Effects of plumbagin on migration and invasion of human hepatoma cell line via J

时间：2024-07-06

CHENG Tao, WEI Yan-fei, LIU Huan, LIU Hong, DENG Shu-ye✉

1.School of Basic Medical Sciences, Guangxi University of Traditional Chinese Medicine, Nanning 530200, China

2.Guangxi Key Laboratory of Molecular Biology of Preventive Medicine of Traditional Chinese Medicine,Nanning 530001,China

Keywords:

ABSTRACT Objective:To study the effect of plumbagin (PL) on the migration and invasion of human hepatocellular carcinoma (HCC) cells and its possible mechanism.Methods:The cell counting kit (CCK-8) was used to detect the effects of different concentrations of plumbagin on the proliferation of human hepatocellular carcinoma Huh-7 and LM3 cells.The effect of plumbagin on the migration ability of Huh-7 and LM3 cells was detected by scratch test and Transwell migration test, and the effect of on the invasion ability of Huh-7 and LM3 cells was detected by Transwell invasion test.Western Blot was used to detect the expression of E-cadherin, N-cadherin, matrix metalloproteinase-2 and related proteins in JAK2/STAT3 signaling pathway in Huh-7 and LM3 cells.Results:Plumbagin could inhibit the proliferation of Huh-7 and LM3 cells in a time-and concentration-dependent manner.Plumbagin inhibited the migration and invasion of Huh-7 and LM3 cells in a concentration dependent manner,and it can down-regulate the expression of N-cadherin and MMP-2 protein, up-regulate the expression of E-cadherin protein, and inhibit the activation of JAK2/STAT3 signaling pathway.Conclusion:Plumbagin can inhibit the migration and invasion of human hepatocellular carcinoma Huh-7 and LM3 cells, and the molecular mechanism of this process may be related to the inhibition of JAK2/STAT3 signaling pathway activation.

1.Introduction

Primary liver cancer (hereinafter referred to as liver cancer) is one of the most common malignant tumors, and China has a high incidence of liver cancer.It is estimated that in 2022, there will be 431 383 deaths from liver cancer and 431 383 new cases of liver cancer [1].Due to the characteristics of liver cancer such as insidious onset, early metastasis, rapid progression and strong invasiveness,early liver cancer has almost no obvious clinical symptoms, and up to 80% of liver cancer patients have entered the middle and advanced stage once diagnosed, thus losing the opportunity for radical treatment [2].The high invasion and metastasis characteristics of liver cancer cells are the key to the rapid proliferation and development of liver cancer cells.Therefore, we urgently need to explore new strategies to inhibit the migration and invasion of liver cancer cells.

Plumbagin (PL) is an active quinone substance isolated from the root of the traditional medicinal plant Plumbagin, and it has been confirmed that Plumbagin has anti-inflammatory, anticancer, anti-diabetes, anti-oxidation, antibacterial, anti-fungal, antiatherosclerosis and other biological activities [3].Current studies have shown that PL can inhibit the invasion and metastasis of breast cancer, lung cancer, tongue squamous cell carcinoma and brain tumor [4-7], but the effect of PL on the invasion and metastasis of liver cancer has not been further studied.Our previous studies have found that PL can inhibit the growth of liver cancer transplanted tumors in nude mice in vivo, and can also inhibit the angiogenesis of liver cancer by inhibiting the migration and invasion of tumorderived vascular endothelial cells [8], but the effect of PL on the invasion and metastasis of liver cancer cells in vitro remains unclear.Therefore, in this study, Huh-7 and LM3 cells of hepatocellular carcinoma were used as research objects to investigate the effects of PL on the migration and invasion of human hepatocellular carcinoma cells and its potential mechanism of action in vitro,providing theoretical and experimental basis for PL in the treatment of hepatocellular carcinoma.

2.Materials and methods

2.1 Materials

2.1.1 Cell lines

Huh-7 and LM3 cells were purchased from Beina Chuanglian Biotechnology Institute in Beijing, and cultured in a 5% CO2incubator at 37 ℃ using DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody.

2.1.2 Drugs and reagents

Plumbagin(Item number: P7262, purity 99%) was purchased from Sigma-Aldrich; Rabbit anti-e-cadherin (E-cadherin,Item number: BM4166), rabbit anti-N-cadherin (N-cadherin,Item number: BM3921), rabbit anti-signal transduction and transcription activator 3(STAT3, Item number:BM4052), rabbit antiphosphorylation STAT3 (p-STAT3,Item number: BM4835) were purchased from Wuhan Bode Biological Engineering Co., LTD.Rabbit anti-matrix metalloproteinase-2 (MMP-2,Item number:66366-1-lg), mouse anti-glyceraldehyde-3-phosphate dehydrogenase(GAPDH, Item number:60004-1-Ig), Goat Anti-mouse secondary antibody (Item number: SA00001-1) and Goat anti-rabbit secondary antibody (Item number: SA00001-2) were all purchased from Wuhan Sanying Biotechnology Co., LTD.Rabbit anti-nonreceptor tyrosine protein kinase 2(JAK2, Item number: BS5769) and rabbit anti-phosphorylation JAK2 (p-JAK2, Item number: BS4109) were purchased from Bio word, USA.

2.1.3 Instrument

Transwell (Item number: 3422) purchased from Corning Company,USA; Fluorescent inverted microscope (Model: IX71) from Olympus Corporation, Japan; Western blot electrophoresis apparatus(Power Model: Pac TM) and Western blot electrophoresis tank(Model: Mini-PROTEAN® Tetra) were purchased from BIO-RAD.

2.2 Methods

2.2.1 CCK-8 assay was used to detect the effect of PL on the proliferation of Huh-7 and LM3 cells

The Huh-7 and LM3 cells of logarithmic growth stage were inoculated into 96-well culture plates (5×103/ well), and the 96-well culture plates were placed in the incubator for culture.After the cells were attached to the wall, the old culture medium was discarded.Fresh medium containing different concentrations (0, 1,2, 4, 8, 10 μmol/L) was added for 24, 48, 72 h, respectively.There were 6 multiple Wells in each concentration group, blank group(adding equal amount of medium) and control group (adding equal amount of medium containing 0.1% DMSO).After the culture was continued in the 37 ℃ incubator for the corresponding time, 100 μL medium containing 10 μL CCK8 was added to each well according to the instructions of the CCK-8 kit.The medium was incubated at 37 ℃ for 1 h in the incubator.The light absorption value of each well at 450 nm wavelength was detected by enzymic marker and the inhibition rate of cell proliferation was calculated.

2.2.2.The effects of PL on the migration ability of Huh-7 and LM3 cells were detected by scratch assay

An appropriate amount of Huh-7 and LM3 cells at logarithmic growth stage were evenly spread in 6-well plates, and the cells were incubated in an incubator for 16-24 h to form monolayer cells.Three parallel lines were drawn in the middle of monolayer cells with the tip of a 10 μL gun and cleaned with PBS.The experimental group was added serum-free medium containing 2, 4, 8 μmol/L quinone,and the control group was added serum-free medium containing 0.1%DMSO.Five fixed sites were selected from each well for realtime monitoring, and photographs were taken with an inverted microscope at 0 h and 24 h respectively, and the cell scratch healing rate was calculated.

2.2.3 Transwell migration assay was used to detect the effects of PL on the migration ability of Huh-7 and LM3 cells

An appropriate amount of Huh-7 and LM3 cells at logarithmic growth stage were taken, and the cell density was adjusted by serumfree medium to 1×105cells /ml.100 μL was added into the upper chamber and different concentrations of quinone (2, 4, 8 μmol/L)were added for intervention.The control group was treated with the medium containing 0.1% DMSO.750 μL culture medium containing 20% fetal bovine serum was added into the room and cultured at 37℃ for 24 h.After that, the Transwell chamber was removed with tweezers, washed with PBS for 3 times, fixed at room temperature with 4% paraformaldehyde for 30 min, and stained with 0.1%crystal violet for 20 min.After that, the unmigrated cells in the upper chamber were wiped with a wet cotton swab.Five random fields were photographed under the microscope and the average number of migrated cells was calculated.

2.2.4 Transwell invasion assay was used to detect the effects of PL on the invasion ability of Huh-7 and LM3 cells

The upper and lower surface of the Transwell chamber was evenly coated with matrix glue and placed in the incubator for solidification for 1 h.An appropriate amount of Huh-7 and LM3 cells at logarithmic growth stage were taken, and the cell density was adjusted by serum-free medium to 1×105cells /mL.The prepared Transwell chamber was taken out and 100 μL serumfree medium was added into the upper chamber and different concentrations of Chrysanthoquinone (2, 4, 8 μmol/L) were added for intervention.The control group was also treated with serum-free medium containing 0.1%DMSO.750 μL medium containing 20%fetal bovine serum was added into the lower chamber and Transwell chamber was placed in 37 ℃ incubator for 24 h.The Transwell chamber was removed, washed with PBS for 3 times, fixed with 4%paraformaldehyde at room temperature and stained with 0.1% crystal violet.Then the uninvaded cells in the upper chamber were wiped with a wet cotton swab, and 5 fields were randomly selected under the microscope to take photos and calculate the average number of invaded cells.

2.2.5 Western blot assay was used to detect the effect of PL on protein expression in Huh-7 and LM3 cells

Huh-7 and LM3 cells were inoculated in 6-well culture plates,treated with different concentrations of quinone, and cultured in the 6-well plates for 24 h.The cells were washed with ice PBS twice and cleaved in ice RIPA lysate for 30 min, then centrifuged at 4 ℃at 12 000 g for 20 min.The protein concentration in the supernatant was determined using a BCA kit, the sample was separated by SDSPAGE gel electrophoresis, and then transferred to PVDF membrane.The membrane was sealed with 5% skim milk powder prepared by TBST at room temperature for 1 h and washed with TBST for 3 times, 10 min each time.After finishing, it was incubated with appropriate concentration of primary antibody at 4 ℃ overnight, and washed with TBST for 3 times, 10 min each time.After washing,the membrane was incubated with appropriate concentration of secondary antibody at room temperature for 1 h, and the membrane was washed again with TBST.Finally, the ECL chemiluminescence system was used to detect the bands on the membrane, and the density was determined by Image J software.

2.3 Statistical Analysis

Data results were statistically analyzed by GraphPad Prism 8.0 software, and measurement data were expressed as (±s)Independent sample t test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups.P＜0.05 indicates statistically significant difference.

3.Results

3.1 Effects of PL on the proliferation of Huh-7 and LM3 cells in HCC

Compared with blank control group, Huh-7 and LM3 cells were treated with quinone for 24, 48 and 72 h, and the cell proliferation inhibition rate was significantly increased with the increase of PL concentration and the prolongation of treatment time (P＜0.05,P＜0.01), indicating that PL can significantly inhibit the proliferation of Huh-7 and LM3 cells, and there is a correlation between concentration and time.The results are shown in Figure 1 and Table 1 and Table 2.

Tab 1 The effect of different concentrations of plumbagin on the proliferation of hepatocellular carcinoma Huh-7 cells (n=6,±s)

Note: Compared with the control group, *P＜0.05 ,**P＜0.01 (The following table is the same)

group Concentration of drug/μmol·L-1Inhibition rate of proliferation (%)24 h 48 h 72 h Blank control group 0 0 0 0 Plumbagin group 1 6.74 ± 4.36 2*5.93 ± 1.98*8.740 ± 3.23*2 25.04 ± 0.71**26.39 ± 4.89**32.28 ± 2.32**4 63.00 ± 2.30**77.46 ± 0.51**87.93 ± 3.01**8 67.47 ± 2.33**79.34 ± 0.81**91.97 ± 0.22**10 70.19 ± 1.58**81.42 ± 0.38**92.38 ± 0.12**F 119.0 297.3 391.4 P＜0.000 1 ＜0.000 1 ＜0.000 1

Tab 2 The effect of different concentrations of plumbagin on the proliferation of hepatocellular carcinoma LM3 cells (n=6,±s)

group Concentration of drug(μmol/L) Inhibition rate of proliferation (%)24 h 48 h 72 h Blank control group 0 0 0 0 Plumbagin group 1 0.61 ± 0.06 0.51 ± 0.05 4.35 ± 1.42*2 6.51 ± 6.51 6.85 ± 1.12**7.35 ± 1.01**4 10.13 ± 3.43*14.22 ± 2.80**23.71 ± 5.07**8 47.85 ± 0.63**79.27 ± 0.11**92.33 ± 0.15**10 57.62 ± 1.03**91.85 ± 0.37**97.47 ± 0.36**F 69.54 110.9 423.1 P＜0.000 1 ＜0.000 1 ＜0.000 1

Fig 1 Effects of different concentrations of plumbagin on proliferation of hepatocellular carcinoma Huh-7 and LM3 cells

3.2 Effects of PL on hepatocellular carcinoma Huh-7 and LM3 cell migration

Compared with the control group, different concentrations of PL could significantly inhibit the migratory cell number of Huh-7 and LM3 cells (P ＜ 0.01), and the effect of 8μmol L-1 group was the most significant, as shown in Figure 2 and Table 3 and 4.Compared with the control group, the scratch healing rate of cells in the intervention group was significantly decreased (P ＜ 0.05, P ＜ 0.01),and the scratch healing rate of cells decreased with the increase of the concentration of PL, indicating that PL could dose-dependent inhibit Huh-7 and LM3 cell migration, as shown in Figure 2 and Table 3 and 4.

Tab 3 Effects of different concentrations of plumbagin on migration of Huh-7 cells(n=3,±s)

Scratch healing rate (%)Blank control group 0 503.0 ± 3.51 87.72 ± 0.07 Plumbagin group 2 310.0 ± 2.89**52.11 ± 0.64**4 233.0 ± 6.81**31.98 ± 0.22**8 191.0 ± 7.55**15.93 ± 1.54**F 616.50 13.55 P＜0.000 1 ＜0.000 1 group Concentration of drug(μmol/L)Number of migrated cells (per cell)

Tab 4 Effects of different concentrations of plumbagin on migration of LM3 cells(n=3,±s)

Scratch healing rate (%)Blank control group 0 124.70 ± 2.40 65.22 ± 1.26 Plumbagin group 2 87.00 ± 1.16**46.62 ± 5.20*4 71.33 ± 0.89**44.55 ± 6.80*8 18.00 ± 1.16**24.37 ± 2.37**F 104.20 13.55 P＜0.001 ＜0.000 1 group Concentration of drug(μmol/L)Number of migrated cells (per cell)

Fig 2 Effect of plumbagin on migration ability of hepatocellular carcinoma Huh-7 and LM3 cells

3.3 Effects of PL on invasion of hepatocellular carcinoma Huh-7 and LM3 cells

Transwell invasion assay results showed that, compared with control group, the number of Huh-7 and LM3 cells passing through the Transwell compartment decreased after treatment with PL(P＜0.01), suggesting that PL could significantly inhibit the invasion ability of Huh-7 and LM3 cells, as shown in Figure 3 and Table 5.

Fig 3 Effect of plumbagin on the invasion ability of hepatocellular carcinoma Huh-7 and LM3 cells

Tab 5 Effects of different concentrations of PL on the invasion of Huh-7 and LM3 cells (n=3,±s)

Number of invasive cells(per cell)Huh-7 cells LM3 cells Blank control group 0 739.7 ± 6.70 102.0 ± 1.16 Plumbagin group 2 567.3 ± 23.69**96.33 ± 3.18 4 333.0 ± 12.66**57.67 ± 0.89**8 274.7 ± 8.11**22.67 ± 1.45**F 222.4 382.3 P＜0.000 1 ＜0.000 1 group Concentration of drug(μmol/L)

3.4 Effects of PL on the expression of Huh-7 and LM3 cell migration and invasion related proteins

Compared with the blank group, the expression level of E-cadherin protein was significantly up-regulated(P＜0.01); The protein expression levels of N-cadherin and MMP-2 in the 8 μmol/L group were significantly down-regulated(P＜0.05, P＜0.01), as shown in Figure 4, Table 6, 7.

3.5 Effects of PL on the activity of JAK2/STAT3 signaling pathway

Compared with blank group, the protein expressions of p-JAK2 and p-STAT3 in PL group (4, 8, 10 μmol/L) were significantly down-regulated (P＜0.05, P＜0.01), and the protein expression of p-JAK2 and p-STAT3 decreased gradually with the increase of the concentration of PL, while the protein expression of JAK2 and STAT3 did not change significantly (P ＞ 0.05), suggesting that PL could inhibit the migration and invasion of Huh-7 and LM3 cells by inhibiting the expression of JAK2/ STAT3 signaling pathway related proteins.as shown in Figure 5, Table 8,9.

Tab 8 Effects of different concentrations of plumbagin on the expression of JAK2/ STAT3 signaling pathway related proteins in hepatocellular carcinoma Huh-7 cells(n=3,±s)

group Concentration of drug(μmol/L) p-JAK2 JAK2 p-STAT3 STAT3 Blank control group 0 1.00 ± 0.01 1.00 ± 0.01 1.00 ± 0.01 1.00 ± 0.01 Plumbagin group 1 0.86 ± 0.068 0.97 ± 0.04 0.97 ± 0.13 1.12 ± 0.04 2 0.73 ± 0.02 1.00 ± 0.07 0.83 ± 0.072 1.16 ± 0.09 4 0.60 ± 0.07*1.12 ± 0.10 0.62 ± 0.04*1.01 ± 0.02 8 0.52 ± 0.07**0.92 ± 0.06 0.53 ± 0.10*1.11 ± 0.15 10 0.30 ± 0.14**0.83 ± 0.08 0.42 ± 0.12**0.91 ± 0.04 F 11.23 2.153 7.130 1.522 P＜0.001 ＞0.05 ＜0.01 ＞0.05

Tab 9 Effects of different concentrations of plumbagin on the expression of JAK2/ STAT3 signaling pathway related proteins in hepatocellular carcinoma LM3 cells(n=3,±s)

group Concentration of drug(μmol/L) p-JAK2 JAK2 p-STAT3 STAT3 Blank control group 0 1.00 ± 0.01 1.00 ± 0.01 1.00 ± 0.01 1.00 ± 0.01 Plumbagin group 1 0.87 ± 0.05 0.90 ± 0.06 0.96 ± 0.11 1.01 ± 0.04 2 0.75 ± 0.08 0.90 ± 0.11 0.85 ± 0.11 0.94 ± 0.03 4 0.66 ± 0.06*0.78 ± 0.02 0.66 ± 0.09 0.90 ± 0.12 8 0.58 ± 0.11*0.78 ± 0.03 0.59 ± 0.10*0.84 ± 0.05 10 0.33 ± 0.11**0.96 ± 0.05 0.37 ± 0.10**0.82 ± 0.08 F 8.784 2.492 6.563 1.582 P＜0.01 ＞0.05 ＜0.01 ＞0.05

Fig 5 Effects of different concentrations of plumbagin on the expression of JAK2/ STAT3 signaling pathway related proteins in hepatocellular carcinoma Huh-7 and LM3 cells

Tab 6 Effects of PL on the expression of E-cadherin, N-cadherin and MMP-2 proteins in hepatocellular carcinoma Huh-7 cells (n=3,±s)

group Concentration of drug(μmol/L) E-cadherin N-cadherin MMP-2 Blank control group 0 1.00 ± 0.01 1.00 ± 0.01 1.00 ± 0.01 Plumbagin group 2 1.71 ± 0.06**0.91 ± 0.04 0.96 ± 0.03 4 1.98 ± 0.15**0.91 ± 0.02 0.65 ± 0.12 8 3.88 ± 0.16**0.68 ± 0.08**0.37 ± 0.07*F 11.18 8.29 32.62 P＜0.000 1 ＜0.001 ＜0.05

Tab 7 Effects of plumbagin on the expression of E-cadherin, N-cadherin and MMP-2 proteins in hepatocellular carcinoma LM3 cells (n=3,±s)

group Concentration of drug(μmol/L) E-cadherin N-cadherin MMP-2 Blank control group 0 1.00 ± 0.01 1.00 ± 0.01 1.00 ± 0.01 Plumbagin group 2 1.14 ± 0.08 0.97 ± 0.04 1.03 ± 0.20 4 1.40 ± 0.15 0.87 ± 0.01 0.60 ± 0.12 8 2.03 ± 0.20**0.55 ± 0.18*0.43 ± 0.07*F 11.71 5.168 6.134 P＜0.01 ＜0.05 ＜0.05

Fig 4 Effect of plumbagin on the expression of E-cadherin, N-cadherin and MMP-2 proteins in hepatocellular carcinoma Huh-7 and LM3 cells

4.Discussion

Due to its high degree of malignancy and rapid progression of liver cancer, only 25%-30% of primary hepatocellular carcinoma cases are diagnosed in the early stage of the disease, and most patients with liver cancer are already in the advanced stage when they see a doctor, which is not suitable for surgical resection [9].The treatment of advanced liver cancer is quite tricky, and there are many adverse reactions in the treatment of western medicine and drug resistance is easy to occur in long-term application.Therefore, it is of great practical significance to search for new drugs to treat liver cancer from the treasure house of traditional Chinese medicine.

The PL is a quinone compound extracted from the root of the Chinese traditional medicine Plumbinis.Its chemical name is 5-hydroxy-2-methyl-1, 4-naphthoquinone.Current studies have found that PL can exert anticancer activity through various mechanisms, such as targeting apoptosis and autophagy pathways,regulating cell cycle, anti-tumor angiogenesis, inhibiting tumor invasion and metastasis, etc.[3].In addition, PL may also play an antitumor role as reactive oxygen species (ROS) inhibitors,glutathione peroxidase inhibitors and proteasome inhibitors [10].

E-cadherin is a kind of transmembrane glycoprotein that mediates intercellular adhesion, and the decrease or loss of its expression can lead to the weakened adhesion between cancer cells, thus making cancer cells prone to abscission and metastasis [11].Studies have found that e-cadherin is closely related to liver metastasis,and its reduced or absent expression can promote the invasion and metastasis of liver cancer cells [12].N-cadherin is a class of calciumdependent transmembrane glycoprotein, which is mainly involved in regulating calcium-mediated intercellular adhesion, aggregation and migration.Its expression can mediate the transformation of tumor cells from epithelium to mesenchyma and promote vascular hyperplasia, thus improving the invasion ability of tumors [13].Matrix metalloproteinase 2 (MMP2), a zinc-dependent enzyme, is a member of the matrix metalloproteinase family (MMPs), which can degrade various protein components in the extracellular matrix and destroy the histological barriers that hinder the invasion of tumor cells, playing a key role in tumor invasion and metastasis [14].

In this study, CCK-8 assay showed that PL could inhibit the proliferation of Huh-7 and LM3 cells in a concentration-dependent manner.Cell scratch assay and Transwell migration assay were used to detect Huh-7 and LM3 cells treated with PL, and it was found that PL could inhibit the migration ability of Huh-7 and LM3 cells.Meanwhile, Transwell invasion assay demonstrated that PL could also inhibit the invasion ability of Huh-7 and LM3 cells.The protein expressions of E-cadherin, N-cadherin and MMP-2 in cells were detected by Western blot.It was found that PL can down-regulate the expression of N-cadherin and MMP-2, and up-regulate the expression of E-cadherin.These results suggest that the inhibition of hepatocellular carcinoma cell migration and invasion may be related to the down-regulation of N-cadherin and MMP-2 expression and up-regulation of E-cadherin expression, and this effect is in a certain concentration-dependent manner.

JAK2/STAT3 signaling pathway is one of the important signaling pathways, which plays an important role in cell migration and invasion as well as the occurrence and development of tumors.Studies have shown that traditional Chinese medicine can affect tumor migration and invasion by regulating JAK2/STAT3 signaling pathway.For example, berberine extracted from Chinese herbal medicine-Chinese Goldthread,inhibits the invasion and metastasis of colorectal cancer cells through COX-2/ PGE2-mediated JAK2/STAT3 signaling pathway [15].phlorizin extracted from Chinese herbal sweet tea inhibits the proliferation, migration and invasion of esophageal cancer cells by antagonizing JAK2/STAT3 signaling pathway [16].The anthraquinone monomers 2-hydroxy-3-methylanthraquinone extracted from Oldenlandia diffusa may partially inhibit the growth and invasion of lung cancer cells by down-regulating the IL-6-induced JAK2/STAT3 pathway [17].In this study, we detected the expression of JAK2/STAT3 signaling pathway related proteins in Huh-7 and LM3 cells treated with different concentrations of PL.It was found that PL could down-regulate the expression of p-JAK2 and p-STAT3 proteins, suggesting that PL may inhibit the activation of JAK2/STAT3 signaling pathway.

In conclusion, PL can inhibit the migration and invasion ability of human hepatocellular carcinoma Huh-7 and LM3 cells in vitro in a concentration-dependent manner, and its mechanism may be through blocking the JAK2/STAT3 signaling pathway.The above study provides theoretical and experimental support for the treatment of hepatocellular carcinoma by PL.

Description of author′s contribution:

Cheng Tao, Wei Yanfei: Experiment design, experiment implementation, paper writing; Liu Huan: Assisted in experimental design and index detection; Liu Hong: Data collection and statistical analysis; Deng Shuye: Experimental design, paper revision,proofreading.

All authors of this article declare no conflict of interest.