Journal of Oncology

Journal of Oncology / 2021 / Article
Special Issue

Prognosis, Diagnosis and Treatments for Digestive System Cancers

View this Special Issue

Research Article | Open Access

Volume 2021 |Article ID 6880473 | https://doi.org/10.1155/2021/6880473

Hua Zhang, Pingling Lin, Lei Fu, Zhijun Li, Yan Ding, "Propofol Suppresses Cell Progression by Inhibiting CCL18 Expression in Hepatoblastoma", Journal of Oncology, vol. 2021, Article ID 6880473, 8 pages, 2021. https://doi.org/10.1155/2021/6880473

Propofol Suppresses Cell Progression by Inhibiting CCL18 Expression in Hepatoblastoma

Academic Editor: Muhammad Wasim Khan
Received25 Jun 2021
Accepted08 Jul 2021
Published27 Jul 2021

Abstract

Background. Propofol is an anesthetic commonly used clinically and has been found to have antitumor activity in various cancers. The purpose of this study was to investigate the role of propofol in hepatoblastoma (HB). Methods. CCK-8 and transwell were used to measure cell proliferation, migration, and invasion in HB cells. Cell apoptosis rate was measured by FCM. The expression of CCL18 in HB tissues and cells was detected by RT-qPCR. Western blotting was used to explore the protein expression of CCK18- and PI3K/AKT-related proteins. Results. The expression of CCL18 in HB tissues and cells was overexpressed compared with control groups. CCL18 knockdown was found to notably block cell proliferation and progression, while enhancing cell apoptosis in HuH-6 and HepT1 cells. Furthermore, propofol suppressed the proliferation of HB cells in a dose-dependent manner. According to the results, we chose 5 μg/mL of propofol-treated cells for 48 hours as the subsequent experimental conditions. We found that propofol (5 μg/mL, 48 h) significantly blocked cell migration and invasion, but induced cell apoptosis in HuH-6 and HepT1 cells. In addition, CCK18 overexpression facilitated cell progression in HB cells, while propofol dramatically suppressed the effect of CCK18. Besides that, propofol suppressed the PI3K/AKT pathway. Conclusion. Propofol suppressed the development of HB cells by inhibiting CCK18 expression and the PI3K/AKT pathway. Therefore, we infer that propofol plays a role in the treatment of HB.

1. Introduction

Hepatoblastoma (HB) is mainly derived from human immature liver precursor cells [1]. HB is the most common primary liver malignant tumor in children, and 90% of patients are affected before the age of 5 years [2]. HB is an increasingly common tumor worldwide, especially in North America and Europe. HB is treated by adjuvant chemotherapy, radiotherapy, and transplantation, and the survival rate for patients with HB has increased dramatically. However, for patients with advanced HB, the prognosis is still poor [3]. Therefore, it is essential to explore the early biomarkers of HB and find effective treatment methods for patients with HB.

Propofol is a short-acting intravenous anesthetic of alkyl phenols, which is widely used in clinical practice [4]. It plays a sedative and hypnotic role by activating the GABA receptor-chloride ion complex [5]. Propofol has the advantages of rapid onset of anesthesia induction, rapid recovery and complete functional recovery, and low incidence of postoperative malignant vomiting [6]. In addition to the anesthetic effect, propofol can also affect the biological process of tumors and have different effects on different tumor cells [7]. Propofol has been reported to affect the epigenetic pathways such as lncRNAs, miRNAs, and cancer-related proteins and regulate the genetic signaling pathways such as MAPK, PI3K/AKT, NF-κB, SLUG, and Nrf2 [8]. A growing number of studies have shown that propofol may exert an antitumor effect in human tumors, such as colon cancer [9], ovarian cancer [10], and lung cancer [11]. At present, propofol is widely used in HB surgery and postoperative sedation. Therefore, the effect of propofol on the biological behavior of HB cells has gradually attracted researchers’ attention.

CCL18 is a member of the chemokines family. CCL18 has been found to play a special role in the development of human tumors. It is reported that the role of CCL18 in tumors appears after TAM involvement into the tumor niche [12]. Lane et al. reported that CCL18 in ascites accelerated cell migration by regulating Pyk2 in ovarian cancer [13]. In oral cancer, CCL18 was found to facilitate cell progression by activating the JAK2/STAT3 pathway [14]. However, the role of CCL18 in HB is not well understood.

In this study, we aimed to investigate the effect of propofol on the progression of HB. Furthermore, propofol was confirmed to suppress HB tumor progression by regulating CCL18.

2. Materials and Methods

2.1. Patient Samples

HB tissues were obtained from 20 children in Yantaishan Hospital (Yantai, China). All patients were diagnosed as HB preoperatively. Twenty paracancer tissues were at least 2 cm from the tumor margin. All specimens were placed in liquid nitrogen within 30 minutes after isolation and then stored in a −80°C refrigerator. This study was approved by the ethics committee of Yantaishan Hospital, and informed consent of the patients was obtained before surgery.

2.2. Cell Lines and Propofol

Human HB cell lines HepG2, HuH-6, and HepT1 and normal liver cells MIHA were collected from Yantaishan Hospital. After resuscitation at 37°C, the cells were resuspended on Dulbecco’s Modified Eagle Medium (DMEM) containing 100 ml/L fetal bovine serum (FBS) and 10 ml/L penicillin and streptomycin. The cells were cultured in a constant-temperature incubator at 37°C and 5% CO2 for 3-4 days. After cell fusion, the cells were subcultured into a cell culture flask (5 ml/L). Cells were cultured for 4-5 generations for subsequent experiments. Propofol was obtained from Qingyuan JiaBo Pharmaceutical Co. Ltd. (China). The final concentrations of propofol were 0, 2, 5, and 10 μg/mL with complete medium.

2.3. Transfection of si-CCL18

Cells (1 × 106/mL) were inoculated into a 6-well plate. HuH-6 and HepT1 cells were transfected with CCL18 si-RNA (5′-ACAAGTTGGTACCAACAAATT-3′), pcDNA3.1-CCL18, and the corresponding negative control oligonucleotides mixed with LipofectamineTM 2000, respectively. After transfection for 48 h, the cells were collected for subsequent experiments.

2.4. Cell Proliferation

Proliferative ability of HuH-6 and HepT1 cells was evaluated by using the cell counting kit-8 (CCK-8) method. Cell suspension was evenly planted in a 96-well plate with a density of 3х 103 cells/well. Cells were cultured in an incubator for 24, 48, and 72 h and then added with 100 μl DMEM medium and 10 μl CCK-8 solution. The cells were replaced in the incubator for further incubation for 1 to 4 hours. A microplate analyzer was used to detect the OD value of cells at 450 nm.

2.5. Cell Migration and Invasion

Transwell assay was performed to measure cell migration and invasion in HuH-6 and HepT1 cells. Cells are routinely digested to prepare cell suspensions. 100 μl cell suspension was inoculated in the upper transwell chamber, and 500 μl DMEM was added to the lower chamber. The cells were cultured in a constant-temperature cell incubator a 37°C and 5% CO2. After 24 hours, 200 μl paraformaldehyde was added to fix cells for 15 min. Then, cells were stained with crystal violet for 20 min. Photographs were taken under a microscope, and the number of migrated and invaded cells was counted.

2.6. Cell Apoptosis

Flow cytometry (FCM) was used to measure cell apoptosis in HuH-6 and HepT1 cells. Cells were seeded into a 24-well plate and incubated in an incubator at 37°C and 5% CO2 for 24 h. After centrifugation at 1500 r/min for 5 min, the supernatant was discarded. Cells were labeled with 1 mL Annexin V and 1 mL Sytox Green. After incubation at 37°C for 15 min, FCM (Beckman Coulter, Germany) was performed to detect cell apoptosis rate within 4 h.

2.7. Real-Time PCR

Total RNA was extracted by Trizol reagent. Reverse transcription system: 5xPrimer Script Buffer 2 μl, RT Enzyme Mix 0.5 μl, primer 0.5 μl, RNA1 μl, and RNAase-free ddH2O 6 μl. The cDNA was obtained by reverse transcription and then diluted to 50 ng/μl. Real-time PCR transcription system: SYBR premix ExTaqTM II (2x) 10 μl, forward primer 1 μl, reverse primer 1 μl, cDNA 1 μl, and ddH2O 7 μl. The primers were CCL18 forward: 5′-TGGCAGATTCCACAAAAGTTCA-3′, reverse 5′-GGATGACACCTGGCTTGGG-3′; GAPDH forward 5′-GCACCGTCAAGGCTGAGAAC-3′, and reverse 5′-TGGTGAAGACGCCAGTGGA-3′.

2.8. Western Blotting

After transfection for 24–48 hours, cell proteins were extracted by RIPA lysate. The BCA kit was used to detect protein concentration and purity. The protein samples were subjected to SDS-PAGE gel electrophoresis at 80 V and for 2 hours. After the protein was transferred to the PVDF membrane, it was sealed with milk powder at room temperature for 1–2 hours. Subsequently, the PVDF membrane was added with primary antibody and incubated overnight at 4°C. Then, the PVDF membrane was incubated with secondary antibody at room temperature for 2 hours. Finally, ECL chemiluminescence solution was added into the gel imaging system to collect images, and the protein bands were quantitatively analyzed with Image J.

2.9. Statistical Analysis

SPSS23.0 statistical software and GraphPadPrism software were used for statistical analysis. The t-test was used to compare the differences between the two experimental groups. Univariate ANOVA analysis was used for comparison between groups. indicated that the comparison results between the experimental groups were statistically significant. All data are mean ± SD from three or more independent trials.

3. Results

3.1. Overexpression of CCL18 Was Observed in HB

According to the RT-qPCR assay, tumor tissues assumed the overexpression of CCL18 compared with normal tissues (Figure 1(a)). Next, western blot results showed that CCL18 was higher expressed in HepG2, HuH-6, and HepT1 cells than in MIHA cells (Figure 1(b)). Therefore, we speculated that CCL18 might play a specific role in HB progression.

3.2. CCL18 Depletion Played an Antitumor Role in HuH-6 and HepT1 Cells

To investigate the specific role of CCL18 in HB, we knock down its expression in HuH-6 and HepT1 cells. As shown in Figure 2(a), the expression of CCL18 was notably reduced by CCL18 si-RNA. CCK-8, transwell, and FCM were used to assess the effect of CCL18 knockdown in HB cells. As we expected, cell proliferation was significantly declined in HuH-6 and HepT1 cells when transfected with CCL18 knockdown (Figure 2(b)). Furthermore, after CCL18 knockdown transfection, the invasiveness of HuH-6 and HepT1 cells was significantly decreased (Figure 2(c)). Similarly, the migration ability of HuH-6 and HepT1 cells transfected with CCL18 knockdown was notably reduced (Figure 2(d)), whereas FCM results verified that CCL18 depletion significantly accelerated cell apoptosis in HB cells (Figure 2(e)). Taken together, we assumed that CCL18 knockdown played a role as a tumor suppressor in HB cells.

3.3. Propofol Suppressed Cell Growth, Migration, and Invasion in HB Cells

To investigate the effect of propofol in HB cells, the concentration and treatment time of propofol were first determined by CCK-8 results. We found that cell viability of HuH-6 and HepT1 cells was notably reduced in a concentration-dependent effect of propofol (0, 2, 5, and 10 μg/mL) (Figure 3(a)). Next, cell viability was suppressed in HuH-6 and HepT1 cells treated with 5 μg/mL propofol for 24, 48, or 96 h (Figure 3(b)). According to the experimental results, 5 μg/mL propofol treatment for 48 hours () was selected for the subsequent experimental condition. CCK-8 assay indicated that propofol (5 μg/mL, 48 h) suppressed cell viability in HB cells (Figure 3(c)). Transwell assay displayed that propofol (5 μg/mL, 48 h) significantly reduced cell invasion (Figure 3(d)). Not surprisingly, cell migration ability was notably reduced by propofol (Figure 3(e)). In addition, propofol dramatically accelerated cell apoptosis in HuH-6 and HepT1 cells (Figure 3(f)).

3.4. Propofol Suppressed Cell Progression by Inhibiting CCL18 Expression and Downregulating the PI3K/AKT Pathway in HB

In order to study the effect of propofol on CCL18 in the progression of HB cells, HuH-6 and HepT1 cells transfected with CCL18 vector were treated with propofol. As shown in Figure 4(a), propofol decreased the expression of CCL18 vector in HuH-6 and HepT1 cells. Next, functional experiments were performed to verify the interaction between propofol and CCL18 in HB cells. We noticed that CCL18 vector accelerated the proliferation of HB cells, while propofol weakened the effect of CCL18 (Figure 4(b)). Likewise, propofol neutralized the stimulative effect of CCL18 on cell migration and invasion (Figures 4(c) and 4(d)).

The PI3K-Akt pathway is a classic antiapoptotic and prosurvival signal transduction pathway, which plays an important role in the occurrence, proliferation, invasion, and metastasis of tumors [15]. Western blot assay was used to study the effect of propofol/CCL18 on the PI3K/AKT pathway. The results displayed that CCL18 vector increased the phosphorylation of PI3K and AKT, while propofol suppressed the phosphorylation of PI3K and AKT (Figure 4(e)).

4. Discussion

The expression and function of CCL18 in HB were evaluated in this study. We found that CCL18 was obviously overexpressed in HB tissues. Additionally, CCL18 was also overexpressed in HB cell lines (HepG2, HuH-6, and HepT1 cells) compared with MIHA cells. CCK-8, transwell, and FCM assay were used to study the effect of CCL18 on tumor progression in vitro. In HuH-6 and HepT1 cells, knockdown of CCL18 significantly reduced cell proliferation, invasion, and migration, but significantly promoted cell apoptosis. Combined with the experimental results, we speculated that CCL18 plays a role as a procancer factor in HB. Many previous studies have confirmed that CCL18 plays an oncogenic role in a variety of cancers. Lin et al. found that CCL18 facilitated cell progression and EMT by regulating the NF-κB pathway in hepatocellular carcinoma [16], which is consistent with our findings. Furthermore, CCL18 promoted cell growth and cell motility in squamous cell carcinoma of the head and neck [17]. In osteosarcoma, Su et al. confirmed that CCL18 promoted tumor progression by increasing UCA1 expression [18].

Propofol is the most widely used intravenous anesthetic in clinical anesthesia. Propofol is characterized by quick action, strong effect, and quick recovery. At present, the role of propofol in human tumors has become a research hotspot. Previous studies have shown that propofol obviously suppresses tumor development. Zhou et al. reported that propofol inhibited tumor growth in mice with liver cancer [19]. Propofol was verified to reduce proinflammatory factors and increase anti-inflammatory factors, so as to effectively improve the immune function of patients with pancreatic cancer [20]. In pancreatic cancer, propofol suppressed cell proliferation and migration by inhibiting the ERK/MMPs pathway [21]. In hepatocellular carcinoma, propofol has been shown to block the progression [2224]. Similarly, we found that propofol inhibited extracellular activity in HB. Unlike the previous results, we studied the effect of propofol on CCL18 expression in HB cells. We noticed that propofol reduced the expression level of CCL18. Above all, the promotional effect of CCL18 in HB was suppressed by propofol.

The PI3K/AKT pathway is a classical signaling pathway involved in the regulation of various cellular functions such as proliferation, differentiation, apoptosis, and glucose transport [25]. In recent years, the PI3K/AKT signaling pathway has been found to be closely related to the occurrence and development of human tumors [26, 27]. This pathway regulates the proliferation and survival of tumor cells and is related to tumor cell migration, adhesion, angiogenesis, and degradation of the extracellular matrix [28, 29]. Furthermore, propofol was found to suppress the phosphorylation of PI3K and AKT. Therefore, the results showed that propofol/CCK18 suppressed the development by inhibiting the PI3K/AKT pathway.

To sum up, we found that propofol notably suppressed the progression in HB cells. In addition, propofol played an inhibitory role by blocking the CCL18 expression and PI3K/AKT pathway in HB. However, the dual effect of propofol in relieving the pain of tumor patients and inhibiting tumor progression needs to be confirmed by animal models and clinical trials.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. D. Sharma, G. Subbarao, and R. Saxena, “Hepatoblastoma,” Seminars in Diagnostic Pathology, vol. 34, no. 2, pp. 192–200, 2017. View at: Publisher Site | Google Scholar
  2. S. Ranganathan, D. Lopez-Terrada, and R. Alaggio, “Hepatoblastoma and pediatric hepatocellular carcinoma: an update,” Pediatric and Developmental Pathology, vol. 23, no. 2, pp. 79–95, 2020. View at: Publisher Site | Google Scholar
  3. K. B. Hooks, J. Audoux, H. Fazli et al., “New insights into diagnosis and therapeutic options for proliferative hepatoblastoma,” Hepatology, vol. 68, no. 1, pp. 89–102, 2018. View at: Publisher Site | Google Scholar
  4. R. Vellinga, L. N. Hannivoort, M. Introna et al., “Prospective clinical validation of the Eleveld propofol pharmacokinetic-pharmacodynamic model in general anaesthesia,” British Journal of Anaesthesia, vol. 126, no. 2, pp. 386–394, 2021. View at: Publisher Site | Google Scholar
  5. M. M. Sahinovic, M. M. R. F. Struys, and A. R. Absalom, “Clinical pharmacokinetics and pharmacodynamics of propofol,” Clinical Pharmacokinetics, vol. 57, no. 12, pp. 1539–1558, 2018. View at: Publisher Site | Google Scholar
  6. S. Kim, S. Hahn, M.-J. Jang et al., “Evaluation of the safety of using propofol for paediatric procedural sedation: a systematic review and meta-analysis,” Scientific Reports, vol. 9, no. 1, Article ID 12245, 2019. View at: Publisher Site | Google Scholar
  7. Y. Xu, S. Pan, W. Jiang, F. Xue, and X. Zhu, “Effects of propofol on the development of cancer in humans,” Cell Proliferation, vol. 53, no. 8, Article ID e12867, 2020. View at: Publisher Site | Google Scholar
  8. Y. Ogawa, M. Irifune, A. Mukai et al., “The indirect γ-aminobutyric acid (GABA) receptor agonist gabaculine-induced loss of the righting reflex may inhibit the descending analgesic pathway,” Pharmacology Biochemistry and Behavior, vol. 198, Article ID 173034, 2020. View at: Publisher Site | Google Scholar
  9. Y. F. Zhang, C. S. Li, Y. Zhou, and X. H. Lu, “Effects of propofol on colon cancer metastasis through STAT3/HOTAIR axis by activating WIF‐1 and suppressing Wnt pathway,” Cancer Medicine, vol. 9, no. 5, pp. 1842–1854, 2020. View at: Publisher Site | Google Scholar
  10. Y. Sun, Y. Peng, L. Ye, L. Ma, M. Zou, and Z. Cheng, “Propofol inhibits proliferation and cisplatin resistance in ovarian cancer cells through regulating the microRNA-374a/forkhead box O1 signaling axis,” Molecular Medicine Reports, vol. 21, no. 3, pp. 1471–1480, 2020. View at: Google Scholar
  11. Y. Huang, L. Lei, and Y. Liu, “Propofol improves sensitivity of lung cancer cells to cisplatin and its mechanism,” Medical Science Monitor: International Medical Journal of Experimental and Clinical Research, vol. 26, Article ID e919786, 2020. View at: Publisher Site | Google Scholar
  12. J. Korbecki, M. Olbromski, and P. Dzięgiel, “CCL18 in the progression of cancer,” International Journal of Molecular Sciences, vol. 21, no. 21, 2020. View at: Publisher Site | Google Scholar
  13. D. Lane, I. Matte, C. Laplante et al., “CCL18 from ascites promotes ovarian cancer cell migration through proline-rich tyrosine kinase 2 signaling,” Molecular Cancer, vol. 15, no. 1, p. 58, 2016. View at: Publisher Site | Google Scholar
  14. X. Jiang, Z. Huang, X. Sun et al., “CCL18-NIR1 promotes oral cancer cell growth and metastasis by activating the JAK2/STAT3 signaling pathway,” BMC Cancer, vol. 20, no. 1, p. 632, 2020. View at: Publisher Site | Google Scholar
  15. N. Jiang, Q. Dai, X. Su, J. Fu, X. Feng, and J. Peng, “Role of PI3K/AKT pathway in cancer: the framework of malignant behavior,” Molecular Biology Reports, vol. 47, no. 6, pp. 4587–4629, 2020. View at: Publisher Site | Google Scholar
  16. Z. Lin, W. Li, H. Zhang et al., “CCL18/PITPNM3 enhances migration, invasion, and EMT through the NF-κB signaling pathway in hepatocellular carcinoma,” Tumor Biology, vol. 37, no. 3, pp. 3461–3468, 2016. View at: Publisher Site | Google Scholar
  17. Y. Qin, J. Wang, G. Zhu et al., “CCL18 promotes the metastasis of squamous cell carcinoma of the head and neck through MTDH‐NF‐κB signalling pathway,” Journal of Cellular and Molecular Medicine, vol. 23, no. 4, pp. 2689–2701, 2019. View at: Publisher Site | Google Scholar
  18. Y. Su, Y. Zhou, Y.-J. Sun et al., “Macrophage-derived CCL18 promotes osteosarcoma proliferation and migration by upregulating the expression of UCA1,” Journal of Molecular Medicine, vol. 97, no. 1, pp. 49–61, 2019. View at: Publisher Site | Google Scholar
  19. Q. Zhou, H. Wu, Y. Liu et al., “Effects of different doses of propofol on the growth and expression of PCNA, CD34 and pAKT proteins in xenografted tumor of BALB/C mice with liver cancer,” Clinical and Translational Oncology, vol. 22, no. 10, pp. 1741–1749, 2020. View at: Publisher Site | Google Scholar
  20. Z. Wang and S. Zhou, “Effect of compound propofol nanoemulsion on immune function in patients with pancreatic cancer,” Journal of Nanoscience and Nanotechnology, vol. 21, no. 2, pp. 1390–1396, 2021. View at: Publisher Site | Google Scholar
  21. X. Yu, J. Shi, X. Wang, and F. Zhang, “Propofol affects the growth and metastasis of pancreatic cancer via ADAM8,” Pharmacological Reports, vol. 72, no. 2, pp. 418–426, 2020. View at: Publisher Site | Google Scholar
  22. Y. Sun and H. Sun, “Retracted: Propofol exerts anticancer activity on hepatocellular carcinoma cells by raising lncRNA DGCR5,” Journal of Cellular Physiology, vol. 235, no. 3, pp. 2963–2972, 2020. View at: Publisher Site | Google Scholar
  23. H. Zheng, Y. Fu, and T. Yang, “Propofol inhibits proliferation, migration, and invasion of hepatocellular carcinoma cells by downregulating twist,” Journal of Cellular Biochemistry, vol. 120, no. 8, pp. 12803–12809, 2019. View at: Publisher Site | Google Scholar
  24. S.-Q. Liu, J.-L. Zhang, Z.-W. Li, Z.-H. Hu, Z. Liu, and Y. Li, “Propofol inhibits proliferation, migration, invasion and promotes apoptosis through down-regulating miR-374a in hepatocarcinoma cell lines,” Cellular Physiology and Biochemistry, vol. 49, no. 6, pp. 2099–2110, 2018. View at: Publisher Site | Google Scholar
  25. H. Sakai, I. Shiina, T. Shinomiya, and Y. Nagahara, “BRAP2 inhibits the Ras/Raf/MEK and PI3K/Akt pathways in leukemia cells, thereby inducing apoptosis and inhibiting cell growth,” Experimental and Therapeutic Medicine, vol. 21, no. 5, p. 463, 2021. View at: Publisher Site | Google Scholar
  26. H. Lin, G. Zhang, X.-C. Zhang et al., “Germline variation networks in the PI3K/AKT pathway corresponding to familial high-incidence lung cancer pedigrees,” BMC Cancer, vol. 20, no. 1, p. 1209, 2020. View at: Publisher Site | Google Scholar
  27. L. Zhang, J. Wu, M. T. Ling, L. Zhao, and K.-N. Zhao, “The role of the PI3K/Akt/mTOR signalling pathway in human cancers induced by infection with human papillomaviruses,” Molecular Cancer, vol. 14, no. 1, p. 87, 2015. View at: Publisher Site | Google Scholar
  28. R. Liu, Y. Chen, G. Liu et al., “PI3K/AKT pathway as a key link modulates the multidrug resistance of cancers,” Cell Death & Disease, vol. 11, no. 9, p. 797, 2020. View at: Publisher Site | Google Scholar
  29. X. Li, D. Dai, B. Chen, H. Tang, X. Xie, and W. Wei, “Efficacy of PI3K/AKT/mTOR pathway inhibitors for the treatment of advanced solid cancers: a literature-based meta-analysis of 46 randomised control trials,” PLoS One, vol. 13, no. 2, Article ID e0192464, 2018. View at: Publisher Site | Google Scholar

Copyright © 2021 Hua Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


More related articles

 PDF Download Citation Citation
 Download other formatsMore
 Order printed copiesOrder
Views136
Downloads190
Citations

Related articles

Article of the Year Award: Outstanding research contributions of 2020, as selected by our Chief Editors. Read the winning articles.