Low‐level gastrokine 2 promoted progress of NSCLC and as a potential biomarker

Abstract Background Gastrokine 2 (GKN2) is significantly downregulated in non‐small cell lung cancer (NSCLC) tissues than in normal tissues (NT), as assessed by mRNA microassay; however, the mechanism and clinical value of GKN2 is unknown in NSCLC. Methods A total of 60 NSCLC samples and corresponding NT samples were prospectively collected GKN2 expression in NSCLC tissues was estimated. Also, the expression level of GKN2 promoter methylation and correlation with clinical data in NSCLC patients from public databases were analyzed. Cytology experiments were also carried out. Results The GKN2 mRNA and protein expression level in NSCLC was significantly lower than that in the NT, and the GKN2 expression level in large tumors NSCLC was significantly lower than that in the small tumor group. Public data showed that expression of GKN2 in LUAD with P53 mutation group was lower than that of the P53 non‐mutation group, and GKN2 promoter methylation level of LUAD was significantly higher than its NT and close to age and clinical stage. Cell migration, invasion, and proliferation ability of GKN2 overexpressed were lower in A549 and PC9 groups than those in GKN2 overexpressed A549 and PC9 negative control groups, while the percentage of apoptotic cells increased in the GKN2 overexpressed A549 and PC9 groups. The DNMT3B mRNA expression levels were higher in PC9 and A549 cells than BEAS‐2B cells. Conclusion The overexpression of GKN2 significantly inhibited cell proliferation, migration, and invasion and promoted apoptosis. Low‐level GKN2 promoted the progression of NSCLC via DNMT3B and is expected to be a biomarker for NSCLC.


| INTRODUC TI ON
Despite continuous improvements in surgical resection, chemotherapy, and radiotherapy techniques, lung cancer is still prone to recurrence and fatality. 2 Non-small cell lung cancer (NSCLC) is the most common lung cancer, accounting for about 80% of the total number of lung cancers, including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Since the biological behavior of NSCLC is highly invasive and metastatic, about 80% of patients are at an advanced stage at the time of diagnosis, which often leads to treatment failure and death. Presently, the overall effect of NSCLC treatment is unsatisfactory, and the 5 years survival rate is only 10%-15%. 3 In recent years, the incidence of NSCLC has been on the rise; however, its pathogenesis is not yet clarified, necessitating further exploration of NSCLC. Therefore, identifying specific molecular targets and early diagnostic biomarkers for NSCLC are the current hot spots in clinical research worldwide.
To further explore the genes of mRNAs associated with lung carcinogenesis, we used high-throughput microarray technology to compare the differential mRNA expression profiles between NSCLC and its adjacent tissues and screen and identify a cohort of NSCLC-associated mRNA molecules. Among these, the mRNA expression of Gastrokine 2 (GKN2) was significantly downregulated (fold-change=−86.9873) among these candidate genes. This gene is located at 2p13.3 and has an mRNA length of 723 bp. Currently, no studies have reported GKN2 expression and lung cancer in China or abroad.
However, the literature reports on GKN2 and cancer-related research mainly focused on gastric cancer. GKN includes three members, GKN1, GKN2, and GKN3. Among these, GKN2 was discovered as a member of GKN in 2002. It is not expressed or underexpressed in gastric cancer tissues and is closely related to the prognosis of gastric cancer patients. The overexpression of GKN2 inhibits the proliferation, migration, and invasion of gastric cancer cells through the interaction with GKN1, indicating GKN2 as a tumor suppressor. [4][5][6] Currently, there is no report on the correlation between CNK2 and lung cancer.
In the present study, GKN2 expression levels were estimated by quantitative polymerase chain reaction (qPCR) in 60 pairs of NSCLC and normal lung tissue (NT) samples, and the correlation between GKN2 and clinical data from NSCLC patients was also analyzed. We also overexpressed GKN2 and observed the changes in the oncological behavior of A549 and PC9 cells.

| qPCR
Total RNA was extracted from frozen NSCLC tissues using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using RT Reagent Kit (Takara,) according to the manufacturer's instructions.
Subsequently, the gene mRNA expression in NSCLC tissues was measured by qPCR using SYBR Premix Ex Taq on ABI 7000. qPCR was used to measure gene mRNA expression in NSCLC tissues.
qPCR was used to measure the gene mRNA expression in NSCLC tissues. The detailed primer sequences for qPCR are shown in  in patient serum were measured by electrochemiluminescence. The reagents, quality control materials, and calibration solutions were obtained from Roche (Germany).

| Cell culture
Normal human bronchial epithelial cells BEAS-2B and three independent NSCLC cell lines (A549 and PC9) were purchased from the cell bank of the Chinese Academy of Sciences and cultured at 37°C in complete medium (containing 10% fetal serum and 90% RPMI1640) at 5% CO 2 . The complete medium was changed at least every two days.

| Transfection
The overexpression plasmid pcDNA-GKN2 and the control vector pcDNA-3.1 were purchased from Suzhou Imax Biotechnology.

| Cell viability assay
Cell viability was assessed using the Cell Counting Kit 8 (CCK-8, Corning Corporation,) according to the manufacturer's protocol.
Briefly, 3000 cells were inoculated into 96-well plates supplemented with 10% FBS. The following day, GKN2-overexpressed cells were incubated with CCK8 for 1 h, and absorbance was measured at 450 nm on days 1, 3, 5, and 7 using a multifunctional enzyme marker (Tecan). The experiments were performed in quadruplicate.

| Cell migration and invasion assays
Migration and invasion assays were performed in 24-well plates (Millipore) with 8.0 μm well inserts. For migration assays, 2 × 10 4 cells were seeded in the upper compartment of the transwell insert.
Invasion assays were performed with a matrix gel-coated filter (Sigma Corporation,). The cells were incubated for 24 and 48 h, respectively, fixed with methanol and stained with 0.1% (w/v) crystal violet, followed by bleaching with 33% acetic acid. The absorbance was measured at 570 nm. Each experiment was performed in triplicate.

| Flow cytometry to detect apoptosis
Apoptosis Detection Kit (KeyGEN,) was used for apoptosis detection. After washing the cells twice with phosphate-buffered saline (PBS), cells were resuspended in 500 μl Binding Buffer, 5 μl Annexin V-APC, and 5 μl 7-AAD dye. Flow cytometry was performed for 5-15 min in the dark (Beckman).

| Public database analyzes the expression level of GKN2 protein and promoter methylation in NSCLC patient tissues and correlation with clinical data
GNK2 mRNA and protein expression levels were analyzed in NSCLC from the GEPIA database (http://gepia.cance r-pku.cn/index.html).
According to http://ualcan.path.uab.edu, we analyzed the GNK2 promoter methylation levels in LUAD and its correlation with clinical data.

| Statistical methods
The two groups were compared using Student's t-test, and enumeration data were analyzed using chi-square test; p < 0.05 was statistically significant.

| Expression level of GKN2 in lung cancer and adjacent tissues and correlation with clinical data
According to Figure 1A,B,C, the GKN2 mRNA expression level in NSCLC was significantly lower than in NT (p < 0.001). We found that the GKN2 levels in large tumors (>2 cm) of NSCLC were significantly lower than those in the small tumors (≤2 cm) (p < 0.001  Table 2).
The expression of GKN2 in NSCLC was analyzed from the GEPIA database (http://gepia.cance r-pku.cn/index.html), and the results showed that the level of GKN2 expression in NSCLC was significantly lower than that of adjacent tissues in LUAD and LUSC ( Figure 1D) and was related to the clinical stage (p = 9.61e-05) ( Figure 1E). Further analysis showed that the expression of GKN2 in LUAD with P53 mutation group was lower than that of the P53 non-mutation group ( Figure 1F p < 0.001), and the prognosis of NSCLC did not differ significantly from the high or low level of GKN2 mRNA expression (p = 0.63) ( Figure 1G). Similarly, the GKN2 protein expression level of NSCLC was significantly lower than its adjacent cancer tissues (http://ualcan.path.uab.edu/cgibin/CPTAC -Result.pl?genen am=GKN2&ctype =LUAD) (p < 0.001, Figure 1H).

| Promoter methylation of GKN2 in LUAD was higher than adjacent tissues and the correlation with clinical stage and age
According to http://ualcan.path.uab.edu/cgi-bin/TCGA-methy l-Result.pl?genen am=GKN2&ctype =LUAD, GKN2 promoter methylation level of LUAD was significantly higher than its adjacent cancer tissues (Figure 2A p < 0.001). Further analysis showed that GKN2 promoter methylation level of stage I, stage II, and stage III was significantly higher than the adjacent cancer tissues (Figure 2B;  Figure 2D). GKN2 promoter methylation level in P53 mutation and P53 non-mutation groups from LUAD patients was higher than the adjacent cancer tissues (p < 0.001, p < 0.001; Figure 2E).

| Construction of A549 and PC9 GKN2 overexpression (OE) groups and GKN2 reduced the proliferation in NSCLC
PC9 and A549 cells were cotransfected with the GKN2 overexpressed plasmid. The qPCR results after transfection showed that the expression levels in the A549 GKN2 OE and PC9 GKN2 OE groups were significantly higher than those in the A549 GKN2 OE NC and PC9 GKN2 OE NC groups ( Figure 3A). This indicated suc-

| GKN2 inhibited cell migration and invasion in NSCLC
Cell migration showed lower cell numbers in GKN2 OE A549 and PC9 than in GKN2 OE A549 and PC9 NC groups (p < 0.001 and p < 0.001, respectively; Figure 4A,B); therefore, GKN2 inhibited cell migration in NSCLC. According to Figure 4C and D, the cell number of GKN2 OE A549 and PC9 was lower than that of GKN2 OE A549 and PC9 NC groups (p < 0.01 and p < 0.01, respectively), suggesting that GKN2 inhibited the cell invasion ability of NSCLC. Figure 5A shows the apoptosis maps of the 4 groups of cell lines.

| GKN2 expression level was associated with cell apoptosis in NSCLC
Furtherly analysis hinted that the percentage of apoptotic cells was significantly increased in the GKN2 OE A549 (17.43 ± 1.12%, p < 0.001) and PC9 (16.91 ± 1.02%, p < 0.001) NC groups compared to the GKN2 OE A549 (7.31 ± 0.001) NC group and PC9 (6.94 ± 0.20%) NC groups, seen in Figure 5B. Figure 6 shows that the DNMT1 mRNA expression was higher than that of BEAS-2B in PC9 (p < 0.001), while that in A549 was lower compared to BEAS-2B (p < 0.01). Compared to BEAS-2B, DNMT3A mRNA expression was increased in PC9 (p < 0.001) but was decreased in the A549 cell line (p < 0.01). Moreover, DNMT3B mRNA expression levels were higher in PC9 and A549 cell lines than BEAS-2B (p < 0.001 and p < 0.001, respectively). Combining the results in Figures 1 and 2, we speculated that DNMT3B plays a critical role in the occurrence of GKN2 methylation.  In this study, we found that the expression level of GKN2 in NSCLC was significantly lower than its adjacent cancer tissues, and the expression in large tumor (>2 cm) NSCLC was significantly lower F I G U R E 2 Analysis of GNK2 promoter methylation in lung cancer and paraneoplastic tissues and the correlation with clinical data. (A) According to http://ualcan.path.uab.edu, the level of GNK2 promoter methylation was significantly higher in LUAD than in the adjacent cancer tissues (p < 0.001). (B) Further analysis showed that GNK2 promoter methylation levels were higher in stage I, II, and III tumors than in the adjacent cancer tissues (p < 0.01, p < 0.05, p < 0.01), while no significant difference was observed in stage IV LUAD. (C) GNK2 promoter methylation levels were higher in men and women with LUAD than in the adjacent cancer tissues (p < 0.001 and p < 0.001).
In summary, GKN2 mRNA expression was downregulated in NSCLC patients and cell lines, and GKN2 promoter hypermethylation was detected in NSCLC. The overexpression of GKN2 significantly inhibited cell proliferation, migration, invasion, and apoptosis.
Thus, it seems a promising biomarker for NSCLC.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The corresponding author should be contacted for any data.