ZHX2 inhibits proliferation and promotes apoptosis of human lung cancer cells through targeting p38MAPK pathway

Abstract

OBJECTIVE:

To investigate the effect of ZHX2 on lung cancer cells proliferation and apoptosis.

MATERIALS AND METHODS:

The mRNA and protein expression of ZHX2 were detected by qRT-PCR and western blot, respectively. The human lung cancer cells were divided into Control, NC, ZHX2, SB, and ZHX2 $+$ Ani groups. The cell proliferation was detected by CCK-8 assay and the cell migration and invasion were detected by Transwell assay. Cell apoptosis was detected by flow cytometry. Apoptosis and p38MAPK signaling pathway related proteins were detected by western blot. The nude mice model of lung cancer xenograft was constructed. The tumor volume and tumor weight were measured. The expression of PCNA protein in tumor tissues was detected by immunohistochemistry. The apoptosis of tumor cells was detected by TUNEL staining. The ZHX2 and p38MAPK signaling pathway related proteins in tumor tissues were detected by western blot.

RESULTS:

The expression of ZHX2 gene and protein in the cancer cell lines were significantly decreased. Compared with control and NC groups, the cells proliferation, migration and invasion were inhibited in ZHX2 and SB groups, while the apoptosis and apoptosis related proteins were increased ( $p<$ 0.05). Meanwhile, compared with ZHX2 group, the tumor growth rate, volume, weight, the percentage of PCNA-positive cells, and p-P38 MAPK/P38 MAPK were increased significantly in ZHX2 $+$ Ani group, while the apoptotic index and the expression of MMP-9 protein were significantly decreased ( $p<$ 0.05).

CONCLUSION:

ZHX2 could inhibit proliferation and promote apoptosis of lung cancer cells by inhibiting p38MAPK signaling pathway.

Keywords

Lung cancer Zinc-fingers and homeoboxes 2 proliferation migration apoptosis

1. Introduction

Lung cancer is one of the most diagnosed malignant tumors, and has been the most frequent cause of cancer-related mortality worldwide[1]. Despite treatment advances, it is still a highly aggressive and fatal disease. Lung cancer is progressed to metastasis, and is not diagnosed until it has progressed to an advanced stage[1]. So, more studies are needed to throw light upon the inherent molecular mechanism and eventually ideal prognostic markers and therapeutic method need to be developed.

Zinc-fingers and homeoboxes (ZHX) is a family of vertebrate transcription factors that contains three members, ZHX1, ZHX2 and ZHX3. These three genes are expressed in many tissues and localized in the nucleus, and have been reported as tumor suppressor genes in many studies[2]. ZHX2 is an important transcriptional repressor, which contains two zinc-finger motifs and five homeodomains[3]. ZHX2 appears to promote cell-cycle repression and involved in tumorigenesis, and the ZHX2 level is associated with the progression and poor outcome of tumor[4]. Furthermore, ZHX2 may be influence drug resistance in cancer cells[5]. Prior study has reported that deficiency of ZHX2 was found in human hepatcellular carcinoma, and ZHX2 deficiency was an early event in the progression of hepatocellular carcinoma[6]. Compared to adjacent none-tumor tissues, the expression of ZHX2 always reduced in human hepatocellular carcinoma tissues[3, 7]. Moreover, decreased ZHX2 expression was associated with hepatocyte proliferation and reduced survival times of the patients[3]. Meanwhile, both in vivo and in vitro experiments, ZHX2 overexpression inhibited the proliferation of hepatocellular carcinoma cells and the growth of tumor xenografts[3]. ZHX2 is an important regulator of hepatic gene regulation in the development of liver cancer, and ZHX2 could suppress the secretion of alpha-fetoprotein (AFP), which suggested that ZHX2 deficiency was associated with AFP reactivation[6, 8].

To the patients with multiple myeloma, the expression of ZHX2 mRNA is low in peripheral blood lymphocytes compared to healthy individuals. Dysregulated ZHX2 levels will increased the risk of multiple myeloma events. The loss of ZHX2 is associated with the poor outcome of this disease, while high ZHX2 expression is associated with a better response and longer survival[9]. After high-dose chemotherapy, high ZHX2 expression heralded a better response and longer survival[10].

As a tumor suppressor, the effect of ZHX2 in the development of tumor has been paid more and more attention. However, there is little research on ZHX2 in lung cancer progression. In the present study, the association between ZHX2 and lung cancer was investigated. We designed a series of experiments to test our hypothesis that ZHX2 may suppress the progress of lung cancer and explore the inherent molecule mechanism.

2. Materials and methods

2.1 Cell culture

Human normal lung epithelial cells BEAS-2B cells, human non-small cell lung cancer A549, H1975, and HCC827 cells were purchased from Shanghai Institute of Cell Biology (Shanghai, China). All cells were grown in RPMI 1640 containing 10% fetal bovine serum (FBS, Gibco, Carlsbad, USA) and 1% penicillin-streptomycin at 37 ${}^{\circ}$ C, 5% CO ${}_{2}$ .

2.2 Transfection and groups

The cells were grown to near confluence and were divided into five group: control group (Control), ZHX2 negative control group (NC), ZHX2 overexpression group (ZHX2), p38MAPK inhibitor control group (SB), and ZHX2 $+$ p38MAPK activator group (ZHX2 $+$ Ani). The replication-incompetent lentivirus (LV) was produced for overexpressing human ZHX2 protein (ZHX2-LV). The negative control (NC) was expression of GFP alone (pGC-GFP-LV) (GeneChem, Shanghai). The cells in Control group receiving no treatment. The cells in NC group were transfected with lentivirus (MOI $=$ 40): GFP-LV for 72 h. The cells in ZHX2 group were transfected with lentivirus (MOI $=$ 40): ZHX2-LV for 72 h. The cells in SB group were treated with 10 $\mu$ M SB203580 (SB203580, Selleck, USA). The cells in ZHX2 $+$ Ani group were transfected with lentivirus (MOI $=$ 40): ZHX2-LV for 72 h, 30 nM Anisomycin.

2.3 Quantitative real-time polymerase chain reaction (qRT-PCR)

qRT-PCR was used to detect the expression of ZHX 2 mRNA in BEAS-2B, A549, H1975 and HCC827 cells. Cell transfection efficiency was also detected by qRT-PCR.

Total RNA was extracted by TRIzol Reagent (Invitrogen, USA) and RNA was then transcribed into cDNA using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, USA). qRT-PCR was performed using Mastercycler ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ nexus X2 (Eppendorf, Hamburg, Germany). Primer sequences used in this study are as follows: ZHX2, forward: 5 ${}^{\prime}$ -CCGGTACCCCCTCCA AAAATAAGCC-3 ${}^{\prime}$ , reverse, 5 ${}^{\prime}$ -CGGAATTCCCTAA GCGTAGTCTGGTACGTCGTAAGGGTAGGCCTGGCCAGCCTCTGCAG-3 ${}^{\prime}$ ; GAPDH, forward: 5 ${}^{\prime}$ -AGGTCGGTGTGAACGGATTTG-3 ${}^{\prime}$ , reverse, 5 ${}^{\prime}$ - TGTAGACCATGTAGTTGAGGTCA-3 ${}^{\prime}$ . The reaction conditions were: 35 cycles of 95 ${{}^{\circ}}$ C for 15 s, 60 ${{}^{\circ}}$ C for 60 s and 72 ${{}^{\circ}}$ C for 40 s. GAPDH mRNA as an internal control and 2 ${}^{-\Delta\Delta{\rm CT}}$ method was used to process the data.

2.4 Cell counting assay Kit-8 (CCK-8) assay

Logarithmic growth cells were plated in 96-well plastic plates at a density of 2 $\times$ 10 ${}^{4}$ /ml cells, 100 $\mu$ l per well. At 24, 48 and 72 h after incubation respectively, 10 ul CCK-8 (Dojindo, Japan) was added, then the plates were incubated at 37 ${}^{\circ}$ C for another 4 h. The microplate reader (Molecular Devices, CA, USA) is zeroed in the Control group, and the optical density (OD) was measured by microplate reader at 450 nm.

2.5 Transwell assay

50 $\mu$ l Matrigel mixture was added to the upper chamber of Transwell inserts (Corning Life Sciences, Corning, NY, USA) and incubated at 37 ${}^{\circ}$ C for 30 min. 100 $\mu$ l cell suspension (5 $\times$ 10 ${}^{4}$ cells/ml) of each group was added into the upper compartment of the chamber while 600 ul RPMI1640 culture media was added to the lower chamber. Membranes were collected after 72 h incubation, fixed in 5% glutaraldehyde for 30 min, stained with 0.1% crystal violet (Suolaibao, Beijing, China) for 30 min and observed under a light microscope (Olympus Corporation) at $\times$ 200 magnification. Images of five random fields were captured for analysis of the number of invading cells.

Matrigel was not coated at the bottom of Transwell chamber in Migration experiment, and other steps were the same as invasive experiment.

2.6 Flow cytometry

Cells were digested by trypsin and collected by centrifugation at 1000 rpm for 5 min at 4 ${}^{\circ}$ C. The cells were washed twice with pre-cooled PBS solution at 4 ${}^{\circ}$ C. After added 300 $\mu$ l 1 $\times$ binding buffer (Beyotime Biotech, Shanghai, China), cells were incubated with 5 $\mu$ l of Annexin V-APC (Beyotime Institute of Biotechnology) for 15 min. Then, 5 $\mu$ l propidium iodide solution (20 $\mu$ g/ml) was added and incubated in the dark for 10 min at room temperature. 200 $\mu$ l 1 $\times$ Binding buffer was added prior to analysis using a flow cytometer (Beckman Coulter, Brea, CA, USA) and Cell Quest 5.1 software (BD Bioscience, San Diego, CA, USA).

2.7 Construction and grouping of lung cancer xenograft model

30 SPF Balb/c female nude mice (weighing 16–18 g, 4 weeks old) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Certificate No. SCXK (Lu) 2014-0007). Animals were housed at 26–28 ${}^{\circ}$ C, 40–60% humidity, and under 12 h light/dark cycles. Feed, drinking water and litter were used after sterilization. All animal experiments were conducted in accordance with National Institutes of Health guidelines (pub. no. 85–23; revised 1996) and all the procedures were reviewed and approved by the Shandong University Animal Care and Use Committee.

30 mice were randomly divided into 5 group ( $n=$ 6): control group (Control), ZHX2 negative control group (NC), ZHX2 overexpression group (ZHX2), p38MAPK inhibitor control group (SB), and ZHX2 $+$ p38MAPK activator group (ZHX2 $+$ Ani). The lung cancer A549 cells in logarithmic growth phase were digested with 0.25% trypsin, and the cells were collected and counted. The cell concentration was adjusted to 2.5 $\times$ 10 ${}^{6}$ cells/ml, and 0.2 ml was inoculated into the soft skin of the right forelimb back of the nude mouse.

2.8 Tumor volume calculation

The tumor long diameter (L) and short diameter (W) were measured every 7 days with vernier caliper to calculate the tumor volume. Tumor volume (V) $=$ (L $\times$ W ${}^{2})/2$ .

Figure 1.

The effect of ZHX2 on cell proliferation. A: The expression of ZHX2 mRNA in each group of cells. B: The expression of ZHX2 mRNA in the transfected cells. C: CCK8 assay. D: Cell migration ( $\times$ 200). E: Cell invasion ( $\times$ 200). Values are mean $\pm$ SD. ${}^{b}$ $p<$ 0.05 vs. BEAS-2B group, * $p<$ 0.05 vs. Control group, ${}^{\#}$ $p<$ 0.05 vs. NC group, ^ $p<$ 0.05 vs. ZHX2 group.

2.9 Immunohistochemistry

After 28 days, the nude mice were anesthetized by intraperitoneal injection of 0.6% sodium pentobarbital (40 mg/kg), and then the neck was removed. The tumor tissue was taken, partially placed in a cryotube, frozen in a $-$ 80 ${}^{\circ}$ C refrigerator, and the other part was fixed in 4% paraformaldehyde for 24 h. The tumor tissues fixed in 4% paraformaldehyde solution were dehydrated and embedded in paraffin, and serially sliced to a thickness of 5 $\mu$ m. The slices were deparaffinized with xylene, and then hydrated in an ethanol gradient solution. Following treatment with 3% H ${}_{2}$ O ${}_{2}$ in methanol for 20 min, the slices were placed in citrate buffer (pH $=$ 6.0) and heated for 10 min for antigen retrieval. The slices were blocked with 5% bovine serum albumin (BSA, Beyotime Biotech, Shanghai, China) at room temperature for 20 min and then incubated with rabbit anti-rat PCNA primary antibody (1:200, ab15497, Abcam, UK) overnight at 4 ${}^{\circ}$ C. Samples were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:1000, ABIN101988, antibodies-online, Germany). 3,3-Diaminobenzidine staining, hematoxylin counterstaining, dehydration and clearing were performed thereafter. Observations were performed at $\times$ 400 magnification under a light microscope (Olympus, Japan) and the positive cells were counted using Aperio Imagescope v11.1 software (Aperio Technologies. Inc, FL, USA). The results were expressed as percentage of positive cells (%).

Figure 2.

The effect of ZHX2 on cell apoptosis. A: Flow cytometry. B: Western blot analysis of apoptosis-related proteins. Values are mean $\pm$ SD. * $p<$ 0.05 vs. Control group, ${}^{\#}$ $p<$ 0.05 vs. NC group, ^ $p<$ 0.05 vs. ZHX2 group.

3. Tunel staining

The sections with a thickness of 4 $\mu$ m were subjected to conventional xylene dewaxing and dehydration with gradient ethanol, and apoptosis was detected by TUNEL method using the apoptosis detection kit (batch number: ZK-8005, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., China). 5 randomly selected fields were observed at x400 magnification (Olympus Corporation, Japan). The brown or brownish yellow cells with apoptotic cell morphology were identified as apoptotic cells. The apoptotic index (AI) was calculated to reflect the degree of apoptosis. AI $=$ (number of apoptotic positive cells $\div$ total cells) $\times$ 100%.

3.1 Western blot

The lung tissues of the Balb/c nude mice, and the cells from 5 groups were prepared for western blot. The total proteins were extracted and protein concentration was measured using bicinchoninic acid (BCA) protein assay kit (Beyotime Biotech, Shanghai, China). 40 $\mu$ g protein sample was separated on a SDS-PAGE 10% gel (Mini-Protean-3 type; Bio-Rad Laboratories) and then transferred to a PVDF membrane (Merck KGaA, Darmstadt, Germany) for 30 min. Membranes were blocked with 5% not-fat milk in TBST solution at 4 ${}^{\circ}$ C for 1 h and followed an incubation with primary antibodies overnight at 4 ${}^{\circ}$ C, including rabbit anti-human anti-Bax (1:500, orb224426, Biorbyt, Cambridge, UK), anti-Bcl-2 (1:500, orb226346, Biorbyt, Cambridge, UK), anti-Cleaved caspase-3 (1:500, orb126597, Biorbyt, Cambridge, UK), anti-Cleaved caspase-9 (1:500, orb227889, Biorbyt, Cambridge, UK), anti-MMP-9 (1:500, orb214260, Biorbyt, Cambridge, UK) , anti-p-P38 MAPK (1:500, orb191822, Biorbyt, Cambridge, UK), anti-P38 MAPK (1:500, orb109098, Biorbyt, Cambridge, UK), anti-ZHX2 (1:500, orb100207, Biorbyt, Cambridge, UK), and anti- $\beta$ -actin (1:2000, orb178 392, Biorbyt, Cambridge, UK). The membrane was incubated with primary antibody overnight at 4 ${}^{\circ}$ C. Following incubation with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1000, ABIN101988, antibodies-online, Aachen, Germany) at room temperature for 1 h, the ECL system (Thermo Fisher Scientific, Inc.) was used to detect the signals. The expression levels were quantified by ImageJ software, and $\beta$ -actin was used as an internal control.

3.2 Statistical analysis

The data are presented as means $\pm$ SD. SPSS 13.0 software (SPSS Inc, Chicago, IL, USA) was used for data analysis. One-way analysis of variance (ANOVA) followed by LSD test was used for multiple groups. $P<$ 0.05 was regarded to be statistically significant

Figure 3.

The effect of ZHX2 on the expression of p38MAPK signaling pathway-related proteins in cells. A: Protein band diagram. B: The relative expression of MMP-9 protein. C: p-P38 MAPK/P38 MAPK. Values are mean $\pm$ SD. * $p<$ 0.05 vs. Control group, ${}^{\#}$ $p<$ 0.05 vs. NC group, ^ $p<$ 0.05 vs. ZHX2 group.

Figure 4.

The effect of ZHX2 on tumor growth and apoptosis of A549 cancer cells. A: Tumor volume. B: Tumor photo. C: Tumor weight. D: Immunohistochemistry ( $\times$ 400). E: TUNEL staining ( $\times$ 400). Values are mean $\pm$ SD. * $p<$ 0.05 vs. Control group, ${}^{\#}$ $p<$ 0.05 vs. NC group, ^ $p<$ 0.05 vs. ZHX2 group.

Figure 5.

Western blot was used to detect the expression of ZHX2 and p38MAPK signaling pathway-related proteins in mice tumor tissues. A: Protein band diagram. B: The relative expression of ZHX2 protein. C: The relative expression of MMP-9 protein. D: p-P38 MAPK/P38 MAPK. Values are mean $\pm$ SD. * $p<$ 0.05 vs. Control group, ${}^{\#}$ $p<$ 0.05 vs. NC group, ^ $p<$ 0.05 vs. ZHX2 group.

4. Results

4.1 Effect of ZHX2 on cell proliferation

As shown in Fig. 1A, compared with BEAS-2B cells, the expression of ZHX2 mRNA in A549, H1975 and HCC827 cells were significantly decreased ( $p<$ 0.05, respectively), which indicating that ZHX2 was lowly expressed in lung cancer A549, H1975 and HCC827 cells. As ZHX2 expression was the lowest in A549 cells, A549 cells were selected for follow-up studies.

Compared with control and NC groups, the expression of ZHX2 mRNA in ZHX2 and ZHX2 $+$ Ani groups were significantly increased ( $p<$ 0.05, respectively). Compared with control and NC groups, the proliferation, migration, and invasion ability in ZHX2 and SB groups were significantly lower ( $p<$ 0.05, respectively). Compared with ZHX2 group, there was no significant change in cell proliferation, migration and invasion ability in SB group ( $p>$ 0.05, respectively), while cell proliferation, migration and invasion ability in ZHX2 $+$ Ani group were significantly increased ( $p<$ 0.05, respectively) (Fig. 1B–E). These results indicated that ZHX2 could inhibit proliferation of human lung cancer cells.

4.2 Effect of ZHX2 on apoptosis

As shown in Fig. 2, compared with control and NC groups, the apoptosis rate and the expression of apoptosis-related proteins in ZHX2 and SB groups were significantly increased ( $p<$ 0.05, respectively). Compared with ZHX2 group, the apoptosis rate and the proteins expression of Bax, Cleaved caspase-3, and Cleaved caspase-9 in SB group had no significant change ( $p>$ 0.05, respectively), while the apoptosis rate and the expression of apoptosis-related proteins in ZHX2 $+$ Ani group were significantly decreased ( $p<$ 0.05, respectively). These results demonstrated that ZHX2 could promote apoptosis of human lung cancer cells.

4.3 Effect of ZHX2 on the expression of p38MAPK signaling pathway related proteins in cells

Compared with control and NC groups, the expression of MMP-9 protein in ZHX2 and SB groups were significantly upregulated, while p-P38 MAPK/P38 MA PK was significantly reduced ( $p<$ 0.05, respectively). Compared with ZHX2 group, the expression of p38MAPK signaling pathway-related protein in SB group had no significant change ( $p>$ 0.05, respectively), whereas the expression of MMP-9 protein in ZHX2 $+$ Ani group was significantly downregulated, p-P38 MAPK/P38 MAPK was significantly increased ( $p<$ 0.05, respectively). The results indicated that ZHX2 could suppress p38MAPK signaling pathway (Fig. 3).

4.4 Effect of ZHX2 on tumor growth and apoptosis in mice

Compared with control and NC group, the tumor growth rate of ZHX2 and SB groups were significantly slowed down, the tumor volume and weight, and the percentage of PCNA positive cells in tumor tissue were significantly reduced, while the apoptotic index was significantly increased ( $p<$ 0.05, respectively). Compared with ZHX2 group, there was no significant change in tumor growth and apoptosis in the SB group ( $p>$ 0.05, respectively), whereas in ZHX2 $+$ Ani group, the tumor growth rate was significantly accelerated, the tumor volume and weight, and the percentage of PCNA-positive cells in the tumor tissue were significantly increased, while the apoptotic index was significantly reduced ( $p<$ 0.05, respectively) (Fig. 4). These results demonstrated that ZHX2 can inhibit tumor growth and promote apoptosis.

4.5 The expression of ZHX2 and p38MAPK signaling pathway related proteins in mice tumor tissues

As shown in Fig. 5, compared with the control and NC groups, the expression of ZHX2 protein in the ZHX2 and ZHX2 $+$ Ani groups were significantly increased ( $p<$ 0.05, respectively). Compared with control and NC group, the expression of MMP-9 protein in ZHX2 and SB groups were significantly upregulated, while p-P38 MAPK/P38 MAPK was significantly reduced ( $p<$ 0.05, respectively). Compared with ZHX2 group, the expression of p38MAPK signaling pathway-related protein in SB group had no significant change ( $p>$ 0.05, respectively), whereas the expression of MMP-9 protein in ZHX2 $+$ Ani group was significantly downregulated, p-P38 MAPK/P38 MAPK was significantly increased ( $p<$ 0.05, respectively). These findings suggest that ZHX2 inhibits proliferation and promotes apoptosis of lung cancer cells by inhibiting p38MAPK signaling pathway.

5. Discussions

Lung cancer is a major health burden with high mortality and poor prognosis. As a member of ZHX family, ZHX2 levels normally enhanced after birth and participated in some types of cancer[11, 12]. In this study, the potential role of ZHX2 in lung cancer was firstly evaluated. We found that the expression of ZHX2 mRNA and protein were significantly reduced in lung cancer tissues than that in adjacent normal tissues. We further detected the effect of ZHX2 overexpression in A549 cells. The results showed that ZHX2 significantly inhibited the proliferation and migration of A549 cells, induced apoptosis of A549 cells, decreased the expression of MMP9, and inhibited p38MAPK signaling pathways.

In order to detect the expression of ZHX2 in lung cancer tissues, we collected specimens of lung cancer tissues and adjacent normal tissues. We first found that the expression level of ZHX2 in lung cancer tissues was significantly lower compared to adjacent normal tissues, which suggested that ZHX2 might participate in the progress of lung cancer. We further detected the exact role of ZHX2 and the mechanisms in human lung cancer cell line (A549) by overexpressing ZHX2 using lentiviral transfection.

Tumors progression is associated with abnormal cells proliferation. Uncontrolled proliferation provides a survival advantage of cancer cells to resist conventional chemotherapeutic agents[13, 14]. It has been documented that ZHX2 inhibited the proliferation of hepatocellular carcinoma cells[13]. In this study, we overexpressed ZHX2 in A549 cells and analyzed its effect on the cell proliferation. MTT and CCK-8 assays showed that ZHX2 overexpression inhibited the proliferation of lung cancer cells, which consistent with the prior study[13]. Tumor cells migration and invasion also played an important role in the progress of lung cancer. Transwell assay showed that ZHX2 overexpression remarkably reduced the migration of A549 cells. These results suggested that ZHX2 could inhibited the progress of lung cancer partly by suppressing the proliferation and migration of lung cancer cells.

Apoptosis is the main cause of tumor cell death and is the core mechanism for preventing tumor growth. TUNEL staining showed ZHX2 overexpression increased the apoptotic level of A549 cells, which suggested that ZHX2 caused a strong programmed cell death in A549 cells. To further investigate the potential mechanisms, the expression of Cleaved caspase-3 and Cleaved caspase-9 were evaluated. We found that overexpression of ZHX2 significantly increased the mRNA and protein expression of Cleaved caspase-3 and Cleaved caspase-9. Bcl-2 and Bax family played an important role in apoptosis, proliferation and invasion of tumor cells. In this study, the protein expression of Bcl-2 and Bax were assessed by western blot. We found that ZHX2 overexpression upregulated the expression of pro-apoptosis protein Bax and downregulated anti-apoptosis protein Bcl-2 expression in A549 cells. Meanwhile, ZHX2 also increased the ratio of the protein expression levels between Bax and Bcl-2. The effect of ZHX2 on the expression of caspases, Bax and Bcl-2 contributed to the apoptosis of tumor cells.

Tumor metastasis was an important manifestation of tumor progression. Among this progression, MMP-9 played a central role by degrading ECM and allowed the cells to reach distant target sites[15]. In this study, we found that ZHX2 could significantly suppress the expression of MMP-9, which participate in the anti-tumor effect of ZHX2. Tumor cells migration and invasion is a complex progress that involved many signaling pathways. Mitogen-activated protein kinases (MAPKs) family participated in the proliferation and migration of lung cancer[16, 17]. MAPK signaling pathway plays an important role in tumor development, including regulation of survival, proliferation, differentiation and apoptosis in tumor cells[18]. Another study showed that inhibition of MAPKs pathway mediated the downregulation of MMP-9 expression and attenuation of invasion in human leukemia U937 cells[19]. We found that ZHX2 significantly inhibited the expression of MMP9.

In conclusion, ZHX2 played anti-tumor role in lung cancer progression through inhibiting the cells proliferation, migration and the expression of MMP-9, and accelerating apoptosis. The possible mechanism is related to suppress p38MAPK signaling pathway. Therefore, ZHX2 maybe a potential therapeutic agent for further treatment of lung cancer.

Footnotes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81672292) and Taishan Scholar Program of Shandong Province (NO. ts201712087).

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