Serum circ_001659 was significantly upregulated in sera of early-stage CRC patients with remarkable diagnostic value
To find potential complementary biomarker of CEA for CRC, we first analyzed the abundance and differential expression of circRNAs in GSE100206 (HC, n = 32) and GSE100063 (CRC, n = 12) datasets (Fig. 1a). Fifty-three circRNAs (26 upregulated and 27 downregulated) with fold change ＞2.0 and FDR < 0.01 were identified as differentially expressed circRNAs (DEcircRNAs) (Fig. 1a-c, Additional file 2: Table S2). Considering that down-regulated circRNAs are not readily associated with tumor progression, we then evaluated the expression levels and diagnostic potentials of these up-regulated circRNAs with serum samples from 20 pairs of age and sex matched CEA-negative early-stage CRC patients and healthy controls (screen cohort). The criteria of screen-level investigation for selected candidates were: (i) the quantification cycle values of each serum sample should be less than 30; (ii) The detectable rate should be more than 95%; (iii) fold change ＞ 2.0 and p-value < 0.01. Eventually, one circRNA (hsa_circ_001659) remained distinct was selected as candidate biomarker (Fig. 1d, Additional file 4: Table S4).
We further verified the expression level of circulating circ_001659 using TaqMan-qPCR method. The results were consistent with the data from SYBR-qPCR method (p < 0.0001; Fig. 1e). ROC analysis showed that circ_001659 was a reliable biomarker in CRC early diagnosis (Fig. 1f, AUC = 0.885 using SYBR-qPCR method; AUC = 0.868 using TaqMan-qPCR method). In addition, a positive correlation of circ_001659 level from SYBR-qPCR method and TaqMan-qPCR method was found which verified the reliability of the experimental data (r = 0.982, p < 0.0001; Fig. 1g). Those data suggest that circ_001659 could be a potential biomarker for diagnosis of early-stage CRC patients.
Finally, we assessed the structure of circ_001659. Sanger sequencing revealed that circ_001659 was 645 bp in length and is spliced by 5-9 exons of the SOX13 gene (RefSeq: NM_005686.3) (Fig. 2a). To determine the stability of circ_001659, total RNA was extracted from two CRC cells SW620 and LS174T and treated with RNase R at 37 ℃ for 30 min. Real-time PCR detected the expression levels of circ_001659 and the host gene SOX13. As shown in Fig. 2b, circ_001659 was more stable than the linear mRNA of the SOX13 gene. Next, we treated SW620 and LS174T cells by actinomycin D (an inhibitor of transcription) for 0, 12, 24, 36, and 48 h. Real-time PCR analysis showed that circ_001659 transcript was stable in comparison to SOX13 mRNA (Fig. 2c), indicating that circ_001659 had a bona fide circRNA structure and possessed the potential as a reliable biomarker.
Serum circ_001659 served as a diagnostic biomarker complementary for CEA
To confirm the diagnostic potential of circ_001659 for early-stage CRC patients, serum samples of a new cohort (validation cohort) were collected. The validation cohort included 98 HC, 66 BID, and 120 CRC. First, we observed that expression level of circ_001659 was similar in BID and HC group (p = 0.1535, Fig. 3a); while circ_001659 was highly expressed in serum of CRC patients compared with BID and HC group (p < 0.0001 , Fig. 3a). We then evaluated differential diagnostic accuracy of circ_001659 and CEA for CRC patients. As we expected, circ_001659 and CEA showed similar diagnostic values for CRC patients compared with control groups (Fig. 3b, Table 1).
Then we tried to demonstrate whether circ_001659 showed better diagnostic potential than CEA in CRC early diagnosis. There are 51 of 120 CRC patients in validation cohort with early-stage disease (Dukes stage A/B). Results showed that circ_001659 had greater AUC, sensitivity, and specificity values than did CEA in CRC early diagnosis (Fig. 3c, Table 1). When we combined two biomarkers together, the diagnostic values were greatly improved compared with either one of them (Table 1).
Further, we divided 51 early-stage CRC patients of validation cohort into CEA-positive and CEA-negative groups to determine the diagnostic value of circ_001659. The ROC curves indicated that circ_001659 showed a compelling diagnostic potential in both CEA-positive and CEA-negative early-stage CRC (Fig. 3d, Table 2). In other words, circ_001659 could be an effective diagnostic biomarker of CRC early detection irrespective of CEA status. Altogether, serum circ_001659 could be used as an independent biomarker for CRC diagnosis or in combination with CEA to improve the diagnostic efficacy of early-stage CRC.
Serum circ_001659 served as a prognostic biomarker for CRC
To evaluate the pathologic and clinical significance of serum circ_001659, we compared the different histologic stages, positive or negative vascular invasion, high or poor pathologic differentiation and AJCC stages between circ_001659 high-expression and circ_001659 low-expression tumors based on the median of serum circ_001659 level. Table 3 listed the association of different clinical characters with circ_001659 high- and low-expression cohorts. There is a tendency that patients with advanced pathological stages have higher levels of circ_001659 in their sera, suggesting that circ_001659 is associated with tumor progression. Additionally, higher level of serum circ_001659 was also correlated with tumor size and metastatic progression.
In patients with preoperative elevated CEA, CEA level will generally return to normal within half a year after surgery [8,13]. If CEA remains high, it indicates that there may be residual tumor or cancer recurrence . For patients with CEA-negative status before operation, the monitoring of CEA level after operation has no significance for evaluation of clinical outcomes. We have demonstrated that serum circ_001659 was increased in CEA-negative CRC patients compared with controls. Thus, we tried to investigate whether circ_001659 can be used as an auxiliary monitoring indicator after treatment in CEA-negative patients. Ninety-eight CRC patients underwent surgical resection were divided into CEA-positive and CEA-negative groups. We compared the levels of circ_001659 in pre- and post-therapeutic serum samples of these cancer patients. Three months after surgery, levels of circ_001659 in serum decreased rapidly in the majority of patients from both CEA-positive (5/72) and CEA-negative group (1/26) (Fig. 4). Interestingly, there were 6 cases showed a similar or elevated trend of serum circ_001659 after operation (Fig. 4). Clinical examination reported that these cases either suffered relapse or had residual tumor in lymph nodes or metastasis during follow-up (Additional file 5: Table S5). Taken together, we conclude that serum circ_001659 may be a novel biomarker in the assessment of successful treatment and remission of cancer tracking. High-level serum circ_001659 after therapy is associated with poor prognosis for CRC patients.
Circ_001659 influenced tumor cell metastasis in vitro and in vivo
To elucidate the potential functions and pathways of circ_001659 involved in tumor progression, RNA sequencing (RNA-seq) analysis was performed to compare the gene expression profiles of circ_001659 siRNA and control siRNA transfectants in SW620 and LS174T cells. A total of 1,985 downregulated genes and 2,258 upregulated genes in SW60 cell and 2,233 downregulated genes and 1,545 upregulated genes in LS174T cell were detected after knockdown of circ_001659 (Fig. 5a-b). The intersection of two gene lists was performed as described in Fig. 5a. Potential functions and pathways that circ_001659 may regulated were pooled using the overlapped gene signature (Additional file 6: Table S6). Gene ontology term enrichment (GO) analysis revealed that the main functions of circ_001659 were tumor metastasis, negative regulation of cell shape, and reorganization of cytoskeleton (Fig. 5c). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that circ_001659 mainly involved in the epithelial-mesenchymal transition (EMT) signaling pathway, small GTPase Rho/Rac signaling pathway, TGF-β signaling pathways, and hypoxia-mediated signaling pathway (Fig. 5d).
To functionally validate the pathway findings, we first detected cell migration and invasion using transwell assays in vitro. The circ_001659 siRNA significantly reduced the migratory activity of CRC cells in transwell assays (Fig. 5e) in both SW620 and LS174T cells. Similarly, in matrigel-coated transwell assays, we showed that downregulation of circ_001659, but not the control siRNA, significantly reduced the invasive ability in colorectal cancer cells (Fig. 5f). To confirm the effect of circ_001659 in vivo, LS174T cells transfected with circ_001659 siRNA or scrambled control siRNA were inoculated into the tail veins of nude mice and assessed the number of metastasized tumor nodules in the lung (Fig. 5g). We found tumor nodules on the lung surface in 100% (5/5) of mice inoculated with LS174T control-siRNA cells, with an average of 10.6 ± 3.53 nodules per lung. However, there were fewer metastatic foci in the lungs of nude mice at 12 weeks after injection of LS174T circ_001659-siRNA cells, with an average of 3.6 ± 2.64 nodules per mouse (Fig. 5h). The weigth of mouse lungs were measured and reflected the decreased metastatic tumor foci size in circ_001659 siRNA group compared with control (Fig. 5i). The data suggest that circ_001659 may promote colorectal cancer progression by regulating cell invasion and metastasis.
Circ_001659 regulated cytoskeleton organization
Microtubules are tightly associated with cell movement and tumor metastasis. Considering the cytoskeleton-related functions of circ_001659 involved in RNA-seq data, we attempted to investigate whether circ_001659 regulated tubulin polymerization and maintained cytoskeleton stability. Immunofluorescence analysis demonstrated that unlike scrambled control-treated SW620 cells, α-tubulin was unable to decorate microtubules in cells after circ_001659 knockdown (Fig. 6), suggesting circ_001659 influenced the content of stable microtubules by regulating tubulin organization.
Circ_001659 regulated EMT, metastasis and cytoskeleton-related genes expression
In order to examine the molecular mechanism in detail of circ_001659, we examined whether circ_001659 influenced the expression of EMT and metastasis-associated genes appeared in the differentially expressed gene signature. Real-time PCR and western blotting results demonstrated that circ_001659 knockdown upregulated expression of the epithelial marker CDH1 (encoding E-cadherin), ZO-1 and downregulated the mesenchymal marker CDH2 (encoding N-cadherin), ZEB1 and metastasis-associated genes MMP2 and MMP9 (Fig. 7a-b). To better understand the relationship between circ_001659 and EMT, we further detected the activation of TGF-β signalling, which is known to be a master regulator of EMT . Expectedly, circ_001659 siRNAs indeed inhibited the activation of TGF-β signaling by reducing SMAD3 protein phosphorylation (Fig. 7b), suggesting circ_001659 may regulate EMT via influencing TGF-β signaling.
Small GTPase Rho/Rac signaling pathway is a well-established regulator of the cytoskeleton reorganization [14-16]. Many GTPase family members, including TALN1 (encoding Talin1), RAC1, RHOC, RHOA, RAC2, Vimentin, LASP1 and RAB10 were significantly enriched in transcriptome sequencing data. We confirmed these results and found that Talin1, RAC1, RHOC, LASP1 and Vimentin were significantly downregulated, whereas RAC2, RHOA and RAB11B were upregulated upon circ_001659 siRNAs treatment (Fig. 7a-b). These data suggest that circ_001659 relies on the regulation of GTPase family members to influence cytoskeleton reorganization and cell migration.