EZH2 axis in colorectal cancer

Biomedicine & Pharmacotherapy 102 (2018) 1188–1194

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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Hsa_circ_0071589 promotes carcinogenesis via the miR-600/EZH2 axis in colorectal cancer Wang Yong, Xuan Zhuoqi, Wang Baocheng, Zhang Dongsheng, Zhang Chuan, Sun Yueming

T

⁎

Department of Colorectal Surgery, The First Affiliated Hospital of Nanjing Medical University, 210029, Nanjing City, Jiangsu Province, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Colorectal cancer hsa_circ_0071589 miR-600 EZH2 Proliferation Invasion Migration

Previous studies have revealed that miRNAs and lncRNAs participate in the pathogenesis of colorectal cancer (CRC); however, whether circular RNAs (circRNAs) are also involved remains unclear. In the present study, qRTPCR was used to examine the expression of hsa_circ_0071589, miR-600, and enhancer of zeste homolog 2 (EZH2) in CRC. MTT assay, colony formation assay, transwell assay, and wound-healing assay were performed to assess the effects of hsa_circ_0071589, miR-600, and EZH2 on CRC cell viability, proliferation, invasion, and migration. Bioinformatics analysis, luciferase reporter assay, and RIP assay were used to explore the correlations among hsa_circ_0071589, miR-600, and EZH2 expression in CRC cells. The results showed that hsa_circ_0071589 expression was significantly higher in CRC tissues than in normal tissues. Blockage of hsa_circ_0071589 in CRC cells inhibited tumor growth, invasion and migration. Hsa_circ_0071589 was able to promote the expression of EZH2 by acting as a sponge of miR-600. In addition, miR-600 expression was negatively correlated to hsa_circ_0071589 expression in CRC tissues. These results demonstrated that the hsa_circ_0071589/miR-600/EZH2 axis may play critical regulatory roles in the pathogenesis of CRC and may serve as a novel therapy target in CRC.

1. Introduction Colorectal cancer (CRC), characterized by high prevalence in elderly people, is one of the most common cancers of digestive tract. CRC severely affects human health and causes enormous social and economic burdens [1–3]. Although public health awareness efforts and preventive measures have achieved tremendous progress in recent decades, the incidence of CRC remains high worldwide, especially in Europe and North America [2]. In 2016, CRC accounted for more than 90,000 new cases and had an almost 50% fatality rate in the United State [3,4]. The prognosis of CRC patients has been improving during the past few years. Data have shown that the 5-year survival rate of CRC patients with regional spread is almost 70%; however, due to distant tumor metastasis, the 5-year survival rate of patients with advanced stages of CRC is only 12% [5]. Therefore, exploring the pathogenesis mechanisms of CRC is urgent and essential for treating CRC patients. It is well known that non-coding RNAs consist of various different RNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and small interfering RNAs (siRNAs) [6,7]. Circular RNAs (circRNAs), which are produced primarily through back-splicing, are a novel subtype of non-coding RNAs that formed a closed loop without 5′3′ ends and poly A tail [8,9]. Previous studies have indicated that

circRNAs may participate in the regulation of gene expression by acting as miRNAs sponges, suggesting that the circRNAs/miRNA/mRNA axis may play an important role in the gene expression associated with diseases, including cancers [10,11]. Although the exact functions of circRNAs remain unclear, accumulating evidence has demonstrated that circRNAs are involved in the progression and development of various tumors, such as gastric cancer, breast cancer, and lung cancer [10,12,13]. Hsa_circ_0071589, formed by back-splicing of exon 24–25 from the FAT1 gene located in chr4, is a novel circRNA that was found to be upregulated in CRC tissues by our microarray assay (data not shown). In this study, we first validated the upregulation of hsa_circ_0071589 in CRC tissues by qRT-PCR. Then, we established an hsa_circ_0071589 knockdown cell model by transfecting CRC cell lines with siRNA, and we found that hsa_circ_0071589 silencing inhibited cell proliferation, invasion and migration. We established an hsa_circ_0071589/miR-600/ EZH2 regulation axis that may contribute to CRC progress and serve as a novel therapy target in CRC.

⁎ Corresponding author at: Department of Colorectal Surgery, The First Affiliated Hospital of Nanjing Medical University, No. 300 Guangzhou Road, Nanjing City 210029, Jiangsu Province, China. E-mail address: [email protected] (S. Yueming).

https://doi.org/10.1016/j.biopha.2018.03.085 Received 8 January 2018; Received in revised form 14 March 2018; Accepted 14 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.

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2. Materials and methods

photographed and used for cell counts under an inverted microscope.

2.1. Cell lines and tissues

2.5. Cell migration assay (wound-healing assay)

The human colon cancer cell line, HCT116, was obtained from the American Type Culture Collection (ATCC) and maintained in Dulbcco’s modified Eagle medium (DMEM, Gibco Laboratories, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Grand Island, NY, USA) and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO2 and 95% O2 at 37 °C. The colorectal cancer tissues and corresponding normal tissues (N = 40) were collected from patients with CRC diagnosed at The First Affiliated Hospital of Nanjing Medical University during 2014–2017. Both cancer tissues and normal tissues were immersed into liquid nitrogen immediately after removal from CRC patients and then stored at −80 °C until further analysis. Written informed consent was obtained from each patient, and all protocols used in this study were approved by the Ethics Committee of The First Affiliated Hospital of Nanjing Medical University.

The ability of cell migration was measured by wound-healing assay. Treated HCT116 cells were cultured in a 6-well plate, and when it reached 100% confluency, a scratch was made by a pipette tip. Then, the cells were gently washed by PBS three times and cultured at 37 °C. Images were captured at 0 h and 48 h under a microscope with a digital camera system (Olympus, Tokyo, Japan). 2.6. Colony formation assay The cell proliferation rate of treated HCT116 cells was determined by colony formation assay. Treated HCT116 cells were seeded into 6well plates at a concentration of 2 × 104 cells/well and cultured at 37 °C under a humidified atmosphere with 5% CO2 and 95% O2 for 14 days. Then, colonies were fixed by 70% ethanol for 10 min and stained with 1% crystal violet for another 10 min at room temperature. Images were captured by a microscope, and the visible colonies were counted using ImageJ software.

2.2. Cell transfection The siRNAs for hsa_circ_0071589 (siRNA1 and siRNA2) and miR600 (anti-miR-600) were all designed and purchased from RiboBio (Shanghai, China). For cell transfection, HCT116 cells were seeded into 6-well plates and cultured for 24 h, and then cells were transfected with 50 nM siRNA1/siRNA2 or anti-miR-600 by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The sequence of siRNA1 was 5′-ACT TTG ACT ATG ACA GTG TCA-3′, and the sequence of siRNA2 was 5′- GAC TAT GAC AGT GTC AGC TTA-3′.

2.7. Cell viability assay (MTT assay) The cell viability of treated HCT 116 cells was assessed by MTT assay. In brief, treated HCT116 cells were cultured in a 96-well plate at a density of 1 × 104 cells/well at 37 °C for 24 h. Then, the cells were treated with MTT solution (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 4 h. Subsequently, the absorbance was detected by a microplate reader at 570 nm at 0, 12, 24 and 48 h.

2.3. RNA extraction, reverse transcription, and quantitative real-time PCR (qRT-PCR)

2.8. Western blot analysis

Total RNAs were extracted from CRC tissues or treated HCT116 cells by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the recommendations of the manufacturers. After quantification by a NanoDrop 2000 kit (Thermo Fisher, Waltham, MA), 10 μl of total RNAs was used to synthesis cDNA through a RevertAid First Strand complementary DNA (cDNA) Synthesis Kit (Thermo Fisher). PCR amplification was performed using FastStart Universal SYBR-Green Master kit (Roche, Indianapolis, IN, USA), and the cycling programs were set as follows: 95 °C for 10 min, then 40 cycles of 95 °C for 50 s, 62 °C for 35 s, and 72 °C for 30 s, followed by a final step at 72 °C, 10 min. GAPDH or U6 was used as the internal reference, and the 2−ΔCt method was applied to calculate the expression. The sequences of the divergent primers for hsa_circ_0071589 were 5′-CAA ACT CCC CTT CTG ACA GC-3′ (Forward) and 5′-CCG AAT CAC ACT GAC AAA CG-3′ (Reverse). The sequences of miR-600 were 5′-ACA CTC CAG CTG GGA CUU ACA GAC AAG AGC C-3′ (Forward) and 5′-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG GAG CAA GG-3′ (Reverse). The sequences of the GAPDH primers were 5′-GTC ACT TTG CGC ATC TTT G-3′ (Forward) and 5′-GCG CCC ACC AAT AGA AAT C-3′ (Reverse). The sequences of U6 were 5′-AGC CCG CAC TCA GAA CAT C-3′ (Forward) and 5′- GCC ACC AAG ACA ATC ATC C-3′ (Reverse).

Treated HCT116 cells were lysed with RIPA buffer (Beyotime Biotechnology, Dalian, China), and the protein concentration was measured by a BCA kit (Sigma) according to the protocols of the manufacturer. Then, total proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). After blocking in low-fat milk for 2 h, the membranes were incubated with primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 h. The signals of membranes were visualized by enhanced chemiluminescence (Thermo-Scientific, Rockford, IL, USA), and the signal intensity was analyzed by ImageJ gel analysis software. Actin was used as an internal control. 2.9. Plasmid construction and dual luciferase activity assay The fragments of hsa_circ_0071589 and EZH2 that bind to miR-600 were amplified by RT-PCR and inserted into the luciferase vector psiCHECK-2 (Promega, Madison, WI, USA). Recombinant mutants of hsa_circ_0071589 and EZH2 were also established. For the luciferase reporter assay, HCT-116 cells were seeded into a 24-well plate and cultured for 24 h; then, the cells were co-transfected with luciferase reporter plasmids and miR-600 mimic using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured using the DualLuciferase Reporter Assay System (Promega) according to manufacturer's instructions. Renilla luciferase activity was normalized to Firefly luciferase activity.

2.4. Cell invasion assay The cell invasive ability of treated HCT116 cells was determined by a 24-well Transwell chamber with Matrigerl coating (Corning, 6.5 mm, 8 μm pore size). Briefly, 100 mL of treated HCT116 cells (1 × 104) was added to the upper chamber, and 200 mL of culture medium containing 15% FBS was added to the lower chamber. After maintaining the cells at 37 °C for 48 h, the cells that remained in the upper surface of the membrane were removed, and the cells in the lower surface of the membrane were fixed with 4% paraformaldehyde followed by staining with 0.5% crystal violet. Six random areas of the membrane were

2.10. RNA immunoprecipitation (RIP) assay An RIP assay was used to confirm the interaction between hsa_circ_0071589 and miR-600 via the EZMagna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) according to 1189

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the manufacturer’s recommendations. HCT116 cells were lysed by RNA lysis buffer and incubated with RIP buffer containing magnetic beads coated with anti-human argonaute 2 (Ago2) antibodies (Millipore). IgG (Millipore, USA) was used as a negative control (input). After maintenance for 2 h on ice, the enrichment of hsa_circ_0071589 and miR-600 was examined by RT-PCR.

Table 1 The relationship between Hsa_circ_0071589 expression and clinicopathological characteristics in 40 patients with CRC. All cases

Age (year) > 60 ≤ 60 Gender Male Female pN status N0 N1-N2 pM status M0 M1 Clinical stage I & II III & IV

2.11. Statistical analysis Data in this study were expressed as the mean ± standard deviation (SD), and all of the statistical analyses were performed with SPSS software ver. 20.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). P values less than 0.05 were considered statistically significant. 3. Results 3.1. Hsa_circ_0071589 was upregulated in CRC tissue First, to confirm that hsa_circ_0071589 was indeed circular, a pair of divergent primers that specifically amplify circular transcripts and a pair of convergent primers that amplify linear transcripts were designed, and cDNA or gDNA was used as templates. The results showed that hsa_circ_0071589 was amplified by the divergent primers on cDNA and not on gDNA (Fig. 1A and B). Then, to investigate the biological functions of hsa_circ_0071589, we measured its expression by qRT-PCR in CRC tissues. The results indicated that hsa_circ_0071589 expression was significantly higher in CRC tissues than in corresponding normal tissues (P < 0.05, Fig. 1C) and that the hsa_circ_0071589 expression in grade IIIeIV was obviously higher than in grade IeII (P < 0.05, Fig. 1D). Moreover, hsa_circ_0071589 expression was significantly increased in CRC patients with lymph node metastasis relative to its

Hsa_circ_0071589 High expression

Low expression

23 17

11 (47.8%) 9 (52.9%)

12 (52.2%) 8 (47.1%)

22 18

9 (40.9%) 8 (44.4%)

13 (59.1%) 10 (55.6%)

11 29

4 (36.4%) 19 (65.5%)

7 (63.6%) 10 (34.5%)

32 8

10 (31.3%) 6 (75.0%)

22 (68.8%) 2 (25.0%)

21 19

7 (33.3%) 13 (68.4%)

14 (66.7%) 6 (31.6%)

P value 0.500

0.538

0.096

0.032

0.028

pN: pathological node; pM: pathological metastasis.

expression in those with negative metastasis (P < 0.05, Fig. 1E). We also found that the expression level of hsa_circ_0071589 was significantly associated with pathological metastasis (P = 0.032) and clinical stage (P = 0.028) of CRC tissues (Table 1). Furthermore, Kaplan-Meier survival analysis and log-rank test indicated that the higher hsa_circ_0071589 expression group had a significantly shorter overall survival time than did the lower hsa_circ_0071589 expression group in CRC patients (Fig. 1F, p < 0.05).

Fig. 1. Hsa_circ_0071589 expression was upregulated in CRC tissue. (A) A schematic diagram of genomic location and splicing pattern of hsa_circ_0071589. (B) Identification of hsa_circ_0071589; it could be amplified by divergent primers in cDNA but could not be produced in gDNA. GAPDH was used as a control. (C) The expression level of hsa_circ_0071589 in 40 paired CRC tissues and corresponding normal tissues was measured by qRT-PCR (P < 0.05). (D) The expression of hsa_circ_0071589 in grade IeII and IIIeIV CRC was examined (P < 0.05). (E) The expression of hsa_circ_0071589 was detected in lymph node metastasis positive or negative CRC tissues (P < 0.05). (F) The Kaplan-Meier method with the log-rank test was applied to assess overall survival in the higher (n = 20) and lower (n = 20) hsa_circ_0071589 expression groups of CRC patients. 1190

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Fig. 2. Effects of hsa_circ_0071589 knockdown on cell viability, proliferation, migration and invasion of CRC cell lines. (A)Graphical representation of targeting sites in hsa_circ_0071589 for siRNA1 and siRNA2 that were designed to block hsa_circ_0071589 expression. (B) Expression of hsa_circ_0071589 in HCT116 cells transfected with siRNA1 or siRNA2 was verified by qRT-PCR (*P < 0.05). (C) MTT assay was used to examine the cell viability. (D and E) The colony formation assay showed the cell proliferation. (F and G) The transwell assay showed the cell invasion. (H and I) The wound-healing assay showed the cell migration in hsa_circ_0071589-silenced HCT116 cells compared to controls (*P < 0.05).

control group (P < 0.05, Fig. 2C). Cell proliferation detected by colony formation assay showed that hsa_circ_0071589 silencing could significantly decrease the relative colony formation rate (P < 0.05, Fig. 2D and E). Transwell assay and wound-healing assay was used to measure the cell invasion and cell migration, respectively, and the results suggested that hsa_circ_0071589 silencing significantly decreased the invasion and migration abilities of HCT116 cells (P < 0.05, Fig. 2F– I). In summary, hsa_circ_0071589 knockdown could inhibit CRC progression.

3.2. Hsa_circ_0071589 silencing inhibited CRC cell proliferation, invasion, and migration To further explore the biological roles of hsa_circ_0071589 in CRC, two specific interfering sequences (siRNA1 and siRNA2) were designed to block the expression of hsa_circ_0071589 (Fig. 2A). The qRT-PCR results showed that hsa_circ_0071589 expression was significantly downregulated in HCT116 cells transfected with siRNA1 or siRNA2 compared with its expression in cells transfected with the siRNA control (P < 0.05, Fig. 2B). MTT assay indicated that hsa_circ_0071589 silencing could markedly decrease the living HCT116 cells compared with 1191

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Fig. 3. Hsa_circ_0071589 was targeted by miR-600. (A)The sequence of binding site in hsa_circ_0071589 or hsa_circ_0071589 mutant for miR-600. (B) Luciferase reported assay was performed to detect the luciferase intensity in HEK cells co-transfected hsa_circ_0071589 or its mutant with miR-600 mimics or its control (*P < 0.05). (C) Expression of miR-600 in HCT116 cells treated with siRNA1 or siRNA2 was verified by qRT-PCR (*P < 0.05). (D) RIP assay was used to validate the interaction between hsa_circ_0071589 and miR-600 (*P < 0.05). (E) Expression of miR-600 in CRC tissues and corresponding normal tissues were measured by qRTPCR (*P < 0.05). (F) Correlation between the expression of miR-600 and hsa_circ_0071589 was assessed by Pearson’s correlation test (r2 = −0.6219, P < 0.001).

3.3. Hsa_circ_0071589 was targeted by miR-600

Table 2 The relationship between miR-600 expression and clinicopathological characteristics in 40 patients with CRC.

As the circRNA-miRNA-mRNA axis has been found to play important roles in various cancers, bioinformatics analysis was performed here to predict binding sites for miRNAs. The results indicated that there was one binding site in hsa_circ_0071589 for miR-600 (Fig. 3A). Luciferase reporter assay showed that the luciferase intensity was significantly attenuated in the cells co-transfected with hsa_circ_0071589 wild-type vector and miR-600 mimics (P < 0.05, Fig. 3B). The qRTPCR results indicated that miR-600 expression was significantly upregulated in HCT116 cells transfected with siRNA1 or siRNA2 (P < 0.05, Fig. 3C). RIP assay indicated that both hsa_circ_0071589 and miR-600 were remarkably enriched in AGO2 containing beads compared to input groups (P < 0.05, Fig. 3D). In addition, we found that miR-600 expression was significantly lower in CRC tumors than in corresponding normal tissues (P < 0.05, Fig. 3E), and we observed a negative correlation between miR-600 and hsa_circ_0071589 expression (P < 0.01, Fig. 3F). In addition, the expression level of miR-600 was significantly associated with the clinical stage (P = 0.005) of the CRC tissues (Table 2).

All cases

Age (year) > 60 ≤ 60 Gender Male Female pN status N0 N1-N2 pM status M0 M1 Clinical stage I & II III & IV

miR-600 High expression

Low expression

23 17

14 (44.4%) 10 (42.9%)

9 (55.6%) 7 (57.1%)

22 18

12 (52.9%) 11 (61.5%)

10 (47.1%) 7 (38.5%)

11 29

8 (33.3%) 11 (77.8%)

3 (66.7%) 18 (22.2%)

32 8

19 (37.5%) 3 (66.7%)

13 (62.5%) 5 (33.3%)

21 19

15 (31.8%) 5 (75.0%)

6 (68.2%) 14 (25.0%)

P value 0.576

0.462

0.053

0.237

0.005

pN: pathological node; pM: pathological metastasis.

3.4. Hsa_circ_0071589/miR-600/EZH2 regulatory pathway in cell proliferation, invasion, and migration of the CRC cell line

proliferation (P < 0.05, Fig. 4F), and attenuated cell invasion (P < 0.05, Fig. 4G) and migration (Fig. 4H) induced by hsa_circ_0071589 knockdown in HCT116 cells were all rescued by treatment with anti-miR-600. Taken together, these results suggested that the hsa_circ_0071589/miR-600/EZH2 regulatory pathway might serve as an effective potential target for the treatment of CRC.

The results present above demonstrate that hsa_circ_0071589 might serve as a sponge of miR-600 in CRC cells (Fig. 3). Similarly, in this study, we revealed that EZH2 was a target gene for miR-600 via bioinformatics analysis, which was further validated by luciferase reporter assay (P < 0.05, Fig. 4A and B). The relative expression levels of EZH2 mRNA and protein showed that hsa_circ_0071589 knockdown significantly downregulated EZH2 expression, and this effect could be abolished by the application of anti-miR-600 (P < 0.05, Fig. 4C and D). Moreover, the decreased cell viability (P < 0.05, Fig. 4E), inhibited cell

4. Discussion Tremendous progress has been achieved in understanding the pathogenic mechanisms of CRC during the past decades; however, 1192

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Fig. 4. Effects of hsa_circ_0071589 knockdown on CRC cells were reversed by miR-600 inhibitor and the interaction between miR-600 and EZH2. (A)The putative binding site in EZH2 for miR-600 was predicted by bioinformatics analysis. (B) Luciferase reported assay was used to examine the luciferase intensity in HEK cells cotransfected EZH2 or its mutant with miR-600 mimics or its control (*P < 0.05). (C) The relative expression level of EZH2 mRNA or (D) EZH2 protein was measured in CRC cells transfected with hsa_circ_0071589 siRNA or miR-600 inhibitor. Effects of hsa_circ_0071589 knockdown on (E) cell viability, (F) cell proliferation, (G) cell invasion, and (H) cell migration were abolished by miR-600 inhibitor (*P < 0.05).

CRC. Accumulating evidence has suggested that the circRNAs/miRNAs/ mRNAs axis is involved in the pathogenesis of many tumors, including bladder cancer, osteosarcoma, and breast cancer [11,17,18]. Therefore, we wonder whether hsa_circ_0071589 exerts its oncogenic effects by miRNA. Studies have demonstrated that a large number of miRNAs are related to the pathogenesis of CRC, such as miR-495, miR-411, miR215-5p, and miR-600 [19–22]. Among them, miR-600 was reported to function as tumor suppressor in human colorectal cancer by targeting p53 [22]. Thus, it was chosen for further research. In our research, the bioinformatics analysis predicted that there was a binding site for miR600 in hsa_circ_0071589, which was validated by the subsequent luciferase reporter assay. The results also showed that miR-600 expression could be regulated by hsa_circ_0071589 in the CRC cell line and revealed a negative correlation between hsa_circ_0071589 and miR-600. Enhancer of zeste homolog 2 (EZH2), which functions as a methyltransferase, is a critical subunit of polycomb repressive complex 2 (PRC2), which has been demonstrated to be involved in the

effective therapeutic measures remain limited [14,15]. Currently, the most effective treatments for CRC continue to be mainly confined to surgical removal and neoadjuvant therapy, which provide limited effects and poor prognosis, especially in patients with distant metastasis [15]. Therefore, it is urgent to identify new and effective molecular targets for the prevention, diagnosis, and treatment of CRC. Previous studies have suggested that miRNAs and lncRNAs might be molecular markers and potential therapeutic targets by acting as gene regulators for the diagnosis and treatment of various cancers. Recently, circRNAs were also reported to be involved in the progression of many tumors, such as gastric cancer, lung cancer, and breast cancer; however, few studies have demonstrated that circRNAs are involved in CRC [12,13,16]. hsa_circ_0071589 is a novel circular RNA that has been found to be upregulated in CRC tissues relative to corresponding normal tissues, suggesting that it may play a role in the progression and development of CRC. The functional experiments showed that hsa_circ_0071589 knockdown in HCT116 cells inhibited cell proliferation, invasion, and migration, implying that it may act as an oncogene in 1193

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development of the embryo [23]. Aberrant expression of EZH2 has been found to be associated with a variety of human cancers due to the gene suppressing activity of EZH2 [24,25]. An increasing number of studies have revealed that miRNAs may produce oncogenic or anti-oncogenic effects by regulating the expression of EZH2 [26–28]. For instance, Cui S et al have reported that miR-137 can inhibit tumor growth of liver cancer by downregulating EZH2 expression [26]. However, whether EZH2 plays a role in the hsa_circ_0071589/miR-600 regulatory axis in colorectal cancer remains undetermined. In this present study, bioinformatics analysis and luciferase reporter assay revealed that miR-600 is able to bind to EZH2, and we found that the downregulation of EZH2 caused by hsa_circ_0071589 knockdown in the CRC cell line could be reversed by anti-miR-600. Further functional experimental results also suggested that the tumor inhibitory effects of hsa_circ_0071589 silencing in the CRC cell line could be abolished by anti-miR-600. In conclusion, hsa_circ_0071589 was increased in CRC tissues and functions as an oncogenic agent via the miR-600/EZH2 axis, providing a novel potential therapeutic pathway for CRC patients.

8981(17)30407-2. [11] Z. Zhong, M. Huang, M. Lv, Y. He, C. Duan, L. Zhang, J. Chen, Circular RNA MYLK as a competing endogenous RNA promotes bladder cancer progression through modulating VEGFA/VEGFR2 signaling pathway, Cancer Lett. 403 (2017) 305–317. [12] H. Sun, W. Tang, D. Rong, H. Jin, K. Fu, W. Zhang, Z. Liu, H. Cao, X. Cao, Hsa_circ_0000520, a potential new circular RNA biomarker, is involved in gastric carcinoma, Cancer Biomark. 21 (2) (2017) 299–306. [13] X. Jin, Y. Guan, H. Sheng, Y. Liu, Crosstalk in competing endogenous RNA network reveals the complex molecular mechanism underlying lung cancer, Oncotarget 8 (53) (2017) 91270–91280. [14] A.P. Sitlinger, S.Y. Zafar, With colorectal cancer treatment, physical toxicity is not the only concern, Dis. Colon. Rectum 61 (1) (2018) 4–5. [15] M. Jalili-Nik, A. Soltani, S. Moussavi, M. Ghayour-Mobarhan, G.A. Ferns, S.M. Hassanian, A. Avan, Current status and future prospective of curcumin as a potential therapeutic agent in the treatment of colorectal cancer, J. Cell. Physiol. (2017), http://dx.doi.org/10.1002/jcp.26368 [Epub ahead of print]. [16] Z. Lai, Y. Yang, Y. Yan, T. Li, Y. Li, Z. Wang, Z. Shen, Y. Ye, K. Jiang, S. Wang, Analysis of co-expression networks for circular RNAs and mRNAs reveals that circular RNAs hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 are candidate oncogenes in gastric cancer, ABBV Cell Cycle (2017) 1–11. [17] N. Deng, L. Li, J. Gao, J. Zhou, Y. Wang, C. Wang, Y. Liu, Hsa_circ_0009910 promotes carcinogenesis by promoting the expression of miR-449a target IL6R in osteosarcoma, Biochem. Biophys. Res. Commun. 495 (1) (2017) 189–196. [18] Y.Y. Tang, P. Zhao, T.N. Zou, J.J. Duan, R. Zhi, S.Y. Yang, D.C. Yang, X.L. Wang, Circular RNA hsa_circ_0001982 promotes breast cancer cell carcinogenesis through decreasing miR-143, DNA Cell. Biol. 36 (11) (2017) 901–908. [19] L. Yan, J. Yao, J. Qiu, miRNA-495 suppresses proliferation and migration of colorectal cancer cells by targeting FAM83D, Biomed. Pharmacother. 96 (2017) 974–981. [20] J. Zhao, J. Xu, R. Zhang, MicroRNA-411 inhibits malignant biological behaviours of colorectal cancer cells by directly targeting PIK3R3, Oncol. Rep. 39 (2) (2017) 633–642. [21] P. Vychytilova-Faltejskova, J. Merhautova, T. Machackova, I. Gutierrez-Garcia, J. Garcia-Solano, L. Radova, D. Brchnelova, K. Slaba, M. Svoboda, J. Halamkova, R. Demlova, I. Kiss, R. Vyzula, P. Conesa-Zamora, O. Slaby, MiR-215-5p is a tumor suppressor in colorectal cancer targeting EGFR ligand epiregulin and its transcriptional inducer HOXB9, Oncogenesis 6 (11) (2017) 5. [22] P. Zhang, Z. Zuo, A. Wu, W. Shang, R. Bi, Q. Jin, J. Wu, L. Jiang, miR-600 inhibits cell proliferation, migration and invasion by targeting p53 in mutant p53-expressing human colorectal cancer cell lines, Oncol. Lett. 13 (3) (2017) 1789–1796. [23] J.K. Alldredge, R.N. Eskander, EZH2 inhibition in ARID1A mutated clear cell and endometrioid ovarian and endometrioid endometrial cancers, Gynecol. Oncol. Res. Pract. 4 (2017) 17. [24] M. Yamagishi, K. Uchimaru, Targeting EZH2 in cancer therapy, Curr. Opin. Oncol. 29 (5) (2017) 375–381. [25] K.S. Yan, C.Y. Lin, T.W. Liao, C.M. Peng, S.C. Lee, Y.J. Liu, W.P. Chan, R.H. Chou, EZH2 in cancer progression and potential application in cancer therapy: a friend or foe? Int. J. Mol. Sci. 18 (6) (2017). [26] S. Cui, Y. Sun, Y. Liu, C. Liu, J. Wang, G. Hao, Q. Sun, MicroRNA137 has a suppressive role in liver cancer via targeting EZH2, Mol. Med. Rep. 16 (6) (2017) 9494–9502. [27] T. Zhi, T. Yu, M. Pan, E. Nie, W. Wu, X. Wang, N. Liu, Y. You, Y. Wang, J. Zhang, EZH2 alteration driven by microRNA-524-5p and microRNA-324-5p promotes cell proliferation and temozolomide resistance in glioma, Oncotarget 8 (56) (2017) 96239–96248. [28] J. Chen, Y. Xu, L. Tao, Y. Pan, K. Zhang, R. Wang, L.B. Chen, X. Chu, MiRNA-26a contributes to the acquisition of malignant behaviors of doctaxel-resistant lung adenocarcinoma cells through targeting EZH2, Cell. Physiol. Biochem. 41 (2) (2017) 583–597.

Conflict of interest The authors declare no conflict of interest. References [1] S.G. Pai, B.A. Carneiro, Y.K. Chae, R.L. Costa, A. Kalyan, H.A. Shah, I. Helenowski, A.W. Rademaker, D. Mahalingam, F.J. Giles, Correlation of tumor mutational burden and treatment outcomes in patients with colorectal cancer, J. Gastrointest. Oncol. 8 (5) (2017) 858–866. [2] M. Navarro, A. Nicolas, A. Ferrandez, A. Lanas, Colorectal cancer population screening programs worldwide in 2016: an update, World J. Gastroenterol. 23 (20) (2017) 3632–3642. [3] F.A. Haggar, R.P. Boushey, Colorectal cancer epidemiology: incidence, mortality, survival, and risk factors, Clin. Colon. Rectal Surg. 22 (4) (2009) 191–197. [4] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2016, CA Cancer J. Clin. 66 (1) (2016) 7–30. [5] R. Siegel, C. DeSantis, K. Virgo, K. Stein, A. Mariotto, T. Smith, D. Cooper, T. Gansler, C. Lerro, S. Fedewa, C. Lin, C. Leach, R.S. Cannady, H. Cho, S. Scoppa, M. Hachey, R. Kirch, A. Jemal, E. Ward, Cancer treatment and survivorship statistics, 2012, CA Cancer J. Clin. 62 (4) (2012) 220–241. [6] E. Anastasiadou, L.S. Jacob, F.J. Slack, Non-coding RNA networks in cancer, Nat. Rev. Cancer 18 (1) (2017) 5–18. [7] B. Narozna, W. Langwinski, A. Szczepankiewicz, Non-coding RNAs in pediatric airway diseases, Genes (Basel) 8 (12) (2017). [8] S. Haque, L.W. Harries, Circular RNAs (circRNAs) in health and disease, Genes (Basel) 8 (12) (2017). [9] Y. Zhang, W. Liang, P. Zhang, J. Chen, H. Qian, X. Zhang, W. Xu, Circular RNAs: emerging cancer biomarkers and targets, J. Exp. Clin. Cancer Res. 36 (1) (2017) 152. [10] W.B. Yin, M.G. Yan, X. Fang, J.J. Guo, W. Xiong, R.P. Zhang, Circulating circular RNA hsa_circ_0001785 acts as a diagnostic biomarker for breast cancer detection, Clin. Chim Acta (2017), http://dx.doi.org/10.1016/j.cca.2017.10.011 PII: S0009-

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