ZCCHC14 regulates proliferation and invasion of non–small cell lung cancer through the MAPK‐P38 signalling pathway

Abstract ZCCHC14 is a CCHC‐type zinc finger protein which is expressed in tissues in human and mouse. The function of ZCCHC14 in tumours remains unclear. In this research, we explored the expression, function and related molecular mechanisms of ZCCHC14 in human non–small cell lung cancer (NSCLC). Immunochemistry staining showed that ZCCHC14 was low‐expressed or absent in NSCLC tissues. In NSCLC patients, the low expression of ZCCHC14 in tumour tissues was significantly correlated with TNM stage, differentiation degree and adverse clinical outcome (P < .05). The proliferation and invasion ability of cancer cells transfected with ZCCHC14 CRISPR/Ca9 KO plasmids was significantly enhanced (P < .05). Immunoblotting analysis indicated that the expression of p‐P38, cyclinD1 and MMP7 were significantly up‐regulated after disabling ZCCHC14 (P < .05). We used MAPK‐P38 pathway inhibitor doramapimod (BIRB 796) to inhibit P38 signalling pathway activity and determined that the agent significantly disrupted the function of ZCCHC14 and hindered the proliferation and invasion of the tumour. The finding revealed that ZCCHC14 can regulate proliferation and invasion of NSCLC through the P38 pathway. ZCCHC14 plays a crucial regulatory role in the development of NSCLC and may become a zinc finger target for clinical treatment.


| INTRODUC TI ON
ZCCHC14 is a CCHC-type zinc finger protein of approximately 100 kDa size and possessing 14 CCHC domains. ZCCHC14 mRNA had been detected in almost all organs such as human brain, lung and liver.
Studies have shown that CCHC-type zinc finger proteins can bind DNA or RNA and play roles in regulating tumour progression by mediating protein interactions. 1 Most CCHC-type zinc finger proteins play biological functions closely related to changes of the MAPK signalling pathway 2-5 composed of P-38, ERK1/2, ERK5 and JNK. 6 It is generally acknowledged that smoking and alcohol are independent risk factors for lung cancer. Using meta-analysis associated with whole genome analysis, researchers determined that the SNP of ZCCHC14 may be caused by susceptibility of nicotine dependence (ND) and a reason for alcohol addiction. 7 Smoking and alcohol intake in turn carry a higher risk of developing tumour. In addition, a Genome Wide Association Study (GWAS) indicated that the leading single-nucleotide polymorphism in small vessel stroke is associated with mRNA expression of ZCCHC14 in arterial tissues, 8 indicating that ZCCHC14 may play a role in cerebrovascular formation and Abstract ZCCHC14 is a CCHC-type zinc finger protein which is expressed in tissues in human and mouse. The function of ZCCHC14 in tumours remains unclear. In this research, we explored the expression, function and related molecular mechanisms of ZCCHC14 in human non-small cell lung cancer (NSCLC). Immunochemistry staining showed that ZCCHC14 was low-expressed or absent in NSCLC tissues. In NSCLC patients, the low expression of ZCCHC14 in tumour tissues was significantly correlated with TNM stage, differentiation degree and adverse clinical outcome (P < .05). The proliferation and invasion ability of cancer cells transfected with ZCCHC14 CRISPR/Ca9 KO plasmids was significantly enhanced (P < .05). Immunoblotting analysis indicated that the expression of p-P38, cyclinD1 and MMP7 were significantly up-regulated after disabling ZCCHC14 (P < .05). We used MAPK-P38 pathway inhibitor doramapimod (BIRB 796) to inhibit P38 signalling pathway activity and determined that the agent significantly disrupted the function of ZCCHC14 and hindered the proliferation and invasion of the tumour. The finding revealed that ZCCHC14 can regulate proliferation and invasion of NSCLC through the P38 pathway. ZCCHC14 plays a crucial regulatory role in the development of NSCLC and may become a zinc finger target for clinical treatment.

K E Y W O R D S
invasion, lung cancer, P38 signalling pathway, proliferation, ZCCHC14 management. ZCCHC14 might be associated with the progress of tumour metastasis, such as brain metastasis, although the function of ZCCHC14 in tumour has not been researched.
In our work, we investigated differences in the expression of ZCCHC14 in normal human lung tissue and NSCLC tissue and addressed the function of ZCCHC14 and its molecular mechanisms in the progression of NSCLC.

| Tissue samples
Paraffin-embedded tissue samples were obtained from 104 NSCLC specimens (79 adenocarcinoma and 25 squamous cell carcinoma), 26 specimens of paired non-tumour portions (>5 cm distance from the edge of the primary tumours) and 48 specimens of brain metastases.
Follow-up data were available for 46 cases. The TNM stages of tumours were revised by the Union for International Cancer Control (UICC). 9,10 All samples were obtained between 2012 and 2015 from surgical resection before patients received radiotherapy, chemotherapy or immunotherapy at the First Affiliated Hospital of China Medical University.
This study was supervised by the Institutional Review Board of China Medical University. All participants provided informed consent.

| Immunohistochemistry
Paraffin sections were dewaxed and antigen repaired.
Immunohistochemistry staining using ZCCHC14 polyclonal antibody Neutral balsam after dehydration. The grades of immunostaining were assessed as described. 10

| Immunofluorescence assay
Cells on coverslips were fixed with cold methanol for 10 minutes.
They were incubated with anti-ZCCHC14 antibody (ab-150591, Abcam, USA) after blocking with 5% BSA. Immunofluorescence assay was performed using secondary antibodies conjugated to tetramethyl rhodamine isothiocyanate (TRITC) for protein staining and DAPI for nuclear staining. The staining was detected using a fluorescence microscope (Olympus IX51, Japan) and images captured with a camera (CoolPIX 5400; Nikon, Japan).

| Immunoblotting analysis
Immunoblotting analysis was performed as described. 9 5% BSA and non-fat milk were used as blocking reagent. The corresponding

| Colony formation assay
The colony forming ability of tumour cells was examined as described. 11 In brief, cell suspensions with 3000 cells were blended evenly and incubated in medium for 10 days following placing in 6-cm cell culture dishes. Haematoxylin was used to stain the cells, and colonies > 0.3 mm were counted. Experiments were repeated three times.

| Transwell assay
Transwell assays were performed as described. 11 Cell suspensions (100 µl containing 1 × 10 4 cells) were placed in the upper chamber and were then incubated for an additional 24 hours. Cells appearing on the lower surface of the filter were stained with crystal violet and five random fields were counted using a high-magnification microscope. Experiments were repeated three times.

| Statistical analysis
SPSS statistical software package version 13.0 (SPSS Inc, Chicago, IL) was used for analysing date. All results were analysed using Pearson's chi-square test. Experiments were repeated three times.
The data were shown as mean ± standard deviation (SD), and significance level was set 5%.

| Aberrant ZCCHC14 expression was significantly correlated with tumour progression and poor survival of patients
Immunochemistry staining was firstly performed to investigate the expression of ZCCHC14. It was found that ZCCHC14 was expressed in normal bronchial epithelial cells and was weaker or absent in lung squamous cancer cells ( Figure 1A). Expression of ZCCHC14 was normal in alveolar cells and low or absent in adenocarcinoma cells of NSCLC patients ( Figure 1B). The ZCCHC14 negative rate was 73% in lung cancer tissues (76/104) and 90% brain metastases tissues (43/48), and both differences were statistically significant (P < .05) ( Table 1). The low expression of ZCCHC14 in cancer tissues was significantly correlated with poor versus good differentiation, TNM advanced stage (III + IV) and male gender (P < .05) ( Table 1)  to the KM Plotter database, patients with NSCLC with high expression of ZCCHC14 mRNA had longer survival probability than that of patients with low levels of ZCCHC14 mRNA expression (P < .001; Figure 1D).

| Low expression of ZCCHC14 in lung cancer cells promoted proliferation and invasion of cancer cells in vitro
To study the function of ZCCHC14 in cancer cells, we conducted in vitro experiments. Firstly, the expression of ZCCHC14 was detected in HBE cells and lung cancer cells A549, H1299, LK2, H460 and H292.
Immunoblotting analysis showed that ZCCHC14 was expressed in HBE and lung cancer cells and was under expressed to varying degrees in LK2, H460 and H292 cells (Figure 2A). Immunofluorescence staining experiments showed that ZCCHC14 was primarily localized on the nucleus of A549 and H1299 cells, and nucleus and cytoplasm of LK2 cells ( Figure 2B). A549 and H1299 cells with appropriate expression of ZCCHC14 were selected for further experiments and transfected with ZCCHC14 CRISPR/Ca9 KO (ZCCHC-KO) plasmids.
Colony formation experiments showed that down-regulation of ZCCHC14 significantly promoted the proliferation of cancer cells (P < .05) ( Figure 3A). The results of transwell assays showed that the invasion ability of cancer cells was significantly increased after down-regulating the expression of ZCCHC14 (P < .05) ( Figure 3B).

| ZCCHC14 regulates molecules downstream of the P38 pathway
We confirmed whether ZCCHC14 regulates the MAPK signalling pathway and the regulatory mechanisms of the subfamily.
Immunoblotting analysis showed that the relative expression of p-P38 significantly increased and P38 expression was not significantly regulated after transfecting ZCCHC14-KO plasmids in A549 and H1299 cells (Figure 4). After ZCCHC14 was knocked out from A549 and H1299 cells, the downstream signalling molecules of the P38 molecular pathway were significantly increased, including cy-clinD1 and MMP7 (Figure 4).

| Addition of a P38 pathway inhibitor abolished the effect of ZCCHC14 to suppress cancer cell proliferation and invasion
To determine whether ZCCHC14 plays a role in lung cancer cells through the P38 signalling pathway, we added the P38 pathway inhibitor doramapimod (BIRB 796). Immunoblotting analysis showed that p-P38 expression significantly decreased after addition of doramapimod compared to cancer cells in the NC group (P < .05) ( Figure 5). Colony formation assays showed that the addition of the inhibitor significantly reduced the proliferation ability of A549 and H1299 cells compared with the NC group (P < .05) ( Figure 6A).
Transwell assays showed that doramapimod significantly reduced the invasion of A549 and H1299 cells in the NC group ( Figure 6B).
Immunoblotting analysis showed that p-P38 decreased sig-

| D ISCUSS I ON
Most lung cancer patients with low survival rates are found in advanced stages. 13 A clinical study revealed that brain metastasis is a crucial reason for disease progression and treatment failure of advanced NSCLC, and patients with brain metastases generally have a poor prognosis. 14 Considering the incidence of NSCLC with brain metastases, and that the control of brain metastases is an important therapeutic target for metastatic NSCLC, in this study we included samples from patients with lung cancer and brain metastases.
It is important to find molecular changes which can help to distinguish lung cancer versus non-lung cancer patients and it will benefit the development of clinically targeted therapies often involved in biological functions. The MAPK signalling pathway participates in multifarious physiological and pathological processes 15 and plays dual roles in different cell types and conditions. 6 Studies have shown that the MAPK signal pathway plays a crucial part in preventing the occurrence and progression of cancer. 16 Recent studies showed that ZCCHC14, a CCHC-type zinc finger protein, is associated with nicotine dependence, alcohol addiction and small blood vessel stroke 7,8 and that ZCCHC14 in plasma has a positive effect on the prognosis of patients with cerebral haemorrhage. 17

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All date generated or used during the study are available from the corresponding author by request.