ZNF488 Promotes the Invasion and Migration of Pancreatic Carcinoma Cells through the Akt/mTOR Pathway.

OBJECTIVE: Studies have demonstrated that zinc finger protein 488 (ZNF488) is highly expressed in pancreatic carcinoma (PC), but its effect on PC and its molecular mechanism remains unclear.
METHODS: Real-time fluorescent quantitative PCR (RT-qPCR) was employed to detect the ZNF488 expression in PC patients' cancer tissues and cell lines. After interfering with or overexpressing ZNF488 in PANC-1 and AsPC-1 cells, respectively, the CCK-8, cell cloning, Transwell, and scratch assays were performed to detect cell proliferation, cell viability, invasion ability, and migration ability. In addition, Western blot was applied to assess the protein expression of Akt, p-Akt, mTOR, and p-mTOR in the Akt-mTOR pathway.
RESULTS: The ZNF488 expression was evidently raised in PC tissues and cell lines, and the starBase V3.0 database indicated that the higher the ZNF488 expression, the lower the survival rate of PC patients. Furthermore, we discovered that overexpressing ZNF488 can markedly promote the proliferation, invasion, and migration of PC cells. At the same time, highly expressed ZNF488 distinctly increased the p-Akt and p-mTOR expressions and the p-Akt/Akt and p-mTOR/mTOR ratios. However, after knocking down the ZNF488 expression, it had the opposite results. In addition, the Akt agonist SC79 can alleviate the effect of ZNF488 knockdown on Akt/mTOR pathway-related proteins, while Akt inhibitor AZD5363 had the opposite effect.
CONCLUSION: ZNF488 could promote the proliferation, invasion, and migration of PC cells, and its mechanism may be related to the activation of the Akt/mTOR pathway. This study demonstrated that ZNF488 could be used as a molecular target for diagnosing and treating PC.

1. Introduction

Pancreatic carcinoma (PC) is a malignant tumor with insidious onset, low survival rate, and high mortality rate. There are 216,000 new cases worldwide each year, causing nearly 200,000 deaths [1]. In recent years, with the higher living standards, longer life expectancy, pollution, and other factors, the incidence of PC has shown an obvious upward trend [2]. Due to the early metastasis of malignant cells to local lymph nodes and the blood which then spread to distant organs, the 5-year survival rate of PC patients is less than 5% [3]. At present, the therapies of this disease mainly include surgery, radiotherapy, chemotherapy, and traditional Chinese medicine, and surgery is often used for tumor resection. However, approximately 80% of tumors cannot be removed at the time of diagnosis [4]. Although the current treatment methods have made significant progress, the overall clinical effect is not ideal, resulting in a poor overall prognosis [5]. Studies have proven that gene deletion and mutation are crucial in tumor growth, spread, and metastasis and tumor recurrence and chemotherapy resistance [6]. Therefore, it is an urgent and major clinical scientific issue to seek suitable markers for PC screening and poor prognosis and thus to improve the early diagnosis rate of PC.

Zinc finger proteins (ZNF) are the most prominent family of sequence-specific DNA binding proteins encoded by 2% of human genes. Recent studies have discovered that the ZNF protein is closely related to human cancer, and its abnormal expression promotes or inhibits the occurrence and progression of tumors in different aspects [7]. Zinc finger protein 488 (ZNF488) is a Kruppel-like zinc finger transcription factor [8], which has been confirmed to be an oncogene and can induce EMT in nasopharyngeal carcinoma by activating the Wnt/β-catenin pathway to promote invasion and tumorigenesis [9]. Besides, ZNF488 can be taken as an independent prognostic indicator for nasopharyngeal carcinoma [10]. What is more, Li et al. predicted that ZNF488 is markedly highly expressed in PC tissues through bioinformatics [11], but its function in PC, as well as its mechanism, is currently unclear.

The AKT/mTOR signaling molecule can be activated by a series of signals at first, leading to its interaction with the receptors, including hormones, growth factors, and extracellular matrix components [12]. The activation of the Akt-mTOR pathway exerts a crucial function in tumor formation, cell cycle, and cell apoptosis and survival [13, 14]. It has been proven that the AKT/mTOR pathway is involved in the apoptosis of PC cells [15] and can inhibit their proliferation [16], suggesting the function of the AKT/mTOR pathway in PC. But its relationship with ZNF488 is currently unclear. Therefore, this study investigated the influence of ZNF488 on the invasion and migration of PC cells using the PC cell line as a model and explored the regulation of the Akt-mTOR pathway in it, aiming to provide a theoretical basis for clinically diagnosing and treating PC.

2. Materials and Methods

2.1. Tissue Specimens and Data Acquisition

A total 30 pairs of PC tissues and adjacent normal tissues were collected from the Sixth People's Hospital of Nantong and The Nantong Tumor Hospital from March 2018 to December 2019. The pathology department confirmed all tissue samples, and none of the patients received chemotherapy or radiation therapy. All patients signed an informed consent form, and the Ethics Committee of the Sixth People's Hospital of Nantong approved this study.

We searched in the starBase V3.0 database (https://starbase.sysu.edu.cn/) to obtain the expression of ZNF488 in PC tissues and normal tissues.

2.2. Cell Culture

Normal pancreatic duct epithelial cells (HPNE) and human PC cell lines (PANC-1, SW1990, AsPC-1, Capan-2) were acquired from the Shanghai Cell Research Institute (Shanghai, China). HPNE and AsPC-1 cells were cultured in 1640 medium (Gibco, USA), and PANC-1, SW1990, and Capan-2 cells were cultured in DMEM medium (Gibco), both of which contained 10% fetal bovine serum (FBS; Gibco) and 1% of penicillin-streptomycin (Gibco), and were incubated in an incubator with 37°C and 5% CO2.

2.3. Cell Transfection

Cells with 2 × 106 cells/ml were planted to a plate with 6 wells and transfected when cultured to a confluency of 50% to 60%. The sh-ZNF488 vector and control vector (sh-NC), ZNF488 overexpression plasmid (ZNF488), and control (vector) were transfected into PANC-1 and AsPC-1 cells using Lipo2000 (Thermo Fisher, USA). sh-ZNF488 and sh-NC, ZNF488, and vector were synthesized by Guangzhou RIBOBIO Biotechnology Co., Ltd. (Guangzhou, China). The sequences were as follows: si-ZNF488, 5′-GCGCCTTTAGCAAACCAAC-3′. And the construction of the znf488 overexpression plasmid and the control are described as previously [10].

2.4. Real-Time Fluorescent Quantitative PCR (qRT-PCR)

Cells and tissues were collected for extracting the total RNA by TRIzol (Invitrogen, USA). Then, the RNA was performed with reverse transcription to synthesize cDNA assisted by reverse transcription PCR kit (Takara, Japan) according to the manufacturer's instructions. The cDNA was taken for a reaction following the real-time PCR instructions. The reaction procedure was 95°C for 1 min; 95°C for 40 s, 58°C for 40 s, and 72°C for 45 s, 40 cycles; and 72°C for 10 min. We applied the 2- method to analyze the data [17]. Table 1 includes the primer sequences.

Table 1

qRT-PCR primer sequence.

Gene name	Primer sequence
ZNF488	F 5′-CTTTCGCCTAACGTCCGA-3′
	R 5′-GCTGTGAGAAGTCATGTGCC-3′
GAPDH	F 5′-CTCCTCCTGTTCGACAGTCAGC-3′
	R 5′-CCCAAT ACGACCAAATCCGTT-3′

2.5. CCK-8 Detection

The transfected cells were planted in 96-well plates with 5000 cells per well. After culturing for 1, 2, 3, and 4 days, 10 μL of CCK-8 reagent (Beyotime Biotechnology, Shanghai, China) and 90 μL of fresh complete medium were added to each well, and then, the plate was settled in the incubator for 1 h. Finally, we evaluated the absorbance at 450 nm with a microplate reader.

2.6. Cell Cloning Experiment

The cells interfered with, or overexpressed, ZNF488 which were collected, counted, and then planted in a plate with 6 wells at a density of 500 cells per well. After culturing for 2 weeks till the clones were visible in the petri dish, the culture was terminated. The supernatant was discarded and rinsed in PBS twice. The cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.5% crystal violet for 3 min. The number of clones with more than 50 cells was counted with a microscope.

2.7. Transwell Invasion Test

Matrigel was taken out from -20°C and placed at 4°C overnight to unfreeze it to liquid, and it was diluted with a serum-free medium at 4°C. 100 μL of Matrigel was transferred into the upper center of the Transwell inserts (BD Biosciences, US), which was then added with 100 μL cell suspension. For the lower chamber, 500 μL medium containing 10% FBS was transferred into it. After 48 h of culture, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Then, the stained cells were counted under an inverted microscope.

2.8. Scratch Test

On the bottom of the 6-well plate, we drew horizontal lines evenly at intervals of 0.5-1 cm with a ruler and a marker, and each well should at least contain five lines. The cells were inoculated to a six-well plate at a density of 1 × 106 cells/well, grown to confluence, and then incubated in a serum-free medium overnight. We made a scratch perpendicular to the horizontal line on the back with a sterile 200 μL micropipette tip, washed the cells in the plate 3 times with PBS to remove the scratched cells, then added the serum-free medium, and put it into an incubator at 37°C and 5% CO2. After culturing for 1, 2, and 3 d, an inverted microscope recorded the cell migration ability. The cell migration ability was calculated [18].

2.9. Western Blot

The tissues or cells were lysed with RIPA Lysis Solution (Gibco, USA) to collect the proteins and measure the protein concentration. After being separated by 12% SDS-PAGE, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocked with 5% skim milk at room temperature for 1 h, the membranes were incubated with primary antibodies against ZNF488 (1 : 1000, ab69933, Abcam, UK), Akt (1 : 500, ab8805, Abcam), p-Akt (1 : 500, ab38449, Abcam), mTOR (1 : 1000, ab32028, Abcam), p-mTOR (1 : 1000, ab109268, Abcam), and GAPDH (1 : 500, ab8245, Abcam) overnight at 4°C. The membrane had been washed twice, then added with the diluted enzyme-labeled secondary antibodies (1 : 3000, ab205718, Abcam), incubated at room temperature for 1 h, and exposed for imaging after washing the membrane. The protein levels were analyzed, taking GAPDH as an internal control.

2.10. Statistical Analysis

SPSS 21.0 software was employed for data analysis. Student's t-test was for comparing two groups, and one-way analysis of variance (ANOVA), followed by Tukey's post hoc test, was for the comparisons among multiple groups. The results were displayed as mean ± standard deviation (SD) of at least three experiments, and P < 0.05 represented the markedly significant data.

3. Results

3.1. ZNF488 Is Highly Expressed in Pancreatic Carcinoma Tissues and Cells

First, we retrieved the ZNF488 expression in 178 PC patients and 4 normal people through the online database starBase V3.0 (https://starbase.sysu.edu.cn/) and the prognostic relationship between ZNF488 expression and PC. The results showed that the ZNF488 expression in PC patients was notably higher than that in the normal group (Figure 1(a)). The survival rate of patients with high ZNF488 expression was distinctly lower than patients with low ZNF488 expression (Figure 1(b)). Furthermore, we verified that the ZNF488 expression in PC tissues and normal tissues (normal) confirmed that ZNF488 mRNA and proteins were upregulated in cancer tissues of PC patients (Figures 1(c) and 1(d)). In addition, the ZNF488 expression was evidently higher in PC cell lines than in normal pancreatic duct epithelial cells (HPME), with the highest expression in PANC-1 cells and the lowest expression in AsPC-1 cells (Figure 1(e)), indicating that ZNF488 is involved in the occurrence and development of PC.

Figure 1

Highly expressed ZNF488 in pancreatic carcinoma. (a) starBase V3.0 database for analyzing the ZNF488 expression in pancreatic carcinoma (PC) patients; (b) starBase V3.0 database for evaluating the relationship between ZNF488 expression and the prognosis of PC patients; (c) qRT-PCR for assessing the ZNF488 expression in cancer tissues and adjacent tissues of PC patients; ∗∗P < 0.01 vs. normal group; N = 30 per group. (d) Western blot for evaluating the protein expression of ZNF488 in cancer tissues and adjacent tissues of PC patients; ∗∗P < 0.01 vs. normal group. (e) qRT-PCR for detecting the ZNF488 expression in PC cell lines (PANC-1, SW1990, AsPC-1, and Capan-2) and normal pancreatic duct epithelial cells (HPME); ∗∗P < 0.01 vs. HPME group. All experiments were repeated three times.

3.2. The Highly Expressed ZNF488 Promotes the Proliferation of Pancreatic Carcinoma Cells

To further explore the function of ZNF488 in PC, we overexpressed or interfered with ZNF488 in PANC-1 and AsPC-1 cells, respectively. RT-qPCR results indicated that after knocking down ZNF488, the ZNF488 expression in PANC-1 cells decreased evidently. After overexpressing ZNF488, the ZNF488 expression in AsPC-1 cells was markedly higher than that in the vector group (Figure 2(a)), indicating that the transfection was successful. Besides, CCK-8 and colony formation experiments exhibited that after interfering with the ZNF488 expression, the PANC-1 cell proliferation rate and colony formation were notably lower. The overexpressing ZNF488 significantly promoted the proliferation rate and colony formation of AsPC-1 cells (Figures 2(b)–2(d)), suggesting that ZNF488 can promote the proliferation of PC cells.

Figure 2

ZNF488 promotes the proliferation of pancreatic carcinoma cells. (a) qRT-PCR for assessing the ZNF488 expression in each group of PANC-1 and AsPC-1 cells; (b) CCK-8 for evaluating the proliferation rate of PANC-1 and AsPC-1 cells; (c) cell cloning experiment for measuring PANC-1 and AsPC-1 cell proliferation ability. ∗∗P < 0.01 vs. sh-NC group, ##P < 0.01 vs. vector group.

3.3. ZNF488 Can Improve the Invasion and Migration Ability of Pancreatic Carcinoma Cells

We further investigated the effect of ZNF488 on the invasion and migration ability of PC cells. The Transwell experiment confirmed that after knocking down ZNF488, the invasion ability of PANC-1 cells was markedly decreased, and after overexpressing ZNF488, the invasion ability of AsPC-1 cells was distinctly improved (Figures 3(a) and 3(b)). What is more, the scratch test demonstrated that after knocking down ZNF488, the cell migration ability in the sh-ZNF488 group was distinctly lower than that in the sh-NC group. However, after overexpressing ZNF488, the ZNF488 group had notably higher cell migration ability than the vector group (Figures 3(c) and 3(d)), proving that ZNF488 can improve PC cell invasion and migration ability.

Figure 3

ZNF488 improves the invasion and migration ability of pancreatic carcinoma cells. (a) Transwell experiment for assessing the invasion ability of each group of cells in PANC-1 cells; (b) Transwell experiment for determining the invasion ability of each group of cells in AsPC-1 cells; (c) scratch experiment for evaluating the migration ability of each group of cells in PANC-1 cells; (d) scratch test for measuring the migration ability of each group of cells in AsPC-1 cells. ∗∗P < 0.01 vs. sh-NC group, ##P < 0.01 vs. vector group.

3.4. ZNF488 Can Activate the Akt/mTOR Signaling Pathway in Pancreatic Carcinoma Cells to Influence the Occurrence and Development of Pancreatic Carcinoma

We further studied the mechanism of ZNF488 in promoting the occurrence and development of PC. Western blot results confirmed that after knocking down ZNF488, the expression levels of p-Akt and p-mTOR were markedly lower than that of the sh-NC group, and the ratios of p-Akt/Akt and p-mTOR/mTOR were distinctly lower (Figure 4(a)). However, after overexpressing ZNF488, the expression levels of p-Akt and p-mTOR in cells increased evidently, as well as the ratios of p-Akt/Akt and p-mTOR/mTOR (Figure 4(b)). To further verify the involvement of the Akt/mTOR pathway, we added the Akt agonist SC79 and Akt inhibitor AZD5363, respectively, to study the function of ZNF488 on the Akt/mTOR pathway. The results suggested that Akt agonist SC79 can relieve the inhibition of ZNF488 knockdown on the Akt/mTOR pathway (Figure 4(c)). Akt inhibitor AZD5363 can inhibit the activation of ZNF488 overexpression on the Akt/mTOR pathway (Figure 4(d)), showing that ZNF488 mediates the PC occurrence by regulating the activity of the Akt/mTOR pathway.

Figure 4

The effect of ZNF488 on Akt/mTOR signaling pathway in pancreatic carcinoma cells. (a, b) Western blot for assessing the expression of Akt, p-Akt, mTOR, and p-mTOR in PANC-1 cells (a) and AsPC-1 cells (b), ∗∗P < 0.01 vs. sh-NC group or vector group; (c) Western blot for evaluating the Akt, p-Akt, mTOR, and p-mTOR expression in PANC-1 cells in the sh-ZNF488 group, SC79 group, and sh-ZNF488+SC79 group cells, ∗∗P < 0.01 vs. sh-ZNF488 group, ##P < 0.01 vs. SC79 group; (d) Western blot for measuring the expression of Akt, p-Akt, mTOR, and p-mTOR in AsPC-1 cells in ZNF488 group, AZD5363 group, ZNF488+AZD5363 group, ∗∗P < 0.01 vs. ZNF488 group, ##P < 0.01 vs. AZD5363 group.

4. Discussion

PC is one of the three most common causes of cancer-related deaths and one of the most destructive cancers in my country. The survival period is only 3 to 5 months, with the 5-year survival rate below 4% [19]. According to statistics, PC incidences and mortality in China from 2004 to 2015 increased at an annual rate of 1.3% and 1.25%, respectively [20]. However, the actual incidence rate is higher than the statistical data due to the statistical data only covering 12% of the national population [19]. High mortality rates are mainly due to late manifestations and suboptimal treatment options. Studies have shown that approximately 10% of PC cases have a familial basis [21]. At present, the clinical prognosis of PC is often poor, partly due to the late identification and treatment resistance, with the current surgical treatment still in the clinical research stage. Therefore, early genetic screening and intervention in patients with PC can affect the diagnosis, treatment, and prognosis.

ZNF488 has been implicated as a glial cell-specific transcriptional repressor [8, 22]. The current study indicates that ZNF488 is involved in the progression of NPC [9]. In the present study, we found that the ZNF488 expression levels in PC patients were significantly increased compared with the normal group by online database search. High expression of ZNF488 was associated with poor patient prognosis. At the same time, we detected significantly elevated expression of ZNF488 in PC tissues and cells. This is consistent with the report by Li et al. [11]. We, therefore, speculated that ZNF488 might be involved in the development of PC. To further clarify the effect of ZNF488 on the function of PC cells, we overexpressed or interfered with the expression of ZNF488 in PC cells and found that ZNF488 overexpression promoted the proliferation of PC cells and significantly increased the viability, migration, and invasion of PC cells. Interfering with the expression of ZNF488 could inhibit the function of PC cells. These results indicate that upregulated ZNF488 can significantly promote the development of PC cells. This is consistent with previous findings that zinc finger proteins promote the malignant development of tumor cells [23, 24].

The Aoki and Fujishita study [25] found that the AKT/mTOR signaling pathway significantly impacts the development and prognosis of malignant tumors. In addition, aberrant activation of the AKT/mTOR pathway can lead to malignant tumor proliferation, blocked apoptosis, accelerated circulatory manipulation, angiogenesis, invasion, and metastasis [26-29]. To further investigate its mechanism of action, we used western blot to detect Akt, p-Akt, mTOR, and p-mTOR protein levels in overexpressed or silenced ZNF488 cells. We found that Akt and mTOR protein phosphorylation levels were significantly increased in overexpressed ZNF488 cells while silencing ZNF488 had the opposite result. The further intervention of cells using Akt agonist SC79 and Akt inhibitor AZD5363, respectively, also revealed that SC79 and AZD5363 reversed the effects on AKT/mTOR signaling pathway-related proteins when silencing ZNF488 or overexpressing ZNF488. The above results indicate that overexpression of ZNF488 can activate the AKT/mTOR signaling pathway to participate in regulating PC development and progression.

5. Conclusions

In summary, ZNF488 is highly expressed in PC, and ZNF488 can promote the proliferation, invasion, and migration of PC cells by activating the AKT/mTOR signaling pathway. This study confirmed that ZNF488 could be used as a new molecular target for preventing and treating PC.