PSAT1 prompted cell proliferation and inhibited cell apoptosis in multiple myeloma through regulating PI3K/AKT pathway

Purpose: To identify the biological function of phosphoserine aminotransferase 1 (PSAT1) in regulating cell proliferation and apoptosis in multiple myeloma (MM). Methods: The mRNA and protein levels of PSAT1 were determined using quantitative real-time polymerase chain reaction (PCR) and western blotting, respectively. Cell proliferation was measured using CCK-8 assay. Results: PSAT1 mRNA and protein expression levels were significantly increased in MM cell lines when compared to control cells. Moreover, downregulation of PSAT1 inhibited MM cell proliferation and induced cell apoptosis, whereas overexpression of PSAT1 promoted MM cell proliferation and suppressed cell apoptosis. Further analysis demonstrated that the underlying mechanism was via regulation of PI3K/AKT pathway. Conclusion: The results identified a novel role for PSAT1 in the progression of MM, which may provide a therapeutic and a new anticancer target for the therapy of MM.


INTRODUCTION
Multiple myeloma (MM) is considered the second most common hematological malignancy worldwide whose clinical manifestations include hypercalcemia, renal insufficiency, anemia, infection and uncontrolled proliferation of monoclonal plasma cells [1]. The incidence of MM is increasing remarkably; accounting for approximately 10 % of all hematological cancers and 2.1 % of all cancer deaths [2]. Over the past few decades, advances in diagnostic and standard therapeutic techniques, have improved the outcomes and prolonged survival of MM patients [3]. Thus, more molecular biomarkers and diagnostic targets for MM, which may be helpful in MM therapy [4,5].
Phosphoserine aminotransferase 1 is an aminotransferase that participates in serine biosynthesis and is highly expressed in various tissues such as breast and prostate [6,7]. Serine biosynthetic pathway has been shown to be crucial in regulating redox status, cell proliferation, and the biosynthesis of cellular components in several types of tumors, such as breast cancer [8,9] and colorectal carcinoma [10,11]. In addition, PSAT1 has been shown to play a role in tumor cell proliferation, migration and chemo resistance, contributing to tumor progression and poor outcomes [12]. However, its biological functions in the development and progression of MM are rarely known.
In this study, the mRNA and protein expression levels of PSAT1 in MM cell lines were measured and its effects on MM cell proliferation were analyzed. The results identified a novel role of PSAT1 in the progression of MM, which may provide a therapeutic and a new anticancer target for the therapy of MM.

EXPERIMENTAL Cell lines
Human bone marrow cell line HS-5 was purchased from COBIOER (China), and used as control cells. Multiple myeloma (MM) cell lines LP-1, NCI-H929, and U266 were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's Modified Eagle's medium containing 10 % fetal bovine serum and penicillinstreptomycin at 37 °C in 5 % CO2 cell incubator.

RNA extraction and real-time PCR
Total RNA was extracted from MM cell lines using the TRIzol reagent (Vazyme, Nanjing). First-strand cDNA was reverse-transcribed from RNA using a reverse transcriptase kit (Vazyme, Nanjing). Quantitative RT-PCR was performed using the SYBR Green PCR Master Mix (Takara, RR047A). The relative expression of target gene transcripts was calculated using the 2 −ΔΔCt method. The sequence of primers for PSAT1 were sense: 5'-GGGTAGGTCCCGTC-3' and antisense: 5'-CCAAAGCCAATTCCA-3'.

Cell proliferation assay
MM cells (1 × 10 6 cells/mL) were seeded onto 96-well plates and cell proliferation was measured using the Cell Counting Kit-8(CCK-8, Dojindo). 10ul/well CCK-8 solution was added and cultured for 1 -4 h. Finally, the optical density (OD) was read using a microplate reader at 450 nm.

Cell transfection
Vectors containing shPSAT1 or pcDNA3.1-PSAT1, and control vectors were transfected into target cells and seeded into 6-well plates using the LP2000 Transfection Reagent (Thermo Fisher Scientific). The screening and subculture of successfully transfected cells were performed for subsequent analysis.

Western blotting
Cultured cells were harvested, and total protein was extracted in lysis buffer (Beyotime, China),separated by SDS-polyacrylamide gel electrophoresis, and blotted onto PVDF membranes [13]. Primary antibodies used were as follows: PSAT1, Bcl-2(CST), Bax (CST), cleaved caspase-3 and GAPDH, which were obtained from Abcam. All antibodies were diluted in 5 % skim milk. The protein bands were visualized using the Tanon 5200 Western Blot System.

Statistical analysis
The data were analyzed using GraphPad 6.0 software and are presented as mean ± SEM. Differences between groups were analyzed using unpaired Student's t test. P < 0.05 was considered statistically significant.

PSAT1 was significantly overexpressed in MM cells
The mRNA and protein expression levels of PSAT1 were measured in MM cell lines (LP-1, NCI-H929, and U266) and control cells by realtime quantitative PCR and western blotting, respectively. Results showed that the expression of PSAT1 mRNA was markedly increased in MM cell lines when compared with control cells. ( Figure 1A). Western blotting analysis showed consistent results (Figure 1 B). Especially, in LP-1 cells, PSAT1 mRNA levels were higher than in the other MM cell lines, NCI-H929, and U266, which was thus used in the subsequent analysis.

Downregulation of PSAT1 inhibited proliferation of MM cells
To explore the function of PSAT1 in the development of MM, the recombinant vectors containing control (shNC) or PSAT1 small hairpin RNA (shPSAT1) were transfected into LP-1 cells, and the transfection efficiency was verified. As expected, PSAT1 was significantly downregulated in LP-1 cells that transfected with shPSAT1 when compared with that transfected with shNC (Figure 2 A). This result was also showed from the western blotting results (Figure  2 B). CCK-8 assay demonstrated that downregulation of PSAT1inhibited the proliferation of LP-1 cells that transfected with shPSAT1 when compared with that transfected with shNC (Figure 2 C).
To determine the effects of PSAT1 on MM cell apoptosis, the protein levels of Bcl-2, Bax, and cleaved caspase-3(all apoptosis-related proteins) in LP-1 cells were investigated. Downregulation of PSAT1, decreased Bcl-2 expression whereas significantly increased the expression levels of apoptosis-associated proteins, Bax and cleaved caspase-3 in LP-1 cells (Figure 2 D).

Upregulation of PSAT1 promotes proliferation of MM cells
The recombinant plasmids, pcDNA3. 1-PSAT1 and pcDNA3.1-NC, were transfected into LP-1cells, and the transfection efficiency was verified by qPCR. The results showed that PSAT1 was significantly upregulated in pcDNA 3.1-PSAT1-transfected cells when compared to pcDNA3.1-NC-transfected cells (Figure 3 A). Western blotting results showed similar results (Figure 3 B). Next, the proliferation of LP-1 cells was assessed using CCK-8 assay, and the results revealed that upregulation of PSAT1 enhanced cell proliferation as compared to shNC group (Figure 3 C). Furthermore, the mRNA level of Bcl-2 was increased significantly, whereas the mRNA levels of Bax and cleaved caspase-3 were significantly decreased (Figure 3 D) in LP-1 cells.

PSAT1 modulated the proliferation of LP-1 cells via PI3K/AKT signaling
To further reveal the underlying mechanism, the protein expression levels of p-PI3K and p-AKT were seriously suppressed by shPSAT1 in LP-1 cells, whereas no obvious differences were observed in the protein levels of PI3K and AKT (Figure 4).
As a downstream component of PI3K/AKT pathway, cyclin D1 is a key intracellular mediator of extracellular mitogens and may promote cell proliferation through the modulation of the G1 phase of the cell cycle [14,15]. Thus, the expression of cyclinD1 in shPSAT1-expressing LP-1 cells was examined, and the results showed that cyclin D1 was downregulated by shPSAT1 (Figure 4).

DISCUSSION
Symptomatic plasma cell myeloma, also known as MM, is considered a plasma cell malignancy. Due to the complex clinical symptoms of MM, its treatment is a great challenge. Recent studies have shown that promising treatments for MM are chemotherapeutic agents that can target and kill cancer cells directly. However, it frequently results in the rapid acquisition of drug resistance [16]. Although the pathogenesis of MM remains poorly understood, therapeutic regimens are considered a promising therapeutic strategy.
In this study, it was found that PSAT1 was overexpressed in MM cell. The upregulation of PSAT1promoted the proliferation of MM cells while downregulation of PSAT1 showed the opposite effect. These results indicate the potential role of PSAT1 in regulating MM cell proliferation. The PI3K/AKT pathway has been found to play a significant role in tumorigenesis and cancer development [17]. Signals from PI3K to the serine/threonine protein kinase AKT mediate cellular processes such as cell cycle, apoptosis, and cell survival via cyclin D1, p21, Bcl-2, and mTOR [18]. During these cellular processes, synthesis of AKT requires PSAT1related serine biosynthesis. Thus, the protein components of PI3K/AKT pathway were examined in this study using western blotting. A significant decrease in the expression levels of p-PI3K and p-AKT in shPSAT1-transfected MM cells were observed. These results supported the hypothesis that PSAT1 exerted an important role in the development of MM by PI3K/Akt signaling pathway. In addition, the levels of the cell cycle biomarker, cyclin D1, were also reduced in shPSAT1-transfected LP-1 cells, suggesting limited cell division cycles. However, additional downstream components of PSAT1, such as JNK/ERK, which are well-known modulators of tumorigenesis [19], have not yet been identified, but this needs to be analyzed in a future study.

CONCLUSION
The results of this study reveal that PSAT1 is significantly upregulated in MM cells and plays a novel role in regulating MM cell proliferation, at least partially via PI3K/AKT/cyclin D1 pathway. Thus, PSAT1 may serve as a molecular biomarker as well as a new potential therapeutic target for MM therapy.