METTL14 Regulates PLAGL2/β-Catenin Signaling Axis to Promote the Development of Nonsmall Cell Lung Cancer

N6-methyladenosine (m6A) is an abundant eukaryotic mRNA modification involved in regulating the formation and metastasis of nonsmall cell lung cancer (NSCLC). We collected clinical NSCLC tissue and paracarcinoma tissue. Then methyltransferase-like 14 (METTL14), pleomorphic adenoma gene like-2 (PLAGL2), and β-catenin expressions were assessed using quantitative real-time PCR and western blot. PLAGL2, and β-catenin (nuclear) expressions were increased in NSCLC tissues. Cell proliferation, migration, invasion, and death were examined. PLAGL2 could activate β-catenin signaling to affect cell proliferation and migration abilities. RNA immunoprecipitation assay was operated to identify m6A modification levels of PLAGL2 after knockdown and overexpression of METTL14. PLAGL2 was regulated by METTL14-mediated m6A modification. Knockdown of METTL14 repressed cell proliferation, migration, and invasion, and promoted cell death. Interestingly, these effects were reversed when PLAGL2 was overexpressed. Finally, tumor formation in nude mice was performed to verify the role of the METTL14/PLAGL2/β-catenin signaling axis. Tumor formation in nude mice demonstrated METTL14/PLAGL2/β-catenin axis promoted NSCLC development in vivo. In brief, METTL14 promoted NSCLC development by increasing m6A methylation of PLAGL2 to activate β-catenin signaling. Our research provided essential clues for in-depth comprehension of the mechanism of NSCLC occurrence and development and also provided the basis for NSCLC treatment.


Introduction
Nonsmall cell lung cancer (NSCLC) accounts for 85% of all lung cancer incidence [1]. While it afects the quality of patients' life, it also increases the global economic burden. Due to a large number of mutations and general heterogeneity in this type of cancer, the use of traditional therapies has been challenging [2]. Small molecule tyrosine kinase inhibitors and immunotherapy have brought unprecedented survival benefts to selected patients. But overall cure rate and survival rate of NSCLC are still low [3]. At present, molecular-targeted therapy has also made great progress in the NSCLC treatment feld [4]. Terefore, it is particularly essential to investigate the internal mechanism of NSCLC and provide potential scientifc clues for subsequent treatment of NSCLC.
N6-methyladenosine (m6A) is an abundant eukaryotic mRNA modifcation and a common transcriptome modifcation in cancer. It was recently found to be involved in the regulation of NSCLC formation and metastasis [5]. Methyltransferase-like 14 (METTL14) is a core component of the m6A methyltransferase complex, and METTL14-induced abnormal m6A levels are related to tumorigenesis, proliferation, metastasis, and invasion [6]. METTL14 regulated various cancer occurrences and development, including breast cancer [7], colorectal cancer [8], and endometrioid epithelial ovarian cancer [9]. In NSCLC, knockdown of METTL14 inhibited Twist-mediated activation of AKT signaling to suppress NSCLC malignancy. Tis revealed METTL14 might be a potential therapeutic target for NSCLC [10]. As a writer of m6A methylation modifcation, METTL14 could promote the methylation modifcation of mRNA, thereby afecting its protein expression [11,12]. Pleomorphic adenoma gene like-2 (PLAGL2) is the zinc fnger protein transcription factor. It is overexpressed in many malignant tumors (gastric cancer, colorectal cancer, and breast cancer) and could facilitate tumor proliferation, migration, and invasion [13][14][15]. Overexpression of PLAGL2 has been implicated in lung cancer [16]. We predicted m6A modifcation of PLAGL2 and METTL14 acting on PLAGL2 by RMBase and m6A2Target. However, METTL14 and PLAGL2 mechanism in NSCLC remains to be investigated.
β-catenin is the complete structural component of cadherin-based and adherent junctions [17]. Abnormal β-catenin activating in nuclear is related to many human cancers [18]. Furthermore, METTL14 attenuated cardiac ischemia-reperfusion injury via activating Wnt/β-catenin in an m6A-dependent manner, which provided a new therapeutic target for ischemic heart disease [19]. In addition, related studies have shown PLAGL2 could regulate β-catenin and its downstream pathways and promote tumor genesis and development [14,20]. However, there have been no studies of PLAGL2 with β-catenin in NSCLC.
In our research, we hypothesized METTL14 promoted PLAGL2 expression by increasing m6A methylation modifcation of PLAGL2, activating β-catenin signaling, thereby afecting NSCLC development. To this end, we collected clinical NSCLC tissue and paracarcinoma tissue for relevant testing. METTL14/PLAGL2/β-catenin role was also investigated in vivo and in vitro to investigate whether METTL14 regulated PLAGL2/β-catenin signaling axis to promote the development of NSCLC. Our research provided essential clues for an in-depth comprehension of the mechanism of NSCLC occurrence and development and also provided the basis for NSCLC treatment.

Collection of Clinical Samples.
Tumor tissue and paracarcinoma tissue samples from NSCLC patients by iconography, serological or histopathological examination in Zhuzhou Central Hospital from November 2020 to August 2021 were collected. Clinical samples were divided into two groups: the NSCLC group (n � 5) and the paracarcinoma tissues group (n � 5). Tis study was approved by the Medical Ethics Committee of Zhuzhou Central Hospital (ZZCHEC2021092-01). Te research was conducted according to the World Medical Association Declaration of Helsinki. All the information about the study will be fully explained to the subjects by the researchers. All the participants provided informed consent before sampling.

Hematoxylin-Eosin (HE)
Staining. HE staining was performed to assess the pathological status of the tumor and paracarcinoma tissues [21]. Te slices were placed in xylene. Each grade of ethanol was placed for 5 min. After dyeing with hematoxylin for 3 min, they were returned to blue with PBS. Eosin was dyed 5 s. Gradient alcohol (95-100%) was dehydrated. After taking them out, they were placed in xylene, sealed with neutral gum, and observed under the microscope (BA210T, Motic).
2.6. RNA Immunoprecipitation (RIP) Assay. RIP lysis was frst prepared. A549 cells were lysed with RIP lysis bufer and then incubated with RIP immunoprecipitation bufer containing magnetic beads coupled to antibodies against Ago2 (CST, USA) or anti-rabbit IgG (negative control, CST, USA). Precipitated RNA was evaluated by qRT-PCR. Total RNA was used as input controls. Te primer sequences were PLAGL2-F: TCAGCTCCCTCCAAAATACCAG, PLAGL2-R: CTCCGCCAGAAAACTATCCAC.

Immunofuorescence (IF)
. Cells were treated with slices, fxed with 4% paraformaldehyde. Slices were added 0.3% triton, permeated at 37°C. Slices were sealed with 5% BSA at 37°C. Cell slices were dropped with the appropriate dilution of primary antibody β-catenin (51067-2-AP, 1 : 100, Proteintech, USA). Ten we added appropriate anti-rabbit-IgGlabeled fuorescent antibody and incubated them at 37°C for 90 min. DAPI working solution was nucleated for 10 min at 37°C, and then cells were sealed with bufer glycerol and observed under the fuorescence microscope.

Plate Clone Formation
Assay. Cells were removed from each group at the exponential growth stage. Cells in each group were inoculated with 200 cells in each well in 6-well plates containing 1 mL culture medium restored to room temperature. Cells were placed in the 5% CO 2 , 37°C, and saturated humidity incubator for 2 to 3 weeks, during which the liquid was appropriately changed. We discarded the culture medium, carefully soaked PBS twice, and added 4% paraformaldehyde (1 mL) to fx the cells. We removed the fxing solution and added 1 mL of dye solution to the working solution to stain. After decolorization, the absorbance (OD) value at 550 nm was assessed by using the enzyme-plate reader.

Trypan Blue
Staining. Cells were collected after digestion with trypsin and EDTA. We centrifuged collected cells at 1500 g for 1 min, discarded the supernatant, and resuspended cells with 1 mL or an appropriate cell suspension depending on the number of cells. Trypan blue staining was performed according to previous reports [22].

In Vivo Tumorigenesis.
Twenty BALB/c nude mice (4-6 weeks) were randomly assigned to sh-NC, sh-METTL14, sh-METTL14+oe-NC, and sh-METTL14+oe-PLAGL2, with 5 mice in each group. Mice were raised under sterile conditions of ambient room temperature of 26-28°C, the humidity of 40-60%, and alternating day and night for 10 h/14 h. Te Mice were fed sterile food and water. After 1 week of adaptive feeding, A549 cells that transfected with sh-NC, sh-METTL14, oe-NC, and oe-PLAGL2 were subcutaneously injected. Te cell concentration was 5 × 10 6 /mL, and 200 μL was injected [24,25]. Te formation of the transplanted tumor was observed, and tumors with a diameter ≥0.5 cm were considered tumor formation. Tumor volume was calculated. After the experiment was completed, we euthanized the mice under animal ethics. Te mice were sacrifced with 150 mg/kg sodium pentobarbital.
2.13. Statistical Analysis. GraphPad Prism 8.0 software was applied for statistical analysis. Data were expressed as mean ± standard deviation (SD). First, normality and homogeneity of variance tests were performed. When the test conformed to the normal distribution and the variance was homogeneous, Student's t-test was used between the two groups. Comparisons among multiple groups were conducted by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. P < 0.05 indicated the diference was statistically signifcant.

PLAGL2 Level Was Elevated in NSCLC Tissues. First, HE
staining assessed the pathological status. Compared with paracarcinoma tissues, NSCLC tissues had larger nuclei, larger nucleoplasmic ratio, more obvious histopathological atypia, and unclear tissue structure (Figure 1(a)). Subsequently, PLAGL2 expression was measured by qRT-PCR. Compared with paracarcinoma tissues, PLAGL2 expression Journal of Oncology 3 was elevated in NSCLC tissues (Figure 1(b)). PLAGL2 expression was further verifed by WB, and WB results were consistent with the trend of qRT-PCR (Figure 1(c)).

PLAGL2 Activated β-catenin Signaling to Afect Cell
Proliferation and Migration. Trough a literature search, we found PLAGL2 regulated β-catenin and its downstream pathways and promoted tumor genesis and development [14,20]. Terefore, we wanted to investigate PLAGL2's role in β-catenin. First, β-catenin (total) and β-catenin (nuclear) expressions were evaluated by WB. β-catenin (nuclear) level was promoted in NSCLC tissues than paracarcinoma tissues, but β-catenin (total) was not signifcantly diferent (Figure 2(a)). Subsequently, PLAGL2 was knocked down and overexpressed in A549 cells. PLAGL2 level in the sh-PLAGL2 group was repressed, and that in the oe-PLAGL2 group was increased than the NC group, indicating that PLAGL2 was successfully knocked down and overexpressed. At the same time, β-catenin expression was repressed in the sh-PLAGL2 group and accelerated in the oe-PLAGL2 group compared with the NC group (Figure 2(b)). WB further verifed that β-catenin (nuclear) expression was inhibited in the sh-PLAGL2 group and accelerated in the oe-PLAGL2 group compared with the NC group. β-catenin (total) showed no signifcant diference (Figure 2(c)). IF further demonstrated β-catenin location. Compared with the NC group, β-catenin in the oe-PLAGL2 group showed nuclear transfer (Figure 2(d)). Ten we performed interference with PLAGL2 and overexpression of β-catenin. PLAGL2 level was repressed in sh-PLAGL2 group than sh-NC. β-catenin expression was elevated in the sh-PLAGL2 + oe-β-catenin group compared with the sh-PLAGL2 + oe-NC group, indicating that we successfully interfered with PLAGL2 and overexpressed β-catenin (Figure 2(e)). Plate clone formation and wound healing assays showed that the number of clones and migration were repressed in the sh-PLAGL2 group than the sh-NC group. After β-catenin was overexpressed, the number of clones and migration increased (Figures 2(f ) and 2(g)). Tis suggested that PLAGL2 activated β-catenin signaling to infuence cell proliferation and migration.

PLAGL2 Signaling Was Regulated by METTL14-Mediated m6A
Modifcation. First, we predicted m6A modifcation of PLAGL2 by RMBase and predicted METTL14 acted on PLAGL2 via m6A2Target. Terefore, we knocked down and overexpressed METTL14 to explore the mechanism of METTL14 and PLAGL2. METTL14 level in the sh-METTL14 group was suppressed and that in the oe-METTL14 group was increased than the NC group, indicating that METTL14 was successfully knocked down and overexpressed. In addition, PLAGL2 expression was repressed in the sh-METTL14 group, and accelerated in the oe-METTL14 group compared with the NC group (Figure 3(a)). Ten we used RIP to identify the m6A modifcation of PLAGL2 after knockdown and overexpression of METTL14. Te results revealed the m6A modifcation of PLAGL2 was repressed in the sh-METTL14 group and accelerated in the oe-METTL14 group than the NC group (Figure 3(b)). Collectively, PLAGL2 signaling was regulated by METTL14-mediated m6A modifcation.

METTL14 Regulated PLAGL2
Signaling to Afect NSCLC Cell Function. Next, to verify METTL14 and PLAGL2's infuence on NSCLC development deeply, METTL14 was knocked down, and PLAGL2 was overexpressed. qRT-PCR showed METTL14 level was repressed in the sh-METTL14 than the sh-NC. In the sh-METTL14 + oe-PLAGL2 group, PLAGL2 expression was increased compared with the sh-METTL14 + oe-NC group. Tis indicated METTL14 was knocked down successfully, and PLAGL2 was successfully overexpressed (Figure 4(a)). WB showed decreased expressions of METTL14, PLAGL2, and β-catenin (nuclear) in the sh-METTL14 than in the sh-NC. PLAGL2 and β-catenin (nuclear) expressions were elevated in the sh-METTL14 + oe-PLAGL2 compared with the sh-METTL14 + oe-NC. β-catenin (total) showed no signifcant diference (Figure 4(b)). Cell function experiments showed knockdown of METTL14 reduced cell proliferation, migration, and invasion and facilitated cell death (cell viability decreased). After PLAGL2 was overexpressed, cell proliferation, migration, and invasion were increased, and cell   Journal of Oncology   (Figures 4(c)-4(f )).

Discussion
NSCLC is one of the most devastating cancers, with high mortality worldwide [26]. New treatment options for NSCLC  Journal of Oncology have become available through extensive in-depth genomic studies to improve preclinical disease patterns and identify specifc toxicity of targeted therapies [27]. In our research, we collected clinical NSCLC and paracarcinoma tissues and investigated METTL14/PLAGL2/β-catenin's role in vivo and in vitro. Our results demonstrated METTL14 promoted NSCLC development by increasing m6A methylation of PLAGL2 to activate β-catenin signaling. Tere has been no reported research on the related mechanism of the METTL14/PLAGL2/β-catenin axis in NSCLC, which is also our innovation of study.
Researchers believed PLAGL2 was overexpressed in diferent malignant tumors, and it could facilitate tumor proliferation, migration, invasion, and self-renewal [28]. We also found that PLAGL2 expression was elevated in NSCLC tissues. Wu et al. reported PLAGL2 facilitated β-catenin expression and nuclear translocation by inhibiting β-catenin phosphorylation level [20]. Liu et al. showed that lncRNA HCP5 could activate Wnt/β-catenin/Cyclin D1 signal through PLAGL2 in multiple myeloma [29]. In addition, PLAGL2 may be a very upstream key molecule regulating epithelial-mesenchymal transition and participated in Wnt/ β-catenin signaling in colorectal adenocarcinoma [30]. Tese studies suggest that PLAGL2 may play an important role in cancer by modulating β-catenin signaling. In this study, we revealed β-catenin (nuclear) level was elevated in NSCLC tissues than paracarcinoma tissues. Moreover, PLAGL2 activated β-catenin signaling to infuence cell proliferation and migration, suggesting that PLAGL2 played an important role in NSCLC by regulating β-catenin signaling. Tis is also the frst time we have reported the study of PLAGL2 and β-catenin signaling in NSCLC.
m6A is the enzyme that plays a vital role in mRNA splicing, translation, and stabilization [31]. It regulates biological metabolism, cell diferentiation and cycle, and responses to heat shock stress and cancer [32]. METTL14 is a well-known RNA m6A that plays a vital role in tumor growth by controlling RNA work [33]. Previous studies have reported that METTL14 can increase m6A modifcation of pri-miR-19a and promote mature miR-19a processing, thereby facilitating atherosclerotic vascular endothelial cell proliferation and invasion [34]. m6A regulator METTL14 has been reported to be diferentially expressed between TP53mutant and wild-type NSCLC [35]. But the mechanism of METTL14-mediated m6A modifcation with PLAGL2 is still unclear. We found that PLAGL2 signaling was regulated by METTL14-mediated m6A modifcation. Furthermore, METTL14 regulated PLAGL2 signaling to afect NSCLC cell function. Tis is also our frst report of METTL14-mediated m6A modifcation and PLAGL2 in NSCLC. Trough in vivo experiments, we further validated METTL14/PLAGL2/ β-catenin axis promoted NSCLC development.
However, our study has some limitations. To verify whether PLAGL2 signaling was regulated by METTL14mediated m6A modifcation, we should frst apply RIP to identify whether PLAGL2 was modifed by m6A and then use RIP to identify the m6A modifcation of PLAGL2 after knockdown and overexpression of METTL14. However, due to time and fnancial constraints, we directly used RIP to identify the m6A modifcation of PLAGL2 after the knockdown and overexpression of METTL14. Although our results also confrmed that PLAGL2 signaling was regulated by METTL14-mediated m6A modifcation, the results need to be proved step by step. In the future, we will further verify whether PLAGL2 was modifed by m6A. Furthermore, the mechanism of METTL14 and the m6A methylation modifcation of PLAGL2 in vivo and the signaling pathways involved need to be further explored.
In conclusion, we demonstrated increased PLAGL2 and β-catenin (nuclear) expressions in NSCLC tissues. Furthermore, we conducted a preliminary exploration of the mechanisms involved in METTL14/PLAGL2/β-catenin. We found METTL14/PLAGL2/β-catenin axis promoted NSCLC development in vitro and in vivo experiments. Our research provides important clues for an in-depth comprehension of the mechanism of NSCLC occurrence and development and also provides a reference for NSCLC treatment.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Te study was approved by the Ethics Committee of Zhuzhou Central Hospital (ZZCHEC2021092-01). Te research was conducted according to the World Medical Association Declaration of Helsinki. All the information about the study will be fully explained to the subjects by the researchers. All the participants provided informed consent before sampling. Tis study was approved by the Animal Ethics Committee of the Second Xiangya Hospital of Central South University (2022760). All experimental procedures were conducted in accordance with institutional guidelines for the use of experimental animals.