Introduction

According to the latest statistics, the incidence and mortality rates of lung cancer rank first in all cancer types throughout the worldwide, and the 5 year overall survival rate is less than 20%1,2.Current treatment strategies such as surgery, chemotherapy, radiotherapy, and molecular targeting therapy as well as immunotherapy are ineffective against advanced tumors. Therefore, there is still an urgent need to search novel approaches to treat lung cancer.

Oncolytic virotherapy has recently been considered as one of the most promising strategies for the treatment of solid tumors due to its tumor-targeting ability and high efficiency3,4. Our group has been working on Cancer-targeting gene-virotherapy (CTGVT) through inserting anti-tumour genes into oncolytic virus vectors, such as adenoviral vector and vaccinia vector5,6, which has obtained better anti-tumour effect compared to oncolytic virus alone and gene therapy alone.

GP73, is a Golgi glycoprotein, also called GOLPH2 and is upregulated in a wide range of cancers7,8. It is an excellent candidate marker for hepatocellular carcinoma, and its specificity is even better than that of the hepatocellular carcinoma marker AFP9. Previously, we constructed a GP73-regulated oncolytic adenovirus (GD55) based on ZD55 of CTGVT, exhibits a stronger oncolytic effect compared to common oncolytic adenovirus in human liver cancer stem-like cells10, human prostate cancer stem-like cells5, and hepatocellular carcinoma11either in vivo or in vitro. It showed that GD55 has an excellent antitumor potential as oncolytic virus vector to deliver tumor suppression gene.

In recent years, phosphoinositide phosphohistidine inorganic pyrophosphate phosphatase (LHPP), a histidine phosphatase, has been shown to be a new tumor suppressor12.Low expression of LHPP has also been found in cancer tissues of bladder cancer13, lung cancer, pancreatic cancer14,15, cervical cancer16, papillary thyroid cancer17, and colorectal cancer18,19. Overexpression of LHPP protein inhibits hepatocellular carcinoma cell growth and metastasis, and also reduces the transcription of CCM1, PKM2, MMP7, and MMP9 in cancer cells, and that high expression of LHPP was negatively correlated with cell cycle and metastasis, among others20. It suggests that LHPP is lung cancer suppressor with potent.

In this study, we constructed a novel oncolytic adenovirus GD55-LHPP in which GD55 was used to carry LHPP gene, and investigated the role of GD55-LHPP on lung cancer cells. The results showed that GD55-LHPP mediated high expression of LHPP in lung cancer cells and inhibited efficiently lung cancer cell growth in vivo and in vitro. Thus, our results implied that GD55-LHPP might act as a promising therapeutic agent for lung cancer.

Result

Low expression of LHPP in lung cancer cells and tissues

The expression of the tumor suppressor LHPP is often absent or lowered in various types of human cancers due to the loss of heterozygosity and promoter hypermethylation21. Here, we first detected LHPP expression in several lung cancer cell lines, and found LHPP expression was down-regulated in detected lung cancer cells except H1975 compared with the human embryonic lung cell line MRC-5 in mRNA level. (Fig. 1a, Supplementray Table 1). Furthermore, it was found that LHPP expression was clearly weakened in five lung cancer tissue specimens compared with paracancer normal tissues (Fig. 1b and c). It is consistent with low expression of LHPP in lung adenocarcinoma derived from CPTAC samples (https://ualcan.path.uab.edu/) (Fig. 1d). The above results suggest LHPP might be a candidate lung cancer suppressor.

Figure 1
figure 1

Low expression of LHPP in lung cancer cells and tissues (a) The expression of LHPP mRNA in normal lung cell lines and different lung cancer cell lines by RT-qPCR . **P < 0.01, ***P < 0.001. (b) LHPP expression in five lung cancer tissue specimens and paracancer normal tissues (Orginal amplification: × 200). (c) The quantitative analysis of the LHPP expression staining images in five lung cancer tissue specimens and paracancer normal tissues. **P < 0.01, ***P < 0.001. (d) The expression of LHPP in lung adenocarcinoma derived from CPTAC samples.

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Construction of recombination oncolytic virus GD55-LHPP and its characteristic

We have previously successfully constructed an oncolytic adenovirus GD55 under the control of GP73 promoter7. GP73 is a potential tumour biomarker, especially in lung, liver, and prostate cancers. Herein, we examined the promoter activity of the GP73 promoter in different cell lines and showed higher promoter activity in four lung cancer cell lines compared with lung normal cells (Fig. 2a). In order to verify whether the higher expression of Coxsackie and Adenovirus Receptor (CAR) and specific for adenoviral infection in lung cancer cells than in normal cells, we examined the expression of CAR in four types of lung cancer cells and normal cells and found that CAR was highly expressed in lung cancer cells (Fig. 2b). Subsequently, we generated a novel oncolytic adenovirus GD55-LHPP by inserting the LHPP gene into E1B 55 K deleting locus (Fig. 2c). Then, we evaluated expression of LHPP and E1A mediated by GD55 as well as progeny replication ability of GD55-LHPP in lung cancer cell lines. The result indicated that the high expression of LHPP gene was observed in GD55-LHPP-infected A549, H460, H1299 and H1975 cells than that of GD55, and whether GD55-LHPP or GD55 can result in adenovirus E1A expression in the above lung cancer cells compared with the normal MRC-5 cells (Fig. 2d). The progeny replication assay indicated that GD55-LHPP exerts higher progeny virus multiplication ability with GD55, implying that insertion of LHPP gene might have the potential to increase the replication of the viral progeny(Fig. 2e).

Figure 2
figure 2

Construction of recombination oncolytic virus GD55-LHPP and its characteristic. (a) The promoter activity of the GP73 promoter in normal lung cell lines and different lung cancer cell lines **P < 0.01, ***P < 0.001. (b) The expression of Coxsackie and Adenovirus Receptor (CAR) protein in normal lung cell lines and different lung cancer cell lines by Western blotting. **P < 0.01, ***P < 0.001. (c) Schematic diagram of the structure of the recombinant oncolytic adenovirus structure. ITR, inverted terminal repeat. (d) The expression of LHPP protein in four lung cancer cell lines (A549, H460, H1299, and H1975) and one human embryonic lung cell (MRC-5) infected with GD55-LHPP or GD55 at the 10 MOI for 48 h by Western blotting. (e) The progeny replication of the virus replicates in four lung cancer cell lines (A549, H460, H1299, and H1975) were infected with 10 MOI of GD55-LHPP or GD55 for 24, 48, 72, 96 h.

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Killing effect of GD55-LHPP on lung cancer cell lines

To evaluate the anti-tumor effect of GD55-LHPP in vitro, four lung cancer cell lines (A549, H460, H1299, and H1975) and one human embryonic lung cell (MRC-5), were infected with various concentrations of GD55-LHPP for 72 h. The MTT assay indicated that GD55-LHPP had a greater inhibitory effect than GD55 (Fig. 3a). The crystal violet assay revealed that GD55-LHPP provoked a greater cytopathic effect on lung cancer cells than GD55(Fig. 3b). In addition, it is noted that whether GD55-LHPP or GD55 showed slight suppression effect to normal cells, suggesting recombinantion oncolytic virus has good targeting and safety.

Figure 3
figure 3

Killing effect of GD55-LHPP on lung cancer cell lines. (a) MTT assay was used to detect the anti-tumor effect of GD55 and GD55-LHPP in one human embryonic lung cell MRC-5 and lung cancer cells NCI-H460, A549, NCI-H1299, NCI-H1975. *P < 0.05, **P < 0.01, ***P < 0.001 (b) Crystal violet assay was employed to determine the cytopathic effect of GD55 and GD55-LHPP in one human embryonic lung cell (MRC-5) and four lung cancer cells (A549, H460, H1299, and H1975). MOI, the multiplicity of infection.

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GD55-LHPP induced cell apoptosis and its mechanism in lung cancer cells

To detect whether the inhibition of cell proliferation by GD55-LHPP was due to the activation of apoptosis, this form of cell death was analyzed. The apoptotic morphological analysis by Hoechst 33342 staining showed that GD55-LHPP triggered a higher level of nuclear fragmentation, the formation of apoptotic bodies, and chromatin condensation, inducing a higher magnitude of cell apoptosis than GD55 (Fig. 4a). Additionally, flow cytometric analysis documented that the rate of cell apoptosis in the GD55-LHPP group was markedly increased compared with the NC group or the GD55 group in NCI-H460 (Fig. 4b).

Figure 4
figure 4

GD55-LHPP induced cell apoptosis and its mechanism in lung cancer cells. (a) After 48 h, nuclear fragmentation (arrows) was observed in MRC-5, A549, NCI-H460, NCI-H1299 cells (0.2 mm fields; original magnification, × 200) in different treatment groups using Hoechst staining under an inverted fluorescence microscope. (b) The percentage of apoptotic cells in NCI-H460 cells treated with PBS, GD55, or GD55-LHPP was detected using flow cytometric analysis. The data are presented as the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. (c) and (d) Expression of apoptosis-associated proteins expression was detected by Western blotting in NCI-H460 after treatment with PBS, GD55, or GD55-LHPP. GAPDH was used as the internal control. (e) Expression of TNF-α, and c-Myc mRNAs in NCI-H460 cells treated with PBS, GD55, or GD55-LHPP. *P < 0.05, **P < 0.01, ***P < 0.001.

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To identify the mechanism of apoptosis activation, the caspase signaling pathway was analyzed in NCI-H460. The result indicated that GD55-LHPP significantly increased the expression of LHPP, Cleaved-Caspase 3, and pro-apoptotic protein Bax, as well as autophagic marker protein p62, and converted more LC3A proteins to LC3B proteins (Fig. 4c and d). Meanwhile GD55-LHPP significantly increased the expression of TNF-α, and demoted the expression of c-Myc (Fig. 4e, Supplementray Table 2). The results showed that recombinant oncolytic adenovirus GD55-LHPP induces tumour cell death by activating the intracellular caspase-related apoptosis and autophagic signaling pathway.

Antitumor effect of GD55-LHPP in mice

To evaluate the antitumor efficacy of intratumoral administration of GD55-LHPP in nude mice carrying human lung cancer xenografts. Based on in vitro cell cytotoxicity results, GD55-LHPP exhibited most effective inhibitory effect in NCI-H460 cells compared with other lung cancer cells, thus NCI-H460 cells was used as further in vivo study. Subsequently, NCI-H460 cells were subcutaneously implanted into nude mice at 5 × 106 cells; a single dose of GD55-LHPP or control is injected intratumorally at 5 × 1011 VP when the tumor volume reached about 80–120 mm3 and a second injection is given two days apart (Fig. 5a). The result shows that compared with the PBS treatment group, the tumor growth rate of nude mice in the virus treatment group was significantly reduced, and the survival time of mice was significantly prolonged. Especially, all mice in the PBS group died after 25 days of treatment and the tumor volume in the GD55-LHPP-treated group was only 1 400.75 mm3 compared that of 1 974.19 mm3 in the GD55-treated group at 29 days (Fig. 5b and c). The results of immunohistochemistry showed that the recombinant oncolytic virus efficiently mediated expression of virus Hexon and E1A protein, and GD55-LHPP mediated obvious LHPP expression at the tumor site (Fig. 6 upper). HE staining showed that our oncolytic virus can induce cell death in tumor tissues, and had no obvious toxic effect on liver and kidney tissues of mice (Fig. 6 lower). It suggests our constructed oncolytic virus can efficiently replicate and induce cell death in tumor tissues with good safety.

Figure 5
figure 5

Antitumor effect of GD55-LHPP in mice. (a) A schematic diagram of building a tumor xenograft model and injecting an oncolytic virus. (b) The tumour volume was measured every 2 days using the formula V (mm3) = 0.52 × length × width2. Data are presented as the mean ± standard deviation, n = 5. *P < 0.05, **P < 0.01, ***P < 0.001. (c) The mouse survival rate in different treatment groups.

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Figure 6
figure 6

Histopathology assay. IHC and HE staining for detection of oncolytic adenovirus amplification and hepatorenal toxicity in different treatment groups. Magnification, × 200.

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Discussion

As an emerging cancer therapeutic strategy, oncolytic virus (OV) therapy has been attracted more and more attention because of its good achievements in preclinical and clinical studies22. OVs are able to target proliferate in tumour cells and ultimately lyse them without harming normal tissues, and simultaneously trigger the host’s innate and adaptive antitumor immune response23,24,25. After the tumour cells are lysed, the viruses within them are also released to infect and kill other tumour cells26,27, which forms a cascade amplification effect. In recent years, several types of OVs have been approved, such as H10128 in China, T-VEC29 in the USA, and the Japanese herpes simplex virus Deltyact30. These marketed OV drugs bring a good prospect and important motivation for the development of OVs. However, the tumour-killing effect of OVs is still insufficient in current research, thus the modification of OVs and delivery of novel tumour suppressor to improve their safety and antitumor effect is a major trend for future research.

Among all used OV vectors, adenovirus (Ad) is the most used viral vector that can efficiently infect almost all types of tumor cells. There are some drawbacks for adenovirus to design as an OV drug, such as the preexisting neutralization antibodies of adenovirus in the human body prone to clear virus and lack of tumor targeting. Thus it is needed to modify adenovirus to overcome these obstacles. To improve tumor-targeting of oncolytic Ad, our group has put forward to the CTGVT strategy and designed an independent intellectual property rights of oncolytic adenovirus ZD55 by deleting Ad E1B 55 kDa gene to target p53-deficient tumor cells10. To further improve transcriptional targeting ability of ZD55, we previously constructed a GP73-regulated OV GD55, and demonstrated that GD55 confers high adenovirus replication, infectivity and antitumor effect9,10. Therefore, recombinant oncolytic adenovirus GD55 has a promising anti-tumor potential especially through delivering novel tumour suppressor genes.

LHPP is a histidine phosphatase, and recently LHPP has been identified to be a tumour suppressor in HCC and other cancers31,32, which can inhibit tumour cell proliferation and metastasis, and induce apoptosis in numerous tumor cells by affecting different target genes33. Mechanically, tumor suppression activity of LHPP may be mediated through inhibiting EGFR34, modulating AKT/mTOR35,36 and TGF-β/smad signaling pathway18 etc. Recent study also proved that loss of LHPP is associated with intestinal inflammation in colitis mice model and inflammatory bowel disease patients21, showing that vital roles of LHPP in maintenance of normal body functions. Because histidine phosphorylation belongs to a hidden phosphoproteome and is a poorly understood and uncharacterized post-translational modification of proteins31, the detailed functions of LHPP as a histidine phosphatase still need to be classified. To be noted, GD55-LHPP showed a certain high progeny replication potential compared with GD55, although some tumour cells showed significant differences, others did not (Fig. 2C). The possible reason is that the oncolytic virus GD55 used here lacks E1B55 kDa gene, but retains the early gene E4orf1. However, E4orf1 restricts the expression of late viral proteins of E1B55 kDa-missing oncolytic adenoviruses such as ONYX-015, thereby reducing progeny viral replication37. The mechanism is that E4orf1 induces the activation of PI3K/Akt signal, leading to phosphorylation of Akt37. Since LHPP could inhibit the Akt signaling pathway, thus GD55-LHPP overexpressing LHPP has the potential to increase viral progeny replication. Moreover, although we have preliminarily found that GD55-LHPP can inhibit the proliferation of tumor cells, the specific mechanism of action and the effect on normal cells have not been clearly classified in this study. Thus, further studies are needed to confirm the roles of LHPP.

Herein, the anti-tumour effect of GD55-LHPP was demonstrated in lung cancer cells and the NCI-H460 xenograft mouse model, which was consistent with our expected results.

However, there are still some shortcomings to be improved in further study. In Fig. 3a, the cell viability was reduced in MRC-5 normal lung cells infected with 40 MOI of GD55-LHPP, implying that its toxicity in normal cells. The possible reason is the effect of LHPP-overexpression mediated by high viral dose in normal cells. Thus, the safety of our constructed oncolytic adenovirus in the clinical application needs to be further evaluated. For example, and further modification of oncolytic adenovirus can enhance its safety in clinical application38,39,40. Meanwhile, its anti-tumour efficacy can also be enhanced by combining with other therapeutic modalities. Studies have shown that combining oncolytic viruses with chemotherapeutic agents41, CAR-T42,43 or immune checkpoint inhibitor molecules, such as CTLA444,45、PD-1/PD-L144、Tim345、Siglec-546, significantly increased their anti-tumour efficacy.

In conclusion, our study confirmed that recombinant oncolytic virus GD55-LHPP could inhibit cell proliferation both in vivo and in vitro in lung cancer cells, and induce tumor cells death by activating the caspase signaling pathway, as well as autophagic pathway. Therefore, this study confirmed the role of LHPP gene and the broad prospect of oncolytic virus GD55-LHPP for lung cancer treatment.

Materials and methods

Real-time quantitative polymerase chain reaction (RT-qPCR)

The treated cells were collected and washed twice with pre-cooled PBS; secondly, each sample was lysed by adding 1 mL of Trizol on ice for 3 min; finally, the lysate was collected into 1.5 mL RNase free EP tubes for mRNA extraction. The extracted mRNA was reverse transcribed by reverse transcription kit to obtain cDNA (Cowin Biotech, China), and the experiments were performed according to the instructions of SYBR Green (Vazyme Biotech, China) in 7500 Fast Real-Time PCR System. The primers used in the experiment were:

  • LHPP qRT-PCR F: 5ʹ-CTGTGTGGTAATTGCAGACGC-3ʹ

  • LHPP qRT-PCR R: 5ʹ-TAGTAACGCCCTTTTCCCAGT-3ʹ

  • GAPDH qRT-PCR F: 5ʹ-ACAACTTTGGTATCGTGGAAGG-3ʹ

  • GAPDH qRT-PCR R: 5ʹ-GCCATCACGCCACAGTTTC-3ʹ

  • c-myc qRT-PCR F: 5ʹ-ACAGCGTCTGCTCCACCT-3ʹ

  • c-myc qRT-PCR R: 5ʹ-ATCCAGCCGCCCACTTTT-3ʹ

  • TNF-α F: 5ʹ-GAGGCCAAGCCCTGGTATG-3ʹ

  • TNF-α R: 5ʹ-CGGGCCGATTGATCTCAGC-3ʹ

Ethics approval

Lung cancer samples were obtained from Zhejiang Xiaoshan Hospital and performed in accordance with relevant guidelines and regulations. The study was approved by the Ethics Committee of Zhejiang Xiaoshan Hospital. Written informed consent was received from all participants prior to inclusion in the study. Animal studies were approved by the Laboratory Animal Ethics Committee of Zhejiang Sci-Tech University (ZSTU) and performed in accordance with the guidelines and regulations of the ZSTU.

Cell lines and culture

The human embryonic lung cell line MRC-5, human lung adenocarcinoma cell lines A549, NCI-H460, NCI-H1299, NCI-H1975 and human embryonic kidney cell lines HEK293A, HEK293T were obtained from Shanghai Institute of Cell, Chinese Academy of Sciences. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, 100 U/mL of penicillin, and 100 mg/mL of streptomycin in a 5% CO2, 37 °C incubator.

Western blot analysis

Western blot analysis was performed according to previous description. Briefly, the cells were harvested and lysed, and protein concentration was determined with Pierce BCA protein assay kit (Thermo Fisher Scientific). Cell proteins were subjected to SDS–polyacrylamide gel electrophoresis. Separated proteins were transferred onto polyvinylidene difluoride membranes and incubated with primary antibodies against procaspase-3, cleaved caspase-3, Bax, p62, LC3A, and LC3B (Cell Signaling Technology, Danvers, MA, USA), LHPP, CAR, GAPDH, and Beclin1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Dual luciferase reporter assay

The luciferase reporter assay for various GP73 promoter truncations was done according to the Dual-glo luciferase assay kit (GeneCopoela, LF002). Briefly, cells were seeded in 6-well culture plate overnight before transfection. The pGL3-L659 (p-677/-19) (which was constructed by previous study9) and the pRL were cotransfected into the lung cancer cell lines A549, NCI-H460, NCI-H1299, NCI-H1975 and lung embryonic fibroblasts MRC-5 at a ratio of 1:50 to test the GP73 promoter activity of GP73 promoter in different cell lines.

Crystal Violet staining

The cells were seeded in 24-well plates (3 ~ 5 × 104 cells/well), treated with GD55-LHPP and GD55 at indicated MOIs (1, 5, 10, 20, and 40) for 72 h, and stained with 0.5% crystal violet solution. Subsequently, the cells were gently washed with PBS twice and dried at 37 ℃ and photographed. Uninfected cells served as a control.

Cell viability assay

The cells were inoculated into 96-well plates at 3 ~ 5 × 103 cells/well, and treated with GD55-LHPP and GD55 via various multiplicity of infection (MOI: 1, 5, 10, 20, 40) after the cells are attached to the well. After 72 h, cell viability was examined by methyl thiazolyl tetrazolium (MTT; Sigma-Aldrich, St. Louis, MO, USA) assay. Briefly, 20 µL MTT (5 mg/mL in PBS) was added into each well, and after 4 h of incubation at 37 ℃, the mediums and MTT were removed, and 150 µL of Dimethyl Sulfoxide was added into each well. The plates were mixed thoroughly for 10 min, and cell viability was assessed by measuring the absorbance at 490 nm using a microplate reader. Each experiment was repeated three times.

Hoechst 33342 assay

The cells were inoculated into 24-well plates (3 × 104 cells/well). After the cells are attached and treated with GD55 and GD55-LHPP at the indicated MOI for 72 h. Subsequently, the cells were incubated in Hoechst 33342 solution (Beyotime, Shanghai, China) for 10 min and then washed twice with PBS. The cells were then observed under the IX71-22FL/PH fluorescence microscope (Olympus Corporation, Japan) at 200 × magnification.

Flow cytometric analysis

Cell apoptosis was determined using Annexin V FITC/PI double staining kit (Biosciences, San Jose, CA, USA) according to our previous description47. Briefly, 5 × 105 cells were inoculated into each well of 6-well plates, cultured for 12 h, and then treated with oncolytic adenovirus GD55-LHPP and GD55 for an additional 48 h. Subsequently, the cells were harvested by trypsinization without EDTA, resuspended in 500 µL of binding buffer, and stained with FITC-labeled annexin V and PI. The staining was immediately evaluated using the flow cytometer (BD Biosciences).

Animal experiments

Animal experiments were performed according to the regulations and standards set by ARRIVE guidelines and were performed according to previous description14. The study was approved by the Laboratory Animal Welfare Ethics Committee of the Zhejiang Sci-Tech University (No. 20220105-05) in January 2022. Briefly, 4 weeks old female BALB/c nude mice were purchased from the Shanghai Experimental Animal Center. A total of 5 × 106 NCI-H460 cells/mice were subcutaneously inoculated into the right flank of the mice. When the xenograft tumors reached 80–120 mm3, the mice were divided randomly into 3 groups (PBS, GD55, and GD55-LHPP). Each mouse was injected twice with PBS (vehicle), GD55 (5 × 108 PFU), and GD55-LHPP (5 × 108 PFU) every third day. After treatment, tumor size was measured with a vernier caliper every two days. Mice were euthanized when tumor volume was more than 2000 mm3. Tumor volume (V) was calculated according to the formula V (mm3) = 0.5 × length (mm) × width (mm2). Then, tumor, liver and kidney tissues were harvested, fixed in 5% paraformaldehyde, dehydrated in a gradient of increasing ethanol concentrations, and embedded in paraffin. 5 µm sections were cut and stained with hematoxylin and eosin for histological analysis. Adenovirus E1A and LHPP expression in tumor tissues were detected with immunohistochemical staining to valuate virus replication.

Histopathology assay

For analysis of histopathology, mice have been randomly selected from each group and were killed after 20 days of the first treatment in different groups. Tumour tissue, heart, liver, kidney and spleen were harvested and fixed in 5% paraformaldehyde, dehydrated with gradient increasing ethanol concentrations and embedded in paraffin wax, which were cut in 5 µm sections. The sections were stained with haematoxylin and eosin for histological analysis. The sections were incubated with anti-E1A and anti-LHPP antibodies and then incubated with the avidin–biotin-peroxidase complex reagent (Vector Laboratories, Burlingame, CA, USA) for the IHC analysis. Haematoxylin was used as a counterstain.

Statistical analysis

All data are presented as the mean ± standard deviation (SD). The differences were assessed by Student’s t-test and the analysis of variance (ANOVA) with GraphPad Prism 6 (GraphPad Software, Inc, CA, USA). p < 0.05 were statistically considered significant.