miR-498 promotes the tumorigenesis of non-small cell lung cancer

Biochemical and Biophysical Research Communications 519 (2019) 518e524

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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

The positive feedback loop FOXO3/CASC11/miR-498 promotes the tumorigenesis of non-small cell lung cancer Ruicheng Yan*, Ying Jiang, Bingyu Lai, Yaqiong Lin, Jingwei Wen Department of Gastrointestinal Surgery East Section, Renmin Hospital of Wuhan University, Wuhan, 430205, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 August 2019 Accepted 24 August 2019 Available online 16 September 2019

An increasing number of studies have indicated that long noncoding RNAs (lncRNAs) are involved in the regulation of non-small-cell lung cancer (NSCLC). Nevertheless, there are still numerous undiscovered mechanisms underlying this molecular regulation. Here, the results illustrated that CASC11 is overexpressed in NSCLC tumor tissues and cell lines, which is closely related to the clinical features of NSCLC and poor survival. In functional experiments, CASC11 was shown to promote proliferation and cycle progression and enhance NSCLC tumorigenesis. In mechanical investigations, CASC11 was shown to target the miR-498/FOXO3 axis via a canonical competing endogenous RNA (ceRNA). In return, the transcription factor FOXO3 targets the CASC11 promoter region, thereby accelerating its transcription. Our findings demonstrate a crucial role for CASC11 as an oncogene in promoting NSCLC. These results reveal that CASC11 might be a potential therapeutic target for NSCLC. © 2019 Published by Elsevier Inc.

Keywords: Non-small-cell lung cancer CASC11 miR-498 FOXO3

1. Introduction Lung cancer is one of the most common cancers and acts as the leading cause of cancer-related mortality worldwide [1]. Among lung cancers, non-small-cell lung cancer (NSCLC) accounts for approximately 80% of all newly reported lung cancer cases [2]. There are several pathological grades for NSCLC, including squamous cell carcinoma, adenocarcinoma and large-cell carcinoma. The development of therapeutic techniques, including surgical therapy, chemotherapy and molecular targeting, has led to an increasing success rate for treating NSCLC. However, the overall 5year survival rate for patients remains as low as 15%. Therefore, a better understanding of the pathogenetic mechanisms of NSCLC progression and metastasis is essential. Long noncoding RNAs (lncRNAs), a subgroup of RNA transcripts longer than 200 nucleotides, are critical regulators of human cancers [3e5]. LncRNAs are mainly located in the cell nucleus or cytoplasm, where they are involved in transcriptional regulation or posttranscriptional regulation [6,7]. Convincing evidence suggests that lncRNAs could interact with their targets to exert regulatory

* Corresponding author. Department of Gastrointestinal Surgery East Section, Renmin Hospital of Wuhan University, Gaoxin Sixth Road, No 17, East Lake Hightech Development Zone, Wuhan, 430205, China. E-mail address: [email protected] (R. Yan). https://doi.org/10.1016/j.bbrc.2019.08.136 0006-291X/© 2019 Published by Elsevier Inc.

roles. For example, lncRNA FEZF1-AS1 is upregulated in NSCLC tissues, and higher lncRNA FEZF1-AS1 expression is associated with lymph node metastasis [8]. DLX6-AS1 is overexpressed in NSCLC tumor tissues and cell lines and promotes the proliferation, invasion, and migration of non-small cell lung cancer cells by targeting the miR-27b-3p/GSPT1 axis [9]. LncRNA CASC11 has been found to be overexpressed in several human cancer tissues and functions as an oncogene [10,11]. In the present study, we noticed that lncRNA CASC11 could target the miR-498/FOXO3 axis to promote NSCLC tumorigenesis. Our findings demonstrate a crucial role for CASC11 as an oncogene in promoting NSCLC. These results reveal that CASC11 might be a potential therapeutic target for NSCLC.

2. Materials and methods 2.1. Tissue sample collection This research was approved by the Institutional Research Ethics Committee of Renmin Hospital of Wuhan University. Before surgery, written informed consent was obtained from each patient. Fresh NSCLC samples and paired adjacent nontumor tissues were obtained from volunteers. Follow-up visits were performed and recorded every month after surgery.

R. Yan et al. / Biochemical and Biophysical Research Communications 519 (2019) 518e524

2.2. Cell culture and cell transformation Normal bronchial epithelial cells (NHBE) and NSCLC cell lines (A549, H460, H1299, H322) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin solution (Gibco, NY, USA) at 37
C in a humidified 5% CO2 atmosphere. 2.3. Transfection of stable cell lines Short hairpin RNAs (shRNAs) against CASC11 (sh-CASC11) and miRNA mimics were synthesized by RiboBio (Guangzhou, China) and subcloned into pLKO.1 plasmids. The miRNA mimics and shRNAs were transfected into cells by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Fortyeight hours later, the transfected cells were harvested for further studies. The target sequence and scramble shRNA for shCASC11 are shown in supplementary Table S1. 2.4. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis The mRNA and mimic levels were determined by qRT-PCR. Total RNA was extracted with TRIzol RNA extraction reagent (Gibco, USA). Five micrograms of total RNA was reverse transcribed into the first strand cDNA, and qRT-PCR analysis was performed (SsoFast TM EvaGreen Supermix, BIO-RAD, USA). GAPDH was used as a qualitative control to normalize gene expression. Data were analyzed using the formula R ¼ 2-[DCt sampleDCt control]. All of the primers used for the RT-PCR are shown in Table S1. 2.5. Western blot analysis Total protein from the tissues and cells was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer with phenylmethanesulfonyl fluoride. The protein samples were centrifuged at 4
C with incubation on ice for 30 min. The protein concentration in each sample was measured by a bicinchoninic acid (BCA) kit with deionized water. Protein extraction was transferred onto 10% SDSPAGE (P0012A, Beyotime, Shanghai, China) and then to a polyvinylidene fluoride (PVDF) membrane (ISEQ00010, Millipore, Billerica, MA, USA), which was treated with Tris-buffered saline tween (TBST) and skim milk for 2 h. Subsequently, the PVDF membranes were treated with the primary antibody rabbit anti-FOXO1 (Abcam, 1:1000). The membranes were imaged with enhanced chemiluminescence (ECL) reaction solution (WBKLS0100, Millipore, Billerica, MA, USA). 2.6. CCK-8 proliferation assay Cell Counting Kit-8 (CCK-8) assays were used to detect cell proliferation as previously described [12]. In brief, A549 and H1299 cells (3  103 cells per well) were seeded in a 96-well plate and then incubated with CCK-8 solution in each well. The supernatant was then removed, and the optical density (OD) was measured at 450 nm.

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overnight and washed with PBS. For apoptosis, cells were washed twice with cold PBS and resuspended in 100 ml binding buffer, 5 ml FITC Annexin V and 5 ml propidium iodide (PI) for 15 min at room temperature in the dark. For the cycle analysis, cells were stained with 0.5 ml propidium iodide (PI) Staining Buffer (BD Biosciences, San Jose, CA, USA) and incubated at room temperature for 15 min. Data were analyzed using CELLQUEST software (BD Biosciences). 2.8. Subcellular fractionation location The nuclear and cytosolic fractions of A549 cells and H1299 cells were extracted using a PARIS kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's manual. 2.9. Luciferase reporter assay Wild-type (WT) and mutant (Mut) sequences, including the CASC11 promoter and the 30 -UTR of FOXO3 or CASC11, were synthesized and inserted into the luciferase reporter vector pGL3-basic (Promega). 293T cells were cotransfected with miR-498 mimics or FOXO3 antibody, as well as the corresponding controls, using Lipofectamine2000 (Invitrogen, California, USA), according to the manufacturer's instructions. Cells were collected 48 h later, and the firefly and Renilla luciferase activities were measured with a DualLuciferase Reporter Assay System (Promega). 2.10. Chromatin immunoprecipitation (ChIP) assay A ChIP assay was performed using an EZ ChIP Chromatin Immunoprecipitation Kit (Millipore, Bedford, MA, USA). In brief, the extracted chromatin was sonicated into 200- to 1000-bp fragments to relieve crosslinking. Anti-FOXO3 antibody (Abcam, USA) was incubated with these fractionations to precipitate the DNA-protein complex. Normal immunoglobulin G (IgG) acted as the negative control. Derived DNA was quantified using qRT-PCR. 2.11. In vivo tumor xenograft experiments All in vivo studies involving mice had been approved by the Ethics Committee of Renmin Hospital of Wuhan University. BALB/c nude mice were raised in a specific pathogen-free (SPF) environment and then subcutaneously injected with 5  106 A549 cells with CASC11 knockdown on the right flank. Tumor volumes were monitored every six days and calculated as 0.5  (short diameter2  longer diameter). Mice were sacrificed to extract the xenograft tumor tissues after 4 weeks. 2.12. Statistical analysis All quantitative data were presented as the mean ± SEM and calculated with SPSS 21.0 (IBM, Armonk, NY, USA) and GraphPad Prism 6.0 software (GraphPad Software, Inc, San Diego, CA, USA). Differences within two groups were compared by t-test, and differences among multiple groups were compared by ANOVA. All experimental data were conducted three times. 3. Results

2.7. Flow cytometry

3.1. CASC11 overexpression predicted the poor outcome of NSCLC patients

NSCLC cells were incubated with a FITC-conjugated specific antibody and analyzed by a Becton Dickinson FACScan flow cytometer (Becton Dickinson, USA). Viable cells (1  104) were analyzed per condition. Cells were collected and fixed using 70% ethanol at 4
C

NSCLC patients were enrolled in this clinical investigation for the tissue specimen analysis. RT-PCR showed that CASC11 expression was upregulated in the NSCLC tissue groups compared to the adjacent controls (Fig. 1A, Table 1). In terms of advanced

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pathological grading (III, IV level), the CASC11 level was higher than that of primary pathological grading (I, II level) (Fig. 1B). As expected, in the TCGA database, the CASC11 level was higher in the lung cancer tissue compared to a healthy group cohort (Fig. 1C). Using the Kaplan-Meier Plotter (http://kmplot.com/analysis), we found that a higher CASC11 expression was closely correlated with a lower survival rate of NSCLC patients, indicating that CASC11 expression was a risk factor for NSCLC (Fig. 1D). In our collected NSCLC patients, the Kaplan-Meier test for survival analysis determined that NSCLC individuals with higher CASC11 expression had a lower survival rate (Fig. 1E). Overall, CASC11 overexpression predicted poor outcomes in NSCLC patients. 3.2. CASC11 motivated NSCLC tumorigenesis in vivo and in vitro LncRNA CASC11 levels were overexpressed in NSCLC cells compared with the normal cells (Fig. 2A). To identify whether CASC11 could motivate NSCLC tumorigenesis, we performed silencing short hairpin RNA (shRNA) transfection into A549 cells and overexpression transfection into H1299 cells (Fig. 2B). Cycle analysis by flow cytometry illustrated that CASC11 silencing induced G0/G1 phase arrest in A549 cells and that CASC11 overexpression accelerated cellular cycle progression in H1299 cells (Fig. 2C and D). Apoptotic analysis by flow cytometry illustrated that CASC11 silencing promoted the apoptotic rate in A549 cells and that CASC11 overexpression accelerated the apoptotic rate in H1299 cells (Fig. 2E and F). The CCK-8 assay indicated that CASC11

Table 1 Correlation within CASC11 level and clinicopathological feature of NSCLC patients. Total

Gender Male Female Age (years) 60 <60 Tumor size 4 cm <4 cm Differentiation well, moderate poor TNM I-II III/IV Lymph metastasis No Yes

CASC11

p value

L(17)

H(23)

23 17

10 7

13 10

20 20

9 8

11 12

18 22

10 7

8 15

15 25

9 8

6 17

18 22

9 8

9 14

17 23

5 12

12 11

0.485

0.588

0.176

0.003*

0.025*

0.317

*P < 0.05 represents statistical difference. Low, L. High, H.

silencing repressed proliferation in A549 cells and that CASC11 overexpression promoted proliferation in H1299 cells (Fig. 2G and H). An in vivo xenograft assay illustrated that CASC11 silencing decreased tumor growth in A549 cells (Fig. 2I and J). In conclusion, CASC11 motivated NSCLC tumorigenesis in vivo and in vitro.

Fig. 1. CASC11 overexpression predicted the poor outcome of NSCLC patients. (A) RT-PCR shows upregulated CASC11 expression in NSCLC tissue groups compared to the adjacent controls. (B) CASC11 expression in advanced pathological grading (III, IV level) and primary pathological grading (I, II level). (C) In the TCGA database, the level of CASC11 was higher in the lung cancer tissue than in the healthy group cohort. (D) Using the Kaplan-Meier Plotter (http://kmplot.com/analysis), a higher CASC11 expression was closely correlated with a lower survival rate for NSCLC patients. (E) The Kaplan-Meier test for survival analysis indicated the NSCLC individuals with higher/lower CASC11 expressions among a group of NSCLC patients. **p < 0.01 vs controls. *p < 0.05 vs controls.

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Fig. 2. CASC11 motivated NSCLC tumorigenesis in vivo and in vitro. (A) The lncRNA CASC11 level was measured using RT-PCR in NSCLC cells. (B) Silencing short hairpin RNA (shRNA) transfection into A549 cells and overexpression transfection into H1299 cells. (C, D) Cycle analysis by flow cytometry illustrates G0/G1 phase arrest in A549 cells and cellular cycle progression in H1299 cells. (E, F) Apoptotic analysis by flow cytometry illustrates the apoptotic rate in A549 cells and H1299 cells. (G, H) CCK-8 assay indicates proliferation in A549 cells and H1299 cells. (I, J) In vivo xenograft assay illustrated tumor growth using A549 cells transfected with CASC11 silencing. **p < 0.01 vs controls. *p < 0.05 vs controls.

3.3. CASC11 targeted the miR-498/FOXO3 axis in NSCLC cells The location of lncRNAs determined their biological roles in NSCLC cells. In the A549 cells and H1299 cells, CASC11 was

primarily located in the cytoplasm rather than the nucleus (Fig. 3A). The potential binding sites within CASC11 and miR-498 were estimated by bioinformatics tools (Starbase, http://starbase.sysu. edu.cn/) and identified by the luciferase reporter assay for

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Fig. 3. CASC11 targeted the miR-498/FOXO3 axis in NSCLC cells. (A) In the A549 cells and H1299 cells, the location of CASC11 in the cytoplasm or nucleus was determined by subcellular fractionation analysis. (B) The potential binding sites within CASC11 and miR-498 were estimated by bioinformatics tools (Starbase, http://starbase.sysu.edu.cn/) and identified by the luciferase reporter assay. (C) miR-498 levels were detected in A549 cells and H1299 cells. (D) miR-498 levels were detected in A549 cells and H1299 cells transfected with CASC11 shRNA or CASC11 overexpression. (E) The potential binding sites within FOXO3 and miR-498 were estimated by bioinformatics tools (TargetScan, http:// www.targetscan.org/) and identified by the luciferase reporter assay. (F) FOXO3 mRNA abundance was determined by RT-PCR following transfection with CASC11 shRNA and CASC11 overexpression. (G) FOXO3 mRNA was determined by RT-PCR following transfection with miR-498 mimics. **p < 0.01 vs controls.

molecular interaction (Fig. 3B). In the A549 cells and H1299 cells, the level of miR-498 was decreased (Fig. 3C). The transfection of CASC11 shRNA upregulated miR-498, and CASC11 overexpression reduced the level of miR-498 (Fig. 3D). The potential binding sites within FOXO3 and miR-498 were estimated by the bioinformatics tools (TargetScan, http://www.targetscan.org/) and identified by the luciferase reporter assay for molecular interaction (Fig. 3E). The transfection of CASC11 shRNA downregulated FOXO3 mRNA, and CASC11 overexpression increased the level of FOXO3 mRNA (Fig. 3F). In A549 cells, miR-498 mimic transfection remarkedly silenced FOXO3 mRNA (Fig. 3G). Overall, we concluded that CASC11 targeted the miR-498/FOXO3 axis in NSCLC cells.

target the promoter region of CASC11 and possibly promote its transcriptional activity (Fig. 4A). Using chromatin immunoprecipitation (ChIP), the results revealed that anti-FOXO3 could interact with the E1 elements of the CASC11 promoter region (Fig. 4B). RTPCR illustrated that CASC11 levels were upregulated in A549 cells when transfected with FOXO3-overexpression plasmids (Fig. 4C). Wild-type and mutant sequences of the CASC11 promoter were constructed for the luciferase reporter assay, illustrating the closely related interaction of the transcription factor FOXO3 and the wildtype sequences of the CASC11 promoter (Fig. 4D and E). Pearson's c2 analysis showed that FOXO3 was positively correlated with CASC11 in NSCLC patients (Fig. 4F). In conclusion, FOXO3 activated the transcription of lncRNA CASC11.

3.4. FOXO3 activated the transcription of lncRNA CASC11 4. Discussion Our finding revealed that CASC11 could target the miR-498/ FOXO3 axis in NSCLC cells. In further research, we found an interesting phenomenon in which the transcription factor FOXO3 could

Given the critical roles of lncRNAs in non-small-cell lung cancer as indicated by increasing evidence, more in-depth studies about

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Fig. 4. FOXO3 activated the transcription of lncRNA CASC11. (A) JASPAR (http://jaspar.genereg.net/) showed that the transcription factor FOXO3 targets the promoter region of CASC11. (B) Chromatin immunoprecipitation (ChIP) reveals the interaction of anti-FOXO3 with E1 elements of the CASC11 promoter region. (C) RT-PCR illustrates the level of CASC11 in A549 cells transfected with FOXO3 overexpression plasmids. (D) Wild-type and mutant sequences were constructed. (E) Luciferase reporter assay illustrates the luciferase activity of FOXO3 targeting the CASC11 promoter. (F) Correlation within FOXO3 and CASC11 was examined by Pearson's c2 analysis in the NSCLC patients. **p < 0.01 vs controls.

lncRNA and NSCLC are required [13e15]. CASC11 has been identified as an oncogene in multiple human cancers; however, the role of CASC11 in NSCLC is still elusive [16,17]. In the field of epigenetic regulation, the ncRNA family exerts its various regulatory functions in transcriptional level regulation and posttranscriptional level regulation [3,18]. For example, LINC00665 markedly reduces the activation of the EGFR and AKT downstream element (PI3K/AKT) pathway in NSCLC, and LINC00665 can interact with EZH2 and regulate this pathway [19]. LncRNAs PVT1 and CARM1 are coexpressed to regulate NSCLC radiosensitivity, and lncRNAs PVT1/CARM1 could combine with miR-424-5p to modulate MMP-2, MMP-9, and Bcl-2 [20]. LncRNA DUXAP9-206 is overexpressed in NSCLC cells and is closely related to NSCLC clinical features, and DUXAP9-206 induces NSCLC cell proliferation and metastasis by interacting with the E3 ubiquitin ligase Cbl-b [21]. In this study, we found that lncRNA CASC11 was significantly upregulated in NSCLC cells and tissue, and the increased CASC11 was closely correlated with a poor prognosis. In the functional experiments of gain or loss function, the results showed that CASC11 could promote cycle arrest and reduce apoptosis. The findings derived from in vitro and in vivo assays suggest the oncogenic roles of CASC11 in NSCLC; in short, the results indicate that CASC11 promotes cancer. CASC11 exerts similar regulations in other cancers. For instance, CASC11 is activated by the transcription factor STAT3 and epigenetically silences PTEN by binding with zeste homolog 2 (EZH2) in hepatocellular carcinoma [22]. In osteosarcoma, CASC11 is correlated with clinical stage and poor prognosis, and CASC11 promotes migration, invasion, epithelial-mesenchymal transition and metastasis of OS cells in vitro and in vivo. CASC11 is associated with the EMT inducer Snail mRNA and increases its stability [23]. We found that lncRNA CASC11 could target miR-498 in the cytoplasm, and then miR-498 could target the 30 -UTR of Forkhead box O3 (FOXO3) mRNA. This evidence suggests the importance of the CASC11/miR-498/FOXO3 axis in NSCLC. This regulation is described as a competing endogenous RNA (ceRNA) regulation in human cancer. FOXO3 functions as a vital transcription factor in

cancer oncogenesis. Further investigations revealed that FOXO3 could target the promoter region of CASC11, increasing its transcription level. In conclusion, these data conclude the regulation of FOXO3/CASC11/miR-498/FOXO3 axis in the NSCLC. In the NSCLC, FOXO3 was found to be involved in the resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) [24]. FOXO3 could promote proliferation and doxorubicin resistance in colon cancer cells via direct binding to the promoter of MDR1, enhancing MDR1 expression [25]. Based on these data, we confirmed that FOXO3 could accelerate NSCLC progression via transcriptional regulation. In conclusion, the data derived from our research point to a crucial role of CASC11 as an oncogene in promoting NSCLC. The positive feedback loop of the FOXO3/CASC11/miR-498 axis promotes NSCLC tumorigenesis. These results reveal that CASC11 might be a potential therapeutic target for NSCLC. Conflicts of interest All authors declare no conflicts of interest. Funding No. Acknowledgement No. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.136. References [1] H.R. Fernandez, S.M. Gadre, M. Tan, G.T. Graham, A. Cheema, R. Varghese, H.W. Ressom, Y. Zhang, C. Albanese, A. Uren, M. Paige, G. Giaccone,

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