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Gene 720 (2019) 144099
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Research paper
Circular RNA circ-PRMT5 facilitates non-small cell lung cancer proliferation through upregulating EZH2 via sponging miR-377/382/498
T
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Yuan Wanga, , Ying Lib, Hangyong Heb, Feng Wangb a b
Department of Respiratory Medicine, Chifeng Hospital, Chifeng 024000, China Department of Respiratory Medicine, Beijing Chaoyang Hospital, Capital Medical University, Chaoyang, Beijing 100026, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Circular RNA NSCLC circ-PRMT5 Proliferation EZH2
Emerging evidence demonstrates that circular RNA (circRNA) is a novel class of non-coding RNA that plays a pivotal role in cancer. Recently, circ-PRMT5 was identified as an oncogene in bladder cancer. Nevertheless, its contribution to non-small cell lung cancer (NSCLC) is unknown. Herein, we aimed to clarify the biological role of circ-PRMT5 in NSCLC. High circ-PRMT5 expression was identified in NSCLC tissues and cell lines and positively correlated with larger tumor size, advanced clinic stage, lymph node metastasis as well as worse prognosis. Stable knockdown of circ-PRMT5 dramatically weakened the proliferative capacities of NSCLC cells both in vitro and in vivo. Mechanically, circ-PRMT5 could simultaneously effectively sponge three miRNAs (miR-377, miR382 and miR-498) and alleviate their repression on the well-known oncogenic EZH2, resulting in increased EZH2 expression, thereby facilitating NSCLC progression. Importantly, a strong positive correlation between circPRMT5 and EZH2 expression was observed in NSCLC tissues. Overall, our data indicate that circ-PRMT5 is an oncogenic circRNA in NSCLC that can promote the growth of NSCLC via regulation of miR-377/382/498-EZH2 axis.
1. Introduction Nowadays, lung cancer is still the leading cause of cancer incidence and mortality worldwide, accounting for 18.4% of cancer-related deaths (Bray et al., 2018). The main subtype of lung cancer is non-small cell lung cancer (NSCLC) that accounts for about 85% of all lung cancers (Postmus et al., 2017). The 5-year survival rate of NSCLC is extremely unfavorable, < 20%, mainly due to the absence of early symptoms in most patients, as well as the metastasis and recurrence (Herbst et al., 2018). Therefore, there is an urgent need to deeply elucidate the nosogenesis of NSCLC, which will provide new approaches for the diagnosis and treatment of NSCLC. Circular RNA (circRNA), as a special type of non-coding RNA, is currently attracting generous attention. It has a covalently closed ring structure without 5′-end cup and 3′-end ploy A tail, enabling it resistant to exonuclease and thus very stable (Li et al., 2018). CircRNA was first discovered 40 years ago, but it was considered to be the “noise” of transcriptional process (Sanger et al., 1976). With the development of high throughput sequencing, it has been found that there are a large number of conserved circRNAs in eukaryotes with cell- or disease-
context-dependent expression manner, and some of which are more abundant than their linear isoforms (Jeck et al., 2013). Up to now, many studies have shown that circRNA is dynamically expressed in human cancers and is capable of regulating gene expression, thereby allowing it closely associated with tumorigenesis and aggressiveness (Ng et al., 2018). Emerging evidence suggests that circRNA participates in a various of cellular biological processes involved in cell proliferation, apoptosis, metastasis, differentiation, senescence and so forth (Wilusz, 2018). It functions mainly through the following four mechanisms, acting as a miRNA molecular sponge, transcriptional regulation, interaction with proteins, and translation of peptides (Kristensen et al., 2018). Among them, the most confirmed one is “miRNA sponge”, in which circRNA is able to abundantly bind to and sequester miRNA to increase the expression of miRNA downstream target (Shang et al., 2019). For instance, circ-HIPK3 was reported as an oncogene that facilitated the progression of colorectal cancer via sponging miR-7 (Zeng et al., 2018a, 2018b). Circ-LAMP1 could increase DDR2 expression by interacting with miR-615-5p, resulting in promoting T-cell lymphoblastic lymphoma growth (Deng et al., 2019). Circ-MTO1 was found to retard
Abbreviations: NSCLC, non-small cell lung cancer; circRNA, circular RNA; qRT-PCR, Quantitative reverse transcription PCR; CCK-8, Cell Counting Kit-8; EdU, 5Ethynyl-2′-deoxyuridine; IHC, Immunohistochemistry; 3′-UTR, 3′-Untranslated Region ⁎ Corresponding author at: Department of Respiratory Medicine, Chifeng Hospital, Chifeng, Inner Mongolia Autonomous Region, China. E-mail address: [email protected] (Y. Wang). https://doi.org/10.1016/j.gene.2019.144099 Received 3 May 2019; Received in revised form 29 August 2019; Accepted 30 August 2019 Available online 31 August 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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miRNAs, respectively.
Table 1 Correlation between circ-PRMT5 expression and clinicopathological characteristics in 90 NSCLC patients. Parameters
All cases
circ-PRMT5 expression
2.3. Cell transfection and establishment of stable circ-PRMT5 knockdown cell lines
P value
Low (n = 45)
High (n = 45)
Gender Male Female
61 29
30 15
31 14
0.822
Age (years) ≤60 > 60
42 48
18 27
24 21
0.205
Smoking status Smokers Non-smokers
50 40
24 21
26 19
0.607
Tumor size ≤3 >3
34 56
24 21
10 35
0.002
Lymph node metastasis No 53 Yes 37
32 13
21 24
0.018
TNM stage I–II III–IV
58 32
34 11
24 21
0.028
Differentiation Well/moderate Poor
57 33
31 14
26 19
0.274
Cell transfection was performed using Lipofectamine 3000 (Invitrogen, MA, USA) based on manufacturer's protocol. All mimics including miR-377, miR-382 and miR-498 (GenePharma, Shanghai, China) were transfected alone or in combination with circ-PRMT5 pCDciR (Geneseed, Guangzhou, China) or EZH2 pcDNA 3.0 (Invitrogen) expression vector into 95-D and H1299 cells. After 48 h of transfection, qRT-PCR was used to measure the transfection efficiency. To establish stable circ-PRMT5 depletion NSCLC cell lines, two shRNA sequences (sh-circ-PRMT5#1: 5′-UCAUCUUCCGGCUCCUCAAGUUCU-3′; sh-circPRMT5#2: 5′-AUCUUCCGGCUCCUCAAGUUC-3′) (Chen et al., 2018) targeting the junction of circ-PRMT5 were synthesized and inserted into pGLV3/H1/GFP/Puro vector, followed by lentiviral packaging and collection, infection into 95-D and H1299 cells and selection by 0.5 μg/ mL puromycin. 2.4. Cell Counting Kit-8 (CCK-8) assay A total of 100 μL 95-D and H1299 cells were plated into 96-well plates at a density of 2 × 103 each well and cultured for 0 h, 24 h, 48 h, and 72 h, followed by treatment with 10 μL CCK-8 solution (DOJINDO, Tokyo, Japan) for 1 h at 37 °C. The absorbance was measured at 450 nm using a microplate reader. 2.5. Colony formation assay
hepatocellular carcinoma initiation and aggressiveness by sponging oncogenic miR-9 to elevate p21 expression (Han et al., 2017). These findings indicate that the circRNA/miRNA/mRNA regulatory network is important for cancer progression. A very recent study showed that circ-PRMT5 was an oncogene, which was closely linked to the development of urothelial carcinoma of the bladder (Chen et al., 2018). However, its role in NSCLC remains undetermined. In the present study, we explored the expression level and biological function of circ-PRMT5 in NSCLC, and the underlying mechanism of action was further elucidated.
A total of 2 mL 95-D and H1299 cells were plated into 6-well plates at a density of 0.5 × 103 each well and cultured for two weeks. Then, the cells were washed by PBS three times and fixed by methanol for 10 min. Lastly, the cells were stained with giemsa dye and counted under a microscope. 2.6. Cell cycle and 5-Ethynyl-2′-deoxyuridine (EdU) assays 95-D and H1299 cells were collected by centrifugation and washed twice with pre-cooled PBS, followed by fixation with pre-cooled 70% ethanol at 4 °C overnight. On the second day, 500 μL PBS containing 50 μg/mL PI, 100μg/mL RNase A, and 0.2% Triton X-100 were added into the cells and incubated at 4 °C for 30 min. Flow cytometry was used to detect 2–3 × 104 cells with standard procedure and the results were analyzed by ModFit software. Besides, EdU proliferation assay was performed using Cell-Light DNA incorporation detection kit (RiboBio, Guangzhou, China) based on manufacturer's instruction with minor change.
2. Materials and methods 2.1. NSCLC patient samples and cell lines In total, we collected 90 pairs of cancer and adjacent normal tissues from patients diagnosed with NSCLC in Chifeng Hospital that had undergone surgical resection. The detailed clinical data of patients are presented in Table 1. Informed consent from each patient was obtained prior to this study. The procedure was approved by the Ethics Committee of Chifeng Hospital. The normal human bronchial epithelial cells (HBE) and NSCLC cell lines including A549, 95-D, HCC827, H1299 and SK-MES-1 were all cultured in RPMI 1640 medium with 10% FBS.
2.7. Xenograft tumor model A total of ten nude mice were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and randomly divided into two groups (n = 5 per group). 2 × 106 control or circ-PRMT5-silenced H1299 cells were subcutaneously injected into nude mice. The volume of subcutaneous tumors in nude mice was measured every one week using a vernier caliper. Five weeks after the injection, all mice were sacrificed and the tumors were photographed and weighed. Then, the tumor tissues were immediately placed at −80 °C for qRT-PCR analysis of RNA expression. All protocols used in animal experiments were approved by the Institutional Animal Care and Use Committee review board of Chifeng Hospital.
2.2. Quantitative reverse transcription PCR (qRT-PCR) Total RNA from NSCLC cells and tissues was extracted using the RNAsimple kit (#DP419, TIANGEN, Beijing, China). In addition, to obtain the cytoplasmic and nuclear RNAs, the NE-PER Reagent (Thermo Scientific) was employed according to manufacturer's standard protocol. The integrity and quality of RNA samples were assessed by the Qubit RNA IQ Assay kit (Thermo Scientific, CA, USA). After that, 1 μg RNA was utilized to reverse transcribed into single-stranded cDNA, followed by amplification and quantification of the product using the SuperReal Color PreMix kit (TIANGEN, Beijing, China). All samples were in triplicate and effectively repeated three times. GAPDH and U3 were employed as the reference controls for circ-PRMT5/EZH2 and
2.8. RNA pull-down assay Total protein from 95-D and H1299 cells was collected using RIPA 2
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PRMT5 expression (Table 1). Moreover, high circ-PRMT5 expression was positively associated with poor overall and progression-free survival, as demonstrated by Kaplan-Meier plotter (Fig. 1C and D). And Cox multivariate proportional hazards model results showed that circPRMT5 is an independent prognostic factor for NSCLC patients (Table 2). These results suggest that circ-PRMT5 is aberrantly expressed in NSCLC and may be linked to NSCLC tumorigenesis.
lysis buffer and then incubated with biotinylated control or circ-PRMT5 probe at 4 °C overnight. Next, the M280 dynabeads magnetic beads (Invitrogen) were added into the above mixture and incubated for another 1 h at room temperature. Lastly, the bead/probe/RNA complex was washed three times and treated with RNAsimple kit (TIANGEN), followed by qRT-PCR analysis. 2.9. Luciferase reporter assay
3.2. Depletion of circ-PRMT5 significantly suppresses NSCLC cell proliferation both in vitro and in vivo
To construct the luciferase reporter vector, the wile-type and mutant full-length sequences of circ-PRMT5 and EZH2 3′-UTR were synthesized and inserted into pmirGLO vectors (Promega, WI, USA), respectively. After that, 95-D and H1299 cells were co-transfected with above luciferase reporter vectors and control or miR-377/382/498 mimics using Lipofectamine 3000 (Invitrogen). The luciferase activity of each group was measured by the GloMax 20/20 Luminescence Detector (Promega).
The protein expression of EZH2 was detected by western blot assay and IHC staining. For western blot, the tissues and cells were cleaved using RIPA lysis buffer, followed by electrophoresis, transfer, blocking, incubation with anti-EZH2 primary (#5246, CST) and HRP conjugated secondary antibodies, and final visualization. For IHC staining, a total of 43 NSCLC tissues were fabricated into tissue microarrays (TMA) in the department of pathology of Chifeng Hospital. The slices were dewaxed, dehydrated, antigen retrieved, blocked, incubation with EZH2 primary and HRP conjugated DAKO secondary antibodies, and final visualization with DAB reagent.
To determine the biological function of circ-PRMT5 in NSCLC, we constructed stable circ-PRMT5 knockdown cell lines using lentiviral vector, and the results showed that two shRNAs significantly reduced circ-PRMT5 expression in 95-D and H1299 cells (Fig. 2A), whereas did not affect the expression of linear PRMT5 as well as the neighboring genes of circ-PRMT5 (Supplementary Fig. 2). Then, we performed a series of functional experiments. The CCK-8 results showed that the absorbance at 450 nm was dramatically reduced after circ-PRMT5 knockdown (Fig. 2B). And compared to the control group, fewer 95-D and H1299 cells in circ-PRMT5-depleted group formed clones (Fig. 2C). Consistently, knockdown of circ-PRMT5 resulted in increased cells in G0/G1 phase, accompanied by decreased cells in S and G2/M phases (Fig. 2D). And circ-PRMT5 depletion significantly reduced EdU-positive 95-D and H1299 cells (Fig. 2E and F). Furthermore, the in vivo animal results showed that less volume and weight of subcutaneous tumors were observed in circ-PRMT5-depleted group in comparison to control group (Fig. 2G and H). Overall, these data reveal that circ-PRMT5 facilitates the growth of NSCLC.
2.11. Statistical analysis
3.3. Circ-PRMT5 physically interacts with miR-377, miR-382 and miR-498
All results are the mean ± SD of at least three independent experiments carried out in triplicate. Student's t and Chi-square test were used for comparison between groups. The survival curves of NSCLC patients based on circ-PRMT5 expression were plotted by Kaplan-Meier method and the difference was calculated by Log-rank test. All figures and statistics are generated by Graphpad Prism v7.0 software. Twotailed P value < 0.05 are considered to be statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.
Through qRT-PCR analysis, we found that circ-PRMT5 was mainly located in the cytoplasm of 95-D and H1299 cells (Fig. 3A). It has been reported that cytoplasmic circRNA functions mainly via acting as a “miRNA sponge” (Shang et al., 2019). We then searched for the CircInteractome online database and found ten miRNAs that might be bound by circ-PRMT5, including miR-1227, miR-1248, miR-335, miR377, miR-382, miR-498, miR-604, miR-605, miR-647 and miR-873. To test this prediction, we conducted RNA pull-down assay and results showed that circ-PRMT5 probe abundantly enriched three miRNAs (miR-377, miR-382 and miR-498), but not other seven miRNAs both in 95-D and H1299 cells (Fig. 3B). Further, luciferase reporter assay was carried out using circ-PRMT5 luciferase vector with wild-type or mutant miR-377/miR-382/miR-498 binding site (Fig. 3C). The results showed that the luciferase activity of wild-type circ-PRMT5 reporter was significantly decreased after miR-377, miR-382 or miR-498 overexpression, whereas this phenomenon was not observed in the mutant circ-PRMT5 reporter (Fig. 3D). In addition, we found that circ-PRMT5 knockdown dramatically increased the expression of these three miRNAs in 95-D and H1299 cells as well as in the subcutaneous tumors (Fig. 3E and F). Moreover, low expression levels of these three miRNAs were observed in NSCLC tissues as compared to normal tissues (Fig. 3G). Collectively, these findings demonstrate that miR-377, miR382 and miR-498 may be the tumor suppressors in NSCLC and are abundantly sponged and inhibited by circ-PRMT5.
2.10. Western blot and Immunohistochemistry (IHC)
3. Results 3.1. Circ-PRMT5 is highly expressed in NSCLC tissues and cell lines We first determined the characteristics of circ-PRMT5. Circ-PRMT5 (circBase ID: hsa_circ_0031250) is located on chromosome 14q11.2 (chr14:23395341-23396023) and its mature length is 327 bp. CircPRMT5 is generated by back-splicing of exons 5 and 7 of PRMT5 gene, which was confirmed by RT-PCR and Sanger sequencing using divergent and convergent primers (Supplementary Fig. 1A). Moreover, circ-PRMT5, but not its linear isoform, was highly resistant to RNase R digestion (Supplementary Fig. 1B), implying that circ-PRMT5 has a ring structure. We then detected the expression of circ-PRMT5 in NSCLC tissues using qRT-PCR method. As shown in Fig. 1A, circ-PRMT5 was significantly overexpressed in NSCLC tissues in comparison to paracarcinoma normal tissues, which was consistent with the microarray data from GSE112214 containing three matched NSCLC and normal tissues (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE112214). Also, the uniformly up-regulated circ-PRMT5 was observed in five NSCLC cell lines as compared with normal human bronchial epithelial HBE cells (Fig. 1B). Next, we analyzed the relationship between circPRMT5 expression and clinicopathological characteristics of NSCLC patients. The results showed that patients with high circ-PRMT5 expression were more likely to develop to larger tumors, lymph node metastasis and later clinical TNM stage than patients with low circ-
3.4. Circ-PRMT5 functions through regulation of miR-377/382/498-EZH2 pathway By the analysis of miRanda online database, we found that the binding sites of miR-377, miR-382 and miR-498 simultaneously existed on the 3′-UTR of the well-known oncogene EZH2 (Fig. 4A). We then performed the luciferase reporter assay and results displayed that overexpression of miR-377, miR-382 or miR-498 significantly reduced the luciferase activity of wild-type EZH2 3′-UTR reporter, while had no 3
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Fig. 1. Circ-PRMT5 is significantly increased in NSCLC. (A) qRT-PCR analysis for circ-PRMT5 expression in 90 pairs of NSCLC and normal tissues. (B) qRT-PCR analysis for circ-PRMT5 expression in human normal bronchial epithelial cells (HBE) and NSCLC cell lines. (C and D) The Kaplan-Meier plotter showing the overall and progression-free survival time of NSCLC patients based on median circ-PRMT5 expression. *p < 0.05, **p < 0.01, ***p < 0.001. Table 2 Uni- and multivariate analysis of prognostic predictors for overall survival in NSCLC patients (n = 90). Variable
Gender Age Smoking status Tumor size Lymph node metastasis TNM stage Differentiation circ-PRMT5 expression
Univariate analysis
Multivariate analysis
HR (95% CI)
P value
HR (95% CI)
P value
1.132 1.036 2.012 3.561 3.082 4.431 2.374 4.863
0.634 0.711 0.058 0.001 0.016 0.004 0.043 < 0.001
3.244 2.613 3.864 1.274 4.331
0.008 0.033 0.005 0.176 0.002
(0.786–2.034) (0.663–1.896) (0.889–4.532) (1.662–5.705) (1.341–4.782) (2.718–9.347) (0.225–4.813) (2.147–8.603)
(1.378–4.697) (1.634–5.893) (2.047–6.307) (0.712–4.178) (1.896–9.774)
circ-PRMT5 in NSCLC. We found that circ-PRMT5 was frequently overexpressed in NSCLC tissues and cells, and closely related to aggressive clinicopathological features and dismal outcome. Functionally, stably knockdown of circ-PRMT5 attenuated the proliferative capacities of NSCLC cells in vitro as well as the tumorigenicity in vivo. Stepwise investigation revealed that circ-PRMT5 could directly bind to and inhibit endogenous miR-377, miR-382 and miR-498, resulting in increased oncogenic EZH2 expression, thereby facilitating the progression of NSCLC. Therefore, our findings underline the biological relevance of circRNA in cancer, and also uncover the importance of circ-PRMT5 in NSCLC carcinogenesis. CircRNA is currently attracting tremendous attention. A growing body of evidence shows that circRNA is dysregulated in human cancers and plays an essential role in cancer initiation, development and progression (Bach et al., 2019). Up to now, some NSCLC-related circRNAs were also identified, such as circ-ABCB10 (Tian et al., 2019), circDDX42 (Qi et al., 2018), and circ-FADS2 (Zhao et al., 2018), they affected the malignant properties of NSCLC via acting as oncogenes or
effect on the mutant one (Fig. 4B). Further, the expression of EZH2 was substantially downregulated in miR-377/382/498-overexpressing 95-D and H1299 cells, however, this decreased effect was almost completely rescued by circ-PRMT5 overexpression (Fig. 4C and D). Importantly, we found that depletion of circ-PRMT5 significantly reduced EZH2 expression in the xenograft tumor model (Fig. 4E and F). And there was a strong positive correlation between circ-PRMT5 and EZH2 expression in NSCLC tissues (Fig. 4G and H). Functionally, miR-377/382/498 overexpression attenuated the proliferative abilities of 95-D and H1299 cells, and this repression was also abolished after ectopic expression of circ-PRMT5 or EZH2 (Fig. 4I). These above data indicate that circPRMT5 upregulates oncogenic EZH2 expression via sponging miR-377, miR-382 and miR-498, leading to promoting the growth of NSCLC (Fig. 4J). 4. Discussion In the current study, we for the first time characterized the role of 4
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Fig. 2. Circ-PRMT5 depletion inhibits NSCLC cell proliferation in vitro as well as tumorigenicity in vivo. (A) qRT-PCR analysis for circ-PRMT5 expression in 95-D and H1299 cells with stable circ-PRMT5 knockdown. (B) CCK-8 assay for the evaluation of cell viability in circ-PRMT5-depleted 95-D and H1299 cells. (C) Colony formation assay in circ-PRMT5-depleted 95-D and H1299 cells. (D) Cell cycle analysis of the percentage of circ-PRMT5-depleted 95-D and H1299 cells in G0/G1, S and G2/M phases. (E and F) EdU incorporation assay in circ-PRMT5-depleted 95-D and H1299 cells. DAPI was used to stain nucleus. (G and H) The image, volume and weight of subcutaneous tumors in nude mice injected with control or circ-PRMT5-depleted H1299 cells (n = 5 per group). *p < 0.05, **p < 0.01, ***p < 0.001.
The most studied mechanism of circRNA is the “miRNA sponge”. Representatively, CDR1as was shown to harbor > 70 conserved binding sites for miR-7 (Memczak et al., 2013). Wealth of studies revealed that circRNA participated in cancer progression by effectively sponging miRNA to regulate gene expression (Arnaiz et al., 2018). However, most researchers focused on studying the interaction of circRNA with a single miRNA, which obviously weakens the biological role of circRNA.
tumor suppressors. Although a small number of circRNAs have been functionally characterized, many members of this category have not yet been studied. Herein, we identified a novel NSCLC-related circRNA, circ-PRMT5, which was highly expressed in NSCLC and had tumorpromoting activity. It will be interesting to explore the crosstalk between circ-PRMT5 and the above reported NSCLC-related circRNAs, which may better elucidate the important role of circRNA in cancer. 5
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Fig. 3. Circ-PRMT5 directly binds to and sequester endogenous miR-377, miR-382 and miR-498 in NSCLC cells. (A) qRT-PCR analysis for nuclear and cytoplasmic circ-PRMT5 expression in 95-D and H1299 cells. GAPDH and U3 were used as reference controls of cytoplasmic and nuclear fragments, respectively. (B) Biotinlabeled RNA pull-down assay in 95-D and H1299 cells, followed by qRT-PCR analysis for the expression of the indicated ten miRNAs predicted by CircInteractome online database. (C) The wild-type and mutant miR-377/382/498 binding site on circ-PRMT5. (D) Luciferase reporter assay in 95-D and H1299 cells co-transfected with wild-type or mutated circ-PRMT5 reporter and control or miR-377/382/498 mimics. (E and F) qRT-PCR analysis for miR-377/382/498 expression in control or circ-PRMT5-silenced NSCLC cells or xenograft tumors. (G) qRT-PCR analysis for miR-377/382/498 expression in 90 pairs of NSCLC and normal tissues. **p < 0.01, ***p < 0.001.
miRNAs. And overexpression of miR-377, miR-382 or miR-489 dramatically reduced the expression of EZH2, whereas this repression was completely abrogated by exogenous circ-PRMT5 expression, implying that the circ-PRMT5-miR-377/382/489-EZH2 axis does exist in NSCLC. This notion was also confirmed by the rescue experiments that overexpression of circ-PRMT5 or EZH2 effectively rescued miR-377/382/
In this study, we found that circ-PRMT5 was able to simultaneously directly bind to three endogenous miRNAs including miR-377, miR-382 and miR-489, as demonstrated by RNA pull-down and luciferase reporter assays. Further, we found that EZH2, a well-known oncogene that promotes NSCLC growth via different signaling pathways (Behrens et al., 2013), was the common downstream target of above three 6
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Fig. 4. Circ-PRMT5 contributes to NSCLC cell proliferation via regulation of miR-377/382/498-EZH2 axis. (A) Microrna.org web tool was used to predict miR-377/ 382/498 targets that uses miRanda algorithm. (B) Luciferase reporter assay in 95-D and H1299 cells co-transfected with wild-type or mutated EZH2 3′-UTR reporter and control or miR-377/382/498 mimics. (C and D) qRT-PCR or western blot analysis for EZH2 expression in 95-D and H1299 cells transfected with miR-377/382/ 498 mimics alone or in combination with circ-PRMT5 expression vector. (E and F) qRT-PCR or western blot analysis for EZH2 expression in control or circ-PRMT5depleted xenograft tumors. (G and H) The correlation between circ-PRMT5 and EZH2 expression in 43 NSCLC tissues. (I) The proliferative assay in 95-D and H1299 cells transfected with miR-377/382/498 mimics alone or in combination with circ-PRMT5 or EZH2 expression vector. (J) The sketch map showing the proliferationpromoting role of circ-PRMT5 in NSCLC via regulation of miR-377/382/498-EZH2 axis. **p < 0.01.
promising prognostic biomarker for NSCLC patients. Recent studies have shown that circRNA is abundant in human body fluids (Hou et al., 2018; Ojha et al., 2018) and further research is needed to determine whether circ-PRMT5 can also be detected in blood, urine, saliva or even in exosomes, which will provide a non-invasive diagnostic biomarker for NSCLC. In conclusion, our study provides robust evidence that circ-PRMT5 is a proliferation-promoting gene as well as a promising prognostic biomarker in NSCLC. Therapeutic targeting of circ-PRMT5 may be an effectual approach for the treatment of NSCLC patients.
489-induced attenuated proliferative abilities of NSCLC cells. Of note, silencing of EZH2 could not completely counteract the enhanced aggressive phenotype of NSCLC cells caused by circ-PRMT5 overexpression, indicating that in addition to EZH2, there may be other target genes of miR-377/382/498 that mediate the function of circPRMT5, which needs further study. The previous study showed that circ-PRMT5 could sponge miR-30c in bladder cancer (Chen et al., 2018), unfortunately, we did not find that miR-30c was capable of interacting with circ-PRMT5 in NSCLC (data not shown), indicating that circ-PRMT5 functions in a cell- or disease-context-dependent manner. The highly conserved and stable property of circRNA enables it to be a promising biomarker.(Zhang et al., 2017) For example, circ-BIRC6 (Yang et al., 2019) circ-BACH2 (Cai et al., 2019), circ-ANKS1B (Zeng et al., 2018a, 2018b) and circ-SHPRH (Jiang et al., 2018) were reported as the effective diagnostic or prognostic biomarkers for hepatocellular carcinoma, papillary thyroid carcinoma, breast cancer and pancreatic ductal adenocarcinoma, respectively. In this study, NSCLC patients with high circ-PRMT5 expression had worse survival time than those with low circ-PRMT5 expression, suggesting that circ-PRMT5 may be a
Declaration of competing interest There is no conflict of interests to declare in the current study.
Acknowledgements None. 7
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2019.144099. References Arnaiz, E., Sole, C., Manterola, L., et al., 2018. CircRNAs and cancer: biomarkers and master regulators. Semin. Cancer Biol. Bach, D.H., Lee, S.K., Sood, A.K., 2019. Circular RNAs in cancer. Mol. Ther.–Nucleic Acids 16, 118–129. Behrens, C., Solis, L.M., Lin, H., et al., 2013. EZH2 protein expression associates with the early pathogenesis, tumor progression, and prognosis of non-small cell lung carcinoma. Clin. Cancer Res. 19, 6556–6565. Bray, F., Ferlay, J., Soerjomataram, I., et al., 2018. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 68, 394–424. Cai, X., Zhao, Z., Dong, J., et al., 2019. Circular RNA circBACH2 plays a role in papillary thyroid carcinoma by sponging miR-139-5p and regulating LMO4 expression. Cell Death Dis. 10, 184. Chen, X., Chen, R.X., Wei, W.S., et al., 2018. PRMT5 circular RNA promotes metastasis of urothelial carcinoma of the bladder through sponging miR-30c to induce epithelialmesenchymal transition. Clin. Cancer Res. 24, 6319–6330. Deng, L., Liu, G., Zheng, C., et al., 2019. Circ-LAMP1 promotes T-cell lymphoblastic lymphoma progression via acting as a ceRNA for miR-615-5p to regulate DDR2 expression. Gene 701, 146–151. Han, D., Li, J., Wang, H., et al., 2017. Circular RNA circMTO1 acts as the sponge of microRNA-9 to suppress hepatocellular carcinoma progression. Hepatology 66, 1151–1164. Herbst, R.S., Morgensztern, D., Boshoff, C., 2018. The biology and management of nonsmall cell lung cancer. Nature 553, 446–454. Hou, J., Jiang, W., Zhu, L., et al., 2018. Circular RNAs and exosomes in cancer: a mysterious connection. Clin. Transl. Oncol. 20, 1109–1116. Jeck, W.R., Sorrentino, J.A., Wang, K., et al., 2013. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 19, 141–157. Jiang, Y., Wang, T., Yan, L., et al., 2018. A novel prognostic biomarker for pancreatic ductal adenocarcinoma: hsa_circ_0001649. Gene 675, 88–93.
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Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Circular RNA circ-PRMT5 facilitates non-small cell lung cancer proliferation through upregulating EZH2 via sponging miR-377/382/498
T
⁎
Yuan Wanga, , Ying Lib, Hangyong Heb, Feng Wangb a b
Department of Respiratory Medicine, Chifeng Hospital, Chifeng 024000, China Department of Respiratory Medicine, Beijing Chaoyang Hospital, Capital Medical University, Chaoyang, Beijing 100026, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Circular RNA NSCLC circ-PRMT5 Proliferation EZH2
Emerging evidence demonstrates that circular RNA (circRNA) is a novel class of non-coding RNA that plays a pivotal role in cancer. Recently, circ-PRMT5 was identified as an oncogene in bladder cancer. Nevertheless, its contribution to non-small cell lung cancer (NSCLC) is unknown. Herein, we aimed to clarify the biological role of circ-PRMT5 in NSCLC. High circ-PRMT5 expression was identified in NSCLC tissues and cell lines and positively correlated with larger tumor size, advanced clinic stage, lymph node metastasis as well as worse prognosis. Stable knockdown of circ-PRMT5 dramatically weakened the proliferative capacities of NSCLC cells both in vitro and in vivo. Mechanically, circ-PRMT5 could simultaneously effectively sponge three miRNAs (miR-377, miR382 and miR-498) and alleviate their repression on the well-known oncogenic EZH2, resulting in increased EZH2 expression, thereby facilitating NSCLC progression. Importantly, a strong positive correlation between circPRMT5 and EZH2 expression was observed in NSCLC tissues. Overall, our data indicate that circ-PRMT5 is an oncogenic circRNA in NSCLC that can promote the growth of NSCLC via regulation of miR-377/382/498-EZH2 axis.
1. Introduction Nowadays, lung cancer is still the leading cause of cancer incidence and mortality worldwide, accounting for 18.4% of cancer-related deaths (Bray et al., 2018). The main subtype of lung cancer is non-small cell lung cancer (NSCLC) that accounts for about 85% of all lung cancers (Postmus et al., 2017). The 5-year survival rate of NSCLC is extremely unfavorable, < 20%, mainly due to the absence of early symptoms in most patients, as well as the metastasis and recurrence (Herbst et al., 2018). Therefore, there is an urgent need to deeply elucidate the nosogenesis of NSCLC, which will provide new approaches for the diagnosis and treatment of NSCLC. Circular RNA (circRNA), as a special type of non-coding RNA, is currently attracting generous attention. It has a covalently closed ring structure without 5′-end cup and 3′-end ploy A tail, enabling it resistant to exonuclease and thus very stable (Li et al., 2018). CircRNA was first discovered 40 years ago, but it was considered to be the “noise” of transcriptional process (Sanger et al., 1976). With the development of high throughput sequencing, it has been found that there are a large number of conserved circRNAs in eukaryotes with cell- or disease-
context-dependent expression manner, and some of which are more abundant than their linear isoforms (Jeck et al., 2013). Up to now, many studies have shown that circRNA is dynamically expressed in human cancers and is capable of regulating gene expression, thereby allowing it closely associated with tumorigenesis and aggressiveness (Ng et al., 2018). Emerging evidence suggests that circRNA participates in a various of cellular biological processes involved in cell proliferation, apoptosis, metastasis, differentiation, senescence and so forth (Wilusz, 2018). It functions mainly through the following four mechanisms, acting as a miRNA molecular sponge, transcriptional regulation, interaction with proteins, and translation of peptides (Kristensen et al., 2018). Among them, the most confirmed one is “miRNA sponge”, in which circRNA is able to abundantly bind to and sequester miRNA to increase the expression of miRNA downstream target (Shang et al., 2019). For instance, circ-HIPK3 was reported as an oncogene that facilitated the progression of colorectal cancer via sponging miR-7 (Zeng et al., 2018a, 2018b). Circ-LAMP1 could increase DDR2 expression by interacting with miR-615-5p, resulting in promoting T-cell lymphoblastic lymphoma growth (Deng et al., 2019). Circ-MTO1 was found to retard
Abbreviations: NSCLC, non-small cell lung cancer; circRNA, circular RNA; qRT-PCR, Quantitative reverse transcription PCR; CCK-8, Cell Counting Kit-8; EdU, 5Ethynyl-2′-deoxyuridine; IHC, Immunohistochemistry; 3′-UTR, 3′-Untranslated Region ⁎ Corresponding author at: Department of Respiratory Medicine, Chifeng Hospital, Chifeng, Inner Mongolia Autonomous Region, China. E-mail address: [email protected] (Y. Wang). https://doi.org/10.1016/j.gene.2019.144099 Received 3 May 2019; Received in revised form 29 August 2019; Accepted 30 August 2019 Available online 31 August 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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miRNAs, respectively.
Table 1 Correlation between circ-PRMT5 expression and clinicopathological characteristics in 90 NSCLC patients. Parameters
All cases
circ-PRMT5 expression
2.3. Cell transfection and establishment of stable circ-PRMT5 knockdown cell lines
P value
Low (n = 45)
High (n = 45)
Gender Male Female
61 29
30 15
31 14
0.822
Age (years) ≤60 > 60
42 48
18 27
24 21
0.205
Smoking status Smokers Non-smokers
50 40
24 21
26 19
0.607
Tumor size ≤3 >3
34 56
24 21
10 35
0.002
Lymph node metastasis No 53 Yes 37
32 13
21 24
0.018
TNM stage I–II III–IV
58 32
34 11
24 21
0.028
Differentiation Well/moderate Poor
57 33
31 14
26 19
0.274
Cell transfection was performed using Lipofectamine 3000 (Invitrogen, MA, USA) based on manufacturer's protocol. All mimics including miR-377, miR-382 and miR-498 (GenePharma, Shanghai, China) were transfected alone or in combination with circ-PRMT5 pCDciR (Geneseed, Guangzhou, China) or EZH2 pcDNA 3.0 (Invitrogen) expression vector into 95-D and H1299 cells. After 48 h of transfection, qRT-PCR was used to measure the transfection efficiency. To establish stable circ-PRMT5 depletion NSCLC cell lines, two shRNA sequences (sh-circ-PRMT5#1: 5′-UCAUCUUCCGGCUCCUCAAGUUCU-3′; sh-circPRMT5#2: 5′-AUCUUCCGGCUCCUCAAGUUC-3′) (Chen et al., 2018) targeting the junction of circ-PRMT5 were synthesized and inserted into pGLV3/H1/GFP/Puro vector, followed by lentiviral packaging and collection, infection into 95-D and H1299 cells and selection by 0.5 μg/ mL puromycin. 2.4. Cell Counting Kit-8 (CCK-8) assay A total of 100 μL 95-D and H1299 cells were plated into 96-well plates at a density of 2 × 103 each well and cultured for 0 h, 24 h, 48 h, and 72 h, followed by treatment with 10 μL CCK-8 solution (DOJINDO, Tokyo, Japan) for 1 h at 37 °C. The absorbance was measured at 450 nm using a microplate reader. 2.5. Colony formation assay
hepatocellular carcinoma initiation and aggressiveness by sponging oncogenic miR-9 to elevate p21 expression (Han et al., 2017). These findings indicate that the circRNA/miRNA/mRNA regulatory network is important for cancer progression. A very recent study showed that circ-PRMT5 was an oncogene, which was closely linked to the development of urothelial carcinoma of the bladder (Chen et al., 2018). However, its role in NSCLC remains undetermined. In the present study, we explored the expression level and biological function of circ-PRMT5 in NSCLC, and the underlying mechanism of action was further elucidated.
A total of 2 mL 95-D and H1299 cells were plated into 6-well plates at a density of 0.5 × 103 each well and cultured for two weeks. Then, the cells were washed by PBS three times and fixed by methanol for 10 min. Lastly, the cells were stained with giemsa dye and counted under a microscope. 2.6. Cell cycle and 5-Ethynyl-2′-deoxyuridine (EdU) assays 95-D and H1299 cells were collected by centrifugation and washed twice with pre-cooled PBS, followed by fixation with pre-cooled 70% ethanol at 4 °C overnight. On the second day, 500 μL PBS containing 50 μg/mL PI, 100μg/mL RNase A, and 0.2% Triton X-100 were added into the cells and incubated at 4 °C for 30 min. Flow cytometry was used to detect 2–3 × 104 cells with standard procedure and the results were analyzed by ModFit software. Besides, EdU proliferation assay was performed using Cell-Light DNA incorporation detection kit (RiboBio, Guangzhou, China) based on manufacturer's instruction with minor change.
2. Materials and methods 2.1. NSCLC patient samples and cell lines In total, we collected 90 pairs of cancer and adjacent normal tissues from patients diagnosed with NSCLC in Chifeng Hospital that had undergone surgical resection. The detailed clinical data of patients are presented in Table 1. Informed consent from each patient was obtained prior to this study. The procedure was approved by the Ethics Committee of Chifeng Hospital. The normal human bronchial epithelial cells (HBE) and NSCLC cell lines including A549, 95-D, HCC827, H1299 and SK-MES-1 were all cultured in RPMI 1640 medium with 10% FBS.
2.7. Xenograft tumor model A total of ten nude mice were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and randomly divided into two groups (n = 5 per group). 2 × 106 control or circ-PRMT5-silenced H1299 cells were subcutaneously injected into nude mice. The volume of subcutaneous tumors in nude mice was measured every one week using a vernier caliper. Five weeks after the injection, all mice were sacrificed and the tumors were photographed and weighed. Then, the tumor tissues were immediately placed at −80 °C for qRT-PCR analysis of RNA expression. All protocols used in animal experiments were approved by the Institutional Animal Care and Use Committee review board of Chifeng Hospital.
2.2. Quantitative reverse transcription PCR (qRT-PCR) Total RNA from NSCLC cells and tissues was extracted using the RNAsimple kit (#DP419, TIANGEN, Beijing, China). In addition, to obtain the cytoplasmic and nuclear RNAs, the NE-PER Reagent (Thermo Scientific) was employed according to manufacturer's standard protocol. The integrity and quality of RNA samples were assessed by the Qubit RNA IQ Assay kit (Thermo Scientific, CA, USA). After that, 1 μg RNA was utilized to reverse transcribed into single-stranded cDNA, followed by amplification and quantification of the product using the SuperReal Color PreMix kit (TIANGEN, Beijing, China). All samples were in triplicate and effectively repeated three times. GAPDH and U3 were employed as the reference controls for circ-PRMT5/EZH2 and
2.8. RNA pull-down assay Total protein from 95-D and H1299 cells was collected using RIPA 2
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PRMT5 expression (Table 1). Moreover, high circ-PRMT5 expression was positively associated with poor overall and progression-free survival, as demonstrated by Kaplan-Meier plotter (Fig. 1C and D). And Cox multivariate proportional hazards model results showed that circPRMT5 is an independent prognostic factor for NSCLC patients (Table 2). These results suggest that circ-PRMT5 is aberrantly expressed in NSCLC and may be linked to NSCLC tumorigenesis.
lysis buffer and then incubated with biotinylated control or circ-PRMT5 probe at 4 °C overnight. Next, the M280 dynabeads magnetic beads (Invitrogen) were added into the above mixture and incubated for another 1 h at room temperature. Lastly, the bead/probe/RNA complex was washed three times and treated with RNAsimple kit (TIANGEN), followed by qRT-PCR analysis. 2.9. Luciferase reporter assay
3.2. Depletion of circ-PRMT5 significantly suppresses NSCLC cell proliferation both in vitro and in vivo
To construct the luciferase reporter vector, the wile-type and mutant full-length sequences of circ-PRMT5 and EZH2 3′-UTR were synthesized and inserted into pmirGLO vectors (Promega, WI, USA), respectively. After that, 95-D and H1299 cells were co-transfected with above luciferase reporter vectors and control or miR-377/382/498 mimics using Lipofectamine 3000 (Invitrogen). The luciferase activity of each group was measured by the GloMax 20/20 Luminescence Detector (Promega).
The protein expression of EZH2 was detected by western blot assay and IHC staining. For western blot, the tissues and cells were cleaved using RIPA lysis buffer, followed by electrophoresis, transfer, blocking, incubation with anti-EZH2 primary (#5246, CST) and HRP conjugated secondary antibodies, and final visualization. For IHC staining, a total of 43 NSCLC tissues were fabricated into tissue microarrays (TMA) in the department of pathology of Chifeng Hospital. The slices were dewaxed, dehydrated, antigen retrieved, blocked, incubation with EZH2 primary and HRP conjugated DAKO secondary antibodies, and final visualization with DAB reagent.
To determine the biological function of circ-PRMT5 in NSCLC, we constructed stable circ-PRMT5 knockdown cell lines using lentiviral vector, and the results showed that two shRNAs significantly reduced circ-PRMT5 expression in 95-D and H1299 cells (Fig. 2A), whereas did not affect the expression of linear PRMT5 as well as the neighboring genes of circ-PRMT5 (Supplementary Fig. 2). Then, we performed a series of functional experiments. The CCK-8 results showed that the absorbance at 450 nm was dramatically reduced after circ-PRMT5 knockdown (Fig. 2B). And compared to the control group, fewer 95-D and H1299 cells in circ-PRMT5-depleted group formed clones (Fig. 2C). Consistently, knockdown of circ-PRMT5 resulted in increased cells in G0/G1 phase, accompanied by decreased cells in S and G2/M phases (Fig. 2D). And circ-PRMT5 depletion significantly reduced EdU-positive 95-D and H1299 cells (Fig. 2E and F). Furthermore, the in vivo animal results showed that less volume and weight of subcutaneous tumors were observed in circ-PRMT5-depleted group in comparison to control group (Fig. 2G and H). Overall, these data reveal that circ-PRMT5 facilitates the growth of NSCLC.
2.11. Statistical analysis
3.3. Circ-PRMT5 physically interacts with miR-377, miR-382 and miR-498
All results are the mean ± SD of at least three independent experiments carried out in triplicate. Student's t and Chi-square test were used for comparison between groups. The survival curves of NSCLC patients based on circ-PRMT5 expression were plotted by Kaplan-Meier method and the difference was calculated by Log-rank test. All figures and statistics are generated by Graphpad Prism v7.0 software. Twotailed P value < 0.05 are considered to be statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.
Through qRT-PCR analysis, we found that circ-PRMT5 was mainly located in the cytoplasm of 95-D and H1299 cells (Fig. 3A). It has been reported that cytoplasmic circRNA functions mainly via acting as a “miRNA sponge” (Shang et al., 2019). We then searched for the CircInteractome online database and found ten miRNAs that might be bound by circ-PRMT5, including miR-1227, miR-1248, miR-335, miR377, miR-382, miR-498, miR-604, miR-605, miR-647 and miR-873. To test this prediction, we conducted RNA pull-down assay and results showed that circ-PRMT5 probe abundantly enriched three miRNAs (miR-377, miR-382 and miR-498), but not other seven miRNAs both in 95-D and H1299 cells (Fig. 3B). Further, luciferase reporter assay was carried out using circ-PRMT5 luciferase vector with wild-type or mutant miR-377/miR-382/miR-498 binding site (Fig. 3C). The results showed that the luciferase activity of wild-type circ-PRMT5 reporter was significantly decreased after miR-377, miR-382 or miR-498 overexpression, whereas this phenomenon was not observed in the mutant circ-PRMT5 reporter (Fig. 3D). In addition, we found that circ-PRMT5 knockdown dramatically increased the expression of these three miRNAs in 95-D and H1299 cells as well as in the subcutaneous tumors (Fig. 3E and F). Moreover, low expression levels of these three miRNAs were observed in NSCLC tissues as compared to normal tissues (Fig. 3G). Collectively, these findings demonstrate that miR-377, miR382 and miR-498 may be the tumor suppressors in NSCLC and are abundantly sponged and inhibited by circ-PRMT5.
2.10. Western blot and Immunohistochemistry (IHC)
3. Results 3.1. Circ-PRMT5 is highly expressed in NSCLC tissues and cell lines We first determined the characteristics of circ-PRMT5. Circ-PRMT5 (circBase ID: hsa_circ_0031250) is located on chromosome 14q11.2 (chr14:23395341-23396023) and its mature length is 327 bp. CircPRMT5 is generated by back-splicing of exons 5 and 7 of PRMT5 gene, which was confirmed by RT-PCR and Sanger sequencing using divergent and convergent primers (Supplementary Fig. 1A). Moreover, circ-PRMT5, but not its linear isoform, was highly resistant to RNase R digestion (Supplementary Fig. 1B), implying that circ-PRMT5 has a ring structure. We then detected the expression of circ-PRMT5 in NSCLC tissues using qRT-PCR method. As shown in Fig. 1A, circ-PRMT5 was significantly overexpressed in NSCLC tissues in comparison to paracarcinoma normal tissues, which was consistent with the microarray data from GSE112214 containing three matched NSCLC and normal tissues (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE112214). Also, the uniformly up-regulated circ-PRMT5 was observed in five NSCLC cell lines as compared with normal human bronchial epithelial HBE cells (Fig. 1B). Next, we analyzed the relationship between circPRMT5 expression and clinicopathological characteristics of NSCLC patients. The results showed that patients with high circ-PRMT5 expression were more likely to develop to larger tumors, lymph node metastasis and later clinical TNM stage than patients with low circ-
3.4. Circ-PRMT5 functions through regulation of miR-377/382/498-EZH2 pathway By the analysis of miRanda online database, we found that the binding sites of miR-377, miR-382 and miR-498 simultaneously existed on the 3′-UTR of the well-known oncogene EZH2 (Fig. 4A). We then performed the luciferase reporter assay and results displayed that overexpression of miR-377, miR-382 or miR-498 significantly reduced the luciferase activity of wild-type EZH2 3′-UTR reporter, while had no 3
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Fig. 1. Circ-PRMT5 is significantly increased in NSCLC. (A) qRT-PCR analysis for circ-PRMT5 expression in 90 pairs of NSCLC and normal tissues. (B) qRT-PCR analysis for circ-PRMT5 expression in human normal bronchial epithelial cells (HBE) and NSCLC cell lines. (C and D) The Kaplan-Meier plotter showing the overall and progression-free survival time of NSCLC patients based on median circ-PRMT5 expression. *p < 0.05, **p < 0.01, ***p < 0.001. Table 2 Uni- and multivariate analysis of prognostic predictors for overall survival in NSCLC patients (n = 90). Variable
Gender Age Smoking status Tumor size Lymph node metastasis TNM stage Differentiation circ-PRMT5 expression
Univariate analysis
Multivariate analysis
HR (95% CI)
P value
HR (95% CI)
P value
1.132 1.036 2.012 3.561 3.082 4.431 2.374 4.863
0.634 0.711 0.058 0.001 0.016 0.004 0.043 < 0.001
3.244 2.613 3.864 1.274 4.331
0.008 0.033 0.005 0.176 0.002
(0.786–2.034) (0.663–1.896) (0.889–4.532) (1.662–5.705) (1.341–4.782) (2.718–9.347) (0.225–4.813) (2.147–8.603)
(1.378–4.697) (1.634–5.893) (2.047–6.307) (0.712–4.178) (1.896–9.774)
circ-PRMT5 in NSCLC. We found that circ-PRMT5 was frequently overexpressed in NSCLC tissues and cells, and closely related to aggressive clinicopathological features and dismal outcome. Functionally, stably knockdown of circ-PRMT5 attenuated the proliferative capacities of NSCLC cells in vitro as well as the tumorigenicity in vivo. Stepwise investigation revealed that circ-PRMT5 could directly bind to and inhibit endogenous miR-377, miR-382 and miR-498, resulting in increased oncogenic EZH2 expression, thereby facilitating the progression of NSCLC. Therefore, our findings underline the biological relevance of circRNA in cancer, and also uncover the importance of circ-PRMT5 in NSCLC carcinogenesis. CircRNA is currently attracting tremendous attention. A growing body of evidence shows that circRNA is dysregulated in human cancers and plays an essential role in cancer initiation, development and progression (Bach et al., 2019). Up to now, some NSCLC-related circRNAs were also identified, such as circ-ABCB10 (Tian et al., 2019), circDDX42 (Qi et al., 2018), and circ-FADS2 (Zhao et al., 2018), they affected the malignant properties of NSCLC via acting as oncogenes or
effect on the mutant one (Fig. 4B). Further, the expression of EZH2 was substantially downregulated in miR-377/382/498-overexpressing 95-D and H1299 cells, however, this decreased effect was almost completely rescued by circ-PRMT5 overexpression (Fig. 4C and D). Importantly, we found that depletion of circ-PRMT5 significantly reduced EZH2 expression in the xenograft tumor model (Fig. 4E and F). And there was a strong positive correlation between circ-PRMT5 and EZH2 expression in NSCLC tissues (Fig. 4G and H). Functionally, miR-377/382/498 overexpression attenuated the proliferative abilities of 95-D and H1299 cells, and this repression was also abolished after ectopic expression of circ-PRMT5 or EZH2 (Fig. 4I). These above data indicate that circPRMT5 upregulates oncogenic EZH2 expression via sponging miR-377, miR-382 and miR-498, leading to promoting the growth of NSCLC (Fig. 4J). 4. Discussion In the current study, we for the first time characterized the role of 4
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Fig. 2. Circ-PRMT5 depletion inhibits NSCLC cell proliferation in vitro as well as tumorigenicity in vivo. (A) qRT-PCR analysis for circ-PRMT5 expression in 95-D and H1299 cells with stable circ-PRMT5 knockdown. (B) CCK-8 assay for the evaluation of cell viability in circ-PRMT5-depleted 95-D and H1299 cells. (C) Colony formation assay in circ-PRMT5-depleted 95-D and H1299 cells. (D) Cell cycle analysis of the percentage of circ-PRMT5-depleted 95-D and H1299 cells in G0/G1, S and G2/M phases. (E and F) EdU incorporation assay in circ-PRMT5-depleted 95-D and H1299 cells. DAPI was used to stain nucleus. (G and H) The image, volume and weight of subcutaneous tumors in nude mice injected with control or circ-PRMT5-depleted H1299 cells (n = 5 per group). *p < 0.05, **p < 0.01, ***p < 0.001.
The most studied mechanism of circRNA is the “miRNA sponge”. Representatively, CDR1as was shown to harbor > 70 conserved binding sites for miR-7 (Memczak et al., 2013). Wealth of studies revealed that circRNA participated in cancer progression by effectively sponging miRNA to regulate gene expression (Arnaiz et al., 2018). However, most researchers focused on studying the interaction of circRNA with a single miRNA, which obviously weakens the biological role of circRNA.
tumor suppressors. Although a small number of circRNAs have been functionally characterized, many members of this category have not yet been studied. Herein, we identified a novel NSCLC-related circRNA, circ-PRMT5, which was highly expressed in NSCLC and had tumorpromoting activity. It will be interesting to explore the crosstalk between circ-PRMT5 and the above reported NSCLC-related circRNAs, which may better elucidate the important role of circRNA in cancer. 5
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Fig. 3. Circ-PRMT5 directly binds to and sequester endogenous miR-377, miR-382 and miR-498 in NSCLC cells. (A) qRT-PCR analysis for nuclear and cytoplasmic circ-PRMT5 expression in 95-D and H1299 cells. GAPDH and U3 were used as reference controls of cytoplasmic and nuclear fragments, respectively. (B) Biotinlabeled RNA pull-down assay in 95-D and H1299 cells, followed by qRT-PCR analysis for the expression of the indicated ten miRNAs predicted by CircInteractome online database. (C) The wild-type and mutant miR-377/382/498 binding site on circ-PRMT5. (D) Luciferase reporter assay in 95-D and H1299 cells co-transfected with wild-type or mutated circ-PRMT5 reporter and control or miR-377/382/498 mimics. (E and F) qRT-PCR analysis for miR-377/382/498 expression in control or circ-PRMT5-silenced NSCLC cells or xenograft tumors. (G) qRT-PCR analysis for miR-377/382/498 expression in 90 pairs of NSCLC and normal tissues. **p < 0.01, ***p < 0.001.
miRNAs. And overexpression of miR-377, miR-382 or miR-489 dramatically reduced the expression of EZH2, whereas this repression was completely abrogated by exogenous circ-PRMT5 expression, implying that the circ-PRMT5-miR-377/382/489-EZH2 axis does exist in NSCLC. This notion was also confirmed by the rescue experiments that overexpression of circ-PRMT5 or EZH2 effectively rescued miR-377/382/
In this study, we found that circ-PRMT5 was able to simultaneously directly bind to three endogenous miRNAs including miR-377, miR-382 and miR-489, as demonstrated by RNA pull-down and luciferase reporter assays. Further, we found that EZH2, a well-known oncogene that promotes NSCLC growth via different signaling pathways (Behrens et al., 2013), was the common downstream target of above three 6
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Fig. 4. Circ-PRMT5 contributes to NSCLC cell proliferation via regulation of miR-377/382/498-EZH2 axis. (A) Microrna.org web tool was used to predict miR-377/ 382/498 targets that uses miRanda algorithm. (B) Luciferase reporter assay in 95-D and H1299 cells co-transfected with wild-type or mutated EZH2 3′-UTR reporter and control or miR-377/382/498 mimics. (C and D) qRT-PCR or western blot analysis for EZH2 expression in 95-D and H1299 cells transfected with miR-377/382/ 498 mimics alone or in combination with circ-PRMT5 expression vector. (E and F) qRT-PCR or western blot analysis for EZH2 expression in control or circ-PRMT5depleted xenograft tumors. (G and H) The correlation between circ-PRMT5 and EZH2 expression in 43 NSCLC tissues. (I) The proliferative assay in 95-D and H1299 cells transfected with miR-377/382/498 mimics alone or in combination with circ-PRMT5 or EZH2 expression vector. (J) The sketch map showing the proliferationpromoting role of circ-PRMT5 in NSCLC via regulation of miR-377/382/498-EZH2 axis. **p < 0.01.
promising prognostic biomarker for NSCLC patients. Recent studies have shown that circRNA is abundant in human body fluids (Hou et al., 2018; Ojha et al., 2018) and further research is needed to determine whether circ-PRMT5 can also be detected in blood, urine, saliva or even in exosomes, which will provide a non-invasive diagnostic biomarker for NSCLC. In conclusion, our study provides robust evidence that circ-PRMT5 is a proliferation-promoting gene as well as a promising prognostic biomarker in NSCLC. Therapeutic targeting of circ-PRMT5 may be an effectual approach for the treatment of NSCLC patients.
489-induced attenuated proliferative abilities of NSCLC cells. Of note, silencing of EZH2 could not completely counteract the enhanced aggressive phenotype of NSCLC cells caused by circ-PRMT5 overexpression, indicating that in addition to EZH2, there may be other target genes of miR-377/382/498 that mediate the function of circPRMT5, which needs further study. The previous study showed that circ-PRMT5 could sponge miR-30c in bladder cancer (Chen et al., 2018), unfortunately, we did not find that miR-30c was capable of interacting with circ-PRMT5 in NSCLC (data not shown), indicating that circ-PRMT5 functions in a cell- or disease-context-dependent manner. The highly conserved and stable property of circRNA enables it to be a promising biomarker.(Zhang et al., 2017) For example, circ-BIRC6 (Yang et al., 2019) circ-BACH2 (Cai et al., 2019), circ-ANKS1B (Zeng et al., 2018a, 2018b) and circ-SHPRH (Jiang et al., 2018) were reported as the effective diagnostic or prognostic biomarkers for hepatocellular carcinoma, papillary thyroid carcinoma, breast cancer and pancreatic ductal adenocarcinoma, respectively. In this study, NSCLC patients with high circ-PRMT5 expression had worse survival time than those with low circ-PRMT5 expression, suggesting that circ-PRMT5 may be a
Declaration of competing interest There is no conflict of interests to declare in the current study.
Acknowledgements None. 7
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Appendix A. Supplementary data
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