Hsa_circ_0046264 up-regulated BRCA2 to suppress lung cancer through targeting hsa-miR-1245

Objective Lung cancer had been leading mounts of deaths worldwide. Advances in genes microarray had helped human further understand genes and identify novel circular RNAs. This study aimed at investigating the biological functions and molecular mechanisms of hsa_circ_0046264 in lung cancer which may be helpful in lung cancer early diagnosis and clinical treatment. Methods Gene microarray data screened the differential gene of hsa_circ_0046264 and its downstream genes were found by bioinformatics analysis and verified by luciferase reporter assay. QRT-PCR and Western blot was used to detect the RNA and protein levels respectively. RNase R digestion confirmed the existences of circular RNA. Cell viability, invasion and apoptosis were determined by MTT assay, flow cytometry and DNA damage assay. Tumor formation in nude mice and immunohistochemistry proved the functions of hsa_circ_0046264 in vivo. Results Hsa_circ_0046264 and BRCA2 were down-regulated in lung cancer tissues while miR-1245 was up-regulated. Hsa_circ_0046264 induced apoptosis but inhibited proliferation and invasion of lung cancer cells through targeting miR-1245 to up-regulate BRCA2. Hsa_circ_0046264 inhibited the tumor growth in vivo. Conclusion Hsa_circ_0046264 was a tumor suppressor in lung cancer. Overexpression of hsa_circ_0046264 could up-regulate BRCA2 expression through down-regulating of miR-1245. Electronic supplementary material The online version of this article (10.1186/s12931-018-0819-7) contains supplementary material, which is available to authorized users.


Introduction
Lung cancer mortality ranks the highest in cancer-related death, and the five-year survival rate is only about 20% [1]. The development of target small molecule inhibitors provides an alternative treatment strategy to chemotherapy for lung cancer patients, with improved survival rate and life quality [2,3]. The exploration of the molecular mechanisms in lung cancer is very important for developing new strategies of diagnosis and treatment to this disease [4]. Novel therapeutic targets together with prognostic biomarkers for lung carcinogenesis have been discovered due to the development of molecular biology techniques [5]. Therefore, detailed understanding of the molecular mechanisms and searching for innovative targets are essential for the improvement of lung cancer treatment.
Circular RNAs (circRNAs) are featured with enormous abundance, evolutional conservation and relative stablity in cytoplasm that confer numerous potential functions, such as acting as competing endogenous RNAs (ceR-NAs) for encoding RNAs, sponging microRNAs (miR-NAs) to regulate miRNAs' downstream genes [6,7]. Hansen et al. identified ciRS-7, a circular RNA, which acts as a designated miR-7 inhibitor/sponge, has conceptually changed the mechanistic understanding of miRNA networks in cancers [8]. With binding capacities to miR-NAs, some circRNAs were demonstrated to have great influence on lung cancer progression. For instance, significant down-regulation of circRNA ITCH was discovered in lung cancer and up-regulating its expression could markedly elevate its parental cancer-suppressive gene ITCH through sponging oncogenic miR-7 and miR-214 [9]. In non-small cell lung cancer, hsa_-circ_0043256 could be up-regulated by cinnamaldehyde and functioned as a miR-1252 sponge to positively affect ITCH expression [10]. Hsa_circ_0013958 was up-regulated in lung adenocarcinoma and identified as a sponge of miR-134, thus up-regulating oncogenic cyclin D1 [11]. Since studies on circRNAs are not sufficient, in-depth researches about their functions in lung cancer are still needed.
As small (up to 21 nucleotides) noncoding RNAs, miR-NAs can regulate their downstream genes through binding to the 3'UTRs of the target genes [12]. MiR-1245 was considered as an oncogene because it was demonstrated as a potent negative regulator of the tumor suppressor protein BRCA2 [13]. MiR-1245 attenuated the expression of NKG2D, an activating receptor involved in tumor immunosurveillance, in natural killer cells [14]. It directly targeted BRCA2 and suppressed its translation in breast cancer [15]. As a tumor suppressor gene, BRCA2 mutation was reported to be associated with breast cancer and ovarian cancer development [16,17]. Its expression was frequently down-regulated in lung adenocarcinomas [18]. Therefore, miR-1245 and its target gene BRCA2 might affect the progression of lung cancers.
In the present study, we evaluated the expression profiles of circRNAs in lung cancer tissues and found the significant downregulation of hsa_circ_0046264. The biological functions of hsa_circ_0046264 and its regulatory relationships with miR-1245 and BRCA2 were investigated. The results indicated the suppressive effect of hsa_circ_0046264 in lung cancer and provided novel insights into the crucial role of circRNA.

Microarray processing
The expression profiles of circRNAs in 10 tissue samples (5 lung cancer tissues and 5 non-tumor adjacent tissues) were analyzed by microarray GSE101586 from GPL19978 platform. Five female lung patients with no smoking history were selected to profile circular RNA expression with microarrays. CircRNAs differentially expressed between tumor and normal tissues were identified via fold change filtering and P value. The screening condition was fold change > 2 and P < 0.05. Hierarchical clustering was performed to show the distinguishable circRNA expression pattern among samples.

Participants and tissue samples
In total, 99 lung cancer tissue specimens, containing 23-paired specimens, were obtained from Affiliated Calmette Hospital of Kunming Medical University (Yunnan, China). The pathological information refered in this paper was provided in Table 1

RNase R digestion
Total RNA (5 μg) was incubated for 15 min at 37°C with 3 U/ug of RNase R (Epicentre Biotechnologies, Madison, WI, USA). The RNase R digestion reaction was performed twice following previously published procedures [19].  Table 2.

Cell proliferation
Lung cancer cells transfected with hsa_circ_0046264 were plated into 96-well plates (

Cell invasion
Invasive capacity was detected using Transwell cell culture inserts (8-mm pores; Corning, NY, USA) in 24-well plates. After transfection, cells were resuspended in

DNA damage assay
After transfected by hsa_circ_0046264 for 48 h, lung cancer cells were digested by pancreatin and fixed on glass slides by polylysine. The cells were incubated with 25 μM Etoposide for 5 h (Treated) or solvent-only for control (Non-treated). Cells were fixed with 4% formaldehyde (10 min) and then blocked in 1% BSA/10% Fig. 1 Hsa_circ_0046264 was down-regulated in lung cancer tissues. a Differentially expressed circRNAs in lung cancer tissues were presented in a volcano plot. The screening condition was fold change > 2 and P < 0.05. b The heat map reflected 50 significantly differentially expressed circRNAs in lung cancer tissues. Hsa_circ_0046264 was lowly expressed in cancer tissues. c The overall survival (OS) of lung cancer patients with high or low circ_0046264 level. A low level predicted a worse prognosis outcome. d The expression level of hsa_circ_0046264 decreased with the deterioration of lung cancer (from stage I to stage IV). * P < 0.05, *** P < 0.001, compared with I (N = 5) group with lung cancer tissues in Stage I. e-f RNase R can digest liner RNA GAPDH but not circRNA hsa_circ_0046264 as the relative expression of GADPH in lung cells decreased after RNase R digestion while that of hsa_circ_0046264 had no significant difference. ** P < 0.01, *** P < 0.001, compared with MOCK group. g The relative expression of hsa_circ_0046264 was lower in lung cancer cells A549 and 59-D than that in normal lung cells. * P < 0.05 compared with HLF-a cells

Xenograft nude mouse model
Male BALB/c nude mice (n = 10, 5-8 weeks old, 18-20 g) were obtained from Experimental Animal Research Center of the Affiliated Calmette Hospital of Kunming Medical University. Empty or recombinant pLO-ciR was transfected into 95-D lung cancer cells, which was then injected into the posterior axilla of nude mice (1 × 10 7 cells / mL, 0.2 mL for each mouse). The mice were randomly divided into two groups: control group injected with empty pLO-ciR (n = 5) and over-circ group injected with recombinant pLO-ciR (n = 5). After 2 weeks, tumors were measured every week and calculated by the formula V = ab 2 /2 (a represents tumor length and b represents tumor width). After 7 weeks, mice were killed and tumors were collected and weighted. Total RNA of tumor tissues was extracted by TRIzol reagent for qRT-PCR. The tumor tissues were embedded by paraffin for immunohistochemistry. All procedures were performed following the ethical standards and under the protocol approved by the Affiliated Calmette Hospital of Kunming Medical University.

Immunohistochemistry
Tumor tissue sections from nude mice were deparaffinized and rehydrated with xylene and ethanol solutions, respectively. They were soaked in citrate buffer (pH = 6.0) and heated by microwave at 95°C for 3 min twice to retrieve antigen and incubated in 3% H 2 O 2 for 10 min after cooling down. Then sections were incubated with nonimmune goat serum for 15 min, followed by primary and secondary antibodies incubation at 4°C for 12 h and 30-40 min respectively. The primary antibody was anti-BRCA2 (ab27976, Abcam, Cambridge, MA, USA) diluted to 1:100 and the secondary one was goat anti-rabbit IgG-HRP (1:1000, ab6721, Abcam). After washing by PBS, each section was added with 1 drop newly prepared diaminobenzidine for coloration (20 min) and hematoxylin for counterstain (30 s). Finally, after being dehydrated with ethanol, sections were transparentized by xylene, sealed with resinene and air dried for observation.

Statistical analysis
Software GraphPad Prism 6.0 was used for data analysis. Values were expressed by mean ± standard deviation. Student's t test and One-Way ANOVA were used to evaluate significance between two groups and among multiple groups, respectively. All experiments were independently repeated three times. P < 0.05 meant statistically significance.

Hsa_circ_0046264 was down-regulated in lung cancer
Bioinformatic analysis screened out differential expressed circRNAs in lung cancer tissues (Fig. 1a) while 50 among them with the most significant differential expressions were displayed in Fig. 1b. Hsa_circ_0046264 was significantly down-regulated in tumor tissues (Fig.  1b). Low hsa_circ_0046264 level predicted a better survival outcome (Fig. 1c). The expression levels of hsa_-circ_0046264 in lung cancer tissues with different tumor stages also proved its low expression, and further indicated that hsa_circ_0046264 expression decreased with the deterioration of lung cancer (Fig. 1d).
To further investigate the regulatory mechanism, 19 potential target miRNAs of hsa_circ_0046264 were screened out by the website circinteractome (Table 3). Furthermore, three binding sites of AGO2 protein (the protein that widely expresses in organisms and serves as a crucial composition of RNA induced silencing complex) on hsa_circ_0046264 were found ( Table 4). The target gene of miR-1245 was determined as BRCA2 through the database miRTarBass. In both HLF-a and MRC-5 cell lines, after RNase digestion, the content of GAPDH significantly decreased, whereas the content of has_circ_0046264 did no significantly differ from that before RNase digestion (Fig. 1e and f). Compared with normal human lung cell lines MRC-5 and HLF-a, the expression of circ_0046264 was much lower in human lung cancer cell lines A549 and 95-D (Fig. 1g).

Hsa_circ_0046264 overexpression down-regulated miR-1245 and up-regulated BRCA2
The expression of miR-1245 was much higher, whereas the expression of BRCA2 mRNA was much lower in III + IV grade cancer tissues than in I + II cancer tissues ( Fig. 2a and  b). We have verified the target relationship between circ_0046264 and miR-1245 as well as miR-1245 and BRCA2. The deletion mutation (MUT1) and point mutation (MUT2) of circ_0046264 and BRCA2 were used to be as the control of wild type vectors (WT) which showed significant suppressions of luciferase activity by miR-1245( Fig. 2c and d). The transfection efficiency of circ264, sicirc, miR-1245 and antimiR-1245 were tested in both A549 and 95-D cell lines ( Fig. 2e and f, h and i). In addition, the influence of modulation of circ_0046264 and miR-1245 expression on BRCA expression was investigated. The results showed that the overexpression of circ_0046264 or the suppression of miR-1245 promoted the expression of BRCA2, whereas the suppression of circ_0046264 led to the suppression of BRCA2 expression (Fig. 2g, j, 3a).

Hsa_circ_0046264 overexpression suppressed lung cancer development in vitro
Overexpression of hsa_circ_0046264 or suppression of miR-1245 significantly suppressed the invasion and viability of lung cancer cells; and the suppression of hsa_-circ_0046264 or the overexpression of miR-1245 significantly suppressed cell invasion and viability (Fig. 3b, 4a and b). On the contrary, the apoptosis of cancer cells was promoted after being transfected with circ_46264 and inhibited after the cells were knocked down of circ_46264. The overexpression of miR-1245 significantly enhanced cell apoptosis as well ( Fig. 4c and  d). Besides, γ-H2AX is a marker protein of DNA double-strand damage. The DNA damage of lung cancer cells was also aggravated as more γ-H2AX was observed in circ264 group (Additional file 1: Figure. S1). In brief, the suppressive effect of hsa_circ_0046246 in lung cancer was proved by the in vitro experiments.

Hsa_circ_0046264 overexpression inhibited tumor growth in vivo
Lung tumors formed in nude mice of hsa_circ_0046264 overexpression group were much smaller than that formed in control group (Fig. 5a). Similarly, hsa_-circ_0046264 overexpression also diminished the tumor weight (Fig. 5b). The tumor tissues obtained from the nude mice had an elevated expression level of hsa_-circ_0046264 in circ264 group, accompanied by lower miR-1245 and higher BRCA2 expression levels ( Fig. 5c  and d). The protein expression level of BRCA2 also (See figure on previous page.) Fig. 2 Hsa_circ_0046264 overexpression down-regulated miR-1245 and up-regulated BRCA2. a The expression level of miR-1245 was higher in advanced lung cancer tissues (stage III and stage IV) than that in early-stage cancer tissues (stage I and stage II). * P < 0.05, compared with I + II (N = 9) group with lung cancer tissues in Stage I + II. b The expression level of BRCA2 was lower in early-stage lung cancer tissues than that in advanced cancer tissues. * P < 0.05, compared with I + II (N = 9) group with lung cancer tissues in Stage I + II. c The dual luciferase activity decreased in HEK293 cells co-transfected with hsa_circ_0046264 WT and miR-1245 mimics, but almost did not change in cells co-transfected with hsa_circ_0046264 MUT and miR-1245 mimics. * P < 0.05, compared with miR-con group. WT: wild type; MUT1: deletion mutation; MUT2: point mutation. d The dual luciferase activity decreased in HEK293 cells co-transfected with BRCA2 3'UTR WT and miR-1245 mimics, but almost not changed in cells co-transfected with BRCA2 3'UTR MUT and miR-1245 mimics. * P < 0.05 compared with miR-con group. (E, H) The relative expression of hsa_circ_0046264 increased in lung cancer cells transfected with the circ-vector pCD2.1-ciR (circ264 group). Control group was transfected with empty pLO-ciR vector. *** P < 0.001, compared with control group. ## P < 0.01, compared with circ264 group. f, i The relative expression of miR-1245 in lung cancer cells decreased in circ264 group and antimir1245 group but increased in sicirc groups and miR-1245 group. *** P < 0.001, compared with control group. ## P < 0.01, compared with circ264 group. g, j The expression of BRCA2 mRNA increased in circ-vector group. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with control group. # P < 0.05, compared with circ264 group Fig. 3 The overexpression of hsa_circ_0046264 promoted BRCA2 expression and cancer cell invasion. a The overexpression of hsa_circ_0046264 and the suppression of miR-1245 promoted BRCA2 expression. The simultaneous transfection of hsa_circ_0046264 and miR-1245 did not result in significant change of BRCA2 expression. * P < 0.05, compared with control group. # P < 0.05, compared with circ264 group. b The overexpression of hsa_circ_0046264 and the suppression of miR-1245 promoted cell invasion. The simultaneous transfection of hsa_circ_0046264 and miR-1245 did not result in significant change of cell invasion. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with control group. # P < 0.05, compared with circ264 group Fig. 4 Hsa_circ_0046264 overexpression suppressed lung cancer development in vitro. a-b Cell viability of A549 and 95-D cells decreased in circ264 group and antimir1245 group but increased in sicirc groups and mir1245 group tested by CCK8 assay. * P < 0.05, compared with control group. c-d Apoptosis of A549 and 95-D cells was promoted in circ264 group and antimir1245 group but inhibited in sicirc groups and mir1245 group. * P < 0.05, compared with control group increased, once again proving the up-regulation effect of hsa_circ_0046264 on BRCA2 expression (Fig. 5e).

Discussion
Significant down-regulation of hsa_circ_0046264 was found in lung cancer tissues. Overexpression of hsa_circ_0046264 effectively suppressed the proliferation and invasion of lung cancer cells, induced apoptosis. MiR-1245 was suppressed by hsa_circ_0046264, while BRCA2, the target gene of miR-1245, was up-regulated. Hsa_circ_0046264 inhibited lung cancer development through acting as a ceRNA which regulated miR-1245 and indirectly activated BRCA2.
Hsa_circ_0046264 and BRCA2 were lowly expressed in lung cancer tissues while miR-1245 was high expressed. Hsa_circ_0046264 could bind miR-1245, and since BRCA2 was the target gene of miR-1245, BRCA2 was indirectly up-regulated by hsa_circ_0046264. The regulatory effect of miR-1245on BRCA2 expression has been reported in previous study as the 3'UTR region of BRCA2 was combined with miR-1245 [15]. We confirmed the target relationship between miR-1245 and BRCA2, and further uncovered their associations with hsa_circ_0046264. Circular RNAs affect cancer processes mainly by serving as ceR-NAs, which cross-talk and co-regulate with other genes by competing for binding of miRNAs [20]. CeRNAs involve in the pathogenesis of various common cancers [21]. Based on the altered genes in patients with lung cancer, Jin et al. established a competing endogenous network with several miRNAs and their interacting circRNAs [5]. Here, hsa_-circ_0046264 and BRCA2 shared common target miRNA, miR-1245, and up-regulation of miR-1245 in lung cancer resulted from the low expression of hsa_circ_0046264 caused the down-regulation of BRCA2.
The biological functions of hsa_circ_0046264 were inspected in detail in this study. Hsa_circ_0046264 expression was decreased with the deterioration of lung cancer. Tumor growth in hsa_circ_0046264 overexpression group (circ264) was slower than that in control group. Tumor volume and weight in circ264 group were smaller than that in control group. ** P < 0.01, compared with control group. c The relative expression of hsa_circ_0046264 was higher in circ264 group than that in control group. * P < 0.05, compared with control group. d The relative expression of miR-1245 decreased while that of BRCA2 increased in tumor tissues of circ264 group. * P < 0.05, *** P < 0.001 compared with control group. e Immunohistochemistry result showed that the expression of BRCA2 protein increased in tumor tissues of circ264 group Overexpression of hsa_circ_0046264 attenuated proliferation, aggravated apoptosis and DNA damage of lung cancer cells as well as inhibited tumor growth. Although circRNAs were suggested to be associated with some kinds of common human cancers, such as circRNAs TCF25 and MYLK in bladder cancer [22,23], hsa_circ_0005986 and hsa_-circ_0004018 in hepatocellular carcinoma [22,24] and hsa_-circ_0000520 in gastric cancer [25], little is known about the roles circRNAs play in lung cancer. Zhao et al. investigated the expression profiles of circRNAs in early-stage lung adenocarcinoma and found that 357 circRNAs were dysregulated and five most significant ones were demonstrated as potential biomarkers [26]. CircRNA ITCH inhibited the activation of Wnt/β-Catenin signaling pathway in lung cancer cells by down-regulating ITCH, thus restraining cancer cell proliferation [9]. In contrast, hsa_circ_0013958 and hsa_-circ_0000064 were up-regulated in lung cancer, and their aberrant expressions promoted migration and invasion activities of lung cancer cells and attenuated apoptosis [11,27]. In this study, we identified a novel tumor suppressive circRNA, hsa_circ_0046264. Both in vitro and in vivo results proved its inhibitory effect on the deterioration of lung cancer, suggesting that hsa_circ_0046264 may be a potential target in lung cancer treatment.
Overexpression of hsa_circ_0046264 could diminish the volume and weight of lung tumors formed in nude mice. Meanwhile, miR-1245 expression was reduced in tumor tissues obtained from mice in hsa_-circ_0046264 overexpression group, and the mRNA and protein expressions of BRCA2 were elevated. These results implied that miR-1245 is a potential tumor promotor and BRCA2 is a suppressor. The influence of miR-1245 in lung cancer has not been identified yet. As for BRCA2, it was considered as a susceptibility gene whose mutation was frequently found in breast cancer and ovarian cancer [28,29]. In lung cancer, Yang et al. suggested that it was associated with a high risk of lung adenocarcinoma [30]. However, according to Lin et al.'s research, polymorphism of rs144848 in BRCA2 could reduce the lung cancer risk [31]. In this study, we found BRCA2 was down-regulated in lung cancer tissues and supposed that it was a suppressive gene.

Conclusion
In summary, hsa_circ_0046264 was proved to be able to inhibit viability and invasion, induce apoptosis of lung cancer cells. It was down-regulated in tumor tissues and successfully restrained tumor growth in vivo. The suppressive effect of hsa_circ_0046264 resulted from its combination with miR-1245, which could indirectly up-regulate miR-1245's target gene, BRCA2. The results demonstrated that hsa_-circ_0046264 may be a novel target in lung cancer.