Pseudogene MSTO2P Interacts with miR-128-3p to Regulate Coptisine Sensitivity of Non-Small-Cell Lung Cancer (NSCLC) through TGF-β Signaling and VEGFC

Background Coptisine has been widely used for treating a variety of cancer types. To date, whether pseudogene is implicated in coptisine resistance of NSCLC remains unknown. Methods We performed MTT to assess the cell viability of A549 and Calu-1 cells. The transwell assay was used to examine the invasion of cells. TUNEL was used to determine apoptosis. Results Our data showed that coptisine treatment suppressed cell viability and invasion of NSCLC cells while contributing to apoptosis. MiR-128-3p negatively regulated MSTO2P. miR-128-3p reverted MSTO2P knockdown-attenuated cell viability and invasion, as well as promoted cell apoptosis of A549 cells. Moreover, we identified TGF-β signaling and VEGFC as key downstream effectors for MSTO2P and miR-128-3p in A549 cells. MiR-128-3p mimic inhibited TGF-β pathway-associated genes (TGFBR1, Smad2, Smad5, and Smad9), whereas miR-128-3p inhibitor exerted opposite effect. MSTO2P knockdown led to attenuated expression levels of TGFBR1, Smad2, Smad5 and Smad9. VEGFC overexpression greatly rescued miR-128-3p-modulated cell viability, invasion, and apoptosis of A549 cells. Conclusion MSTO2P plays a role in coptisine therapy of NSCLC through miR-128-3p. The findings will advance our understanding of NSCLC treatment.


Introduction
Lung cancer remains one of the most malignant cancer types. About 85% of lung cancers are non-small-cell lung cancers (NSCLC) [1]. e 5-year overall survival rate is around 16% [2]. Although surgery, radiotherapy, and chemotherapy are widely used approaches for treating cancer patients, drug resistance will limit the use of these therapies [3].
us, more effective chemotherapeutic agents for NSCLC need to be developed.
In recent years, researchers have found that natural products such as plant extracts can induce apoptosis in NSCLC and have good potential for anti-NSCLC therapeutic applications. Coptisine is one of the components extracted from the Chinese medication Rhizoma coptidis (RC) [4]. In previous reports, coptisine was involved in different biological processes, including antibacterial, antitumor, and lipid-lowering [5,6]. In NSCLC, coptisine triggered mitochondrial-dependent cell apoptosis and cell cycle arrest [7]. However, the majority of antitumor drugs are eventually resistant to cancer. e present study studied the molecular mechanism concerning coptisine drug resistance.
Pseudogene derives from protein-coding genes, but it has no ability to translate into proteins [8]. Hence, pseudogenes were initially believed to be nonfunctional RNAs [9]. Recently, increasing evidence has shown that pseudogenes could regulate the progression of various tumors [10][11][12]. e Misato family member 2 (MSTO2P) pseudogene was found to be implicated in gastric cancer [13], lung cancer [14] and HCC [15]. Moreover, Shi et al. demonstrated that MSTO2P enhanced osteosarcoma progression through PD-L1 [16]. In our study, we attempted to investigate the detailed mechanism of MSTO2P-induced NSCLC.
MiRNAs are short noncoding RNAs which have the ability to play roles in cell proliferation, migration, invasion, and metastasis of tumor cells [17]. Commonly, miRNAs exert their effects via targeting 3′-UTRs of downstream genes [18]. Han et al. showed miR-128 induced apoptosis of pancreatic cancer cells by targeting MDM4 [19]. Some groups revealed that miR-128-3p suppressed breast or colorectal cancer [20,21]. Besides, miR-128-3p could also promote lung cancer cell apoptosis. However, whether miR-128-3p could be regulated by MSTO2P participating in NSCLC development was obscure.

Cell Culture.
e human embryonic kidney cell line 293T and human NSCLC cell lines (A549 and Calu-1) were from the Cell Bank of the Chinese Academy of Sciences. All cells were cultured in complete medium. e complete medium was composed of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C, 5% CO 2 . 25 μg/mL of coptisine (MedChemExpress, HY-N0430) was used to treat the NSCLC cells [26].

MTT Assay.
We seeded A549 and Calu-1 (∼8000 cells) cells into 96-well plates. en, the seeded plates were incubated for indicated time. MTT was added and subsequently incubated for 4 h at 37°C. 150 µL DMSO was added into each well and incubated for 10 min. OD570 nm was then measured using a microplate reader.

Invasion Assay.
We suspended A549 and Calu-1 cells (2 × 10 5 cells) in 200 µL of medium. en, the cell suspensions were placed into the top chamber with an 8 μm pore membrane precoated with Matrigel. Media plus 20% FBS was added to the bottom chamber. About 24 h postinvasion, invaded cells were stained with 0.01% crystal violet. All pictures were captured under a microscope (400x).

TUNEL.
We seeded appropriate A549 and Calu-1 cells on the coverslips. e TUNEL reagent was used to stain the cells and the apoptosis rate was determined. DAPI dye was used to stain cell nuclei. All pictures were captured under a microscope (200x).

Reverse Transcription-Quantitative PCR (RT-qPCR).
RNAs of NSCLC cells were extracted with TRIzol reagent (Invitrogen). e total RNAs were dissolved in DEPC ddH 2 O. After that, about 1 µg of total RNA was employed to generate cDNAs using the PrimeScript RT reagent kit (Takara). Real-time PCR was performed using SYBR Green.

Statistical Analysis.
We performed all experiments in triplicate to show the mean ± SD and statistical analysis using Graphpad 6.0. Two or multiple group comparisons were carried out by unpaired Student's t-test and ANOVA (Tukey's post hoc test), respectively. P < 0.05 was considered as significant.

Coptisine Inhibited Tumorigenesis of NSCLC Cells.
To determine the effect of coptisine on NSCLC tumorigenesis, A549 and Calu-1 cells were treated with or without coptisine. First, we examined the cell viability of these two cell lines.
e results showed that coptisine significantly inhibited the cell viability of A549 and Calu-1 cells (Figure 1(a)). e TUNEL assay demonstrated that coptisine could increase apoptosis of both A549 and Calu-1 cells (Figures 1(b) and 1(c)). Besides, we performed a transwell assay to determine the invasion ability of NSCLC cells. Coptisine reduced the invaded number of A549 and Calu-1 cells compared to control (Figures 1(d) and 1(e)). Interestingly, we found that coptisine remarkably suppressed pseudogene MSTO2P expression in A549 and Calu-1 cells (Figure 1(f )). Taken together, coptisine markedly inhibited NSCLC tumorigenesis.

Discussion
Advances in chemotherapy of NSCLC have been achieved by using other targeted drugs, such as crizotinib, cetuximab, and bevacizumab [26]. However, few studies reported that natural bioactive components were used to treat NSCLC. In our research, we evaluated the cell viability, invasion, and apoptosis of A549 and Calu-1 cells under coptisine treatment.
e results showed coptisine significantly inhibited cell viability and invasion. Based on these, we reckoned that coptisine might be a useful strategy for NSCLC treatment.
Pseudogene has been shown to regulate tumor phenotypes via affecting transcriptional or post-transcriptional processes [27]. Unfortunately, the precise mechanisms of this regulation have not been uncovered. Liao et al. demonstrated that pseudogene LGMN sponged miR-495-3p to promote glioblastoma progression [28]. DUXAP8 promoted pancreatic cancer cell growth by epigenetically regulating CDKN1A and KLF2 [29]. In our paper, we believed that MSTO2P acted as a sponge for miR-128-3p in NSCLC cells.
MiR-128-3p was regarded as a potential target for cancer, because it has been investigated in many cancers [20,21,30]. MiR-128-3p played an inhibitory role in EMT of osteosarcoma and induced glioma cell apoptosis [31,32]. In agreement with these data, we also confirmed miR-128-3p reverted NSCLC cell tumorigenesis-affected by MSTO2P. miR-128-3p inhibited cell viability and invasion of NSCLC cells. Hence, it is possible to use miR-128-3p as a diagnostic and therapeutic marker for NSCLC.
is axis regulates NSCLC response to coptisine. Our conclusions will facilitate the development of combination treatments of coptisine and other targeted therapies.

Data Availability
Data included in this study are available from the corresponding author upon reasonable request.