circ_SMAD2 regulate colorectal cancer cells proliferation through targeting miR-1258/RPN2 signaling pathway

Circular RNAs (circRNAs) are associated with various diseases, including cancers. However, their roles in colorectal cancer (CRC) have not been established. Hsa_circ_0000847 (circ_SMAD2) is a novel circRNA that was found to be elevated in CRC cell lines and tissues. High circ_SMAD2 levels were positively correlated with CRC clinicopathological features. Functional assays revealed that circ_SMAD2 enhanced CRC cell invasion, proliferation, and tumor growth. Mechanistically, circ_SMAD2 elevated Ribophorin II (RPN2) levels by inhibiting miR-1258. Therefore, circ_SMAD2 is a potential indicator for CRC progression.


Introduction
Globally, colorectal cancer (CRC) is the 3 rd most common malignancy. It is highly associated with cancer related mortalities [1,2]. Therapeutic options for CRC include surgical resection, radiotherapy, chemotherapy, and vital adjuvant treatment. These options significantly improve survival outcomes [3,4]. However, the 5-year survival of patients with distal metastasis is low (12.5%) [5]. To identify novel biomarkers and therapeutic targets for CRC, more studies aimed at elucidating its molecular basis are needed.
Circular RNAs (circRNAs) modulate multiple biological processes in CRC [6,7]. Ge et al reported that down-regulation of circMTO1 suppresses the proliferative and invasive abilities of CRC through the Wnt/beta-catenin signaling pathway [8]. In addition, circITGA7 inhibits CRC cell proliferation and metastasis by suppressing the Ras signaling pathway while enhancing ITGA7 expression [9]. Elevated hsa_circ_0136666 levels promote the invasion and proliferation of CRC by molecularly sponging miR-136 [10]. However, the roles of hsa_circ_0000847 (circ_SMAD2) in the progression of CRC have not been established. circ_SMAD2 have been shown to be elevated in CRC, therefore, we hypothesized that circ_SMAD2 may be oncogenic. miRNAs promote CRC development and progression [11]. Li et al documented that miR-205 suppresses CRC metastasis by targeting CREB1 [12]. The down-regulation of miR-143 in CRC has been shown to suppress cell progression by regulating the expression of MMP7 [13]. miR-1258 is significantly down-regulated in CRC [14,15]. However, the relationships between circ_SMAD2 and miR-1258 have not been determined.
This study aimed at determining the roles of circ_SMAD2 in CRC progression and metastasis, as well as its mechanism of action.

Clinical specimens
This study was approved by the ethical committee of Henan Provincial People's Hospital. The participants who agreed to be enrolled in this study were required to sign an informed consent. A total of 53 pairs of CRC tissue and adjacent normal tissue (ANT) specimens were collected between 2015 and 2016. Before the surgical procedures, none of the study participants had undergone radiotherapy, chemotherapy, or targeted therapy. After surgical resection, the specimens were immediately frozen in liquid nitrogen.

CCK-8 assay
Cells were seeded in 96-well plates (2×10 3 cells/well) and allowed to adhere. 10μL of Cell Counting Kit-8 solution (CCK-8, Dojindo, Japan) were added into each well and cultured for 2 h. Optical density was recorded at 450nm by a microplate reader. Proliferation rates were measured at 24, 48, 72 and 96 h after transfection.

Flow cytometry assay
The transfected cells were dissociated with trypsin and twice rinsed with PBS 1X. Cells were then fixed in 70% ethanol at 4 °C overnight. Cell cycle analyses were done using a FACS Calibur flow cytometer (BD Biosciences) after propidium iodide (PI) staining for 30 min in the presence of RNase A (KeyGEN Biotech, Nanjing, China).

Colony formation assay
After transfection, cells were cultured in 6-well plates for 2 weeks in media containing 10% FBS. They were fixed in methanol and stained with 0.5% crystal violet. The number of colonies formed were then counted.

Transwell invasion assay
Cell invasion was explored using the Transwell chambers with Matrigel (Millipore, Billerica, MA, USA). Briefly, the upper chambers of Transwell inserts were covered with 1×10 5 transfected cells resuspended in 200 μL FBS-free DMEM medium. A volume of 500 μL DMEM+10% serum was added to the lower chamber. After 48 h, cells seeded in the upper and lower chambers were obtained using cotton swabs, fixed in 4% PFA and stained with 0.1% crystal violet (Beyotime, China) for 30 min. Stained cells were counted using an upright microscope (Nikon, 200× magnification).

Animal studies
BALB/c female nude mice, weight 16-20 g, aged 6-8 weeks, were bought from Laboratory Animal Co., Ltd. (Beijing Vital River; Beijing, China). 5×10 6 transfected cells were subcutaneously injected into mice right flanks. During the experimental period, tumor volumes were calculated every 7 days by the equation: volume = 0.5 × length × width 2 . 35 days after inoculation, mice were euthanized. Mice experiments were approved by the ethics committee of Henan Provincial People's Hospital and we performed following the guidelines of the National Institute of Health.

Actinomycin D, RNase R and Western blot assays
All these assays were performed as previously described [16,17].

Statistical analysis
All study data were analyzed using SPSS software (version 20.0) and presented as mean ± SD. Differences between experimental groups were analyzed Student's t-test or one-way ANOVA. Statistical significance was set at p≤0.05.

Circ_SMAD2 is significantly upregulated in CRC
Analysis of circRNA expression in the CRC microarray datasets (GSE121895 and GSE142837) revealed an aberrant expression of Hsa_circ_0000847 and hsa_circ_0006667 ( Figure 1A). Figure 1B-D shows the expression levels of hsa_circ_0000847 and hsa_circ_0006667 in CRC cell lines and tissues. Figure  1E-F shows that hsa_circ_0000847 was significantly upregulated in metastatic and advanced CRC.
Hsa_circ_0000847 is located on chromosome 18, and is comprised of exon 2-5 of its host gene, SMAD2 ( Figure 2A). Circ_SMAD2 was found to be resistant to RNase R digestion ( Figure 2B). The amplification of circ_SMAD2 with divergent primers revealed its amplification in cDNA, but not in gDNA ( Figure 2C). Additionally, circ_SMAD2 was shown to be mainly localized in the cytoplasmic fraction. However, it was also present in the nuclear fraction ( Figure 2D).

Circ_SMAD2 knockdown inhibits CRC cell proliferation and invasion
To determine the role of circ_SMAD2 in CRC, 2 siRNAs against its unique back splicing junction were used. The antisense spliced junction siRNAs significantly suppressed circ_SMAD2 expression without affecting linear SMAD2 mRNA levels ( Figure  3A-B).
The CCK-8 assay revealed that down-regulation of circ_SMAD2 suppressed the proliferation of SW480 and LoVo cells ( Figure 3C-D). Figure 3E-F shows that upon circ_SMAD2 suppression, the cell cycle was arrested at G1/S phases in SW480 and LoVo cells ( Figure 3E-F). In addition, circ_SMAD2 knockdown significantly suppressed in vitro CRC cell invasion ( Figure 3G-H).
The roles of miR-1258 in CRC were also evaluated. Figure 5A-C shows that miR-1258 was significantly down-regulated in CRC tissues. qRT-PCR analysis revealed that miR-1258 levels were significantly low in CRC cell lines compared to normal cells ( Figure 5D). Low miR-1258 levels were positively correlated with poor CRC prognosis ( Figure 5E). Moreover, colony formation and wound healing assays confirmed that the over-expression of miR-1258 suppressed the in vitro migration and proliferation of CRC cells (Figure 5F-G). Therefore, circ_SMAD2 directly binds miR-1258 in CRC cells.

RPN2 is a downstream target of miR-1258
Next, we used Targetscan, Starbase, miRTarBase and MircoT-CDS to predict miR-1258 targets and found that it could bind to RPN2 (Figure 6A-B). Luciferase reporter assays indicates that miR-1258 markedly suppressed luciferase activity of WT-RPN2 group ( Figure 6C). TCGA dataset analysis revealed high RPN2 expression in most tumors, including CRC ( Figure 6D). IHC analysis revealed high RPN2 levels in CRC tissues (T) relative to normal tissues (N) ( Figure 6E). Kaplan-Meier analysis showed that CRC patients with high RPN2 levels exhibited poor overall survival ( Figure 6F). Moreover, RT-qPCR analysis revealed that miR-1258 overexpression significantly reduced RPN2 levels in CRC cells ( Figure 6G).

circ_SMAD2 promotes CRC progression by regulating miR-1258/RPN2 axis
Next, we investigated whether circ_SMAD2 promotes CRC proliferation and invasion via the miR-1258/RPN2 axis. SMAD2 knockdown upregulated RPN2 expression, while miR-1258 suppression or RPN2 upregulation significantly reversed these effects ( Figure 7A-B). Moreover, rescue experiments showed that down-regulation of miR-1258 or the up-regulation of RPN2 partially abolished the circ_SMAD2 silencing-induced reduced cell proliferative and invasive abilities of CRC cells (Figure 7C-E).

circ_SMAD2 modulates progression of CRC cells in vivo
To determine the effects of circ_SMAD2 on in vivo tumorigenesis, CRC cells were stably transfected with sh-NC, or sh-circ_SMAD2 and subcutaneously xenografted into nude mice. Compared to the sh-NC CRC cells, the sh-circ_SMAD2 CRC cells exhibited markedly small tumors ( Figure 7F-G). Circ_SMAD2 knockdown significantly elevated miR-1258 levels and suppressed RPN2 expression ( Figure 7H). These findings imply that circ_SMAD2 depletion impedes in vivo tumor growth. Therefore, circ_SMAD2 enhances CRC progression through the miR-1258/RPN2 axis ( Figure 7I).

Discussion
Recent developments in high-throughput sequencing technology have generated a lot of interest in the role of circRNAs in cancer [18]. CircRNAs influence tumorigenesis by modulating various biological processes, including glycolysis, metastasis, and cell cycle [19]. In hypoxic conditions, circ_MAT2B promotes liver cancer glycolysis and malignancy through the miR-338-3p-PKM2 axis [20]. In addition, circRNA_0025202 regulates tamoxifen sensitivity in breast cancer by targeting the miR-182-5p-FOXO3a axis [21]. The circ100284-miR-217-EZH2 axis influences arsenite-accelerated cell cycle in human keratinocytes during carcinogenesis [22]. In this study, hsa_circ_0000847 (circ_SMAD2), a novel circRNA, was found to be markedly upregulated in CRC tissues and cell lines. Elevated circ_SMAD2 levels were correlated with advanced clinical features and poor CRC prognosis. Moreover, circ_SMAD2 knockdown significantly suppressed the in vitro proliferative and invasive rates of CRC cells and reduced the tumor growth in vivo, suggesting that circ_SMAD2 influences CRC progression.
Bioinformatic analyses identified 5 putative circ_SMAD2 miRNA targets and miR-1258 for further characterization. miR-1258 modulates multiple biological processes. The overexpression of miR-1258 suppresses osteosarcoma cell proliferation and G0/G1 by targeting AKT3 [23]. In addition, it suppresses lung cancer proliferation and induces apoptosis by modulating the GRB2/Ras/Erk pathway [24]. Hsa_circ_0101432 enhances the proliferative and invasive ability of liver cancer by adsorbing mirR-1258 and miR-622 [25]. In this study, miR-1258 expression was suppressed in CRC cell lines and tissues. miR-1258 mimics inhibited the invasive and proliferative abilities of CRC cells. Therefore, miR-1258 confers anti-CRC effects. Circ_SMAD2 sponges miR-1258 in CRC.
Ribophorin II (RPN2) is an endoplasmic reticulum glycoprotein that modulates cell functions and signal transductions [26,27]. Elevated RPN2 levels have been documented to exhibit oncogenic functions in various cancers. Down-regulated RPN2 levels have been correlated with enhanced esophageal squamous cell carcinoma responses to docetaxel [28]. RPN2 promotes liver cancer metastasis and reduces autophagy by regulating STAT3 and NF-κB signaling pathways [29]. The in vitro invasive and proliferative abilities of colon carcinoma cells are hampered by the down-regulation of RPN2 [30]. However, the role of RPN2 in CRC progression has not been established. In this study, elevated RPN2 levels in CRC patients confirmed that RPN2 is a downstream target of miR-1258 in CRC cells. The overexpression of RPN2 reverses the effects of circ_SMAD2 silencing in CRC cells. This implies that circ_SMAD2/miR-1258/RPN2 is involved in CRC progression.
In conclusion, up-regulated circ_SMAD2 levels are associated with invasion and poor prognosis of CRC. In addition, circ_SMAD2 enhances malignancy by targeting miR-1258 to upregulate RPN2 expression in CRC. This is a potential therapeutic avenue against CRC.