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Circular BANP, an upregulated circular RNA that modulates cell proliferation in colorectal cancer
Biomedicine & Pharmacotherapy 88 (2017) 138–144
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Original article
Circular BANP, an upregulated circular RNA that modulates cell proliferation in colorectal cancer Mingchen Zhua,c,1, Yijun Xub,1, Yun Chena,* , Feng Yanc,** a
School of Pharmacy, Nanjing Medical University, Nanjing 211166, China Department of Gastroenterology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China Department of Clinical Laboratory, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nanjing Medical University Affiliated Cancer Hospital, Nanjing 210009, China b c
A R T I C L E I N F O
Article history: Received 1 November 2016 Received in revised form 19 December 2016 Accepted 22 December 2016 Keywords: Colorectal cancer Circular RNA BANP Proliferation siRNA
A B S T R A C T
Circular RNAs (circRNAs) are recently identified as widespread and diverse endogenous noncoding RNAs that may harbor vital functions in human and animals. However, the role of circRNAs in the process of tumorigenesis and development of colorectal cancer (CRC) remains vague. Here we characterized the circRNA expression profile from three paired CRC cancerous and adjacent normal tissues by human circRNA array, and identified 136 significantly overexpressed circRNAs and 243 downregulated circRNAs in CRC cancerous tissues (>2-fold changes). We further validated one circRNA generated from Exon 5–11 of BANP gene, termed circ-BANP. In addition, RT-PCR result showed that circ-BANP was overexpressed in 35 CRC cancerous tissues. Knockdown of circ-BANP with siRNA significantly attenuate the proliferation of CRC cells. In summary, our findings demonstrated that dysregulated circ-BANP appears to have an important role in CRC cells and could serve as a prognostic and therapeutic marker for CRC. © 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Colorectal cancer (CRC) is one of the most frequent cancers worldwide, leading to 694,000 deaths in 2012 [1]. 5-year survival rate of patients with CRC was 90.1% for patients at early stage and only 11.7% for patients with distant metastasis [2]. To improve the prognosis and treatment response of patients, a better understanding of molecular mechanisms underlying the development of CRC is clinically important. Non-coding RNA (ncRNA), including miRNA, siRNA, piRNA and lncRNA, has been reported to be involved in epigenetic modification. For example, miRNAs play a vital role in the tumorigenesis and progress of CRC through direct interaction with the 30 -UTR of
Abbreviations: CircRNAs, Circular RNAs; CRC, Colorectal Cancer; ncRNA, Noncoding RNA; circ-BANP, Circular BANP; qRT-PCR, Quantitative RT-PCR; DAPI, 4,6diamidino-2-phenylindole; siRNA, Small interfering RNA; CCK-8, Cell Counting Kit8; FDR, False Discovery Rate. * Corresponding author at: School of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing, 211166, China. ** Corresponding author at: Department of Clinical Laboratory, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nanjing Medical University Affiliated Cancer Hospital, Baiziting 42, Nanjing, 210009, China. E-mail addresses: [email protected] (Y. Chen), [email protected] (F. Yan). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2016.12.097 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
their target mRNA and affecting the functions of oncogenes and tumor suppressor gene [3–5]. Recently, circular RNAs (circRNAs) have been reported as a novel type of non-coding RNA that formed by a junction of the 5’ end and 3’end [6,7]. Increasing evidence indicates that circRNA could serve as miRNA sponge and regulate gene expression [6–8]. It has been proved that circular RNA ciRS-7 could function as a miR-7 sponge, leading to reduction of miR-7 activity and upregulaion of miR-7-targeted genes in nervous system [9,10]. In esophageal squamous cell carcinoma, circular RNA cir-ITCH was downregulated in cancerous tissue and functions as sponge of miR-7, miR-17, and miR-214. Therefore, it could enhance the level of ITCH and then inhibit the Wnt/b-catenin pathway [11]. More recently, it has been demonstrated that circRNA could regulate the proliferation and survival of cancer cell [12,13]. To date, the validated functions of circRNA are the tip of the iceberg and thousands of circRNAs have been found with unknown functions in different tissues and cells [7]. It has been reported that circRNA was also involved in the CRC onset and progression. Circ_001569 was confirmed to promote proliferation and invasion via functioning as a sponge of miR-145 [14]. Even so, there is little evidence of the expression profile of circRNA in CRC and the role of circRNA in the process of CRC tumorigenesis remains vague. To address this question, we employed the circRNA array analysis to detect differential expression profiles of circRNAs in CRC cancerous tissues and
M. Zhu et al. / Biomedicine & Pharmacotherapy 88 (2017) 138–144
adjacent tissues. We found that circular BANP (circ-BANP) was upregulated in CRC cancerous tissues compared with adjacent normal tissues and contributed to the proliferation of colorectal cancer cells. 2. Materials and methods 2.1. Cells HCT116 and HT29 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were cultured in McCoy’s 5A (modified) medium supplemented with 10% FBS and 50 U/ mL penicillin and streptomycin sulfate. 2.2. Tissue specimens The study was approved by the Clinical Research Ethics Committee of Nanjing medical university and written informed consent was obtained from all the patients. The paired tissue samples from cancerous and adjacent normal tissues were collected from 35 patients with CRC who underwent surgical resection between 2013 and 2015 at Jiangsu cancer hospital (Nanjing, China). All of the specimens were subsequently verified by histology. Patients who received preoperative chemotherapy were excluded. The samples were freshly frozen in liquid nitrogen and stored at 80 C. 2.3. Microarray analysis Total RNA from each sample was quantified using the NanoDrop ND-1000. The sample preparation and microarray hybridization were performed based on the Arraystar’s standard protocols. Briefly, total RNAs were digested with Rnase R (Epicentre, Inc.) to remove linear RNAs and enrich circular RNAs. Then, the enriched circular RNAs were amplified and transcribed into fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array (8 15 K, Arraystar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C.
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Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the R software package. Differentially expressed circRNAs with statistical significance between two groups were identified through Volcano Plot filtering. Differentially expressed circRNAs between two samples were identified through Fold Change filtering. Hierarchical Clustering was performed to show the distinguishable circRNAs expression pattern among samples. 2.4. Total RNA extraction, and RNase R digestion Total RNA was extracted using TRIzol reagent (Invitrogen, California, USA) according to the manufacturer’s recommendations. Extracted RNA was quantified using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). Total RNA was incubated for 15 min at 37 C with 3 units of RNase R (Epicentre) per 1 mg RNA. RNA was subsequently purified by phenol–chloroform extraction and reprecipitated in three volumes of ethanol. 2.5. Quantitative real-time RT-PCR cDNA was synthesized with the Reverse Transcription System (Promega, Madison, USA) using random primers. Quantitative RTPCR (qRT-PCR) analyses were carried out to detect circ-BANP expression using SYBR Premix Ex Taq (TaKaRa, Dalian, China), and b-actin was utilized as an internal control. Primers used in this experiment were: 50 - CAGGACGGTCAGCGTCGT-30 (forward), and 50 GGCACAGCGTTGCTAATGAC-30 (reverse) for circ-BANP, yielding a product of 138 bp; and 50 - GCATGGGTCAGAAGGATTCCT-30 (forward) and 50 -TCGTCCCAGTTGGTGACGAT-30 (reverse) for b-actin, yielding a product of 106 bp. 2.6. In situ hybridization We employed in situ hybridization to investigate the expression and intracellular location of circ-BANP in CRC tissue and cell lines. Tissue slides were deparaffinized with xylene, rehydrated through a graded series of ethanol. HT29 cells were grown on cover slips and fixed 48 h later. In situ hybridization was carried out using the
Fig. 1. circRNA array analysis of CRC tissues. (A) Hierarchical clustering revealed circRNA expression profile in 6 samples of cancerous and adjacent tissues. (B) The scatter plot indicated the variation in circRNA expression between normal (group-N) and CRC (group-D) samples. (C) The volcano plots suggested the fold-change values and p-values of the array data. The red point represents the significantly upregulated or downregulated circRNAs.
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Fig. 2. Validation of the circular transcript of BANP. (A) Total RNA were treated with RNase R. Circ-BANP was stably expressed in both RNase R() and RNase R(+) total RNA samples. (B) The result of 1% agarose gel electrophoresis of RT-PCR product displays a single lane. (C) RT-PCR was utilized to validate the expression of circ-BANP in cancerous and adjacent normal tissues from 35 CRC patients. Consistent with the array results, expression of circ-BANP in cancerous was upregulated significantly. (D) Sanger sequencing of the RT-PCR product. The sequence of product is consistent with the sequence of circ-BANP in GenBank. The red arrow indicates the backsplicing site of circBANP. (E) RNA in situ hybridization for circ-BANP (green) in paired colorectal cancer tissues ( 200). Nuclei were stained with DAPI (blue).
cirRNA Hybridization Kit (Foco, Guangzhou, China) according to the manufacturer’s instructions. Briefly, the slides were deparaffinized with 100% xylene and rehydrated in series alcohol, followed by incubating with 4% (v/v) paraformaldehyde for 30 min at room temperature. Afterwards, the slides were washed and incubated with prehybridization solution for 1 h at 55 C. Digoxin-labeled circ-BANP probe was denatured at 85 C for 5 min, subsequently added to each tissue section and hybridized for 24 h at 37 C. After washing with washing buffer, blocking was performed for 1 h at 37 C, and then the anti-digoxigenin HRP conjugate was added to
the sections at 37 C for 1 h in the humidified chamber. After washing with PBS, TSA-488 solution was added to the slides. The sections were counterstained with 20 mL 4,6-diamidino-2-phenylindole (DAPI) to stain the cell nucleus. 2.7. RNA interference Cell transfection was carried out by small interfering RNA (siRNA, GenePharma) into HCT116 and HT29 using LipofectamineTM 2000 (Invitrogen, MA, USA) in accordance with the
M. Zhu et al. / Biomedicine & Pharmacotherapy 88 (2017) 138–144 Table 1 The relationship between relative expression levels of circ-BANP in CRC cancerous tissues with clinical characteristics. Characteristics
Number
Median (Percentiles 25 75)
P value
Age 60 >60
22 13
2.39(0.72 8.29) 1.62(0.82 10.89)
0.669
Gender Male Female
24 11
2.22(1.44 10.77) 1.32(0.47 7.99)
0.27
Location Colon Rectum
18 17
1.87(0.55 7.21) 2.14(1.36 11.29)
0.32
Differentiation Well Moderate Poor
5 19 11
5.65(1.4 25.06) 1.51(0.52 4.63) 4.62(1.31 10.47)
0.4
Tumor invasion 1+2 3+4
5 30
2.64(1.14 4.63) 1.98(0.59 10.68)
0.15
Lymph node metastasis N0 N1-N2
21 14
1.51(0.45 6.11) 5.08(1.55 10.68)
0.1
TNM stage I II III IV
5 16 9 5
2.63(1.14 4.63) 1.46(0.29 10.82) 7.99(1.87 41.94) 1.86(0.89 6.79)
0.19
Serum CEA Positive Negative
18 17
1.68(0.52 8.2) 2.29(1.42 10.88)
0.23
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2.9. Colony formation assay HCT116 and HT29 cells (500 per well) were seeded in 6-well plates for 2 weeks. Subsequently, the colonies were stained with 1% crystal violet for 10 min after fixation with 4% paraformaldehyde for 5 min. Colonies were microscopically examined and counted. The assays were repeated 3 times. 2.10. Western blot assay Cells were lysed in lysis buffer (KeyGEN BioTECH, Nanjing, China), and the protein samples were subject to western blot as described [4]. Antibodies recognizing Akt (1:1000) and PhosphoAkt (Ser473) (1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). 2.11. Statistical analysis Statistical analyses were carried out using SPSS 16.0 for Windows (SPSS, Chicago, IL, USA). To examine significant differences of expression of circ-BANP in cancerous and adjacent normal tissues, Wilcoxon test was utilized. The association between circBANP expression and clinicopathological characteristics of colorectal cancer was analyzed by Mann-Whitney U test. Paired and independent T test were also used to determine the differences. Statistical differences were declared significant at P < 0.05. 3. Results 3.1. Expression profiles of circRNAs
manufacturer’s procedure. The siRNA sequences are as follows: circ-BANP: 50 -GAAUCAAGCAGAGCAUCGATT-30 (sense), 50 0 UCGAUGCUCUGCUUGAUUCTT-3 (antisense); negative control: UUCUCCGAACGUGUCACGUTT-30 (sense), 50 -ACGUGACACGUUCGGAGAATT-30 (antisense). 2.8. CCK-8 Cell proliferation rates were detected with Cell Counting Kit-8 (CCK-8, DOJINDO, Japan). 1 104 cells were cultured in each 96-well plate for 24, 48, 72, and 96 h, respectively. Subsequently, the absorbance at 450 nm was measured after incubation with 10 mL CCK-8 solution at 37 C for 1.5 h. All detections were carried out in triplicate.
CircRNA expression profiles were analyzed using a microarray to determine differentially expressed circRNAs in CRC cancerous and adjacent normal tissues (Fig. 1A). The scatter and volcano plots revealed the variation of circRNA expression between CRC cancerous tissues and adjacent normal tissues (Fig. 1B and C). Of the 2608 human circRNA analyzed, a total of 136 exhibited significantly overexpression and 243 circRNAs decreased in CRC cancerous tissues (>2-fold changes, respectively), compared with adjacent normal tissues. We found that one circRNA, circ-BANP, was remarkably upregulated in CRC cancerous tissues (>25.9-fold changes). Additionally, both p-value and FDR (False discovery rate) of circ-BANP were very low. The raw intensity of circ-BANP expression in tumor group and normal group was also homogeneous. Hence, circ-BANP was selected as a candidate circRNA for the further researches. 3.2. Expression of circ-BANP in CRC Subsequently, real-time PCR was utilized to validate the profile results. CircRNA was validated by RNase R digestion and RT-PCR.
Fig. 3. Expression and location of circ-BANP in CRC cell lines. (A) Expression of circ-BANP in CRC cell lines. (B) RNA in situ hybridization for circ-BANP (green) in HT29 ( 1000). Nuclei were stained with DAPI (blue).
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Fig. 4. Function of circ-BANP in CRC cells. (A, C) After transfection with siRNA and negative control for 48 h in HCT116 and HT29 cells, expression of circ-BANP was detected by RT-PCR. (B, D) Downregulation of circ-BANP repressed cell proliferation as determined by CCK-8 assays. OD, optical density. (E, F) Colony formation was performed to analyze cell proliferation. *P < 0.05. (G) The expression of Akt and p-Akt was detected by western blotting assay.
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We observed that circ-BANP was resistant to RNase R, whereas the linear control was not stable with RNase R treatment (Fig. 2A). The specificity of the RT-PCR was validated by 1% agarose gel electrophoresis and sanger sequencing (Fig. 2B and D). Using the divergent primers, we investigated the expression of circ-BANP in cancerous and adjacent normal tissues from 35 CRC patients by RTPCR. Similarly, the expression of circ-BANP in cancerous tissues was overexpressed, compared with adjacent normal tissues (P = 0.037, Fig. 2C). The potential association between circ-BANP expression and clinicopathologic features in 35 patients with CRC was also investigated. However, no correlation was observed between circ-BANP expression with age, gender, location, differentiation, TNM staging, and serum CEA (Table 1). In agreement with the PCR results, fluorescence in situ hybridization results showed that circ-BANP expression level was remarkably upregulated in CRC tissues, as compared to matched normal tissues (Fig. 2E). Taken together, our results show that circ-BANP is an abundant and stable circRNA expressed in CRC cancerous tissues. 3.3. Expression and location of circ-BANP in CRC cell lines We further used RT-PCR to determine the expression of circBANP in different CRC cell lines. The expression of circ-BANP was most overexpressed in HT29 and HCT116, especially in HT29 (Fig. 3A). Hence, we intend to select HT29 and HCT116 for the functional researches. Additionally, the location of circ-BANP in cellular localization was also examined via in situ hybridization. We observed that circ-BANP was mainly localized in cytoplasm (Fig. 3B). 3.4. Function of circ-BANP Next, we intended to explore the biological function of circBANP in CRC cells. Using siRNA targeting circ-BANP [15,16], the expression of circ-BANP was attenuated as expected (P = 0.01 for HCT116, P = 0.025 for HT29, Fig. 4A and C). We found that the proliferation of si-circ-BANP-group was repressed significantly, compared with negative control group in HCT116 and HT29 cells (Fig. 4B and 4D). To further confirm the impact of circ-BANP on CRC proliferation, the plate colony formation assay was performed. As expected, colony formation was significantly reduced in HCT116 and HT29 cells by siRNA transfection (P < 0.01; Fig. 4E and F). Moreover, Fig. 4G displays that knockdown of circ-BANP reduced the protein expression of p-Akt, whereas the total protein levels of Akt stayed unchanged. This result suggested that Akt pathway was also involved in the process of circ-BANP-induced cell proliferation. Together, these results confirmed that circ-BANP may contribute to the proliferation of CRC cells. 4. Discussion In this study, circRNA expression profiles in colorectal cancerous and adjacent normal tissues were determined by circRNA microassay, and a number of circRNAs were revealed to be dysregulated. Most importantly, we found that the expression of circ-BANP was significantly overexpressed in CRC cancerous tissues. The relationship between circ-BANP expression and CRC clinicopathologic features was also investigated. However, no correlation was observed. Knockdown using siRNA targeted to circBANP could suppress CRC cells proliferation. Collectively, circBANP was indicated to be involved in the biological processes of CRC cells. Previously, circRNAs were regarded as the result of splicing errors or by-products of mis-splicing until extensive deregulations of circRNAs were identified with high-throughput deep
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sequencing recently [17–22]. Currently, circRNAs were reported mainly as miRNA sponge and function as the regulators of miRNA activity, such as CDR1as, the testes-specific Sry gene, circHIPK3 and EIciRNAs [10,15,23]. With approximately 70 binding sites for miRNAs, CDR1as was considered to be an extreme case; however, most natural miRNA sponges contain only 1-2 miRNA binding sites [24]. Hence, circRNAs could have other potential functions besides miRNA sponges, such as regulation of transcription [25] and interactions with RNA binding proteins [8]. To our knowledge, this is the first report about circ-BANP in CRC and whether the circBANP could function as a miRNA sponge is still unknown. Evolving information suggests that circRNAs could serve multiple biological functions, and be related with pathogenesis of various diseases [12,26–29]. In the process of carcinogenesis and cancer progression, circHIPK3 RNA was demonstrated to promote cell growth in human cells [15]. circ-Foxo3 could regulate cell cycle progression and repress cell proliferation [30]. Similarly, in our study we also found circ-BANP could enhance CRC cells proliferation. Circ-BANP is produced by back-splicing reactions of parent BANP mRNA that covalently link the 30 end of exon 11 to the 50 end of an upstream exon 5. BANP encodes a protein that binds to matrix attachment regions. It has been demonstrated that BANP (also known as SMAR1) interacts with HDAC1 associated repressor complex at Cyclin D1 promoter and allows histone deacetylation to cause its transcriptional repression [31]. Therefore, BANP could function as a tumor suppressor and cell cycle regulator. It has been demonstrated that PI3 K/Akt pathway plays a critical role in regulating colorectal cancer cell cycle and cell proliferation [32,33]. In this study, we found that si-circ-BANP transfection impeded the expression of p-Akt. Hence, we speculated that circ-BANP could enhance the growth of colorectal cancer cell by PI3 K/Akt pathway. Further more studies are desired to validate the mechanism of circBANP on cell growth. In conclusion, we conducted circular RNA profiles to identify circ-BANP as being frequently upregulated in CRC cancerous tissues. The biological function of circ-BANP was convinced to be related with cell proliferation. Hence, circ-BANP could serve as a potential diagnostic and therapeutic marker for CRC. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21475063, 81601843, 81602126, 21675089, 21175071, 21605086), the Jiangsu Health Department (H201325), Program of Jiangsu Provincial Medical Innovation Teams, the Foundation of Jiangsu Cancer Hospital (ZM201501), the Project sponsored by SRF for ROCS, SEM (39), the Jiangsu Six-type Top Talents Program (D), the Open Foundation of Nanjing University (SKLACLS1102) and the Foundation of Nanjing medical university (2015NJMU050). References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics 2012, CA. Cancer J. Clin. 65 (2015) 87–108. [2] 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 (2012) 220–241. [3] S. Liu, X. Sun, M. Wang, Y. Hou, Y. Zhan, Y. Jiang, Z. Liu, X. Cao, P. Chen, Z. Liu, X. Chen, Y. Tao, C. Xu, J. Mao, C. Cheng, C. Li, Y. Hu, L. Wang, Y.E. Chin, Y. Shi, U. Siebenlist, X. Zhang, A microRNA 221- and 222-mediated feedback loop maintains constitutive activation of NFkappaB and STAT3 in colorectal cancer cells, Gastroenterology 147 (2014) 847–859 (e11).
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Original article
Circular BANP, an upregulated circular RNA that modulates cell proliferation in colorectal cancer Mingchen Zhua,c,1, Yijun Xub,1, Yun Chena,* , Feng Yanc,** a
School of Pharmacy, Nanjing Medical University, Nanjing 211166, China Department of Gastroenterology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China Department of Clinical Laboratory, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nanjing Medical University Affiliated Cancer Hospital, Nanjing 210009, China b c
A R T I C L E I N F O
Article history: Received 1 November 2016 Received in revised form 19 December 2016 Accepted 22 December 2016 Keywords: Colorectal cancer Circular RNA BANP Proliferation siRNA
A B S T R A C T
Circular RNAs (circRNAs) are recently identified as widespread and diverse endogenous noncoding RNAs that may harbor vital functions in human and animals. However, the role of circRNAs in the process of tumorigenesis and development of colorectal cancer (CRC) remains vague. Here we characterized the circRNA expression profile from three paired CRC cancerous and adjacent normal tissues by human circRNA array, and identified 136 significantly overexpressed circRNAs and 243 downregulated circRNAs in CRC cancerous tissues (>2-fold changes). We further validated one circRNA generated from Exon 5–11 of BANP gene, termed circ-BANP. In addition, RT-PCR result showed that circ-BANP was overexpressed in 35 CRC cancerous tissues. Knockdown of circ-BANP with siRNA significantly attenuate the proliferation of CRC cells. In summary, our findings demonstrated that dysregulated circ-BANP appears to have an important role in CRC cells and could serve as a prognostic and therapeutic marker for CRC. © 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Colorectal cancer (CRC) is one of the most frequent cancers worldwide, leading to 694,000 deaths in 2012 [1]. 5-year survival rate of patients with CRC was 90.1% for patients at early stage and only 11.7% for patients with distant metastasis [2]. To improve the prognosis and treatment response of patients, a better understanding of molecular mechanisms underlying the development of CRC is clinically important. Non-coding RNA (ncRNA), including miRNA, siRNA, piRNA and lncRNA, has been reported to be involved in epigenetic modification. For example, miRNAs play a vital role in the tumorigenesis and progress of CRC through direct interaction with the 30 -UTR of
Abbreviations: CircRNAs, Circular RNAs; CRC, Colorectal Cancer; ncRNA, Noncoding RNA; circ-BANP, Circular BANP; qRT-PCR, Quantitative RT-PCR; DAPI, 4,6diamidino-2-phenylindole; siRNA, Small interfering RNA; CCK-8, Cell Counting Kit8; FDR, False Discovery Rate. * Corresponding author at: School of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing, 211166, China. ** Corresponding author at: Department of Clinical Laboratory, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nanjing Medical University Affiliated Cancer Hospital, Baiziting 42, Nanjing, 210009, China. E-mail addresses: [email protected] (Y. Chen), [email protected] (F. Yan). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2016.12.097 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
their target mRNA and affecting the functions of oncogenes and tumor suppressor gene [3–5]. Recently, circular RNAs (circRNAs) have been reported as a novel type of non-coding RNA that formed by a junction of the 5’ end and 3’end [6,7]. Increasing evidence indicates that circRNA could serve as miRNA sponge and regulate gene expression [6–8]. It has been proved that circular RNA ciRS-7 could function as a miR-7 sponge, leading to reduction of miR-7 activity and upregulaion of miR-7-targeted genes in nervous system [9,10]. In esophageal squamous cell carcinoma, circular RNA cir-ITCH was downregulated in cancerous tissue and functions as sponge of miR-7, miR-17, and miR-214. Therefore, it could enhance the level of ITCH and then inhibit the Wnt/b-catenin pathway [11]. More recently, it has been demonstrated that circRNA could regulate the proliferation and survival of cancer cell [12,13]. To date, the validated functions of circRNA are the tip of the iceberg and thousands of circRNAs have been found with unknown functions in different tissues and cells [7]. It has been reported that circRNA was also involved in the CRC onset and progression. Circ_001569 was confirmed to promote proliferation and invasion via functioning as a sponge of miR-145 [14]. Even so, there is little evidence of the expression profile of circRNA in CRC and the role of circRNA in the process of CRC tumorigenesis remains vague. To address this question, we employed the circRNA array analysis to detect differential expression profiles of circRNAs in CRC cancerous tissues and
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adjacent tissues. We found that circular BANP (circ-BANP) was upregulated in CRC cancerous tissues compared with adjacent normal tissues and contributed to the proliferation of colorectal cancer cells. 2. Materials and methods 2.1. Cells HCT116 and HT29 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were cultured in McCoy’s 5A (modified) medium supplemented with 10% FBS and 50 U/ mL penicillin and streptomycin sulfate. 2.2. Tissue specimens The study was approved by the Clinical Research Ethics Committee of Nanjing medical university and written informed consent was obtained from all the patients. The paired tissue samples from cancerous and adjacent normal tissues were collected from 35 patients with CRC who underwent surgical resection between 2013 and 2015 at Jiangsu cancer hospital (Nanjing, China). All of the specimens were subsequently verified by histology. Patients who received preoperative chemotherapy were excluded. The samples were freshly frozen in liquid nitrogen and stored at 80 C. 2.3. Microarray analysis Total RNA from each sample was quantified using the NanoDrop ND-1000. The sample preparation and microarray hybridization were performed based on the Arraystar’s standard protocols. Briefly, total RNAs were digested with Rnase R (Epicentre, Inc.) to remove linear RNAs and enrich circular RNAs. Then, the enriched circular RNAs were amplified and transcribed into fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array (8 15 K, Arraystar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C.
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Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the R software package. Differentially expressed circRNAs with statistical significance between two groups were identified through Volcano Plot filtering. Differentially expressed circRNAs between two samples were identified through Fold Change filtering. Hierarchical Clustering was performed to show the distinguishable circRNAs expression pattern among samples. 2.4. Total RNA extraction, and RNase R digestion Total RNA was extracted using TRIzol reagent (Invitrogen, California, USA) according to the manufacturer’s recommendations. Extracted RNA was quantified using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). Total RNA was incubated for 15 min at 37 C with 3 units of RNase R (Epicentre) per 1 mg RNA. RNA was subsequently purified by phenol–chloroform extraction and reprecipitated in three volumes of ethanol. 2.5. Quantitative real-time RT-PCR cDNA was synthesized with the Reverse Transcription System (Promega, Madison, USA) using random primers. Quantitative RTPCR (qRT-PCR) analyses were carried out to detect circ-BANP expression using SYBR Premix Ex Taq (TaKaRa, Dalian, China), and b-actin was utilized as an internal control. Primers used in this experiment were: 50 - CAGGACGGTCAGCGTCGT-30 (forward), and 50 GGCACAGCGTTGCTAATGAC-30 (reverse) for circ-BANP, yielding a product of 138 bp; and 50 - GCATGGGTCAGAAGGATTCCT-30 (forward) and 50 -TCGTCCCAGTTGGTGACGAT-30 (reverse) for b-actin, yielding a product of 106 bp. 2.6. In situ hybridization We employed in situ hybridization to investigate the expression and intracellular location of circ-BANP in CRC tissue and cell lines. Tissue slides were deparaffinized with xylene, rehydrated through a graded series of ethanol. HT29 cells were grown on cover slips and fixed 48 h later. In situ hybridization was carried out using the
Fig. 1. circRNA array analysis of CRC tissues. (A) Hierarchical clustering revealed circRNA expression profile in 6 samples of cancerous and adjacent tissues. (B) The scatter plot indicated the variation in circRNA expression between normal (group-N) and CRC (group-D) samples. (C) The volcano plots suggested the fold-change values and p-values of the array data. The red point represents the significantly upregulated or downregulated circRNAs.
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Fig. 2. Validation of the circular transcript of BANP. (A) Total RNA were treated with RNase R. Circ-BANP was stably expressed in both RNase R() and RNase R(+) total RNA samples. (B) The result of 1% agarose gel electrophoresis of RT-PCR product displays a single lane. (C) RT-PCR was utilized to validate the expression of circ-BANP in cancerous and adjacent normal tissues from 35 CRC patients. Consistent with the array results, expression of circ-BANP in cancerous was upregulated significantly. (D) Sanger sequencing of the RT-PCR product. The sequence of product is consistent with the sequence of circ-BANP in GenBank. The red arrow indicates the backsplicing site of circBANP. (E) RNA in situ hybridization for circ-BANP (green) in paired colorectal cancer tissues ( 200). Nuclei were stained with DAPI (blue).
cirRNA Hybridization Kit (Foco, Guangzhou, China) according to the manufacturer’s instructions. Briefly, the slides were deparaffinized with 100% xylene and rehydrated in series alcohol, followed by incubating with 4% (v/v) paraformaldehyde for 30 min at room temperature. Afterwards, the slides were washed and incubated with prehybridization solution for 1 h at 55 C. Digoxin-labeled circ-BANP probe was denatured at 85 C for 5 min, subsequently added to each tissue section and hybridized for 24 h at 37 C. After washing with washing buffer, blocking was performed for 1 h at 37 C, and then the anti-digoxigenin HRP conjugate was added to
the sections at 37 C for 1 h in the humidified chamber. After washing with PBS, TSA-488 solution was added to the slides. The sections were counterstained with 20 mL 4,6-diamidino-2-phenylindole (DAPI) to stain the cell nucleus. 2.7. RNA interference Cell transfection was carried out by small interfering RNA (siRNA, GenePharma) into HCT116 and HT29 using LipofectamineTM 2000 (Invitrogen, MA, USA) in accordance with the
M. Zhu et al. / Biomedicine & Pharmacotherapy 88 (2017) 138–144 Table 1 The relationship between relative expression levels of circ-BANP in CRC cancerous tissues with clinical characteristics. Characteristics
Number
Median (Percentiles 25 75)
P value
Age 60 >60
22 13
2.39(0.72 8.29) 1.62(0.82 10.89)
0.669
Gender Male Female
24 11
2.22(1.44 10.77) 1.32(0.47 7.99)
0.27
Location Colon Rectum
18 17
1.87(0.55 7.21) 2.14(1.36 11.29)
0.32
Differentiation Well Moderate Poor
5 19 11
5.65(1.4 25.06) 1.51(0.52 4.63) 4.62(1.31 10.47)
0.4
Tumor invasion 1+2 3+4
5 30
2.64(1.14 4.63) 1.98(0.59 10.68)
0.15
Lymph node metastasis N0 N1-N2
21 14
1.51(0.45 6.11) 5.08(1.55 10.68)
0.1
TNM stage I II III IV
5 16 9 5
2.63(1.14 4.63) 1.46(0.29 10.82) 7.99(1.87 41.94) 1.86(0.89 6.79)
0.19
Serum CEA Positive Negative
18 17
1.68(0.52 8.2) 2.29(1.42 10.88)
0.23
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2.9. Colony formation assay HCT116 and HT29 cells (500 per well) were seeded in 6-well plates for 2 weeks. Subsequently, the colonies were stained with 1% crystal violet for 10 min after fixation with 4% paraformaldehyde for 5 min. Colonies were microscopically examined and counted. The assays were repeated 3 times. 2.10. Western blot assay Cells were lysed in lysis buffer (KeyGEN BioTECH, Nanjing, China), and the protein samples were subject to western blot as described [4]. Antibodies recognizing Akt (1:1000) and PhosphoAkt (Ser473) (1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). 2.11. Statistical analysis Statistical analyses were carried out using SPSS 16.0 for Windows (SPSS, Chicago, IL, USA). To examine significant differences of expression of circ-BANP in cancerous and adjacent normal tissues, Wilcoxon test was utilized. The association between circBANP expression and clinicopathological characteristics of colorectal cancer was analyzed by Mann-Whitney U test. Paired and independent T test were also used to determine the differences. Statistical differences were declared significant at P < 0.05. 3. Results 3.1. Expression profiles of circRNAs
manufacturer’s procedure. The siRNA sequences are as follows: circ-BANP: 50 -GAAUCAAGCAGAGCAUCGATT-30 (sense), 50 0 UCGAUGCUCUGCUUGAUUCTT-3 (antisense); negative control: UUCUCCGAACGUGUCACGUTT-30 (sense), 50 -ACGUGACACGUUCGGAGAATT-30 (antisense). 2.8. CCK-8 Cell proliferation rates were detected with Cell Counting Kit-8 (CCK-8, DOJINDO, Japan). 1 104 cells were cultured in each 96-well plate for 24, 48, 72, and 96 h, respectively. Subsequently, the absorbance at 450 nm was measured after incubation with 10 mL CCK-8 solution at 37 C for 1.5 h. All detections were carried out in triplicate.
CircRNA expression profiles were analyzed using a microarray to determine differentially expressed circRNAs in CRC cancerous and adjacent normal tissues (Fig. 1A). The scatter and volcano plots revealed the variation of circRNA expression between CRC cancerous tissues and adjacent normal tissues (Fig. 1B and C). Of the 2608 human circRNA analyzed, a total of 136 exhibited significantly overexpression and 243 circRNAs decreased in CRC cancerous tissues (>2-fold changes, respectively), compared with adjacent normal tissues. We found that one circRNA, circ-BANP, was remarkably upregulated in CRC cancerous tissues (>25.9-fold changes). Additionally, both p-value and FDR (False discovery rate) of circ-BANP were very low. The raw intensity of circ-BANP expression in tumor group and normal group was also homogeneous. Hence, circ-BANP was selected as a candidate circRNA for the further researches. 3.2. Expression of circ-BANP in CRC Subsequently, real-time PCR was utilized to validate the profile results. CircRNA was validated by RNase R digestion and RT-PCR.
Fig. 3. Expression and location of circ-BANP in CRC cell lines. (A) Expression of circ-BANP in CRC cell lines. (B) RNA in situ hybridization for circ-BANP (green) in HT29 ( 1000). Nuclei were stained with DAPI (blue).
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Fig. 4. Function of circ-BANP in CRC cells. (A, C) After transfection with siRNA and negative control for 48 h in HCT116 and HT29 cells, expression of circ-BANP was detected by RT-PCR. (B, D) Downregulation of circ-BANP repressed cell proliferation as determined by CCK-8 assays. OD, optical density. (E, F) Colony formation was performed to analyze cell proliferation. *P < 0.05. (G) The expression of Akt and p-Akt was detected by western blotting assay.
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We observed that circ-BANP was resistant to RNase R, whereas the linear control was not stable with RNase R treatment (Fig. 2A). The specificity of the RT-PCR was validated by 1% agarose gel electrophoresis and sanger sequencing (Fig. 2B and D). Using the divergent primers, we investigated the expression of circ-BANP in cancerous and adjacent normal tissues from 35 CRC patients by RTPCR. Similarly, the expression of circ-BANP in cancerous tissues was overexpressed, compared with adjacent normal tissues (P = 0.037, Fig. 2C). The potential association between circ-BANP expression and clinicopathologic features in 35 patients with CRC was also investigated. However, no correlation was observed between circ-BANP expression with age, gender, location, differentiation, TNM staging, and serum CEA (Table 1). In agreement with the PCR results, fluorescence in situ hybridization results showed that circ-BANP expression level was remarkably upregulated in CRC tissues, as compared to matched normal tissues (Fig. 2E). Taken together, our results show that circ-BANP is an abundant and stable circRNA expressed in CRC cancerous tissues. 3.3. Expression and location of circ-BANP in CRC cell lines We further used RT-PCR to determine the expression of circBANP in different CRC cell lines. The expression of circ-BANP was most overexpressed in HT29 and HCT116, especially in HT29 (Fig. 3A). Hence, we intend to select HT29 and HCT116 for the functional researches. Additionally, the location of circ-BANP in cellular localization was also examined via in situ hybridization. We observed that circ-BANP was mainly localized in cytoplasm (Fig. 3B). 3.4. Function of circ-BANP Next, we intended to explore the biological function of circBANP in CRC cells. Using siRNA targeting circ-BANP [15,16], the expression of circ-BANP was attenuated as expected (P = 0.01 for HCT116, P = 0.025 for HT29, Fig. 4A and C). We found that the proliferation of si-circ-BANP-group was repressed significantly, compared with negative control group in HCT116 and HT29 cells (Fig. 4B and 4D). To further confirm the impact of circ-BANP on CRC proliferation, the plate colony formation assay was performed. As expected, colony formation was significantly reduced in HCT116 and HT29 cells by siRNA transfection (P < 0.01; Fig. 4E and F). Moreover, Fig. 4G displays that knockdown of circ-BANP reduced the protein expression of p-Akt, whereas the total protein levels of Akt stayed unchanged. This result suggested that Akt pathway was also involved in the process of circ-BANP-induced cell proliferation. Together, these results confirmed that circ-BANP may contribute to the proliferation of CRC cells. 4. Discussion In this study, circRNA expression profiles in colorectal cancerous and adjacent normal tissues were determined by circRNA microassay, and a number of circRNAs were revealed to be dysregulated. Most importantly, we found that the expression of circ-BANP was significantly overexpressed in CRC cancerous tissues. The relationship between circ-BANP expression and CRC clinicopathologic features was also investigated. However, no correlation was observed. Knockdown using siRNA targeted to circBANP could suppress CRC cells proliferation. Collectively, circBANP was indicated to be involved in the biological processes of CRC cells. Previously, circRNAs were regarded as the result of splicing errors or by-products of mis-splicing until extensive deregulations of circRNAs were identified with high-throughput deep
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sequencing recently [17–22]. Currently, circRNAs were reported mainly as miRNA sponge and function as the regulators of miRNA activity, such as CDR1as, the testes-specific Sry gene, circHIPK3 and EIciRNAs [10,15,23]. With approximately 70 binding sites for miRNAs, CDR1as was considered to be an extreme case; however, most natural miRNA sponges contain only 1-2 miRNA binding sites [24]. Hence, circRNAs could have other potential functions besides miRNA sponges, such as regulation of transcription [25] and interactions with RNA binding proteins [8]. To our knowledge, this is the first report about circ-BANP in CRC and whether the circBANP could function as a miRNA sponge is still unknown. Evolving information suggests that circRNAs could serve multiple biological functions, and be related with pathogenesis of various diseases [12,26–29]. In the process of carcinogenesis and cancer progression, circHIPK3 RNA was demonstrated to promote cell growth in human cells [15]. circ-Foxo3 could regulate cell cycle progression and repress cell proliferation [30]. Similarly, in our study we also found circ-BANP could enhance CRC cells proliferation. Circ-BANP is produced by back-splicing reactions of parent BANP mRNA that covalently link the 30 end of exon 11 to the 50 end of an upstream exon 5. BANP encodes a protein that binds to matrix attachment regions. It has been demonstrated that BANP (also known as SMAR1) interacts with HDAC1 associated repressor complex at Cyclin D1 promoter and allows histone deacetylation to cause its transcriptional repression [31]. Therefore, BANP could function as a tumor suppressor and cell cycle regulator. It has been demonstrated that PI3 K/Akt pathway plays a critical role in regulating colorectal cancer cell cycle and cell proliferation [32,33]. In this study, we found that si-circ-BANP transfection impeded the expression of p-Akt. Hence, we speculated that circ-BANP could enhance the growth of colorectal cancer cell by PI3 K/Akt pathway. Further more studies are desired to validate the mechanism of circBANP on cell growth. In conclusion, we conducted circular RNA profiles to identify circ-BANP as being frequently upregulated in CRC cancerous tissues. The biological function of circ-BANP was convinced to be related with cell proliferation. Hence, circ-BANP could serve as a potential diagnostic and therapeutic marker for CRC. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (21475063, 81601843, 81602126, 21675089, 21175071, 21605086), the Jiangsu Health Department (H201325), Program of Jiangsu Provincial Medical Innovation Teams, the Foundation of Jiangsu Cancer Hospital (ZM201501), the Project sponsored by SRF for ROCS, SEM (39), the Jiangsu Six-type Top Talents Program (D), the Open Foundation of Nanjing University (SKLACLS1102) and the Foundation of Nanjing medical university (2015NJMU050). References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics 2012, CA. Cancer J. Clin. 65 (2015) 87–108. [2] 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 (2012) 220–241. [3] S. Liu, X. Sun, M. Wang, Y. Hou, Y. Zhan, Y. Jiang, Z. Liu, X. Cao, P. Chen, Z. Liu, X. Chen, Y. Tao, C. Xu, J. Mao, C. Cheng, C. Li, Y. Hu, L. Wang, Y.E. Chin, Y. Shi, U. Siebenlist, X. Zhang, A microRNA 221- and 222-mediated feedback loop maintains constitutive activation of NFkappaB and STAT3 in colorectal cancer cells, Gastroenterology 147 (2014) 847–859 (e11).
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