Hypoxia-induced hsa_circ_0000826 is linked to liver metastasis of colorectal cancer

2.1 Cell culture and in vitro experiments

Human CRC cell lines (HCT116 and SW620) and 293T cells were purchased from American Type Culture Collection (ATCC). All the CRC cells were authenticated, and no mycoplasma contamination was detected. Cell was cultured at DMEM containing 10% FBS, 100 units penicillin and streptomycin per m in a humidified atmosphere of and 95% air at 37°C. For hypoxia treatments, CRC cells were cultured in a hypoxia chamber (with 1% O₂ at 37°C) (I-Glove, BioSpherix) for 24 hours.

2.2 RNAscope

Expression of Hsa_circ_0000826 in CRC cells was assayed with the BaseScopeTM Reagent Kit v2-RED (Advanced Cell Diagnostics) according to the manufacturer's instructions. Briefly, samples were incubated with Hsa_circ_0000826 probes at 40°C for 2 hours. After hybridization, a series of single amplification procedures were performed and finally visualized with Fast Red.¹¹ The number of puncta was calculated in 200 cells/group.

2.3 Lentivirus stable transduction

Hsa_circ_0000826 KO and Hsa_circ_0000826 OEX adenovirus particles with luciferase labeling were thawed at room temperature, mixed gently, and added to 2 × 10⁵ HCT116 or SW620 cells per well. After gently mixing, cells were incubated overnight. And cells treated with negative control lentivirus (LV-NC) were as the control. After 12 hours, culture medium was replaced with 2 mL of complete medium containing 10 µg/mL blasticidin S (Cat. No. ant-bl, Invitrogen) and replaced every 3-4 days until resistant cells can be identified. The remaining cells were collected, expanded, and then frozen down until usage. For experiments, cells were thawed and allowed to grow for three passages before use.

2.4 Cell migration and cell invasion assays

HCT116 and SW620 cells were serum-starved by cultivation in 0.5% FBS/DMEM media for 24 hours. To analyze migration capability, 1.5 × 10⁵ HCT116 or SW620 cells were seeded in the upper chamber in serum-free medium. To analyze invasion capability, chamber membranes were first coated with Matrigel (BD Bioscience) at a dilution of 3.3 ng/mL in medium without serum. Then, 1.5 × 10⁵ cells were seeded on the Matrigel in the upper chamber in serum-free medium. 10% FBS was placed as a chemo-attractant in the lower chamber. For cells cultured under normoxia, cells were incubated with complete media and exposed to 21% oxygen for 4 hours. For cells cultured under hypoxia, cells were incubated with complete media and exposed to 1% oxygen for 4 hours. After that, cell was detected in triplicate for each treatment after crystal violet staining. The absorbance was recorded at 560 nm with a microplate reader.

2.5 Animals

All animal protocols were conducted in accordance with the guiding principles for the care and use of animals approved by the animal ethics committee of Nanjing Medical University. The APC^Min/+ mice, 5-week-old C57BL/6J mice, and nude mice were purchased from the Model Animal Research Center of Nanjing University. The animals were housed in standard pathogen-free (SPF) conditions at 24 ± 2°C, with 40%-70% relative humidity, and on a 12-hours/12-hours light/dark cycle. Water and a basal diet were given ad libitum.

2.6 Tumor-bearing nude mice model

SW620 cells or HCT116 cells (1 × 10⁶ cells/100 µL PBS) were subcutaneously injected into the flanks of nude mice (3 mice/group), and the mice were sacrificed 4 weeks later. The tumor, liver, and lung tissues were then harvested for further analysis.

2.7 AOM-DSS models

To induce colitis-associated colon cancer, 5-week-old C57BL/6J mice received a single treatment of mutagen azoxymethane (AOM, Sigma-Aldrich) (10 mg/kg body weight) through intraperitoneal injection and then administered three cycles of 2% dextran sulfate sodium (DSS) in drinking water for 5 days, followed by regular drinking water for 14 days. For hypoxia treatment, mice were placed into an animal hypoxia model box and subjected to 10% O₂ from 8:00 am to 8:00 pm every day during the model construction.^{16, 17} Mice were killed after 11 weeks.

2.8 APC mice models

APC^Min/+ mice were exposed to normoxia or hypoxia. For hypoxia group, APC^Min/+ mice were placed into an animal hypoxia model box and subjected to 10% O₂ from 8:00 am to 8:00 pm every day for 6 consecutive weeks. For normoxia group, APC^Min/+ mice were placed into an animal hypoxia model box and subjected to clean air from 8:00 am to 8:00 pm every day. A total of 1 × 10⁷ PFU mmu_circ_0000807 KD adenovirus were injected by tail vein per mice every week during the model construction. All mice were killed after 6 weeks.

2.9 HE staining and miRNA in situ hybridization (RNA-ISH)

HE staining was performed by using Hematoxylin and Eosin Staining Kit (Beyotime) according to the manufacturer's protocol. Briefly, after deparaffinization and rehydration, tissue sections were stained with hematoxylin solution for 5 minutes followed by 5 dips in 1% ethanol hydrochloride (1% HCl in 70% ethanol) and then rinsed in distilled water. Then, the sections were stained with eosin solution for 3 minutes and followed by dehydration.

The RNA-ISH was conducted as described.^{18, 19} Briefly, tissue sections were deparaffinized, rehydration, and treated with 10 µg/mL proteinase K at 37°C for 20 minutes. Slides were hybridized with 20-60 nmol/L double-DIG-labeled circRNA probes (Focofish) overnight at 42°C. Slides were washed in 5 × SSC, 1 × SSC, and 0.2 × SSC for 10 minutes at room temperature. Staining was performed with DAB Substrate kit (Thermo) for 30 minutes at room temperature according to the manufacturer's protocol, and then, nuclear was staining by hematoxylin for 10 minutes at room temperature. An NC-circRNA probe with scrambled sequence was used as a negative control.

2.10 Liver metastasis mice model

A total of 5 × 10⁵ WT or hsa_circ_0000826 overexpressed SW620 cells were injected through sub-spleen to male C57BL/6 mice to construct liver metastasis model. For hypoxia group, mice were placed into an animal hypoxia model box and subjected to 10% O₂ from 8:00 am to 8:00 pm every day. For normoxia group, mice were placed into the same model box but supplied with filtered room air. 21 days later, mice were sacrificed for macroscopic examination, and liver sections were subjected to H&E staining.

2.11 Patients and specimens

All of the samples used in this study were in compliance with the informed consent and agreement of patients as well as the institution's ethical regulations. The serum specimens and corresponding medical record information were obtained from CRC patients (n = 100) undergoing surgery in the Jiangsu Tumor Hospital in 2009. And sex- and age-matched healthy donors (n = 100) enrolled from the Nanjing Hospital of Chinese Medicine from 2016 to 2017 as control subjects, and each healthy donor had a negative diagnosis of any malignancy tumors and infectious diseases. Tumor stage was defined according to the criteria of the sixth edition of the American joint Committee on Cancer (AJCC). Detailed information of healthy donors and CRC patients is presented in Table 1.

2.12 Tissue/serum RNA isolation

Serum was separated from the venous blood within 1 hour by centrifugation at 3000 g for 10 minutes, followed by a 15-minutes high-speed centrifugation at 12 000 g to completely remove the cell debris. The total RNA from tissues and serum was extracted using TRIzol reagent (Invitrogen) and an miRNeasy Serum/Plasma Kit (Qiagen) according to the manufacturers’ instructions.

2.13 Quantitative real-time PCR analysis

For circRNA expression analysis, 1 µg total RNA was reverse-transcribed into cDNA using Quantitect RT Kits (Qiagen). And then, cDNA was analyzed using either a SYBR green-based quantitative fluorescence method (Invitrogen). GAPDH was used as a normalization control for all of the samples.

2.14 Statistical analysis

Results are represented as mean ± SEM. RT-qPCR results were analyzed by 2^−ΔΔCt method. Differences in miRNA expression levels were analyzed by Wilcoxon rank-sum test. Probability of difference in overall survival (OS) was ascertained by Kaplan-Meier curves, and the significance was detected by a log-rank test. Multivariate Cox regression analysis was used to estimate the crude HRs, adjusted HRs, and corresponding 95% CI. Statistical analyses were performed using R, GraphPad Prism, or Stata software, and significance was set at P < .05.

3.1 Expression of hsa_circ_0000826 is upregulated in CRC cells under hypoxia condition

According to the previous study, we further analyzed the sequence of hsa_circ_0000826 and indicated its generation from back-splicing of exons 2 and 8 of its pre-mRNA ANKRD12 (Figure 1A). In addition, a murine circRNA mmu_circ_0000807 has been identified with 91% homology to hsa_circ_0000826 (Figure 1B).

To explore the role of hypoxia condition on the hsa_circ_0000826 expression, the WT and hsa_circ_0000826 knockout (KO) SW620 and HCT116 cells were exposed to normoxia and hypoxia conditions, respectively. As shown in Figure 1C, hypoxia, a common tumor microenvironment, can significantly increase the hsa_circ_0000826 expression in HCT116 NC cells. Once the expression of hsa_circ_0000826 was ablated, the hypoxia did not exert any effects on it. The similar condition has also been observed in SW620 cells (Figure 1D). Thus, it is found that hypoxia could induce the expression levels of hsa_circ_0000826 in CRC cells.

3.2 Hypoxia promotes the migration and invasion capacities of CRC cells in vitro through hsa_circ_0000826 induction

The migration and invasion capabilities of HCT116 and SW620 cells were evaluated by Transwell assays. As shown in Figure 2A, hypoxia condition significantly increased the migration and invasion of HCT116 cells in NC group. However, hsa_circ_0000826 KO could block the effects of hypoxia and rescue the migration and invasion of HCT116 cells to control levels. Besides, the same results were also confirmed by the results of SW620 cells in vitro (Figure 2B). These results suggested hypoxia can increase the malignant phenotypes of CRC cells in vitro via induction of hsa_circ_0000826 expression.

3.3 Hypoxia upregulated hsa_circ_0000826 expression is associated with tumorigenesis and metastasis of CRC cells

To further evaluate the function of hsa_circ_0000826 in vivo, luciferase-labeled HCT116 cells were subcutaneously injected into nude mice. As shown in Figure 3A, xenografts in the hypoxia-treated group showed higher tumorigenesis capability than those in the NC group. Notably, this effect was markedly reversed upon the deletion of hsa_circ_0000826. Further, the luciferase signals in tumor from the hypoxia-treated group were the highest among the three groups (Figure 3B). Besides, the luciferase in the liver and lung tissues was also detected to explore the metastasis capacities of CRC cells. Compared with the NC group, significantly higher and significantly lower luciferase activities were observed in the liver tissues from mice bearing the hypoxia-treated HCT116 cells and bearing the hsa_circ_0000826 KO HCT116 cells, respectively (Figure 3C). However, there was no obvious difference in luciferase activity was found in lung tissues among the three groups (Figure 3D). Meanwhile, the same results were also confirmed by the results of SW620 cells in vivo (Figure 3E-H). Taken together, our results suggest that hypoxia treatment of CRC cells can promote tumorigenesis and liver metastasis.

3.4 Hypoxia-induced mmu_circ_0000807 expression was linked to CRC development in AOM-DSS model

To investigate the relationship between hypoxia and CRC in vivo, we firstly established a pharmacological model that C57BL/6J mice were subjected to azoxymethane-dextran sulfate sodium (AOM-DSS) treatment and hypoxia treatment. As shown in Figure 4A, hypoxia and AOM-DSS treatment can significantly decrease the overall survival of mice and increase colon tumor formation ratio and the number of tumors each mouse, relative to the normoxia condition. Further, hypoxia accelerated the pathological grade of tumors in murine colon than normoxia (Figure 4B). The expression levels of mmu_circ_0000807 in tumor tissues were also increased following hypoxia treatment (Figure 4C). Further analysis revealed that hypoxia condition markedly promoted mmu_circ_0000807 expression in colorectal tissues and serum and liver tissues in control and AOM-DSS murine models (Figure 4D). Taken together, our results suggest that the hypoxia microenvironment upregulated expression levels of mmu_circ_0000807, which is involved in colitis-associated colon cancer formation.

3.5 Hypoxia upregulated expression of mmu_circ_0000807, which promote CRC tumorigenesis in APC^Min/+ mice

To further explore the relationship among hypoxia, mmu_circ_0000807 expression, and CRC development in vivo, a spontaneous colorectal adenoma murine model was used in our study. APC mutant (APC^Min/+) mice develop spontaneous CRC due to mutation in a tumor suppressor gene encoding adenomatous polyposis coli (APC).^{20, 21} The mmu_circ_0000807 knockdown or control virus was injected through the tail vein of APC^Min/+ mice to inhibit the expression of mmu_circ_0000807 in vivo. As shown in Figure 5A, the incidence of intestinal tumors and the number of tumors in each APC^Min/+ mouse were markedly increased upon hypoxic treatment. Figure 5B showed the images of pathology of colonic tumor and hypoxia with mmu_circ_0000807 NC-treated group showed severe pathological alteration. The expression levels of mmu_circ_0000807 in tumor tissues were induced in hypoxia group, but reduced by mmu_circ_0000807 KD (Figure 5C).

The qPCR analysis revealed that the tail vein injection of mmu_circ_0000807 KD adenovirus significantly decreased mmu_circ_0000807 expression both under normoxic and hypoxic conditions (Figure 5D). Furthermore, the relative abundance of mmu_circ_0000807 in the colorectal tissues and serum and liver tissues of APC^Min/+ mice under hypoxic conditions was significantly increased compared to normoxic condition (Figure 5D). Taken together, our results suggest that the hypoxia-induced mmu_circ_0000807 promoted CRC development in APC^Min/+ mice.

3.6 hsa_circ_0000826 is a potential biomarker of CRC metastasis to liver

To investigate the relationship among hypoxia, hsa_circ_0000826 expression and CRC liver metastasis, we constructed the liver metastasis mice model by sub-spleen injection. Results showed that hypoxia treatment can facilitate the colonization of CRC cells in liver, by showing increased macroscopic neoplasm in liver than normoxia group. Based upon the pathological evaluation by H&E staining verified tumor formation in liver (Figure 6A). However, hsa_circ_0000826 KD in SW620 cells can block this effect (Figure 6A). Therefore, elevated hsa_circ_0000826 plays a role in the liver metastasis of CRC cells.

Given the association between hsa_circ_0000826 and liver metastasis of CRC revealed by in vitro and in vivo studies, we next sought to determine its potential role in human. Serum samples of CRC patients and healthy donors were used to verify the expression levels of the hsa_circ_0000826. The qPCR results confirmed serumal hsa_circ_0000826 levels were extremely higher in CRC patients (n = 100) compared to healthy donors (n = 100) (Figure 6B, P < .001). Meanwhile, ROC analysis results showed that hsa_circ_0000826 can accurately discriminate CRC from healthy donors (Figure 6B, P < .0001, AUC = 0.7778, 95% Cl: 0.7157-0.8399). Furthermore, stratified analysis according to the medical record information of patients showed that hsa_circ_0000826 levels in serum samples from CRC patients with liver metastasis (n = 8) are significantly high than CRC patients without liver metastasis (n = 92) (Figure 6C, P < .001). And hsa_circ_0000826 could be a potential biomarker for CRC liver metastasis (Figure 6C, P < .001, AUC = 0.9035, 95% Cl: 0.8123-0.9948). According to those results, we found that the expression level of hsa_circ_0000826 could use for auxiliary diagnosis and prognosis prediction of CRC and its liver metastasis.