, migration and invasion in pituitary adenoma

Background: Pituitary adenoma (PA) is one of the most common intracranial neoplasms. Tissue inhibitors of metalloproteinases (TIMPs) are prognostic biological markers, but their biological roles remains largely unclear in invasive PA. Methods: The promoter methylation status of TIMP2 and TIMP3 genes in invasive PA tissues and cells was measured by methylation-specic polymerase chain reaction (MSP). The expression of TIMP1-3 was validated by quantitative real time PCR and western blot analysis. Overexpression and knockdown of TIMP2 and TIMP3 in GH3 cells were created by transfection of pcDNA3.0 and siRNA against TIMP2 and TIMP3, respectively. Functional experiments in GH3 cells were performed with CCK-8 assay, wound healing assay and transwell assay. Effects of 5-Azacytidene (5-AzaC) on the methylation of TIMP2 and TIMP3 gene, and DNA methyltransferase 1 (DNMT1), DNMT3a and DNMT3b were determined by western blot analysis. Results: We found the expression of TIMP1, TIMP2 and TIMP3 was down-regulated in invasive PA tissues and cells. Moreover, decreased TIMP2 and TIMP3 expression was closely associated with their promoter methylation. The in vitro experiments showed that overexpression of TIMP2 and TIMP3 exerted suppressive effects, while knockdown of TIMP2 and TIMP3 presented enhanced effects on cell proliferation, migration and invasion in PA cells. Furthermore, 5-AzaC treatment concomitantly up-regulated the protein levels of TIMP2, TIMP3, DNMT1, DNMT3a and DNMT3b. Conclusions: In conclusion, our results support that DNA methylation at least partly accounts for TIMP2 or TIMP3 silencing in invasive PA, which will provide new insights into the mechanisms underlying the function of TIMPs in PA


Introduction
Pituitary adenoma (PA), accounting for 10 to 15% of intracranial tumors, is one of the most common intracranial neoplasms with an overall prevalence of about 20% in the general population (1)(2)(3).Two major symptoms (endocrine-related and tumor occupying symptoms) have been reported to be associated with PA, which could cause some side effects, including mood disorders, sexual dysfunction, obesity, visual disturbances, and diabetes mellitus (4,5).It was estimated that invasive PA is composed of approximately 30% of PA, which is characterized as tumor invading the adjacent sphenoid sinus and cavernous sinus (5,6).Since complete resection is di cult for extensive local invasion, better understanding the molecular mechanisms underlying invasive PA is urgently needed for early diagnosis and treatment (7) Matrix metalloproteinase (MMPs), a family of zinc-containing endopeptidases that can degrade the natural extracellular matrix and basement membrane, allows for the growth and mobility of tumor cells, thereby easily enabling tumor cell proliferation and invasion, including into the PA (8-10).Tissue inhibitors of metalloproteinases (TIMPs) can non-selectively but effectively inhibit the active MMPs (8-10) which have been known to act as tumor suppressor genes in several tumors.TIMP2, a possible antagonist of MMP2, is a metastasis suppressor in the process of invasion of cervical cancer cells (11).TIMP3 is a potential biomarker for predicting the tumor stage and T-status in patients with oral squamous cell carcinoma (12).Overexpression of TIMP3 suppresses cell proliferation, induces apoptosis, and inhibits migration and invasion in hepatocellular carcinoma (13).Additionally, TIMPs also act as functional tumor suppressors in non-small cell lung cancer (14,15), prostate cancer (16), and colorectal cancer (17).
Notably, our interests were aroused by TIMP1-3 which have been reported to be prognostic biological markers in invasive PA (18-20).However, the exact biological functions of TIMP1-3 and underlying mechanisms in invasive PA remains largely unclear.
In recent times, aberrant DNA methylation by repressing some tumor suppressor genes is the most widely investigated cancer epigenetics.This seems to be a common feature of all human tumors (24)(25)(26) Changes of TIMPs expression by methylation have been signi cant in tumor growth, invasion and metastasis.For example, EZH2 inhibits TIMP2 expression via H3K27me3 and DNA methylation, which relieves the repression of MMP and facilitates ovarian cancer invasion and migration (21).A study by Cao et al showed that TIMP3 promoter methylation is positively correlated with gastric cancer risk and could be used as a molecular marker for gastric cancer (22).In addition, female patients exhibited lower methylation levels of MMP-9 but higher methylation levels of TIMP-1 compared to male patients, and the methylation levels of TIMP-1 gradually decreased with age (23).These evidences prompted us to make an assumption that aberrant expression of TIMPs participated in invasive PA cellular function might be associated with transcriptional silence by DNA methylation.
Based on these facts, we rst investigated the expression pattern of TIMPs in PA tissues and the methylation status in clinical samples and cell lines.Moreover, we explored the biological function of TIMPs in PA cell proliferation, migration and invasion by RNA interference or 5-AzacytidineAzacytidine treatment.These preliminary results will give us better insights into the effects of TIMPs on progression of invasive PA and help provide new approaches for the prevention and diagnosis of invasive PA.

Tissues samples
Human tissue specimens were collected from PA patients, including 20 cases of invasive and 20 cases of non-invasive pituitary tumor.Meanwhile, 20 cases of normal pituitary tissues were obtained from the thin layer of pituitary tissue around the ACTH microadenocarcinoma as control group.All samples were obtained from the Second A liated Hospital of Guilin Medical University (Guilin, Guangxi, China).After surgical resection, all the tissue samples were immediately frozen in liquid nitrogen and stored at -80 °C until further analysis.Before tissue collection, all patients were administered the informed consent and this study was approved by the Research Ethics Committee of the Second A liated Hospital of Guilin Medical University.

Quantitative real time PCR
Total RNA was extracted from tissue samples or cell lines using TRIzol reagent (Invitrogen) and reverse transcribed using M-MLV reverse transcriptase (Bio-Rad, Minneapolis, MN, USA)by extension of oligo primers (TaKaRa).Quantitative real time PCR was performed with DBI Bestar ® SybrGreen qPCR Master Mix on a Stratagene Real time PCR (Mx3000P, Agilent).The mRNA expression was calculated by using the 2 −ΔΔCt method and normalized to the expression of GAPDH mRNA.The primer sequences are shown in Table 1.
The DNA samples were treated with sodium bisul te using the EpiTect Bisul te Kit (QiaGen, Hilden, Germany), followed byDNA modi cation by EZ DNA Methylation-Gold Kit (Zymo Research).Bisul te DNA was ampli ed with methylated or unmethylated speci c primer sequences designed by Methyl primer Express v1.0, as listed in Table 2. MSP was used to amplify methylated gene and MSP products were analyzed by 2% agarose gel electrophoresis.Methylated and non-methylated human DNAs were used as positive and negative controls, respectively.

CCK-8 assay
Treated or transfected GH3 cells at a density of 3,000 cells per well were seeded in a 96-well plate and incubated overnight at 37°C.Then, cell proliferation was determined using Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan).Brie y, 10 μL CCK-8 solutions was added to each well at 24, 48, and 72 h, respectively, followed by incubation for 2 h at 37°C.The optical density values were measured using a microplate reader (Bio-Tek, USA) at 450 nm.

Wound healing assay
Wound healing assay was performed to evaluate cell migration ability.Brie y, GH3 cells at a density of 4, 0000 cells per well were seeded in six-well plates and cultured until 90% con uence.Subsequently, a 100-μL sterile pipette tip was used to arti cially create a straight scratch through the monolayer.The images of the same location at 0 and 48 h after wounding in ve randomly selected elds were observed under a microscope (Olympus, Tokyo, Japan).The relative average migration distance was calculated as follows: (Width 0h − Width 48h )/Width 0h ×100%.

Cell invasion assay
For cell invasion assay, approximately 3 × 10 5 cells in serum free medium were added to the upper chamber coated with Matrigel (Millipore) in 24-well transwell insert.Then, 500 μL serum-containing medium was added to the lower chamber.After 48 h incubation, cells that had invaded to the lower chamber were xed and stained with 0.5 % crystal violet.Stained cells were photographed and counted in ve randomly selected elds under a light microscope.

Statistical analysis
Quantitative data were analyzed with SPSS 21.0 software package and expressed as mean ± standard deviation (SD) of at least three repetitive experiments.Comparisons of continuous data were performed using two-tailed t-test for two groups or one-way analysis of variance for multiple comparisons data.The values of p less than 0.05 were considered to be statistically signi cant differences.

Results
The expression and methylation status of TIMP1-3 in PA tissues Quantitative real time PCR was rst performed to determine the expression of TIMP1, TIMP2, and TIMP3 in human PA samples, including 20 cases of invasive, 20 cases of non-invasive pituitary tumor and 20 matched controls.As shown in Figure 1A, the results showed that the expression of TIMP1-3 was downregulated in invasive pituitary tumor and non-invasive pituitary tumor compared with matched controls.Notably, the expression of TIMP1-3 was obviously decreased in invasive pituitary tumor compared with non-invasive pituitary tumor, indicating their associated with tumor metastasis.Representative three pairs of tissue samples were applied to the western blot analysis.Consistently, all the protein levels of TIMP1-3 were presented the same change trend as results from quantitative real time PCR (Figure 1B).To better explain the aberrant expression of TIMP1-3 in PA tissues, the methylation status of CpG sites of TIMP1-3 promoter was predicted online.The predicted results showed only TIMP2 and TIMP3 contained typical CpG islands, which indicated the potential role of promoter CpG methylation in TIMP2 and TIMP3 silencing.Next, we focus the methylation status of TIMP2 and TIMP3 by quantifying the degree of their methylation.MSP assay showed that the methylation percentage of TIMP2 (Figure 1C) and TIMP3 (Figure 1D) in representative three cases of invasive pituitary tumors was 100%, but 0% in non-invasive tumors and matched controls.Taken together, both TIMP2 and TIMP3 expression were decreased, which might be associated with their promoter methylation in invasive pituitary tumors.

Both TIMP2 and TIMP3 exerted suppressive effects on cell proliferation, migration and invasion in PA cells
To further investigate the biological function of TIMP2 and TIMP3, their expression levels were rst determined in several PA cell lines by quantitative real time PCR and western blotting.The results revealed that the expression of TIMP2 and TIMP3 mRNA (Figure 2A-B) and protein (Figure 2C) levels were signi cantly down-regulated in PA cell lines (MMQ, GH1, GH3 and RC-4B/C), in comparison with normal rat pitultary cells.It is noteworthy that TIMP2 and TIMP3 expression was the lowest in GH3 cells among all the PA cell lines.Subsequently, GH3 cells were transfected with siTIMP2, siTIMP3 or pcDNA3.0-TIMP2,pcDNA3.0-TIMP3 to construct TIMP2 and TIMP3 silenced or overexpressed cells.As illustrated in quantitative real time PCR, the mRNA expression of TIMP2 and TIMP3 were both signi cantly reduced after the corresponding siRNA transfection, but elevated after pcDNA3.0transfection in GH3 cells (Figure 2D).Consistently, western blotting showed the same expression levels (Figure 2E).A series of functional experiments, including CCK-8 (Figure 2F), wound healing (Figure 3A) and transwell invasion assays (Figure 3B) showed that knockdown of TIMP2 or TIMP3 signi cantly promoted cell proliferation, migration and invasion.Meanwhile, overexpression of TIMP2 or TIMP3 remarkably suppressed cell proliferation, migration and invasion in GH3 cells.

Demethylation of TIMP2 or TIMP3 inhibited cell proliferation, migration and invasion in PA cells
To analyze whether promoter methylation affected the expression of TIMP2 and TIMP3 in vitro, GH3 cells were treated with 5-AzaC.As demonstrated by MSP, 5-AzaC treatment obviously reduced the methylation status of TIMP2 and TIMP3 in GH3 cells (Figure 4A).As expected, 5-AzaC treatment signi cantly upregulated the expression of TIMP2 (p< 0.001) and TIMP3 (p< 0.01) mRNA levels (Figure 4B) and protein levels (Figure 4C).In addition, western blot demonstrated that 5-AzaC treatment repressed the expression of DNMT1, DNMT3a and DNMT3b (Figure 4C).Subsequently, we investigated whether demethylation of TIMP2 or TIMP3 by 5-AzaC treatment presented similar results with TIMP2 or TIMP3 overexpression.The in vitro functional assay showed 5-AzaC treatment signi cantly inhibited cell proliferation, migration and invasion, as determined by CCK-8 assay (Figure 4D), healing (Figure 4E) and transwell invasion assays (Figure 4F).Collectively, these results demonstrated that ectopic expression of TIMP2 and TIMP3 by demethylation also exerted tumor suppressive effects on PA cell proliferation, migration and invasion.

Discussion
In the present study, we evaluated the expression patterns of TIMP1-3 in patients with PA using quantitative real time PCR and demonstrated that TIMP1-3 was signi cantly down-regulated in invasive PA tissues.Moreover, we rst revealed that downregulation of TIMP2 or TIMP3 was partly due to the promoter DNA methylation status in invasive PA tissues.Similarly, demethylation could reduce methylation of the TIMP2 promoter and nally reactivate TIMP2 transcription to promote ovarian cancer invasion and migration (21).TIMP3 hypermethylation was found in HPV-positive oropharyngeal squamous cell carcinoma (24).A recent study by Maleva Kostovska et al. showed that TIMP3 promoter methylation is an epigenetic marker of BRCA1 ness tumors (25).We focused on the role and methylation status of TIMP2 and TIMP3 in the subsequent experiments because only TIMP2 and TIMP3 contained typical CpG islands in PA tissues.
The in vitro functional assays demonstrated that PA cell proliferation, migration, and invasion were signi cantly enhanced by knockdown TIMP2 or TIMP3, but remarkably suppressed by TIMP2 or TIMP3 overexpression.TIMPs are endogenous regulators of MMPs by inhibiting MMP activity, which are downregulated in many solid tumors, and act as repressors of tumor metastasis (26, 27).For instance, TIMP3 functions as a tumor suppressor in melanoma and negatively regulates several aspects of the metastatic cascade (28).Furthermore, TIMPs are important in the maintenance of extracellular matrix in the human pituitary (29).Invasive PA less signi cantly expresses TIMP-1 and TIMP-2 (36) Invasive PA were signi cantly less expressing TIMP-1 and TIMP-2 (30).Therefore, we speculated that TIMP2 and TIMP3 might act as tumor suppressors in invasive PA cells.
For further con rmation, decreased TIMP2 or TIMP3 levels were correlated with DNA methylation, we used an agent of DNA demethylation, 5-AzaC to treat GH3 cells and analyzed its effects on cell proliferation, migration, and invasion.Consistent with TIMP2 or TIMP3 overexpression, 5-AzaC signi cantly suppressed GH3 cell proliferation, migration, and invasion.We also showed that the expression of DNMT1, DNMT3a and DNMT3b was decreased in 5-AzaC treated GH3 cells accompanied increased TIMP2 and TIMP3.It has been previously reported that inhibition of promoter hypermethylation could cause a marked increase in TIMP3 expression via decreased DNMT1 expression in lung cancer (31).Except for 5-AzaC, a natural secolignan, peperomin E also exhibits potential as a DNA methylation inhibitor to down-regulates DNMT1, 3a, and 3b expression and induces metastatic-suppressor genes, including E-cadherin and TIMP3 promoter hypomethylation in gastric cancer cells (32).Ma

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Figure 2 TIMP2Figure 3 TIMP2Figure 4
Figure 2 (33)l.have support a possible role of DNMT1 and DNMT3a in tumor suppressor gene promoter methylation leading to PA invasion and suggest that inhibition of DNMTs has the potential to become a new therapeutic approach against invasive PA(33).These data further suggest that DNA methylation might be a major cause for downregulation of TIMP2 or TIMP3 and that the inhibition of promoter hypermethylation could effectively promote the tumor suppressive effects of TIMPs in PA cells.In summary, this study, for the rst time, described that the loss of TIMP2 or TIMP3 in invasive PA is partly due to epigenetic hypermethylation at the promoter regions.Restored TIMP2 or TIMP3 by RNA interference or 5-AzaC treatment could promote its suppressive role in PA cell proliferation, migration, and invasion.These ndings will provide new insights into the mechanisms underlying the function of TIMPs in PA.SL conceived and designed research, drafted and revised manuscript; YY, FH, XW and CH performed experiments; FH and XW analyzed data and prepared gures; All authors approved nal version of manuscript;YY edited andrevised manuscript.