Introduction: Growth hormone (GH) secreting pituitary adenoma is considered one of the most harmful types of Pituitary Neuroendocrine Tumors (PitNETs). Our previous research has found that high expression of Lysine methyltransferase 5A (KMT5A) is closely related to the proliferation of PitNETs. The aim of this study was to investigate the role and molecular mechanism of KMT5A in the progression of GH PitNETs. Methods: Immunohistochemistry, qRT-PCR, and Western blot (WB) were used to assess the expression levels of KMT5A in human normal pituitary and GH PitNETs, as well as in rat normal pituitary and GH3 cells. Additionally, we utilized RNA interference technology and treatment with a selective KMT5A inhibitor to decrease the expression of KMT5A in GH3 cells. CCK-8, EdU, flow cytometry (FCM), clone formation, and WB assay were further employed to evaluate the impact of KMT5A on the proliferation of GH3 cells in vitro. A xenograft model was established to evaluate the role of KMT5A in GH PitNETs progression in vivo. Results: KMT5A was highly expressed in GH PitNETs and GH3 cells. Moreover, the reduction of KMT5A expression led to inhibited growth of GH PitNETs and increased apoptosis of tumor cells, as indicated by the findings from CCK-8, EdU, clone formation, and FCM assays. Additionally, WB analysis identified the Wnt/β-catenin signaling pathway as a potential mechanism through which KMT5A promotes GH PitNETs progression. Conclusion: Our research suggests that KMT5A may facilitate the progression of GH PitNETs via the Wnt/β-catenin signaling pathway. Therefore, KMT5A may serve as a potential therapeutic target and molecular biomarker for GH PitNETs.

GH PitNETs, accounting for about 12% of all pituitary adenomas, are common functional pituitary adenomas that pose a significant threat to human health [1]. As the tumor grows, it can exert space-occupying effects, such as optic chiasm compression leading to visual field loss, decreased vision, or even blindness, and third ventricle compression resulting in obstructive hydrocephalus [2, 3]. Moreover, patients with GH PitNETs who are exposed to prolonged excessive GH secretion may lead to physical deformities and extensive systemic manifestations, including gigantism, acromegaly, goiter, osteoarthritis, colon polyps, sleep apnea, reproductive disorders, diabetes, hypertension, and cardiovascular diseases, which seriously impact the quality of patients’ life and lifespan [4‒6].

Surgical resection of tumors is the primary treatment for GH PitNETs, which can effectively reduce serum GH levels and alleviate the effects of the tumor’s space-occupying impact. Pharmacological therapy is often employed as a secondary option for patients with persistent GH secretion post-surgery involving somatostatin receptor ligands and dopamine agonists. Radiotherapy is a potential alternative if medical treatment is unavailable or unsuccessful. However, a minority of GH PitNETs grow aggressively and rapidly, cannot be controlled even after standardized surgical, pharmacological, and radiological treatments, and have early recurrence [7, 8]. Therefore, it is essential to explore new molecular biomarkers and therapeutic targets for GH PitNETs to enhance the clinical diagnosis and treatment of GH PitNETs.

Our previous research has revealed that LINC00473 promotes the progression of PitNETs by up-regulating KMT5A through a ceRNA-mediated evasion of miR-502-3p [15]. KMT5A, also known as SET8, SETD8, PR-Set7, is a member of the SET gene family, located on the long arm of human chromosome 12 (12q24.31). In normal physiological conditions, KMT5A plays a critical role in regulating gene transcription and replication initiation, maintaining genome stability, and controlling the cell cycle [9, 10]. Recently, overexpression of KMT5A has been observed in various tumors, including esophageal squamous cell carcinoma [11], prostate cancer [12], and breast cancer [13]. KMT5A exerts its tumor promoting effects through regulating signaling pathways such as Wnt/β-catenin, p53, and TWIST [14].

However, the role of KMT5A in GH PitNETs and its specific molecular mechanisms remain unclear. The objective of this study is to ascertain the involvement of KMT5A in the progression of GH PitNETs and elucidate its potential mechanisms through in vitro and in vivo experiments.

Tissues Collecting

Normal human pituitary tissues were obtained from cadaver specimens without any endocrine diseases at the Anatomy Department of Kunming Medical University. Pathologically confirmed GH PitNET tissues were obtained from intraoperative specimens from the Department of Neurosurgery, the First Affiliated Hospital of Kunming Medical University. The study was approved by the Ethics Committee of the First Affiliated Hospital of Kunming Medical University (No. 2019-0816).

Cell Line and Cell Culture

The GH3 cell line (N0.1101RAT-PΜMC000008) was obtained from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. All GH3 cells were cultivated in DMEM medium (Hyclone, Logan Utah, USA) supplemented with 10% fetal bovine serum (BI, Kibbutz Beit Haemek, Israel) and 1% penicillin/streptomycin (Gibco, NY, USA) in a 37°C, 5% CO2 incubator.

Lentiviral Transfection

The KMT5A gene was specifically knocked down in GH3 cells using the vector GV493 (hU6-MCS-CBh-gcGFP-IRES-puromycin) through services provided by GenePharma Company (Shanghai, China). Prior to transfection, GH3 cells were seeded in 6-well plates at a confluence of 60%. Transfected cells were cultured for 48 h and then examined for transfection efficiency using an inverted fluorescent microscope. Concurrently, the shKMT5A stably transfected cell line was generated by screen with complete medium supplement with 1 μg/mL puromycin, as recommended by the lentiviral stable transfection instructions. The knockdown efficiency of KMT5A in GH3 cells was assessed using WB and qRT-PCR. The stably transfected cell lines were subsequently constructed and cryopreserved for future use.

qRT-PCR

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, CA, USA). Subsequently, cDNA was synthesized employing a reverse transcription kit (Thermo Scientific, MA, USA). qRT-PCR was conducted using the FastStart Universal SYBR Green (Roche, Basel, Switzerland) with GAPDH utilized as the reference gene. Amplification was detected using the TaqMan® Universal PCR Master Mix II machine (Applied Biosystems, CA, USA). qRT-PCR experiments were performed in triplicate for each primer set. The primer sequences are shown in Table 1.

Table 1.

Primer sequences for qRT-PCR

Gene namePrimer sequences (5′→3′)
Rat-KMT5A 
 F ACC​ATT​GGC​CGG​AAT​CTA​CA 
 R ATG​ACG​GGG​GTG​GAG​TTT​TT 
Rat-GAPDH 
 F ACT​CCC​ATT​CTT​CCA​CCT​TTG 
 R CCC​TGT​TGC​TGT​AGC​CAT​ATT 
Gene namePrimer sequences (5′→3′)
Rat-KMT5A 
 F ACC​ATT​GGC​CGG​AAT​CTA​CA 
 R ATG​ACG​GGG​GTG​GAG​TTT​TT 
Rat-GAPDH 
 F ACT​CCC​ATT​CTT​CCA​CCT​TTG 
 R CCC​TGT​TGC​TGT​AGC​CAT​ATT 

WB Analysis

Total proteins were extracted using RIPA lysis buffer containing PMSF (Solarbio, Beijing, China) and quantified with BCA protein assay kit (Beyotime, Shanghai, China). The proteins were separated by SDS-PAGE (Beyotime, Shanghai, China) and transferred on PVDF membranes (Millipore, MA, USA). The membranes were then incubated with the primary antibodies overnight at 4°C. The primary antibodies include: anti-KMT5A (Abcam, ab111691, Cambridge, UK), anti-cyclin D1 (Abcam, ab16663, Cambridge, UK), anti-β-catenin (Abcam, ab32572, Cambridge, UK), anti-TCF1 (Sigma-Aldrich, ZRB1460, St. Louis, MO, USA), anti-c-Myc (Abcam, ab19312, Cambridge, UK), anti-cleaved caspase-3 (Abcam, ab32043, Cambridge, UK), anti-GAPDH (Abcam, ab128915, Cambridge, UK). Next, the membranes were incubated with the corresponding secondary antibody (Abcam, ab205718, Cambridge, UK) for 90 min at room temperature. Proteins were detected using the super ECL-Plus reagent (Millipore, MA, USA) and quantified using Image J software.

CCK-8

Cells were counted and diluted a concentration of 1 × 105 cells/mL. Cells were seeded into 96-well plates at a density of 1 × 104 cells per well, with 5 duplicate wells assigned to each experimental group. Subsequently, the plates were placed in an incubator for continuous incubation. Following a culture period of 24 h, 48 h, 72 h, and 96 h respectively, 10 µL CCK-8 solution (Dojindo, Kumamoto, Japan) was added to each well and further incubated at 37°C for 2 h. Finally, the absorbance at 450 nm was measured using a SpectraMax M5 (Molecular Devices, CA, USA) microplate reader, and the measurement results were used to generate a proliferation curve.

EdU

A total of 1 × 105 cells were seeded per well in 12-well plates. The cells were then treated with DMSO, UNC0379 (5 µm) and UNC0379 (10 µm) respectively. EdU reagent was prepared following the instructions provided in the EdU cell proliferation detection kit (Ruibo Biotechnology Co., LTD., Guangzhou, China). Subsequently, 500 µL prepared EdU reagent was added to each well, and the cells were immediately fixed and stained after incubation for 2 h. Finally, the cells were observed and photographed using an inverted fluorescence microscope. Image analysis was performed by Image J software.

Colony Formation Assay

GH3 cells in the logarithmic phase were counted, and 1,000 cells were incubated in each well of a 6-well plate. After cell adherence, the medium was changed every 3 days for 14 days, and cell growth was observed under a microscope. Afterward, the cells were fixed using 4% paraformaldehyde and stained with 1% crystal violet. Subsequently, the cells were observed under a microscope, and the rate of clone formation rate was determined by calculating the number of observed cell clones, clone formation rate = (clone number/number of incubated cells) × 100%.

Cell Apoptosis

The cells were digested with trypsin without EDTA and collected. The collected medium was combined with the cell suspension solution in a 10 mL centrifuge tube, and the cells were harvested through centrifugation. After counting, the cells were resuspended in 100 µL 1×Annexin V binding buffer (Solarbio, Beijing, China) at a concentration of 1 × 106 cells/mL. Following the instructions of the Annexin V 633 Apoptosis Detection Kit (Dojindo, Kumamoto, Japan), the stained cells were examined by FCM within 1 h, and the data were analyzed using FlowJo V 10.6.2.

Immunohistochemical Analysis

Tissues were fixed using 4% paraformaldehyde, and subsequently embedded in paraffin blocks, and then dewaxed and rehydrated sequentially. After antigen repair and endogenous peroxidase inactivation, the primary antibody anti-KMT5A (Abcam, ab111691, Cambridge, UK) was added and incubated at 4°C overnight. Next, the slices were washed with PBS and incubated with secondary antibody (Abcam, ab205718, Cambridge, UK) at 37°C for 30 min. The developed DAB signal was used for visualization, and the samples were counterstained with hematoxylin. Following dehydration, images were captured under microscope. KMT5A expression was quantified by measuring the average absorbance of the positive (brown) and negative (blue) staining using Image J.

Nude Mouse Xenograft Tumor Assay

Female BALB/c nude mice (n = 6 per group), aged 6–8 weeks and weighing approximately 18–20 g were utilized. The axillary subcutis of the nude mice served as the site for tumor formation. The logarithmically growing GH3 cells were harvested and resuspended in PBS at a concentration of 1 × 107 cells/mL. Subsequently, each nude mouse’s axillary region was injected subcutaneously with 0.2 mL cell suspension (2 × 106 cells/mouse). Tumor volume was measured every 7 days, and the tumor growth curve was plotted. On the 7th day after subcutaneous injection of GH3 cells, the UNC0379 group received a daily intraperitoneal injection of 1 mg/kg of UNC0379 (a selective KMT5A inhibitor). After 42 days, the nude mice were euthanized, and the transplanted tumors were harvested for subsequent qRT-PCR, WB, and immunohistochemistry experiments. All procedures were conducted in accordance with the guidelines and regulations of the Animal Ethics Committee of Kunming Medical University (No. kmmu20190718).

Statistical Analysis

The statistical analysis was performed using SPSS version 25.0 software. The data were presented as mean ± standard deviation. Intergroup comparisons were conducted using an independent sample t test, while multigroup comparisons were conducted using the one-way analysis of variance. p < 0.05 was considered statistically significant.

Expression Level of KMT5A in GH PitNET Tissue Is Higher than That in Normal Pituitary Tissues

Three cadaver-derived normal pituitary tissues and three GH PitNET tissues were obtained for comparative analysis of KMT5A expression levels using immunohistochemistry. The results showed that KMT5A expression in GH PitNETs group was significantly higher than that in the normal pituitary tissue group (shown in Fig. 1a).

Fig. 1.

Expression of KMT5A in normal human pituitary tissues, GH PitNET tissues, Wistar rat pituitary tissues and GH3 cells. a Immunohistochemical analysis of the expression level of KMT5A in human normal pituitary tissues and GH PitNET tissues. b Detection of KMT5A mRNA expression levels in Wistar rat pituitary tissues and GH3 cells by qRT-PCR. c Detection of KMT5A protein expression levels in Wistar rat pituitary tissues and GH3 cells by WB. ***p < 0.001, ****p < 0.0001. Bars represent mean ± SD.

Fig. 1.

Expression of KMT5A in normal human pituitary tissues, GH PitNET tissues, Wistar rat pituitary tissues and GH3 cells. a Immunohistochemical analysis of the expression level of KMT5A in human normal pituitary tissues and GH PitNET tissues. b Detection of KMT5A mRNA expression levels in Wistar rat pituitary tissues and GH3 cells by qRT-PCR. c Detection of KMT5A protein expression levels in Wistar rat pituitary tissues and GH3 cells by WB. ***p < 0.001, ****p < 0.0001. Bars represent mean ± SD.

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Pituitary tissues from 12 Wistar rats were obtained to assess the expression levels of KMT5A in both Wistar rat pituitary tissues and GH3 cells using qRT-PCR and WB analysis. The findings demonstrated a notably elevated expression of KMT5A in GH3 cells compared with that in Wistar rat pituitary tissues (shown in Fig. 1b, c).

Knockdown of KMT5A Suppresses the Proliferation of GH3 Cells

To establish the KMT5A knockdown GH3 cell line, shRNA was constructed and transduced into GH3 cells. The transfection efficiency was assessed by qRT-PCR and WB analyses. The data revealed a notable decrease in the expression level of KMT5A in knockdown groups #1, #2, and #3 compared with shCtrl groups. Notably, group #2 displayed the most significant reduction (shown in Fig. 2a, b). Consequently, group #2 was selected as the shKMT5A group for subsequent experiments.

Fig. 2.

Effect of KMT5A knockdown on GH3 cell proliferation. a qRT-PCR detection of the shRNA transfection efficiency in GH3 cells. b Detection of shRNA transfection efficiency of GH3 cells by WB. c Detection of the effect of KMT5A knockdown on GH3 cell viability by CCK-8 assay. d Clonal formation evaluation of the effect of KMT5A knockdown on GH3 cell proliferation. *p < 0.05, ***p < 0.001, ****p < 0.0001. Bars represent mean ± SD.

Fig. 2.

Effect of KMT5A knockdown on GH3 cell proliferation. a qRT-PCR detection of the shRNA transfection efficiency in GH3 cells. b Detection of shRNA transfection efficiency of GH3 cells by WB. c Detection of the effect of KMT5A knockdown on GH3 cell viability by CCK-8 assay. d Clonal formation evaluation of the effect of KMT5A knockdown on GH3 cell proliferation. *p < 0.05, ***p < 0.001, ****p < 0.0001. Bars represent mean ± SD.

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The CCK-8 assay was utilized to assess the impact of KMT5A knockdown on the proliferation of GH3 cells. The results showed that compared with the shCtrl group, the proliferation ability of GH3 cells in the shKMT5A group was decreased 24 h after transfection. Furthermore, this reduction in proliferation became significant as the experimental duration progressed to 72 h post-transfection (shown in Fig. 2c). Subsequently, these outcomes were validated by clone formation assay, which demonstrated a significant reduction in the number of cell clones in the shKMT5A group compared with the shCtrl group (shown in Fig. 2d).

Knockdown of KMT5A Induced GH3 Cell Apoptosis and Inhibited Cell Cycle Progression

FCM was employed to detect the impact of KMT5A knockdown on GH3 cell apoptosis. The results showed that the shKMT5A group exhibited a higher percentage of apoptotic cells (11.67%) compared with the shCtrl group (8.92%) (shown in Fig. 3a). WB was utilized to investigate the effect of the KMT5A knockdown on the expression of cleaved caspase-3, an apoptosis related protein, as well as Cyclin D1, a cell cycle related protein, in GH3 cells. The results showed that compared with the shCtrl group, the expression level of cleaved caspase-3 in the shKMT5A group increased (shown in Fig. 3b), while the expression level of Cyclin D1 decreased (shown in Fig. 3c).

Fig. 3.

Effect of knockdown of KMT5A on the apoptosis and cell cycle of GH3 cells. a FCM results showed that knockdown of KMT5A promoted GH3 cell apoptosis. b WB assay showed that the expression level of cleaved caspase-3 was elevated in the shKMT5A group compared with the shCtrl group. c WB assay showed that the cell cycle protein cyclin D1 expression level was decreased in the shKMT5A group compared with the shCtrl group. **p < 0.01, ***p < 0.001. Bars represent mean ± SD.

Fig. 3.

Effect of knockdown of KMT5A on the apoptosis and cell cycle of GH3 cells. a FCM results showed that knockdown of KMT5A promoted GH3 cell apoptosis. b WB assay showed that the expression level of cleaved caspase-3 was elevated in the shKMT5A group compared with the shCtrl group. c WB assay showed that the cell cycle protein cyclin D1 expression level was decreased in the shKMT5A group compared with the shCtrl group. **p < 0.01, ***p < 0.001. Bars represent mean ± SD.

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KMT5A Inhibitor UNC0379 Suppresses the Proliferation of GH3 Cells

The aforementioned experimental results indicate that the knockdown of KMT5A in GH3 cells exerts inhibitory effects on cell proliferation and induces cell apoptosis. Subsequent studies were conducted to ascertain whether the KMT5A inhibitor UNC0379 exhibits the same effects. The qRT-PCR results showed that the expression of KMT5A decreased with the increase of UNC0379 concentration (shown in Fig. 4a). CCK-8, clone formation and EdU assay results showed that treatment with UNC0379 could inhibit the proliferation of GH3 cells, and this inhibitory effect became more pronounced with the increase in treatment duration and UNC0379 concentration (shown in Fig. 4b–d).

Fig. 4.

The effect of UNC0379 on the proliferation of GH3 cells. a qRT-PCR analysis of the expression level of KMT5A mRNA in GH3 cells treated with UNC0379. b CCK-8 detection of the effect of UNC0379 on GH3 cell viability. c Clonal formation evaluation of the effect of UNC0379 on GH3 cell proliferation. d Cell proliferation was analyzed by EdU assay. The nucleus is shown in blue and EdU-staining positive cells are shown in green. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent mean ± SD.

Fig. 4.

The effect of UNC0379 on the proliferation of GH3 cells. a qRT-PCR analysis of the expression level of KMT5A mRNA in GH3 cells treated with UNC0379. b CCK-8 detection of the effect of UNC0379 on GH3 cell viability. c Clonal formation evaluation of the effect of UNC0379 on GH3 cell proliferation. d Cell proliferation was analyzed by EdU assay. The nucleus is shown in blue and EdU-staining positive cells are shown in green. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent mean ± SD.

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UNC0379 Induces GH3 Cell Apoptosis and Inhibit Cycle Progression

The effect of UNC0379 on apoptosis of GH3 cells was detected by FCM. The results showed that compared with the DMSO group, the apoptosis rate of the UNC0379 group was higher, and the apoptosis rate increased with the increase of UNC0379 concentration (shown in Fig. 5a). WB analysis was conducted to assess the effect of UNC0379 on the expression levels of cleaved caspase-3 and cyclin D1 in GH3 cells. Compared with the DMSO group, the expression of cleaved caspase-3 was increased in the UNC0379 group (shown in Fig. 5b), while the expression of cyclin D1 was decreased (shown in Fig. 5c), and these changes were more significant with the increase of UNC0379 concentration.

Fig. 5.

Effect of UNC0379 on the GH3 cell proliferation. a The apoptotic effect of UNC0379 on GH3 cells was demonstrated by FCM. b WB assay showed that the expression level of cleaved caspase-3 was higher in the UNC0379 group than in the DMSO group. c WB assay showed that the expression level of cyclin D1 was lower in the UNC0379 group compared with the DMSO group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars represent mean ± SD.

Fig. 5.

Effect of UNC0379 on the GH3 cell proliferation. a The apoptotic effect of UNC0379 on GH3 cells was demonstrated by FCM. b WB assay showed that the expression level of cleaved caspase-3 was higher in the UNC0379 group than in the DMSO group. c WB assay showed that the expression level of cyclin D1 was lower in the UNC0379 group compared with the DMSO group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars represent mean ± SD.

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KMT5A May Affect GH3 Cell Proliferation and Apoptosis through the Wnt/β-Catenin Signaling Pathway

Based on previous experimental results, the knockdown of KMT5A or treatment with UNC0379 inhibited the cell cycle progression and promoted the apoptosis in GH3 cells. KMT5A may play a significant role in the proliferation of GH3 cells. To further investigate the potential molecular mechanism by which KMT5A affects the proliferation of GH3 cells, we performed WB analyses. The results demonstrated that the expressions of β-catenin, TCF1, c-Myc and Cyclin D1 were significantly decreased following the KMT5A knockdown (shown in Fig. 6a). Similarly, UNC0379 also inhibited the expression levels of KMT5A, β-catenin, TCF1, c-Myc and Cyclin D1 proteins in GH3 cells, and the inhibitory effect was more obvious with the increase of UNC0379 concentration (shown in Fig. 6b).

Fig. 6.

Effects of KMT5A on the expression levels of Wnt/β-catenin signaling pathway and its downstream target genes. a WB showed that the expression levels of β-catenin, TCF1 as well as their target proteins c-Myc and cyclin D1 were decreased after KMT5A knockdown. b WB results showed that UNC0379 treatment inhibits the expression levels of β-catenin, TCF1, c-Myc, and cyclin D1 protein in GH3 cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars represent mean ± SD.

Fig. 6.

Effects of KMT5A on the expression levels of Wnt/β-catenin signaling pathway and its downstream target genes. a WB showed that the expression levels of β-catenin, TCF1 as well as their target proteins c-Myc and cyclin D1 were decreased after KMT5A knockdown. b WB results showed that UNC0379 treatment inhibits the expression levels of β-catenin, TCF1, c-Myc, and cyclin D1 protein in GH3 cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars represent mean ± SD.

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Knockdown of KMT5A or UNC0379 Treatment Inhibits GH3 Cell Proliferation in vivo

The xenograft model of GH PitNETs was constructed by subcutaneous injection of GH3 cells into nude mice. Tumor volume was measured every 7 days after injection, and a tumor growth curve was plotted. The results showed that tumor growth in the shKMT5A and UNC0379 groups was slower than that in the control group (shown in Fig. 7a). After 42 days, the nude mice were euthanized; the transplanted tumors were harvested and photographed. Compared with the control group, the tumor volume in shKMT5A and UNC0379 groups was significantly decreased (shown in Fig. 7b). qRT-PCR and immunohistochemistry results showed that compared with the control group, the expression level of KMT5A in shKMT5A and UNC0379 groups was decreased (shown in Fig. 7c, d).

Fig. 7.

Effect of KMT5A on the growth of GH PitNETs. a The tumor growth curve. b The images and volume of the tumors when harvested. c qRT-PCR detection of the expression level of KMT5A in tumor tissues. d Immunohistochemical detection of the expression level of KMT5A in tumor tissues. **p < 0.01, ***p < 0.001. Bars represent mean ± SD.

Fig. 7.

Effect of KMT5A on the growth of GH PitNETs. a The tumor growth curve. b The images and volume of the tumors when harvested. c qRT-PCR detection of the expression level of KMT5A in tumor tissues. d Immunohistochemical detection of the expression level of KMT5A in tumor tissues. **p < 0.01, ***p < 0.001. Bars represent mean ± SD.

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KMT5A May Affect the Proliferation of GH3 Cells through the Wnt/β-Catenin Pathway

The effects of KMT5A on the growth of GH PitNET xenografted model tumors were analyzed by WB detection. The results showed that knockdown of KMT5A or UNC0379 treatment resulted in down-regulation of KMT5A protein expression levels, and the expression levels of β-catenin, TCF1 and downstream target proteins c-Myc and Cyclin D1 in Wnt/β-catenin signaling pathway were also down-regulated accordingly. These results are consistent with in vitro cellular experimental findings (shown in Fig. 8a), suggesting that KMT5A may influence the proliferation of GH3 cells and promote tumor progression through the Wnt/β-catenin pathway (shown in Fig. 8b).

Fig. 8.

WB detection of molecular mechanism of KMT5A affecting GH PitNETs in vivo. a The expression levels of β-catenin, TCF1, as well as their downstream target genes, c-Myc and Cyclin D1 were significantly decreased after knockdown of KMT5A or UNC0379 treatment. b The schematic diagram shows that KMT5A promotes GH PitNETs tumor progression through the Wnt/β-catenin signaling pathway. **p < 0.01, ***p < 0.001. Bars represent mean ± SD.

Fig. 8.

WB detection of molecular mechanism of KMT5A affecting GH PitNETs in vivo. a The expression levels of β-catenin, TCF1, as well as their downstream target genes, c-Myc and Cyclin D1 were significantly decreased after knockdown of KMT5A or UNC0379 treatment. b The schematic diagram shows that KMT5A promotes GH PitNETs tumor progression through the Wnt/β-catenin signaling pathway. **p < 0.01, ***p < 0.001. Bars represent mean ± SD.

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N-lysine methyltransferase KMT5A is a methyltransferase containing a SET domain that catalyzes the monomethylation of histone H4K20me1. KMT5A has been implicated in various pathways, including Wnt, histone, p53, PCNA, and TWIST, and is primarily involved in DNA repair, gene transcription, cell cycle, and apoptosis, as well as other essential physiological processes. It plays a crucial role in maintaining genomic integrity and controlling cell cycle progression and development [16‒18]. The loss of KMT5A function in Drosophila, as demonstrated by Huang et al. [19], leads to a delay in reactivation of neural stem cells (NSCs) and a deficiency in H4K20 monomethylation in the brain. Further investigations have confirmed that KMT5A promotes NSCs reactivation by regulating Wnt signaling and cell cycle progression. The potential mechanism is that when Wnt is activated, β-catenin accumulates and translocates the nucleus. Then KMT5A promotes the transcription of downstream target genes such as Cyclin D1, c-Myc, matrix metalloproteinases (MMPs) by replacing the inhibitory protein Groucho and binding to lymphocyte enhancer factor (LEF)/T cell factor (TCF) transcription factors [20].

KMT5A’s effects and regulatory mechanisms in human tumors have been extensively reported. For instance, one study demonstrated that KMT5A promotes Numb protein methylation in breast cancer, leading to the inhibition of p53-dependent apoptosis. Down-regulation of KMT5A expression induces apoptosis through the KMT5A/Numb/p53 pathways [21]. Other research has revealed that miR-335 significantly inhibits the expression of KMT5A through the Wnt/β-catenin pathway, thereby suppressing the expression of its downstream genes cyclin D1 and c-Myc in breast cancer. In addition, the ectopic expression of miR-335 or the deletion of its target gene KMT5A can enhance the sensitivity of paclitaxel-resistant breast cancer cells to paclitaxel [22]. Similarly, An et al. [23] found that KMT5A promotes the expression of downstream molecules by activating the β-catenin signaling pathway, thereby increasing the proliferation and metastasis of osteosarcoma cells. In our previous studies, we discovered that LINC00473 down-regulates miR-502-3p and up-regulates KMT5A through the ceRNA mechanism, thereby regulating the cell cycle through the LINC00473/miR-502-3p/KMT5A signaling axis, and affecting the proliferation and invasion of pituitary adenomas [15]. In this study, we found that both KMT5A knockdown and the application of the inhibitor UNC0379 could inhibit the proliferation of GH PitNETs by affecting the process of cell cycle and promoting apoptosis.

The mechanism underlying the promotion of GH PitNETs proliferation by KMT5A was further investigated in this study. Our findings revealed that a decrease in KMT5A expression leads to apoptosis of GH PitNETs cells, which is accompanied by an increased expression of caspase-3. Caspase-3 is a member of the caspase family and exists in the form of zymogen. When the promoter caspase-8/9 releases pro-apoptotic signals and activates caspase-3, caspase-3 cleaves key proteins within the cell through proteolysis, executing the function of apoptosis [24]. Germline mutation of AIP gene is a known cause of familial solitary pituitary adenomas, especially GH tumors [25]. In adult male rats, AIP alterations induce IGF-1 elevation and gigantism along with pituitary hyperplasia by blocking the RET/caspase-3/PKCδ apoptotic pathway [26]. These findings suggest that caspase-3-related apoptosis is closely related to the occurrence and development of GH PitNEts.

In addition to the effect of apoptosis, KMT5A plays a more significant role in the regulation of the cell cycle. Research indicates that the abundance of KMT5A varies at different stages of the cell cycle: it is lower in the G1 phase, and inhibition of KTM5A can lead to down-regulation of E2F expression; during the S phase, KMT5A is degraded by the CRL4cdt2 ubiquitin ligase to avoid chromatin fixation and abnormal DNA replication; in the G2/M phase, the abundance of KMT5A first increases and then decreases, ensuring the normal progression of mitosis [27]. This demonstrates that the precise regulation of KMT5A is indispensable for the cell cycle. Our findings indicate that KMT5A knockdown or UNC0379 treatment can diminish the expression of Cyclin D1. Cyclin D1 is encoded by the proto-oncogene CCND1, expressed in early G1, peaking in late G1, and is ubiquitinated and degraded in the S phase. It acts as a cell cycle initiator or growth factor sensor, accelerating the transition from G1 to S phase, and facilitating progression through the G1/S checkpoint when its expression is increased [28]. In our previous research [15], as well as in this study and in drug-resistant breast cancer research [22], it has been observed that KMT5A promotes tumor cell proliferation and invasion by up-regulating the expression of Cyclin D1.

As previously mentioned, KMT5A has the ability to activate the Wnt/β-catenin signaling pathway through monomethylated H4K20. β-Catenin aids KMT5A in competitively binding transcription factors LEF/TCF to further activate downstream target genes like cyclin D1 and c-Myc [20]. As a conserved signal transduction pathway, the Wnt/β-catenin pathway regulates cell proliferation and differentiation [29]. Dysregulation of this pathway often arises from mutations in various components, particularly mutations or silencing of Wnt tumor suppressors, with mutations in β-catenin being the most commonly observed alterations in cancer [30]. Aberrant Wnt/β-catenin signaling is closely associated with the development of numerous tumors, including PitNETs [31]. Gaston-Massuet et al. [32] found that the expression of mutated β-catenin leads to a significant increase in the total number and proliferative potential of pituitary progenitor/stem cells. Furthermore, β-catenin mutations in undifferentiated Rathke’s pouch progenitor cells are closely associated with the occurrence of craniopharyngiomas in both mice and humans. Studies on non-functioning PitNETs have revealed that phosphorylation at serine 552 of β-catenin is correlated with tumor invasion and recurrence [33]. In this study, we observed that both KMT5A knockdown and UNC0379 treatment resulted in reduced the expression of β-catenin and TCF1, which suggests that KMT5A is involved in the activation of Wnt/β-catenin pathway in GH PitNETs.

In conclusion, our research findings indicate that KMT5A is overexpressed in GH PitNETs, influencing the cell cycle and apoptosis of GH PitNETs, thereby promoting tumor proliferation. Down-regulation of KMT5A or the application of inhibitor UNC0379 can reverse this effect. Further studies indicate that KMT5A promotes the progression of GH PitNETs through activating Wnt/β-catenin signaling pathway, suggesting that KMT5A is a potential molecular marker and therapeutic target for GH PitNETs.

We express our gratitude to Dr. Lu Di and Jiazhi Guo for their valuable technical support.

The study involving human participants was conducted in accordance with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. All procedures were approved by the Research Ethics Committee of the First Affiliated Hospital of Kunming Medical University (Approval No. 2019-0816) and written informed consent was obtained for participation in this study. In addition, for the animal experiments, all procedures adhered to the guidelines outlined in the Care and Use of Laboratory Animals in cancer research and received approval from the Animal Ethics Committee of Kunming Medical University (Approval No. kmmu20190718).

The authors have no conflicts of interest to declare.

This study was supported by the Applied Basic Research Joint Special Fund project of Science and Technology Department of Yunnan Province and Kunming Medical University (Nos. 202101AY070001-017 and 202101AY070001-081).

X.L.D. and Y.Q. designed research. J.J.L, H.S., S.X.Z., C.Z., and T.C. performed research. C.M., L.Y.Z., and T.F.W. analyzed the data. J.J.L. and H.S. wrote the paper.

Additional Information

Junjun Li and Hao Song contributed equally to this work.

All data generated or analyzed during this study are included in this published article. Further inquiries can be directed to the corresponding author.

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