UBTF facilitates melanoma progression via modulating MEK1/2-ERK1/2 signalling pathways by promoting GIT1 transcription

Background UBTF is an HMGB-box DNA binding protein and a necessary Pol I/Pol II basal transcription factor. It has been found that UBTF involves in carcinogenesis and progression of a few cancers. Nevertheless, the the biological function and potential molecular mechanism of UBTF in melanoma are still not clear and need to be clarified. Methods UBTF and GIT1 expressions in melanoma specimens and cell lines were examined by quantitative real-time PCR (qRT-PCR) and Western blot. MTT and colony formation assays were used to investigate the effects of UBTF and GIT1 on melanoma cell proliferation. Cell cycle and apoptosis assays were detected by flow cytometry. Tumor formation assay was used to analyze the effect of UBTF on melanoma growth. Bioinformatics predicting, chromatin immunoprecipitation (ChIP)-qRT-PCR and reporter gene assay were fulfilled for verifing GIT1 as UBTF targeting gene. Results Here we reported that UBTF mRNA and protein expressions were upregulated in primary melanoma specimens and cell lines. UBTF overexpression facilitated melanoma cell proliferation and cell cycle progression and restrained. Silencing UBTF suppressed cell multiplication, cell cycle progression and tumor growth, and promoted apoptosis. UBTF expression was positively related with GIT1 expression in human melanoma tissues. It was verified that UBTF promoted GIT1 transcription in melanoma cells through binding to the promoter region of GIT1. Furthermore, GIT1 overexpression promoted melanoma cell growth and suppressed apoptosis. Knockdown of GIT1 inhibited cell multiplication and induced apoptosis. Overexpression of GIT1 eliminated the effects of silencing UBTF on melanoma cells. Importantly, UBTF activated MEK1/2-ERK1/2 signalling pathways by upregulating GIT1 expression. Conclusions Our study demonstrates that UBTF promotes melanoma cell proliferation and cell cycle progression by promoting GIT1 transcription, thereby activating MEK1/2-ERK1/2 signalling pathways. The findings indicate that UBTF plays a crucial function in melanoma and may be a potential therapeutic target for the treatment of this disease. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-021-02237-8.


91,270 new melanoma patients and 9320 deaths in the United States
. The statistics data of China reveals that there are 8000 new sufferers and 3200 deaths [5]. In China, the melanoma morbidity is lower, however, the mortality is observably higher. The therapy of melanoma contains surgery, radiotherapy, chemotherapy, gene therapy and immunotherapy. Although significance advances in melanoma therapy have been acquired, the therapeutic effect remains still dissatisfied. The 5-year survival rate of sufferers is only 30-40% [6]. The ultimate causes of the unsatisfactory are insufficient on molecular mechanism of melanoma oncogenesis and development. Hence, it is imminently needed to confirm the molecular pathogenesis of melanoma for achieving better therapeutic effect.
Upstream binding transcription factor (UBTF) is a multiple HMGbox architectural protein. It is found that Pol I transcription factor UBTF has a double function in the modulation of Pol I and Pol II mediated transcription. UBTF is not only considered to play a role in RNA Pol I-specific transcription of the ribosomal genes, but also enrich at Pol II-transcribed genes throughout human genomes [7,8]. UBTF participates in DNA damage and repair containing mediators of ATR/ATM-regulated DNA damage response [8]. UBTF also involves in the responds of growth factor stimulation. UBTF can regulate differentiation, proliferation, and cell growth through different signalling pathways [9]. The role of UBTF is damaged via reciprocity with repressive proteins of cell multiplication, such as Rb, p130 and p53 [10]. The function of UBTF in cancers has not been extensively studied. It was reported that UBTF expression upregulated in lung cancer specimens [11]. Recently, studies found that UBTF involved in oncogenesis and development in a few tumor [12,13]. Nevertheless, the function of UBTF in many kinds of tumors, including melanoma, hasn't been investigated exactly. Especially, the potential mechanisms underlying the role of UBTF are still not clear.
In this study, we detected the UBTF expression level in 66 melanoma sufferers, and explored the function and corresponding mechanisms of UBTF in modulating melanoma progression. The data showed that UBTF expression was observably increased in melanoma and the high UBTF expression was associated with clinical pathological characteristics. UBTF facilitated melanoma cell proliferation and suppressed apoptosis. Through chromatin immunoprecipitation-qRT-PCR (ChIP-qRT-PCR) and reporter gene assay, we further confirmed that UBTF promoted melanoma cell growth and cell cycle progression by facilitating G-protein-coupled receptor kinaseinteracting protein 1 (GIT1) transcription, thereby activating MEK1/2-ERK1/2 signalling pathways.

Melanoma clinical specimens
Human melanoma and adjacent non-tumor tissues were gathered from 66 sufferers at the Department of Pathology, the First Affiliated Hospital of Medical College, Xi'an Jiaotong University, PR China. These sufferers hadn't been treated before surgery. According to the sufferers' pathology records, clinical pathological features such as age, sex, anatomic site, tumor thickness, lymph node metastasis and initial stage were collected. Before collection of specimens, the informed consents were acquired from each sufferer. This research was authorized by the Ethical Committee of the First Affiliated Hospital of Medical College, Xi'an Jiaotong University.

Animals
Male BALB/c nude mice (4 week-old) were purchased from Yangzhou University and were fed under aseptic condition. The researchers were blinded to the group allocation in animal experiment. Institutional Animal Care and Use Committee of the First Affiliated Hospital of Medical College, Xi'an Jiaotong University authorized these animal experiments. On the basis of institution's guidelines of laboratory animals, we fulfilled animal experiments.

Cell culture
Melanoma cell lines (Mel-RM, A375 and SK-MEL-28) and human keratinocyte HaCaT cell were bought from the Cell Bank (Shanghai institutes for Biological Sciences). These cell lines have been identificated and detected by the Cell Bank. Cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, MD, USA), supplemented with 10% fetal calf serum (Gibco), and incubated in 5% CO 2 incubator at 37 °C.

siRNA synthesis and transfection
Small interfering RNAs (siRNAs) were designed and synthesized for silencing UBTF and GIT1 expressions by GenePharma (SGC, Shanghai, China). These sequences of siRNA were showed in Additional file 4: Table S1. After cultivating A375/SK-MEL-28 cells for 24 h, UBTF siR-NAs and GIT1 siRNAs were instantaneously transfected into melanoma cells with Jet Prime (Polyplus-transfection, Ilkirch, France) according to the manufacturer's protocol.

Lentiviral construction and transfection
UBTF shRNA and control were inserted into the lentiviral vectors (Genechem Company Ltd.), UBTF shRNA lentiviral vector was constructed for silencing UBTF. These sequences were showed in Additional file 4: Table S2. Lentiviral vector including invalid short hairpin RNA was known as negative control (sh-Ctrl). A375 cells were planted in 12-well plates and infected by using 0.5 ml viral stock for 10 h. Next, the solution was substituted with DMEM supplemented with fetal calf serum.

MTT assay
A375/SK-MEL-28 cells were planted at 3000 cells/well in 96-well plates. Cell activity was detected through performing MTT assay (Sigma, MO, USA) after treatment. After the intervention, these cells was added by 20 µl MTT diluent/well and cultured for 4 h in incubator. Next, suspension liquid was thrown away and 150 µl dimethylsulfoxide (Sigma Louis, USA) was used to dissolve formazan crystals. The assay value was determined through using multi-microplate test system (CLARIOstar, BMG LABTECH, USA).

Colony formation assay
A375/SK-MEL-28 cells were concentrated in 12-well plates in the number of 500 per well 24 h after intervention. Then, these cells were cultivated with ordinary medium for 11 days. Cell colonies were fixed by Methanol for 10 min, stained with 0.2% crystal violet for 30 min. Four parallel wells were performed for each group.

Cell cycle assay
Melanoma cells were planted in 12-well plates and intervented for 24 h. A375/SK-MEL-28 cells were washed, digested, and resuspended in phosphate-buffered saline (PBS). Then, ice-cold ethanol (70%) was applied to immobilize these treated cells at 4 °C overnight. The cells were gathered as single cell suspensions, washed twice with PBS, and resuspended in PBS. RNase A (0.1 mg/ml) and propidiumiodide (0.05 mg/ml) were appended to these cells and incubated for 20 min. The proportion of cell cycle was determined by using flow cytometry (Beckman, Miami, USA).

Apoptosis assay
A375/SK-MEL-28 cells were intervented for 48 h and harvested. The cells were washed thrice with PBS and resuspended as single cell suspensions. Then, these cells were stained with an Annexin-V-FITC Apoptosis Detection Kit (Sigma-Aldrich, USA) according to the instructions. Apoptosis level was measured and analyzed by using flow cytometry (Beckman, Miami, USA).

Tumor formation assay
A375 cells were infected with sh-Ctrl or UBTF shRNA and were then resuspended with DMEM. Male 5-weekold BALB/c nude mice were applie to measure tumor formation. Institutional Committee of Animal Use at Xi'an Jiaotong University authorized this animal experiment. The infected A375 cells (2 × 10 6 ) inoculated subcutaneously into both posterior flanks of the mice. Tumor volume was measured through using vernier caliper every 3 d after injection. At 31 days, the nude mice were anesthetized through inhalation of 3% isofluorane and given one subcutaneous dose of carprofen (8 mg/kg) for euthanasia. The length (L) and width (W) of tumor were applied to figure up the volume (V). Formula: V = (L ×W 2 )/2. The tumors were stripped and detected weight 31 d after injection. These tumor specimens were frozen storage for following experiment.

Chromatin immunoprecipitation (ChIP)-qRT-PCR and -RT-PCR
According to previous research methods [14], we fulfilled ChIP assay. Formaldehyde (1%) was used to crosslink for fifteen min in A375/SK-MEL-28 cells and glycine was applied to quench. Ultrasonic processor was used to sonicate melanoma cells and extract nuclear lysates. The chromatin was broken into approximately 200 bp DNA fragments with ultrasonic processing. These DNA fragments were incubated through using UBTF or IgG antibodies for 12 h 4 °C, respectively (Additional file 4: Table S3). Then, Dynabeads Protein A (Thermo Fisher Scientific, USA) was applied to obtain DNA-protein complexes and TE buffer was used to elute the complexes at 65 °C. Next, these complexes were treated for reversing crosslinking 8 h at 65 °C. DNA was purified with the QIA quick PCR purification kit (QIAGEN, Germany). These DNA products were verified by applying qRT-PCR and RT-PCR. The correlative gene-specific primers were revealed in Additional file 4: Table S4.

Reporter gene assay
A375/SK-MEL-28 cells were cultivated in 96-well plates. There were five parallel wells each group. Melanoma cells were transfected with pGL3-luc or pGL3-GIT1-luc, and were co-transfected with pGL3-GIT1-luc and UBTF siR-NAs or GIT1 overexpression vector for 48 h. Trypan blue was used to stain these cells for detecting the luciferase activity. Next, we examined and analyzed the luciferase activity through applying the Dual-Luciferase Reporter System (Promega, USA).

Quantitative RT-PCR (qRT-PCR)
TRIzol was applied to extract RNA from melanoma specimens or cell lines on the basis of specified methods (Invitrogen, CA, USA). These RNA specimens were examined with Nanodrop 2000 (Thermo Scientific, USA) and the OD260/OD280 specific value ranging from 1.9 to 2.0 were regarded as good quality. cDNA was reversed transcription in the light of the specification (Takara, Dalian, China). Quantitative polymerase chain reaction (qRT-PCR) assay was carried out with the SYBR Green qRT-PCR kit (Applied Biosystems, CA, USA). These correlative primers were showed in Additional file 4: Table S4. The qRT-PCR reactions were fulfilled by applying the IQ5 Multicolor qRT-PCR Detection System (Bio-Rad, USA). GAPDH was regarded as internal control for gene mRNAs.

Western blot
Melanoma specimens or cell lines were use to extract protein through applying RIPA buffer (KenGEN, China). The protein was quantified with BCA Protein Assay Kit (Beyotime, China). Equal amounts of protein solution were separated in 10% SDS-PAGE gels followed by transfer onto PVDF membranes. Nonfat milk (5%) was used to block the membranes. Next, these membranes were blotted with relevant primary antibodies for 12 h at 4 °C. These primary antibodies were showed in Additional file 4: Table S3. After that, these membranes were incubated by using the homologous secondary antibodies for 2 h. Proteins were measured through using ECL (GE Healthcare, Beijing, China). Chemiluminescence examination was performed with Syngene GBox (Syngene, UK).

Statistical analysis
SPSS24.0 software was applied to analyze the data. Mean ± SD represented the data. All data came from at least three independent trials. The data was analyzed by Student's t-test or one-way ANOVA. Pearson's correlation analysis was used to evaluate the association between UBTF and GIT1. The relationships between UBTF or GIT1 and clinical pathological features were evaluated by Chi-square test. Value of p < 0.05 is statistically significant.

UBTF is evidently upregulated in melanoma and is related to clinicopathologic characteristics
To investigate the function of UBTF in melanoma progression, we analyzed and detected UBTF expression in melanoma specimens and cell lines. The Cancer Genome Atlas (TCGA) data revealed that the UBTF expression was observably upregulated in melanoma samples compared with normal skin tissues ( Fig. 1a; p < 0.001). The UBTF expression was also remarkable higher in metastatic melanoma tissues than in tumor and normal skin tissues ( Fig. 1b; p < 0.001). The qRT-PCR results showed that UBTF mRNA level was significantly increased in 80.3% (53/66) of the melanoma tissues compared to the normal tissues ( Fig. 1c; p < 0.001). The UBTF mRNA expression was also markedly upregulated in melanoma cell lines (Mel-RM, A375 and SK-MEL-28) than in human keratinocyte HaCaT cells ( Fig. 1d; p < 0.001). The UBTF protein expression was significantly upregulated in melanoma tissues and cell lines (Fig. 1e). Moreover, the high UBTF expression was associated with melanoma thickness, lymph node metastasis and initial stage. Nevertheless, the expression was not related to age, gender and anatomic site (Additional file 4: Table S5). The results indicated that UBTF might be involved in human melanoma progression.

UBTF facilitates melanoma cell growth in vitro and in vivo
To explore the function of UBTF in human melanoma, A375/SK-MEL-28 cells were transfected with UBTF siR-NAs, negative control, UBTF overexpression vector, or control empty vector, independently. qRT-PCR results showed that UBTF siRNAs remarkably decreased UBTF mRNA expression in A375/SK-MEL-28 cells ( Fig. 2a; p < 0.01), and UBTF overexpression vector evidently enhanced UBTF mRNA expression ( Fig. 2b; p < 0.01). Results from MTT assays revealed that silencing UBTF significantly restrained melanoma cell proliferation at 48 and 72 h after transfection ( Fig. 2c; p < 0.01), whereas UBTF overexpression observably promoted melanoma cell multiplication ( Fig. 2d; p < 0.01). Colony formation assays also showed the similar results (Fig. 2e, f ). Cell cycle analysis discovered that silencing UBTF led to the accumulation of cells at the G1/G0 phase and reduced the ratio of S and G2/M phase ( Fig. 2g; p < 0.01); nevertheless, UBTF overexpression remarkably decreased the proportion of G0/G1 phase cells and enhanced the the proportion of S and G2/M phase cells ( Fig. 2h; p < 0.01). In addition, analysis of cell apoptosis confirmed that silencing UBTF clearly increased the proportion of apoptosis cells, while UBTF overexpression observably reduced the proportion of apoptosis (Fig. 2i, j; p < 0.01). It was also found that silencing UBTF downregulated Bcl-2 protein expression and upregulated Bax protein expression in melanoma cell (Fig. 2k), whereas UBTF overexpression increased Bcl-2 expression and decreased Bax expression (Fig. 2l).
To further detect the role of UBTF in melanoma progression in vivo, artificial UBTF shRNA lentiviral vector was constructed and obtained a steady A375 cell clone. UBTF expression was significantly decreased at both mRNA and protein levels in A375 cell (Additional file 1: Fig. S1a, b; p < 0.01). UBTF shRNA remarkably suppressed melanoma cell growth, as evidenced by MTT and colony formation assays (Additional file 1: Fig. S1c, d; p < 0.01). UBTF shRNA resulted in an increase in cells of G1 phase and a decrease in cells of S and G2/M phases (Additional file 1: Fig. S1e; p < 0.01). UBTF shRNA also induced apoptosis (Additional file 1: Fig. S1f; p < 0.01). Next, sh-Ctrl-infected and UBTF shRNA-infected A375 cells were injected subcutaneously and tumor growth was observed. The data of tumor size and weight revealed that tumor growth was evidently restrained by UBTF shRNA as compared with that seen in sh-Ctrl group (Fig. 3a-c). At day 31, the mean volume of tumors with sh-Ctrl was about 2.87-fold higher than that of tumors with UBTF shRNA (Fig. 3b; n = 5, p < 0.01). The mean weight of tumors was 0.51 g in sh-Ctrl group and 0.15 g in UBTF shRNA group (Fig. 3c; p < 0.01). The downregulation of UBTF in tumors derived from UBTF shRNA-transduced A375 cells was verified at mRNA and protein expressions (Fig. 3d, e; p < 0.01). These findings demonstrated that UBTF facilitated human melanoma cell proliferation and suppressed apoptosis. Nevertheless, many factors and molecular networks participate in the occurrence and development of melanoma. Hence, we further research the molecular mechanism of UBTF modulation in human melanoma.

UBTF accelerates GIT1 gene transcription through binding to its promoter
In order to further analyze the molecular mechanism of UBTF regulating melanoma progression, UBTF target gene was predicted by bioinformatics analysis (UCSC Genome Browser). It was found that UBTF could bind to the promoter region of GIT1 (Fig. 4a).

GIT1 is frequently increased in melanoma and relevant to pathological features
TCGA data showed that the GIT1 expression was markedly upregulated in melanoma tissues compared with normal skin tissues ( Fig. 5a; p < 0.001). The GIT1 expression was significant higher in metastatic melanoma tissues than in tumor and normal skin tissues ( Fig. 5b; p < 0.001). The high GIT1 expression was correlated to the overall survival and disease specific survival of melanoma patients (Fig. 5c, d; p < 0.01). The qRT-PCR assay revealed that GIT1 mRNA expression was remarkably upregulated in 75.8% (50/66) of the melanoma tissues ( Fig. 5e; p < 0.001). The GIT1 protein expression was observably increased in melanoma tissues (Fig. 5f ). Furthermore, the high GIT1 mRNA expression was relevant to melanoma thickness, lymph node metastasis and initial stage. But the high expression was not associated with age, gender and anatomic site (Additional file 4: Table S6).

Discussion
UBTF is an HMGB-box DNA binding protein and a necessary Pol I/Pol II basal transcription factor [15,16]. UBTF can modulate binding of the pre-initiation factor SL1/TIF1B and assemble pre-initiation complex on ribosomal DNA promoter region. Moreover, UBTF displaces histone chromatin on transcribed regions of ribosomal DNA by forming a nucleosome like structure, and modulates RNA polymerase I transcription elongation responding growth factor signalling [17][18][19]. Some studies have reported that UBTF, as a regulator of gene expression, involves in carcinogenesis and progression of a few cancers, such as colorectal cancer, breast cancer, lung cancer, cervical cancer and liver cancer [12,[20][21][22]. It is found that S6K-mediated UBTF promotes colorectal cancer cell proliferation by facilitating the transcription ribosomal DNA [12]. Peng et al. reported that UBTF facilitated osteosarcoma cell proliferation through binding the ribosomal DNA promoter and interacting with 1A6/DRIM at the promoter [23]. Another study demonstrates that loss of UBTF suppresses mouse embryonic fibroblasts proliferation and DNA replication, resulting in cell cycle arrest [24]. Nevertheless, the function of UBTF in other cancers is still unknown. This study revealed that UBTF was upregulated in melanoma and was related to clinicopathologic characteristics. UBTF facilitated melanoma cell proliferation in vitro and in vivo, and the promotion was achieved through promoting cell cycle G1-S phase transition. Furthermore, loss of UBTF also induced nuclear disruption and a rapid and highly penetrant apoptosis, particularly in cells subjected to oncogenic stress [24]. Our results showed that UBTF could restrain melanoma cell apoptosis. The findings indicate that UBTF play a crucial function in melanoma.
To explore the possible molecular mechanism of UBTF regulation in melanoma, we fulfilled bioinformatics predicting, ChIP-qRT-PCR and reporter gene assay, through which we ascertained GIT1 as UBTF targeting gene. It was demonstrated that UBTF promoted GIT1 expression in melanoma cells through binding to the promoter region of GIT1. GIT1 is a multifunctional scaffold protein and has multiple domains, containing paxillin-binding domains, three ankyrin repeat, Spa2 homology, ARFGAP and coiled-coil, so it can interact with various signalling molecules [25,26]. GIT1 is involved in many biological processes, including cell migration, cell adhesion, cell growth, apoptosis, turnover of focal adhesions, angiogenesis, spine morphogenesis, lamellipodia formation, and synapse formation [27][28][29][30]. It is reported that GIT1 expression is upregulated and is a biomarker for tumor prognosis in lung, oral, gastric, breast, colorectal, esophagus and prostate cancers [31,32]. Recently, it is found that GIT1 plays a crucial function in regulating tumorigenesis and progression. For example, GIT1 promotes the migration of lung cancer cells through activating the activity of Rac1/Cdc42 [33]. Silencing GIT1 inhibits the metastasis of oral squamous cell carcinoma cells [31]. GIT1 can promote gastric cancer cell proliferation and metastasis [34]. In the present study, we found that GIT1 expression was upregulated in melanoma. GIT1 promoted melanoma cell proliferation and suppressed apoptosis in vitro. The rescue studies demonstrated that UBTF facilitated melanoma cell proliferation, cell cycle progression, and inhibited apoptosis through promoting GIT1 transcription.

Conclusions
In summary, this study demonstrates that UBTF functions as an oncogene in melanoma. We found that UBTF is overexpressed and related to the clinicopathologic characteristics of melanoma patients. UBTF promotes melanoma cell proliferation and cell cycle progression by facilitating GIT1 transcription, thereby activating MEK1/2-ERK1/2 signalling pathways. This study suggests that UBTF plays a crucial function in melanoma and may be a potential therapeutic target for the treatment of this disease.