PCAT19 Regulates the Proliferation and Apoptosis of Lung Cancer Cells by Inhibiting miR-25-3p via Targeting the MAP2K4 Signal Axis

Both PCAT19 and miR-25-3p have been reported in lung cancer studies, but whether there is a correlation between the two and whether they jointly regulate the progress of lung cancer have not been reported yet. Therefore, this study carried out a further in-depth research. The expression of PCAT19 was detected in lung cancer (LC) tissues and cells by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of PCAT19 on tumor growth was detected in a tumor-bearing model of nude mice. PCAT19-transfected cells were treated with Honokiol and anisomycin. The effects of PCAT19 on proliferation, apoptosis, and cycle of LC cells were investigated by biomolecule experiments. The effects of PCAT19 on the expressions of mitogen-activated protein kinase- (MAPK-) related proteins were evaluated by western blotting. The expression of PCAT19 was decreased in LC tissues and related to patient survival, tumor size, and pathology. In addition, upregulation of PCAT19 hindered LC cell proliferation, miR-25-3p expression, and the activation of extracellular regulated protein kinases (ERK) 1/2, p38, and c-Jun N-terminal kinase (JNK), while facilitating LC cell apoptosis. Furthermore, upregulation of PCAT19 reversed the effects of Honokiol and anisomycin on promoting cell proliferation and inhibiting cell apoptosis. Collectively, our findings show that upregulated PCAT19 suppresses proliferation yet promotes the apoptosis of LC cells through modulating the miR-25-3p/MAP2K4 signaling axis.


Introduction
According to the latest statistics, there are approximately 235,760 cases of lung and bronchial cancer being diagnosed and 131,880 deaths in 2021 (Siegel et al. [1]). Lung cancer (LC) is a frequently diagnosed malignant tumor, with the highest morbidity and mortality worldwide (Torre et al. [2], Goubran et al. [3]). At present, the treatment of early lung cancer is often based on comprehensive treatments such as surgery, radiotherapy, and chemotherapy and Chinese medicine (Wangari-Talbot and Hopper-Borge [4]).
Although advances have been made in the treatment of lung cancer, the 5-year survival rate of LC patients has not been improved greatly, and the mortality rate of LC remains the highest as compared with that of other cancer deaths (Baghdadi et al. [5]). Though the etiology of LC has not yet been fully clarified, there are data indicating that the occurrence of is closely related to assorted factors such as living habits, family inheritance, and endocrine disorders (Avci et al. [6], Granger et al. [7], Dreher et al. [8]). Molecular biology studies have revealed that the occurrence of LC is caused by abnormal proliferation, differentiation, and apoptosis of cells, and these abnormal biological behaviors are associated with abnormal molecular expression, activation, and mutation in vivo (Zhao et al. [9], Jiang et al. [10]). Thus, comprehensive discovery and further exploration on the functions and regulatory mechanisms of these related molecules could help us to understand the molecular mechanism of LC and provide a more effective target for LC treatment.
It is widely recognized that long noncoding RNAs (lncRNAs) make profound impacts on the regulation of gene expressions, and their abnormal expressions are associated with various diseases. As previously reported, lncRNAs are also involved in the occurrence and development of tumors through acting as protooncogenes or tumor suppressor genes (Engreitz et al. [11], Liu and Zhao [12], Wang et al. [13]). Several studies have found that lncRNA PCAT19 plays a carcinogenic role in prostate cancer and laryngeal cancer (Hua et al. [14], Xu, Guo, and Zhang [15]), but its role in LC is less reported. Therefore, the current study was aimed at fathoming out the mechanism of action of PCAT19 in LC.
A mutual regulatory relationship has been corroborated between lncRNA and microRNA (miRNA) (Chen et al. [16], Wang, Jia, et al. [17]), where lncRNAs can interact with miRNA through acting as a competitive endogenous RNA and participate in the regulation of target gene expressions (Yoon, Abdelmohsen, and Gorospe [18], Hao et al. [19]). miRNAs produce their biological effects via an RNAinduced silencing complex to regulate lncRNA. Moreover, miRNA and lncRNA both participate in the occurrence of various diseases (He et al. [20]). A previous study has indicated that miR-25-3p is highly expressed in LC tissues (Giordano et al. [21]). However, whether there is a targeting relationship between PCAT19 and miR-25-3p and whether such a relationship participates in the development of LC remain to be further determined. Thus, this study was dedicated to probing into the potential mechanisms of PCAT19 and miR-25-3p in LC. In addition to the fact that activated mitogen-activated protein kinase (MAPK) is involved in physiological processes such as cell proliferation, differentiation, migration, apoptosis, and stress response, the present studies also found that MAPK is implicated in the occurrence and development of diversified tumors, including those in LC (Hu et al. [22], Wang, Jia, et al. [17]), and that activation of protein kinase B (AKT) and extracellular regulated protein kinase (ERK-) MAPK signaling pathways could mediate the downstream effects of miR-25-3p, which in turn promotes tumor cell proliferation (Chen et al. [23]). In the current study, LC cells were subjected to the treatment with MAPK signaling pathway-associated agonists for further investigating whether PCAT19 regulated the progression of LC through modulating the MAPK signaling pathway.

Material and Methods
2.1. Biological Information Analysis. Gene Expression Profiling Interactive Analysis (GEPIA) was applied to predict the expression of PCAT19 in LC. The binding sites between PCAT19 and miR-25-3p were predicted by using LncBase Predicted v.2, and those between miR-25-3p and MAP2K4 were analyzed by using TargetScan 7.2.

Clinical
Specimens. LC tissue and adjacent tissue samples were obtained from 74 cases of LC patients who attended the Second Affiliated Hospital of Chongqing Medical University for treatment from January 2018 to May 2019. Patients with lung cancer included in the study had not undergone any treatment before. Their tissue samples were kept in liquid nitrogen and maintained at -80°C. All subjects signed the written informed consent, and the current study was approved by the Ethics Committees of the Second Affiliated Hospital of Chongqing Medical University.

Cell
Culture and Transfection. BEAS-2B was used as the human normal lung epithelial cell line, while A549, H1299, and SK-MES-1 were LC cell lines. The cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 (Gibco, USA) containing 10% fetal bovine serum (FBS, Gemini Bio-Products, Sacramento, CA) at 37°C with 5% CO 2 . The cells at the logarithmic growth stage were selected for experiments.
The cells were digested, thoroughly mixed, seeded into the 6-well plates at a concentration of 1 × 10 6 cells/mL, and then evenly distributed in an orifice plate to reach 80-90% confluence. 20

Establishment of the Tumor-Bearing Model in Nude
Mice. Sixteen athymic nude mice (6 weeks old, 20 ± 2 g, male) were purchased from Shanghai Lab, Animal Research Center (license no. SCK (Shanghai) 2008-0016, China) and housed under specific disease-free (SPF) conditions. Ethical and legal approval of animals was obtained from Zhengzhou University. All experiments were performed following institutional and national guidelines and regulations of Zhejiang University. The mice were raised under the temperature of 18-25°C and the humidity of 40-70%. Animals were lived in the day-night cycle of 12 to 12 h with the supplement of regular feed and fertilized water. The mice were divided into 3 Disease Markers two groups, namely, NC group (n = 8) and PCAT19 group (n = 8). 200 μL 1× PBS was used to resuspend the A549 cells transfected with PCAT19 or NC (1 × 10 5 /mL). In the NC group, A549 cells transfected with NC were injected into the right flank of each mouse (weighing 25 g). In the PCAT19 group, A549 cells transfected with PCAT19 were injected into the right flank of each mouse (weighing 25 g).
After being fed for 27 days, all the mice were sacrificed and their tumors were weighed. During the experiment, length and width of the mouse tumor were measured by using Vernier calipers every 3 days, and tumor volumes were measured according to the formula as follows: the V ðmm 3 Þ = largest diameter × perpendicular diameter 2 /2. Tumor tissues of the mice from the two groups were extracted for immunohistochemistry and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay.
2.12. Immunohistochemical Staining. The subcutaneous tumor tissues of nude mice were fixed with formaldehyde (SF877503, Sinopharm Chemical Reagent Beijing Co., Ltd., China), dehydrated with gradient alcohol (80%, 90%, 95%, and 100%), and transparentized with xylene (10023418, Sinopharm Chemical Reagent Beijing Co., Ltd., China). Next, the tissues were wax-immersed, paraffin-embedded, and finally sectioned into tissue slices. Then, the tissue slices were dewaxed with xylene, dephenylated with ethanol at the concentrations of 100%, 95%, 80%, and 70% for 2 min, and washed with PBS twice. After that, 30 mL EDTA antigen repair buffer (P0086, Beyotime Biotechnology, China) was diluted with 1,500 mL distilled water and then boiled in a microwave box. Next, the tissue slices were completely immersed in the buffer to repair antigen, heated for 10 min, naturally cooled, and rinsed in PBS for three times. Following these, each tissue slices were added with 3 drops of 3% H 2 O 2 , incubated under light at room temperature for 15 min to eliminate the activity of endogenous peroxidase, and then washed with PBS three times. Subsequently, the tissue slices were incubated with a primary antibody (anti-Ki-67, ab15580, Abcam, USA) at 4°C overnight and washed with PBS for three times. Afterwards, the tissue slices were ulteriorly cultivated with a secondary antibody at 37°C for 30 min and rinsed with PBS again for three times, followed by being stained with the DAB developing kit (P0202, Beyotime Biotechnology, China) and washed with distilled water for 1 min. Later, the cells were stained with hematoxylin for 1 min, washed under tap water for 30 min, and then sealed with gum. Eventually, the red and granulated cells were observed under an optical microscope (BX40, Olympus, Japan).
2.13. TUNEL Assay. Apoptosis was detected by using the TUNEL kit (Promega, USA). In brief, the tumor tissue samples were embedded with paraffin, sectioned (4 μm), and digested with protease K for 30 min. Next, the tissue sections were incubated with TUNEL reaction fluid at 37°C for 1 h. Then, the streptavidin-HRP working solution (SNN1004, Thermo Fisher Scientific) was added to further incubate the sections at room temperature for 30 min. After that, the sections were stained with DAB chromogen, counterstained with hematoxylin, and dehydrated with ethanol at the concentrations of 70%, 80%, 90%, and 95%, respectively. Finally, the sections were observed under an optical microscope.
2.14. Statistical Analysis. Prism 6 (version 6.01, GraphPad Software, Inc., San Diego, CA, USA) was used for data analysis. The results were presented as mean ± standard deviation (SD) of at least 3 independent experiments. Student's t-test was performed to compare the differences in the mean between the continuous variables, while those among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by the Bonferroni post hoc test. The Kaplan-Meier plotter was employed to predict the relationship between PCAT19 and LC survival. The Spearman correlation was used to analyze the relationships among PCAT19, miR-25-3p, and MAP2K4. P < 0:05 was considered to be statistically significant.

PCAT19 Was Lowly Expressed in LC Tissues and Cells.
The GEPIA website was used to analyze the expression of   BRCA  CESC  CHOL  COAD  DLBC  ESCA  GBM  HNSC  KICH  KIRC  KIRP  LAML  LGG  LIHC  LUAD  LUSC  MESO  OV  PAAD  PCPG  PRAD  READ  SARC  SKCM  STAD  TGCT  THCA  THYM  UCEC  UCS (Figure 1(a)). Besides, PCAT19 presented a low expression in LUAD and LUSC tissues compared with paracancer tissues (ANT) (P < 0:001, Figure 1(b)). Moreover, the relationship between PCAT19 and lung cancer survival was predicted by using the Kaplan-Meier plotter, and the result uncovered that LC patients with a high expression of PCAT19 had a higher survival rate (P < 0:001, Figure 1(c)).

Relationship between PCAT19 Expression Level and
Clinical Characteristics of LC Patients. qRT-PCR was performed to detect the expression of PCAT19 in cancer tissues and ANT collected from 74 LC patients, and the results exhibited that PCAT19 was conspicuously downregulated in tissues of LC patients (P < 0:001, Figure 2(a)), with the mean PCAT19 expression level being 4 ± 1:2 (Figure 2(B)). In this research, the LC patients with PCAT19 expression lower than the average level were defined as having low expression of PCAT19 (n = 34), while those with the expression higher than the average level were identified as having high expression of PCAT19 (n = 40). In addition, the relationship between PCAT19 and clinical characteristics of the patients was analyzed. As illustrated in Table 2, we found that PCAT19 expression was related to tumor size and pathology (P < 0:05), but not associated with age (P = 0:859), gender (P = 0:719), clinical stage (P = 0:423), or lymph node metastasis (P = 0:370). Furthermore, the expression of PCAT19 in the human normal lung epithelial cell line (BEAS-2B) and LC cell lines (A549, H1299, and SK-MES-1) was also detected by qRT-PCR, and the result mirrored that PCAT19 level was downregulated in LC cell lines (P < 0:001, Figure 2(c)).

Upregulated PCAT19 Inhibited LC Cell Proliferation.
PCAT19 and siPCAT19 vectors were transfected into A549 and SK-MES-1 cells to further investigate the effect of PCAT19 on proliferation of LC cells. As depicted in Figures 3(a) and 3(b), PCAT19 and siPCAT19 vectors were To further confirm that, two pmirGLO-dual-luciferase reporter vectors, MAP2K4-3′-UTR wt and MAP2K4-3′-UTR mut (hereafter MAP2K4-WT and MAP2K4-MUT), were constructed. Subsequently, these two vectors were cotransfected with the miR-25-3p mimic into A549 and SK-MES-1 cells, respectively. The results uncovered that the luciferase activity of cells cotransfected with MAP2K4-WT and miR-25-3p mimic was remarkably suppressed, and that

Upregulated PCAT19
Inhibited the Activation of the MAPK Signaling Pathway. We detected the effects of PCAT19 on the MAPK signaling pathway by western blotting, and the results unraveled that the expressions of MAPK signaling pathway-related proteins (p-ERK1/2, p-p38, and p-JNK) were markedly inhibited in A549 and SK-MES-1 cells transfected with the PCAT19 overexpression vector (P < 0:001, Figures 7(a)-7(d)), while PCAT19 silencing augmented the expressions of MAPK signaling pathway-related proteins (p-ERK1/2, p-p38, and p-JNK) in A549 and SK-MES-1 cells (P < 0:001, Figures 7(a)-7(d)). In addition, the ratios of MAPK signaling pathway-related proteins were calculated in A549 and SK-MES-1 cells, and we found that the ratios of p-ERK1/2/ERK1/2, p-p38/p38, and p-JNK/JNK were visibly reduced in A549 and SK-MES-1 cells transfected with the overexpressed PCAT19 vector (P < 0:001, Figures 7(e) and 7(f)), while the ratios of p-ERK1/2/ERK1/   3.8. PCAT19 Regulated LC Cell Growth via Inhibiting the MAPK Signaling Pathway. After A549 and SK-MES-1 cells were treated with 10 μM Honokiol (ERK agonist) or 0.1 μM anisomycin (p38 and JNK agonist) and transfected with the PCAT19 overexpression vector, the viability of A549 and SK-MES-1 cells was detected by the MTT assay, and the results revealed that the cell viability in the PCAT19 group was lower than that in the NC group, the cell viability of Honokiol and anisomycin groups was higher than that of the NC group, and the cell viability of PCAT19+Honokiol and PCAT19+anisomycin groups was lower than that of Honokiol and anisomycin groups, respectively (P < 0:001, Figures 8(a) and 8(b)). In addition, the colony numbers in PCAT19+Honokiol and PCAT19+anisomycin groups were lower than those in Honokiol and anisomycin groups, respectively (P < 0:001, Figures 8(c) and 8(d)). Furthermore, the results of flow cytometry showed that apoptosis in PCAT19+Honokiol and PCAT19+anisomycin groups was overtly higher than that in Honokiol and anisomycin groups, respectively (P < 0:001, Figures 8(e) and 8(f)).

Discussion
The pathogenesis of LC has been affirmed to be closely related to malignant proliferation and differentiation disorders of cells (Hu et al. [25]). Therefore, investigating the molecular mechanism of LC is of great significance for understanding the occurrence and development of LC, so as to make effective therapeutic regimens for LC patients. Increasing studies have corroborated that the abnormal expressions of lncRNAs are involved in the occurrence and development of tumor cells, and much attention has been focused on the modulating the expressions of lncRNArelated genes to reverse the growth of tumor cells (Engreitz et al. [11], Liu and Zhao [12]).
Studies conducted on PCAT19 in prostate cancer and laryngeal cancer (Gao, Xia, et al. [26], Xu, Guo, and Zhang [15]) demonstrated that aggressiveness of prostate cancer is positively associated with increased expression of PCAT19 Gao, Xia, et al. [26], and upregulation of PCAT19 in laryngeal tumor tissue is associated with shorter overall survival time (Xu,Guo,and Zhang [15]). In this study, we observed that PCAT19 was lowly expressed in 11 types of cancers, especially in lung cancer tissues. Meanwhile, we found that PCAT19 level was appreciably downregulated in cancer tissues derived from LC patients, which was similar to the results of Acha-Sagredo et al. (Acha-Sagredo et al. [27]). Moreover, our survival analysis uncovered that the survival rate of LC patients with high expression of PCAT19 was much higher, indicating that the overall survival rate of LC patients was positively related to PCAT19 level. Furthermore, we analyzed the clinical features of LC patients with different expression levels of PCAT19 and discovered that tumor size and pathology were related to the expression of PCAT19.
Regulating gene expressions is effective in treating cancer (Belguise et al. [28], Sui et al. [29], Amirsaadat et al. [30]). At present, the roles of various genes in the regulation of LC progression have been increasingly investigated. For example, Cui et al. confirmed that SNHG1 was highly expressed in LC patients, and downregulation of SNHG1 could inhibit the growth of non-small-cell lung cancer (Cui et al. [31]). PCAT19 expression was downregulated in lung adenocarcinoma patient tissues, and overexpression of PCAT19 could signally hamper the proliferation, migration, and invasion of lung adenocarcinoma cells (Tang et al. [32]). In the current study, we obtained similar results, the PCAT19 overexpression vector and silencing vector were transfected into LC cells to investigate the effects on LC cells, and we observed that overexpression of PCAT19 repressed cell viability, reduced the cell colony number, and promoted apoptosis. Moreover, tumor formation experiments also verified that overexpression of PCAT19 suppressed tumor growth while promoting tumor cell apoptosis, indicating that regulation of PCAT19 expression could regulate the progression of LC. miR-25-3p has been proven to be highly expressed in LC tissues (Giordano et al. [21]). Consistently, the current study also found the similar results by detecting the expression of miR-25-3p in LC tissues, but whether miR-25-3p was involved in the development of LC and whether it was related to PCAT19 have not been elucidated yet. In addition, a prior study pointed out that inhibition of BTG2 expression in breast cancer could indirectly activate AKT and ERK-MAPK signaling pathways, thereby mediating the downstream effects of miR-25-3p and controlling disease progression (Chen et al. [23]). On this basis, the current study found that MAP2K, an important factor in the MAPK cascade, had a binding site with miR-25-3p and that MAP2K4 level was apparently reduced in LC patients.
Studies have indicated that activation of the MAPK signaling pathway promotes the progression of gastric cancer cells (Mi et al. [33]), and increasing MAPK signaling in lung adenocarcinoma can promote rapid progression of adenomas to malignant adenocarcinoma (Cicchini et al. [34]). Therefore, we explored the effects of PCAT19 on the MAPK signaling pathway in LC cells and confirmed the targeted relationship between PCAT19 and miR-25-3p by in vitro experiments. ERK1/2, p38, and JNK pertain to the MAPK family (Gao, Shan, et al. [35], Bubici and Papa [36,37]), and activation of ERK1/2 protein can promote the development of lung cancer in mice (Yamakawa et al. [38]), activation of p38 phosphorylation can enhance the production of tumor factors (Alam et al. [39]), and JNK can regulate a variety of cell biological functions. Additionally, the JNK protein expression has been reported to strengthen the activity of cancer cells (Wang, Ni, et al. [40]). In the current study, we found that overexpression of PCAT19 could downregulate the phosphorylation levels of MAPK signaling pathway-related proteins ERK1/2, p38, and JNK, thereby inhibiting the activation of the MAPK signaling pathway. However, PCAT19 silencing generated the opposite results. As evidenced by a prior study, the inhibition on the activation of the MAPK signaling pathway could repress the growth of H460 and H1299 LC cells (Liu et al. [41]). Furthermore, in this research, MAPK signaling pathway agonists were applied to treat the cells transfected with the PCAT19 overexpression vector, and the results signified that overexpression of PCAT19 hindered the progression of agonist-induced LC cells. Moreover, PCAT19 overexpression could hamper the viability and proliferation while promoting the apoptosis of LC cells through regulating the MAPK signaling pathway.
In previous reports, PCAT19 was proven to be able to facilitate tumor cell growth in non-small-cell lung cancer cells H1993 with the characteristic of lymphatic metastasis (Zhang et al. [42]) and in laryngeal cancer (Cossu et al. [43]). However, similar to TCGA data and the report of Acha-Sagredo et al. (Acha-Sagredo et al. [27]), data from this study suggested that PCAT19 may be a tumor suppressor in non-small-cell lung cancer cells A549 and lung squamous cells SK-MES-1. The discrepancy on the different functions of PCAT19 needs to be further deciphered in the future. In our study, the overexpressed PCAT19 markedly impacted the tumor cell behaviors.

Conclusions
In conclusion, the study reveals that upregulated PCAT19 suppresses the proliferation yet promotes the apoptosis of A549 and SK-MES-1 cells through modulating the miR-25-3p/MAP2K4 signaling axis.

Data Availability
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.