LncRNA AFAP1-AS1 Supresses miR-139-5p and Promotes Cell Proliferation and Chemotherapy Resistance of Non-small Cell Lung Cancer by Competitively Upregulating RRM2

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. This study aims to understand the underlying mechanism of lncRNA, actin filament-associated protein 1 antisense RNA 1(AFAP1-AS1) in mediating chemotherapeutic resistance in NSCLC. The levels of AFAP1-AS1 in NSCLC tissues and cells were determined using RT-PCR. The protein levels of RRM2, EGFR, and p-AKT were analyzed using Western blotting. Binding between AFAP1-AS1 and miR-139-5p was confirmed using dual luciferase reporter and RNA immunoprecipitation (RIP) assays, and binding between miR-139-5p and RRM2 was confirmed by a dual luciferase reporter assay. NSCLC cell proliferation, apoptosis, and colony formation were examined using MTT, flow cytometry, and colony formation assays, respectively. It was found that AFAP1-AS1 expression was upregulated in NSCLC tissues and cells. In addition, AFAP1-AS1 bound to and downregulated the expression of miR-139-5p, which was reduced in NSCLC tissues. Knockdown of AFAP1-AS1 and overexpression of miR-139-5p inhibited NSCLC cell proliferation, colony formation and chemotherapy resistance and increased cell apoptosis. Additionally, AFAP1-AS1 upregulates RRM2 expression via sponging miR-139-5p. Furthermore, AFAP1-AS1 enhanced NSCLC cell proliferation and chemotherapy resistance through upregulation of RRM2 by inhibiting miR-139-5p expression. Moreover, RRM2 promoted cellular chemotherapy resistance by activating EGFR/AKT. Finally, knockdown of AFAP1-AS1 significantly suppressed tumor growth and chemoresistance in nude mice. In conclusion, AFAP1-AS1 promoted chemotherapy resistance by supressing miR-139-5p expression and promoting RRM2/EGFR/AKT signaling pathway in NSCLC cells.


INTRODUCTION
Lung cancer is the leading cause of cancer-related death worldwide (1)(2)(3). Non-small cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancer cases and comprises two histological subtypes, adenocarcinoma (AD) and squamous cell cancer (SCC) (4,5). The current overall 5-year survival rate for NSCLC is <15% due to both limited therapeutic options and recurrence (4). The prognosis of NSCLC is affected by chemotherapy resistance (6,7). Thus, a better understanding of carcinogenesis and chemotherapy resistance is critical for developing novel therapies to treat NSCLC patients.
Ribonucleoside-diphosphate reductase subunit M2 (RRM2) is the catalytic subunit of ribonucleotide reductase and modulates the enzymatic activity, which is essential for DNA replication and repair (24). RRM2 has been reported to be involved in the progression of various cancers, including gliomas (25), colorectal cancer (26), bladder cancer (27) and NSCLC (28)(29)(30)(31). In addition, RRM2 is a prognostic biomarker for NSCLC (28)(29)(30)(31). Interestingly, AKT-induced tamoxifen resistance is reversed by RRM2 inhibition in breast cancer (32), suggesting that RRM2 may participate in the chemotherapy resistance of cancer cells. The abnormal overexpression or activation of AKT has been observed in cancers including lung, ovarian and pancreatic cancers (33), and AKT could be activated by epidermal growth factor receptor (EGFR) (34), implying that targeting EGFR or AKT could offer important approaches for cancer prevention and therapy. Subsequently, we investigated the effect of RRM2 on EGFR/AKT signaling.
In this study, we investigate the role of AFAP1-AS1 in NSCLC cell proliferation and chemotherapy resistance to DDP (Cisplatin) and 5-FU (fluorouracil), which are commonly used for countering progression of cancers in clinic. We also explore the function of RRM2 in the chemotherapy resistance of NSCLC cells. Our data indicate that AFAP1-AS1 expression was elevated in patients with NSCLC and that AFAP1-AS1 acts as a competing endogenous RNA for miR-139-5p, which is an important suppressor in several tumors (35)(36)(37)(38)(39). Knockdown of AFAP1-AS1 or overexpression of miR-139-5p inhibited the proliferation, increased the apoptosis, and attenuated the chemotherapy resistance of lung cancer cells by upregulating RRM2. In addition, knockdown of AFAP1-AS1 reduced tumor volume and weight in vivo. Taken together, AFAP1-AS1 supresses miR-139-5p and promotes cell proliferation and chemotherapy resistance of NSCLC cells by competitively upregulating RRM2 expression.

Tissue Collection
This study was approved by the ethics committee of first affiliated hospital of Chengdu Medical College. From Feb. 2018 to Apr. 2019, a total of 44 NSCLC patients were recruited from Department of Respiratory Medicine, the First Affiliated Hospital of Chengdu Medical College Chengdu. All participants signed an informed consent form. NSCLC tissues and adjacent normal lung tissues were collected and stored at −80 • C until used. The drugs cisplatin (DDP), 5-fluorouracil (5-FU), adriamycin, and paclitaxel were used for NSCLC treatment in all patients. In accordance with the Response Evaluation Criteria in Solid Tumors, we grouped the patients with a complete or partial response as responders and defined those with stable or progressive disease as non-responders. The clinicopathological characteristics of the patients with NSCLC are summarized in Table 1.

Cell Culture
The NSCLC cell lines H1975, PC-9, A549, and SPCA-1, and a human normal lung epithelial cell line BEAS-2B were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The H1975 and SPCA-1 cells were maintained in RPMI 1640 basic medium (GIBCO, Carlsbad, CA), and the PC-9 and A549 cells were cultured in DMEM (GIBCO) in a humidified incubator at 37 • C with 5% CO 2 . All media were supplemented with heatinactivated 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) (GIBCO).

Luciferase Reporter Assay
The luciferase reporter vector pGLO-basic (Promega, Beijing, China) containing the wild-type (WT) or mutant AFAP1-AS1 sequences 1/2 (Mut 1/2) were transfected into A549 cells. The pGLO plasmids containing the full-length RRM2 3 ′ UTR or its corresponding mutant were co-transfected with an miR-139-5p mimic or inhibitor into A549 cells. After 48 h of incubation, the cells were harvested and luciferase activity was determined using a dual luciferase assay kit (Promega).

Colony Formation Assay
NSCLC cells were seeded in fresh six-well plates in a 5% CO 2 incubator at 37 • C and were then transfected with the indicated siRNAs. Following incubation for 2 weeks, NSCLC cells would grow into colonies, and they were fixed with methanol and stained with 0.1% crystal violet. Visible colonies were manually counted and recorded.

Apoptosis Analyses
Treated cells were collected, centrifuged at 2,000 rpm for 5 min and washed with PBS three times. The cells were resuspended in 100 µL of PBS, and annexin V/FITC (5 µL) and propidium iodide (PI) (1 µL) were added to each sample. After 15 min incubation at room temperature in the dark, the apoptosis of the cancer cells was analyzed on an S3e flow cytometer (Bio-Rad, Shanghai, China). Cells stained with either annexin V or PI were counted as apoptotic cells. Caspase-3 activity was also checked in cancer cells by Caspase-3 Activity Assay Kit (Beyotime, Shanghai, China) followed the instruction.

RNA Immunoprecipitation (RIP) Assays
Rip experiments were performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer's instructions. Antibodies against EZH2 and AGO-2 were obtained from Sigma. The AGO2 expression level was determined by immunoprecipitation and Western blotting, and the AFAP1-AS1 expression level was determined by real-time PCR.

Generation of Drug-Resistant Cell Lines
The DDP-or 5-FU-resistant cell lines were generated by incubating NSCLC cells with increasing concentration of the indicated drugs. NSCLC cells were plated into plates and maintained in medium containing 0.2 µM DDP. After 48 h incubation, the 0.2 µM DDP-containing medium was discarded, and medium containing gradually increasing concentrations of DDP was added. Finally, cells resistant to 10 µM DDP were obtained and named A549/DDP or SPCA-1/DDP cells.

Pull Down Assay With Biotinylated AFAP1-AS1 DNA Probe
The biotin-labeled ABHD11-AS1 DNA probe was designed (Thermo), dissolved in binding and washing buffer and mixed with M-280 streptavidin magnetic beads (Thermo) to generate probe-coated beads according to the manufacturer's instruction. The A549 cell lysates were incubated with the probe-coated beads. Then, we used real-time PCR analysis to determine the beads-binding RNAs. The AFAP1-AS1 pull-down probe sequence was 5 ′ -Bio-AGT AAA CAC GCA GTT GCA CAT GGC TGG GGA GGC CTC AGA ATC ATG GCG GGA GGC GAA AGA CAC TTC TTA CGT GGC AGC AGC-3 ′ ; and random pulldown probe sequence used as negative control was 5 ′ -Bio-TGC ATC CAA GCC GAT TGC GGT AAC GTG CAT CCA AGC CGA TTG CGG TAA CG-3 ′ .

Xenograft Tumor Assay
Male athymic nude BALB/c mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). At 5 weeks of age, the mice were randomly divided into four groups. The animal procedure was approved by the Ethics Committee of Animal Experiments of Chengdu Medical College (Chengdu, China). A549 cells were transfected with Lvscramble or Lv-AFAP1-AS1 KD. 2 × 10 6 cells were inoculated subcutaneously into the mice. After 10 days, the mice were administered 3 mg/kg (body weight) DDP or PBS every 4 days for 28 days. During this period, the tumor lengths and widths were measured, and tumor volumes were calculated as follows: tumor volume = (length × width 2 )/2. Finally, the tumors were harvested and weighed.

Bioinformatic Analyses
In this study, LNCipedia version 5.2, lncBase version 2, and starBase were used to predict the potential binding sites between AFAP1-AS1 and miR-139-5p. miRBase and miRDB were applied to analyse the binding sites between miR-139-5p and the RRM2 3 ′ UTR. In addition, we predicted the RNA-binding activity by examining previous studies (40).

Statistical Analysis
Statistical analyses were performed using SPSS software (version 19.0). The data are expressed as means ± standard deviations (S.D.). A two-tailed Student's t-test was used to analyse difference between two groups. For multi-group comparisons, we used one-way analysis of variance (ANOVA) with a post-hoc Tukey's honestly significant difference (HSD) test. P-value of < 0.05 were considered statistically significant.

AFAP1-AS1 Is Overexpressed in NSCLC Tissues and Cells
Firstly, NSCLC tissues and adjacent tissues were collected from hospital. The tissues were analyzed by H&E staining, and the results showed that abnormal cell over-growth appeared in tumors ( Figure 1A). RT-PCR was performed to determine the expression of AFAP1-AS1 in NSCLC tissues and cells. It was found that the expression of AFAP1-AS1 was significantly higher in NSCLC tissues than in normal tissues ( Figure 1B). In addition, AFAP1-AS1 was overexpressed in NSCLC tissues of patients in the chemotherapy non-response group compared to the chemotherapy response group (Figure 1C). Moreover, Additionally, the expression of AFAP1-AS1 was higher in NSCLC cells than in BEAS-2B cells and was highest in SPCA-1 cells and lowest in H1975 cells ( Figure 1D).

Knockdown of AFAP1-AS1 or Overexpression of miR-139-5p Decreases the Chemotherapy Resistance of NSCLC Cells
To analyse the effect of AFAP1-AS1 and miR-139-5p on the chemoresistance of NSCLC cells, scramble-or siAFAP1-AS1-transfected A549 and SPCA-1 cells were incubated with DDP or 5-FU. It was found that the drug-induced growth inhibition increased in a dose-dependent manner, and AFAP1-AS1 knockdown increased the inhibitory activity of DDP and 5-FU in NSCLC cells (Figures 4A-D), implying that suppression of AFAP1-AS1 alleviated the chemotherapy resistance of NSCLC cells. In addition, we also observed that AFAP1-AS1 was significantly overexpressed in DDP-resistant A549 and SPCA-1 cells compared with canonical A549 and SPCA-1 cells (Figure 4E). Furthermore, interfering with AFAP1-AS1 expression significantly increased the DDP-induced apoptosis in the drug-resistant cancer cells (Figures 4F,G). Similarly, AFAP1-AS1 was obviously increased in 5-FU-resistant A549 and SPCA-1 cells, and knockdown of AFAP1-AS1 also promoted 5-FUtriggered cell apoptosis (Figures 4H-K).

Cooperation of miR-139-5p and AFAP1-AS1 Regulates RRM2 Expression by Targeting Its 3 ′ UTR
To investigate the interaction between miR-139-5p and RRM2, a luciferase reporter gene assay was performed. The binding sites between miR-139-5p and the RRM2 3 ′ UTR were predicted by bioinformatics (Figure 6A). The RRM2 3 ′ UTR sequences were sub-cloned into the pGLO plasmid, and A549 and SPCA-1 cells were co-transfected with the RRM2 3 ′ UTR plasmid and the miR-139-5p mimic or inhibitor. The results showed that the miR-139-5p mimic or inhibitor significantly decreased or increased, respectively, the luciferase activity driven by RRM2 WT; however, the miR-139-5p mimic or inhibitor did not affect the luciferase activity driven by the mutated RRM2 3 ′ UTR (termed RRM2 MUT) (Figures 6B,C). The miR-139-5p mimic or inhibitor noticeably decreased or increased RRM2 mRNA expression ( Figure 6D) and protein expression (Figures 6E,F), respectively. Interestingly, the miR-139-5p mimic or inhibitor also modulated the protein levels of EGFR and p-AKT (Figures 6E,F). RRM2 was found to be overexpressed in NSCLC tissues ( Figure 6F) and multidrug resistant NSCLC cells (Figures 6G,H). RRM2 was also found has higher mRNA level in NSCLC cells than normal.

DISCUSSION
LncRNAs are involved in many aspects of cancer development and chemotherapy resistance (8)(9)(10)(11). To investigate whether there is an abnormal expression of lncRNA AFAP1-AS1 in NSCLC tissues and cancer cells, real-time PCR was performed.
We assessed the effect of AFAP1-AS1 on the proliferation, apoptosis and chemotherapy resistance of lung cancer cells. AFAP1-AS1 could perform as a sponge of miR-139-5p in cancer progression. Suppression of AFAP1-AS1 or overexpression of miR-139-5p significantly repressed the proliferation, increased the apoptosis, and ameliorated the chemotherapy resistance of NSCLC cells by downregulating RRM2. Furthermore, downregulation of AFAP1-AS1 decreased xenograft tumor volume and weight. These findings suggested that AFAP1-AS1 could be an oncogene and induce chemotherapy resistance by modulating miR-139-5p/RRM2 signaling in NSCLC.
Accumulating evidence demonstrated that dysregulated lncRNAs are major contributors to tumourigenesis and cancer development. For example, HOTAIR plays an important role in cellular proliferation, invasion, and clinical relapse in small cell lung cancer (41). HOTAIR also mediates chemoresistance in NSCLC by regulating HOXA1 methylation and could be a potential target for new adjuvant therapy against chemoresistance (42). In addition, the p53-regulated lncRNA TUG1 affects NSCLC cell proliferation in part by epigenetically controlling HOXB7 expression (43). MEG3 acts as a tumor suppressor in NSCLC cell proliferation and induces p53-mediated cancer cell apoptosis (15). Moreover, downregulation of AFAP1-AS1 results in growth inhibition and apoptosis promotion in lung adenocarcinoma cells, indicating that this lncRNA participates in tumourigenesis (44). In NSCLC, AFAP1-AS1 increases tumourigenesis by epigenetically repressing p21 expression. AFAP1-AS1 recruits EZH2 to the p21 promoter region, resulting in downregulation of p21, which is a tumor suppressor (22,23). Consistent with these previous findings, we found that AFAP1-AS1 was upregulated in NSCLC tissues and cells, and it was overexpressed in chemotherapy-resistant tissues, indicating that AFAP1-AS1 is a positive regulator of NSCLC development and chemoresistance. Previous studies have shown that lncRNAs can act as competing endogenous RNAs of miRNAs (45). Thus, in the present study, we predicted binding sites between AFAP1-AS1 and miR-139-5p, which is a tumor suppressor in colorectal cancer and endometrial cancer (38,46). However, the role of miR-139-5p in NSCLC has not been explored. Luciferase reporter assays, RIP assays, and real-time PCR were performed, and the data showed that AFAP1-AS1, as a sponge, directly bound to miR-139-5p, leading to downregulation of miR-139-5p expression, and that miR-139-5p expression was Knockdown of AFAP1-AS1 downregulated RRM2 protein expression and reduced the protein levels of EGFR and p-AKT, while miR-139-5p reversed these effects in A549, and SPCA-1 cells. All data shown as means ± S.D. # P < 0.05 indicates a significant difference between the two indicated groups. decreased in chemotherapy-resistant tissues. We also found that the miR-139-5p inhibitor reversed AFAP1-AS1-induced biological effects, indicating that the interaction of AFAP1-AS1 and miR-139-5p is involved in NSCLC progression and chemotherapy resistance.
In addition, we found that AFAP1-AS1 participated in positively modulating luciferase activity of RRM2 3 ′ UTR and RRM2 level by acting as a sponge of miR-139-5p in cancer cells, suggesting that RRM2, as AFAP1-AS1, is a oncogenic regulator. This finding is in consistent with previous reports that RRM2 is an oncogene in certain cancers (25)(26)(27)(28)(29)(30)(31)(32). It was reported that silencing RRM2 suppresses glioblastoma cell invasion and migration by reducing the expression of metalloproteinase-2 (MMP-2) and MMP-9 (25). The abnormal overexpression or activation of AKT has been observed in many cancers, including lung, ovarian, and pancreatic cancers, and is associated with increased cancer cell proliferation and survival (33). AKT could be activated by epidermal growth factor receptor (EGFR) (34). Consequently, targeting EGFR or AKT could offer important approaches for cancer prevention and therapy. More importantly, overexpression of RRM2 in gastric cancer cells promotes their invasiveness by regulating the AKT/NF-κB signaling pathway (47), and RRM2 increases tumor angiogenesis and growth by modulating the expression of thrombospondin-1 (TSP-1) and vascular endothelial growth factor (VEGF) (48). Subsequently, we investigated the effect of AFAP1-AS1/miR-139-5p/RRM2 signaling on EGFR expression and phosphorylation of AKT. Expectedly, our data showed that overexpression of RRM2 promoted the proliferation, inhibited the apoptosis, and increased the chemotherapy resistance of NSCLC cells through upregulating EGFR expression and AKT phosphorylation. The EGFR inhibitor AST1306 reversed the RRM2-induced effects on cancer cells, indicating that the function of RRM2 is associated with EGFR/AKT signaling pathway. These findings suggested that miR-139-5p inhibited the proliferation and promoted the apoptosis of NSCLC cells by upregulating RRM2/EGFR/AKT signaling pathway. The mutation of EGFR was thought main driver in NSCLC (49,50), our data shown the mutation of EGFR in H1975 (L858R+T790M) and PC-9 (del19) has little affect on AFAP1-AS1. The underlying mechanism needs more research to lighten.
Taken together, our study demonstrates that AFAP1-AS1 expression is upregulated and miR-139-5p expression downregulated in NSCLC tissues and cells. AFAP1-AS1 promotes NSCLC development and increased chemotherapy resistance by modulating miR-139-5p/RRM2/EGFR/AKT pathway. Suppression of AFAP1-AS1 expression reduced tumor growth and attenuated chemotherapy resistance in vivo. Therefore, AFAP1-AS1 could be a promising and therapeutic target of NSCLC.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the manuscript/supplementary files.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the ethics committee of first affiliated hospital of Chengdu Medical College. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee of Animal Experiments of Chengdu Medical College.