miR-199a-3p plays an anti-tumorigenic role in lung adenocarcinoma by suppressing anterior gradient 2

ABSTRACT Previous studies have explored the association between protein-coding genes and microRNAs (miRNAs) in lung adenocarcinoma (LUAD). However, the influence of the miR-199a-3p/anterior gradient 2 (AGR2) axis in LUAD has not yet been fully explored. Therefore, this study aimed to examine the underlying roles of AGR2 and miR-199a-3p in the development of LUAD. The expression levels of miR-199a-3p and AGR2 in LUAD tissues and cells were detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A luciferase assay was also performed to identify the interaction between AGR2 and miR-199a-3p. Moreover, the cell counting kit 8 (CCK-8), 5ʹ-bromo-2ʹ-deoxyuridine (BrdU), and adhesion assays were used along with flow cytometry to verify the malignancy of LUAD in vitro, while a xenograft tumor assay was performed to confirm the tumor growth in vitro. The findings showed a decrease in the expression of miR-199a-3p in LUAD. Additionally, miR-199a-3p overexpression inhibited the growth of LUAD cells in vitro and in vivo, while elevating the apoptosis rate of the cells. AGR2 knockdown had the same effect in the cells as that of miR-199a-3p overexpression. It was also found that miR-199a-3p directly targeted AGR2 in LUAD cells to suppress tumorigenesis. In conclusion, this study suggests that miR-199a-3p plays an anti-tumorigenic role in LUAD by targeting AGR2. Moreover, our study provides insights into the development of novel therapeutic targets for the treatment of LUAD.


Introduction
Lung cancer is a malignancy that originates in the lungs, which accounted for 1.5 million incident cases in 2017 [1]. There are many types of lung cancer, and lung adenocarcinoma (LUAD) is the most frequent type of lung cancer, accounting for almost 40% of all lung cancer cases [2]. Due to its insidious symptoms, LUAD tends to be diagnosed at a late stage, resulting in a dismal clinical outcome of patients [3]. This disease can be treated by several methods, including surgery, chemotherapy, targeted drug therapy, radiotherapy, and medications. The antitumorigenic potential of fungiassociated materials has also been explored in clinical practice [4]. Although satisfactory clinical outcomes have been achieved with these treatment methods, the 5-year overall survival rate of patients with LUAD remains poor (11%) [5].
Moreover, patients with LUAD often experience a relapse of the disease within the first five years of treatment [5]. By investigating the underlying mechanism of LUAD, it may be possible to improve the strategies for the diagnosis and treatment of LUAD.
Located on chromosome 7q21.1, the anterior gradient 2 (AGR2) consists of eight exons. AGR2, a member of the protein disulfide isomerase (PDI) family of endoplasmic reticulum (ER) proteins, regulates the growth and metastasis of malignant tumors [21,22]. AGR2 was also identified as an oncogene in multiple cancers, including the pancreatic, colorectal, and gastric cancers [22][23][24]. Upregulation of AGR2 is associated with the initiation of cancer, and this protein can induce cell apoptosis and enhance the sensitivity of pancreatic cancer cells to chemotherapy via the extracellular signal-regulated kinase (ERK)/serine-threonine kinase (AKT) pathways [25]. Furthermore, AGR2 overexpression predicts the poor survival rate in lung cancer [26]. So far, only one study has demonstrated the ability of miR-342-3p to prevent cell growth and migration by inhibiting AGR2 in NSCLC [27]. Furthermore, AGR2 activates the mammalian target of rapamycin complex 2 (mTORC2) pathway and promotes cancer metastasis [28]. Meanwhile, miR-199a-3p retards tumorigenesis in a hepatocellular carcinoma transgenic mouse model by modulating the mTORC2 pathway [29]. Therefore, we hypothesized that AGR2 may be the key regulator in LUAD and the target gene of miR-199a-3p, which regulates the development of LUAD. The results of this study can help to increase our understanding of the strategies that can be used for the diagnosis and treatment of LUAD.

LUAD specimens and cell lines
We obtained 32 paired LUAD tissues and adjacent normal tissues (5 cm from the tumor) from patients with LUAD who consented to participate in this experiment. The collected samples were stored in the TRIzol reagent (cat#: 15,596,026; Invitrogen, USA) at −80°C for RNA extraction. The clinical characteristics of the 32 patients are listed in Supplementary Table S1. All cell lines were purchased from the American Type Culture Collection (USA) and included human LUAD cell lines (PC9, A549, H1975, DV-90, and Calu-3) as well as the normal lung epithelial cell line (BEAS-2B). H1975 and DV-90 cells were maintained in the Roswell Park Memorial Institute (RPMI)-1640 medium, while A549, PC9, BEAS-2B, and Calu-3 cells were transferred to Dulbecco's modified Eagle medium (DMEM). Then, fetal bovine serum (FBS, 10%) was added to the DMEM or RPMI-1640 media, and the cells were cultured at 37°C in air containing 5% carbon dioxide (CO 2 ). All reagents, including the culture medium and FBS, were purchased from Life Technologies Corporation (USA).

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA from the LUAD tissues, adjacent tissues, LUAD cells, and BEAS-2B cells was isolated using the TRIzol reagent (cat#: 15,596,026; Invitrogen, USA). Reverse transcription was conducted using the PrimeScript First Strand cDNA Synthesis Kit (Cat#: 6210A; Takara, Japan). qRT-PCR was performed using SYBR Premix Ex Taq (Cat#: DRR420A; Takara, Japan). AGR2 expression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), while the expression of miR-199a-3p was normalized to U6. Finally, data analysis was performed using the 2 −∆∆CT method [30]. The primers used in this study are listed in Supplementary Table S2.

Cell counting kit 8 (CCK-8) assay
The CCK-8 kit (Cat#: K1018) purchased from APExBIO (China) was used to investigate the cell viability. PC9 and Calu-3 cells at a density of 5,000 cells/well were added to 96-well plates. The CCK-8 solution was diluted to a ratio of 1:10 using the medium. At 0, 12, 24, 48, and 72 h, the medium was changed with 100 µL CCK-8 and incubated for 3 h at 37°C. Finally, the optical density (OD) was measured at 450 nm using a microplate reader (Tecan, Switzerland).

5ʹ-bromo-2ʹ-deoxyuridine (BrdU) assay
The BrdU Cell Proliferation Assay Kit (Cat#: 6813; Cell Signaling Technology, USA) was used to perform the BrdU assay. Briefly, PC9 and Calu-3 cells with a density of 2 × 10 4 cells/well were plated on 96-well plates for 12 h, and an FBS-free medium was used to culture the cells for 12 h. Next, the medium was changed with the BrdU working solution for 4 h, and the mixture was incubated at 37°C. After removing the labeling medium, the cells were fixed and denatured with a fixation/ denaturation solution. Next, the BrdU mouse monoclonal antibody (mAb) was added to the solution, and the mixture was cultured for 2 h. Anti-mouse IgG and horseradish peroxidase (HRP)-linked antibodies were subsequently used to detect the binding antibody. Tetramethylbenzidine (TMB), a HRP substrate, was then added to color the mixture. Finally, the optical density (OD) value at 450 nm was measured using a microplate reader (Tecan, Switzerland).

Cell adhesion assay
The cell adhesion plate was first prepared by adding 30 μL/well of the 40 μg/mL collagen I solution (Cat#: C7661; Sigma-Aldrich, USA) to the 96-well plate, and it was stored at 4°C for 12 h. Then, PC9 and Calu-3 cells (2 × 10 4 cells/well) were plated on the 96-well plates for 24 h. Next, 10 mM ethylenediaminetetraacetic acid (EDTA) (solute in DMEM) was added for 10 min to dissociate the cells. After the cells were suspended in DMEM with 0.1% bovine serum albumin (BSA), 100 μL of the cell suspension was added to the collagen I solution, and the mixture was incubated at 37°C. Non-adherent cells were then washed with PBS for three times, and the cells were cultured at 37°C for 1 h. After 1 h of incubation, the cells were incubated with 10 μL of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) substrate (Cat#: CT01; Sigma-Aldrich, USA) at 37°C for 2 h. Finally, the OD of each well was measured at 570 nm using a microplate reader (Tecan, Switzerland).

Apoptosis assay
The apoptosis rates of PC9 and Calu-3 cells were assessed using the Cell Apoptosis Detection Kit (BD, USA). Cells (6 × 10 4 ) were harvested at logarithmic growth phase and suspended in 100 µL of the binding buffer. After that, the cells were incubated with a mixture containing 5 µL/ well Annexin V-fluorescein isothiocyanate (FITC) and 5 µL/well propidium iodide (PI) in the absence of light for 15 min at 25°C. After incubation, the cells were washed three times with PBS and suspended in 400 µL of the binding buffer. Flow cytometry was used to detect the cells. The cell apoptosis rate was analyzed by summing the percentage of upper-right and lower-right quadrants.

Xenograft tumor assay
miR-199a-3p agomir and NC were purchased from RiboBio Co., Ltd. (China). BALB/c nude mice (4 weeks old) were purchased from Charles River (Wilmington, MA, USA) and were allocated into two groups, NC and miR-199a-3p agomir. After transfection for 48 h, 2 × 10 6 Calu-3 cells transfected with miR-199a-3p agomir or its corresponding NC were collected and resuspended using 0.2 mL phosphate-buffered saline (PBS). The transfected cells were then subcutaneously injected into the nude mice. The xenografts were observed after cell inoculation. Five weeks after injection, the nude mice were sacrificed, and the xenografts were removed and photographed. The tumor weight was determined and the tumor samples were stored in liquid nitrogen.

Luciferase assay
The pmiRGLO-AGR2 3ʹ-untranslated region (UTR) wild-type (WT) and mutant (MUT) plasmids were designed and constructed by Tuoran Co., Ltd (Shanghai, China). Next, 5 × 10 5 PC9 and Calu-3 cells were added to 24-well plates and co-transfected with pmiRGLO-AGR2 WT or MUT plasmids and miR-NC or miR-199a-3p mimic using Lipofectamine 3000. The dual-luciferase reporter assay system was then used to detect the firefly and Renilla luciferase activities after 72 h of transfection. This detection was performed using the Luciferase Assay Kit (Cat#: #16,185; Thermo Scientific, USA), and the firefly luciferase activity was normalized to the results of relative luciferase activity.

RNA pull-down assay
Two biological materials were purchased from Thermo Fisher Scientific (USA). They included biotin-labeled miR-199a-3p (bio-miR-199a-3p) and its negative control (bio-NC). PC9 and Calu-3 cells were first treated with Bio-miR-199a-3p and Bio-NC for 48 h. Subsequently, the cells were collected in a lysis buffer containing RNase and inhibitors. After washing, the streptavidin beads (Cat#: #88,817) from Thermo Fisher (USA) were added to the cell lysates. After the mixture was incubated overnight at 4°C, the RNAs in the beads were eluted and purified using the RNeasy Mini Kit (Cat#: 74,104; QIAGEN, Germany). Finally, qRT-PCR analysis was performed to verify the enrichment of AGR2 in the bio-miR-199a-3p and bio-NC groups.

Statistical analysis
The mean ± standard deviation (SD) of all the data was obtained using the SPSS v. 19.0 software (IBM Corp., USA). The paired or unpaired Student's t-test was applied to compare the statistical difference between two groups, while the one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test was applied to compare the statistical differences among multiple groups. All experiments were performed at least thrice to eliminate errors. Statistical significance was set at P < 0.05.

MiR-199a-3p inhibited the malignancy of LUAD cells both in vitro and in vivo
To examine the function of miR-199a-3p in LUAD cells, the expression levels of miR-199a-3p in LUAD cell lines (PC9, Calu-3, H1975, A549, and DV-90) and normal lung epithelial cells (BEAS-2B) were identified. Experimental results showed that miR-199a-3p expression decreased in LUAD cell lines compared to that in BEAS-2B cells. More specifically, miR-199a-3p expression declined by more than 50% in PC9 and Calu-3 cell lines compared to that in BEAS-2B cells ( Figure 2a); therefore, these two cell lines were selected for further research. After transfection with mimics negative control (NC), miR-199a-3p mimics, inhibitor-NC, or miR-199a-3p inhibitor into PC9 and Calu-3 cell lines, results displayed that miR-199a-3p mimics increased by more than 4-fold in miR-199a-3p expression compared to mimic-NC groups; however, miR-199a-3p expression declined by 80% in the miR-199a-3p inhibitor groups compared to the inhibitor NC groups in both PC9 and Calu-3 cell lines (Figure 2b). Furthermore, cell viability was downregulated in the miR-199a-3p mimic group compared to that in the mimic-NC group. However, in both PC9 and Calu-3 cell lines, the miR-199a-3p inhibitor increased cell viability compared to the inhibitor-NC group (Figure 2c).
While cell proliferation in the miR-199a-3p mimic groups decreased by 50% compared to the mimic NC groups, cell proliferation in the miR-199a-3p inhibitor groups increased by 1.5-fold compared to the inhibitor NC groups in both PC9 and Calu-3 cell lines (Figure 2d). Cell adhesion in the miR-199a-3p mimic groups declined by 40% compared to the mimic NC groups; nonetheless, cell adhesion in the miR-199a-3p inhibitor groups increased by 1.5-fold in contrast to the inhibitor NC groups in both PC9 and Calu-3 cell lines (Figure 2e). Moreover, the cell apoptosis rate increased by 2-fold in the miR-199a-3p mimic groups, but it declined by 50% in both PC9 and Calu-3 cell lines after transfection with miR-199a-3p inhibitor (figure 2f). Moreover, the effect of miR-199a-3p on LUAD cells was observed in nude mice, showing that miR-199a-3p overexpression inhibited tumor growth (Figures 3a and 3b), and miR-199a-3p agomir enhanced miR-199a-3p expression in tumor sections compared to the NC group (Figure 3c). These results suggest that miR-199a-3p is a tumor suppressor in LUAD.
AGR2 expression was enriched in the Bio-miR-199a-3p group in both PC9 and Calu-3 cell lines (Figure 4c). AGR2 expression increased 2-fold in tumor tissues compared to normal tissues ( Figure 4d). As for the outcome of the correlation analysis, a negative correlation between miR-199a-3p expression and AGR2 expression was found in LUAD tissues (Figure 4e). Taken together, these results suggest that miR-199a-3p targets AGR2.

MiR-199a-3p played an anti-tumorigenic role in LUAD by targeting AGR2
To investigate the mechanism of miR-199a-3p in LUAD, si-NC, inhibitor-NC, Si-AGR2, miR-199a-3p inhibitor, and Si-AGR2+ miR-199a-3p inhibitor were transfected into PC9 and Calu-3 cells. The qRT-PCR results showed that AGR2 expression in the miR-199a-3p inhibitor group increased 5-fold in PC9 cells and 7-fold in Calu-3 cells, whereas AGR2 expression in the Si-AGR2 group decreased by more than 70% in both PC9 and Calu-3 cells. Nonetheless, AGR2 expression in the Si-AGR2+ miR-199a-3p inhibitor group was similar to that in the NC groups (si-NC or inhibitor-NC) (Figure 5a). The cell viability of the Si-AGR2 groups was significantly lower than that of the Si-NC groups; however, this effect was reversed in the Si-AGR2 + miR-199a-3p inhibitor groups in both PC9 and Calu-3 cells (Figure 5b). Similarly, the cell proliferation of the Si-AGR2 groups was lower than that of the Si-NC groups; however, this change was restored in the Si-AGR2 + miR-199a-3p inhibitor groups in both PC9 and Calu-3 cells (Figure 5c). In addition, the Si-AGR2 groups showed a 30% downregulation of cell adhesion ability compared to the Si-NC groups; however, the effect was abrogated in the Si-AGR2 + miR-199a-3p inhibitor groups in both PC9 and Calu-3 cells (Figure 5d). The results of the cell apoptosis assay indicated that Si-AGR2 enhanced the apoptosis rate by 2-fold compared to the si-NC group; however, the effect was reversed in the Si-AGR2+ miR-199a-3p inhibitor groups in both PC9 and Calu-3 cells (Figure 5e). Overall, the data indicated that by targeting AGR2, miR-199a-3p repressed cell proliferation but induced cell apoptosis in LUAD.

Discussion
In the present study, we found that miR-199a-3p was less expressed in LUAD tissues and cells, and miR-199a-3p mimic retarded tumor growth in vitro and in vivo, while miR-199a-3p inhibitor strengthened the proliferative capacity of LUAD cells. Mechanistically, miR-199a-3p targeting argonaute 2 (AGO2) inhibited the LUAD progression. These results suggest that targeting the miR-199a-3p/AGO2 axis might be an effective approach for LUAD management.
MiR-199a-3p can regulate cell growth, differentiation, and death in various cancer types, such as esophageal, gastric, and prostate cancers [16,33,34]. MiR-199a-3p significantly inhibits the cell proliferation by interacting with p21-activated kinase 4 in esophageal cancer [35]. MiR-199a-3p also promotes cell invasion and migration in gastric cancer cells, and the upregulation of miR-199a-3p predicts poor prognosis [15]. Moreover, there is increased expression of miR-199a-3p in patients with lung cancer, and this miRNA could serve as a biomarker for diagnosing lung cancer [36,37]. Bai et al. demonstrated that the upregulation of miR-199a-3p inhibited ZEB1 inhibited lung cancer growth in vitro and in vivo [20]. Therefore, miR-199a-3p plays a tumor-suppressing role during cancer progression. Consistently, this study showed that miR-199a-3p expression obviously decreased in LUAD, and miR-199a-3p was found to impair cell growth and promote cell apoptosis in vivo and in vitro. Our findings support previous findings that miR-199a-3p plays a tumorsuppressive role.

Conclusion
Our findings demonstrate that miR-199a-3p is downregulated in LUAD tissues and cells, and its overexpression reduces the proliferation of LUAD cells, while triggering the apoptosis of cells. Mechanical research has shown that miR-199a-3p influences the malignancy of LUAD by targeting AGR2. The outcome of this research could provide more insights into the development of novel therapeutic targets for the treatment of LUAD. However, this study has several limitations such as Lack of study on downstream signaling pathway. In the future, our study will explore other potential signaling pathways that influence the miR-199a-3p-AGR2 axis in LUAD cells. In addition, the results of this research should be verified with more clinical samples and in vivo assays in the future.

Highlight
1. MiR-199a-3p overexpression inhibited the growth of the LUAD cells in vitro.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Ethics approval and informed consent
The present study was approved by the Ethics Committee of the Affiliated Cancer Hospital of Zhengzhou University. The processing of clinical tissue samples is in strict compliance with the ethical standards of the Declaration of Helsinki. All patients signed written informed consent. The processing of animals is in strict compliance with the Ethics Committee of the Affiliated Cancer Hospital of Zhengzhou University. Animal licenses are included in the supplementary files.

Consent for publication
Consent for publication was obtained from the participants.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.