Abstract
Background. Colorectal cancer (CRC) is one of the most common cancers, and its progression is regulated by several factors, including circular RNA (circRNA).
Objectives. The objective of this study was to determine the role, or roles, of circ_0000673 in CRC.
Materials and methods. We used quantitative real-time polymerase chain reaction (qPCR) to detect the expression of circ_0000673, miR-548b-3p and cleavage and polyadenylation specific factor 6 (CPSF6) in DLD-1 and RKO cells. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2’-deoxyuridine (EdU) assays were used to determine circ_0000673 roles in proliferation. Wound healing and transwell assays were used to detect cell migration and invasion abilities. Expression of CPSF6 protein and stem cell-associated proteins were examined using western blot. The putative relationship between miR-548b-3p and circ_0000673 or CPSF6 was verified with dual-luciferase reporter assay. The role of circ_0000673 in CRC was also investigated in a tumor xenograft assay in nude mice.
Results. Circ_0000673 expression was increased in CRC tissues and cancer cells. Silencing circ_0000673 reduced tumor cell proliferation, migration and invasion, while also decreasing cell stemness. MiR-548b-3p was found to be a target of circ_0000673, while CPSF6 was a downstream target of miR-548b-3p. The tumor-regulatory effects of si-circ_0000673, anti-miR-548b-3p and oe-CPSF6 were partially reversed by anti-miR-548b-3p, si-CPSF6 and si-circ_0000673, respectively, in rescue assays. Downregulation of circ_0000673 reduced solid tumor growth in vivo.
Conclusions. Circ_0000673 inhibition reduced CPSF6 expression by targeting miR-548b-3p, thereby blocking proliferation, migration and invasion of CRC tumor cells.
Key words: colorectal cancer, CPSF6, circ_0000673, miR-548b-3p
Background
Progression of colorectal cancer (CRC) begins with abnormal transformation of epithelial cells in the colon or rectum and has the 3rd highest incidence and 2nd highest mortality rate of all malignancies.1, 2 In developing countries, the incidence of CRC is quickly growing and is now more than 4 times that of industrialized countries.3 Moreover, the prognosis for individuals with advanced CRC remains poor, with recurrence and distant metastases being the primary causes of death following surgery.4, 5 The mechanism of CRC carcinogenesis is complex and entails numerous steps in the disease evolution. Numerous genes and signaling pathways have been implicated in tumor cell invasion and metastasis, yet the underlying mechanisms of CRC are not thoroughly understood. Thus, exploring the molecular pathways integral to the pathogenesis of CRC and identifying novel biomarkers are pivotal for enhancing early diagnosis and prognosis for patients.
Circular RNA (circRNA), a type of non-coding RNA, possesses a covalently closed-loop structure at the 3’ and 5’ untranslated regions (UTRs), resulting from the reverse splicing of precursor messenger RNA (pre-mRNA).6, 7, 8 Circular RNA is more stable than linear RNA in cells due to its unique structure, which prevents cleavage from exonucleases. Circular RNA is involved in various aspects of cancer biology and can play a crucial role in regulating gene expression by acting as a sponge for microRNAs (miRNAs).9, 10, 11 It is also linked to malignant tumor characteristics, including cell proliferation, the cell cycle and invasion. For instance, silencing of circ_RNF121 can reduce cell proliferation, metastasis and glycolysis through the miR-1224-5p/FOXM1 axis, which plays a significant role in CRC progression.12 Circ-LECRC functions as a competitive endogenous RNA (ceRNA) to regulate KLF4 expression and acts as a “brake signal” to reduce the over-activation of oncogenic YAP signaling, thereby inhibiting CRC tumor growth.13 However, whether circ_0000673 plays a role or participates in particular mechanisms in CRC requires further research.
Objectives
By conducting an extensive review of existing literature and conducting meticulous bioinformatics analysis, our goal was to explore the potential significance of circ_0000673 as a pivotal circRNA in the regulation of CRC progression, specifically through the miR-548b-3p/CPSF6 pathway. Unraveling these intricate molecular mechanisms in the context of CRC tumorigenesis not only enhances our comprehension but also sets the stage for potential clinical applications in this domain.
Materials and methods
Tissues collection and study approval
A cohort of 34 CRC patients was enlisted from the Second Affiliated Hospital of Harbin Medical University, China, for this study. The resected tissues were immediately preserved in liquid nitrogen for RNA analysis. Pathological sections confirmed that all tumor tissues belonged to CRC, and the patients’ clinicopathological information was fully documented. Informed consent forms were signed by all patients or their family members, indicating their awareness and approval of sample usage. The collection of clinical specimens was conducted in accordance with the ethical standards set forth by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (approval No. KY2021-230).
Cell culture
The CRC cell lines DLD-1 and RKO, alongside the normal colorectal epithelial cell lines NCM460, were procured from the Institute of Type Culture, Chinese Academy of Sciences, Shanghai, China. After retrieving the frozen storage tube from liquid nitrogen, it was immediately thawed in a water bath at 37°C to ensure rapid lysis. The contents were then transferred to an ultra-clean operating table for further processing. Initially, the cell suspension was pipetted from the freezing tube into a 15 mL centrifuge tube, and 1 mL of complete culture medium was added and mixed. The mixture was then centrifuged at 1,000 rpm for 5 min, and the supernatant was carefully decanted. Subsequently, 5 mL of complete medium was added to the centrifuge tube, and the cells were gently mixed and transferred to cell culture dishes or flasks. The cells were finally incubated in an incubator at 37°C with 5% CO2. All cells were maintained for less than 6 months.
Quantitative real-time polymerase chain reaction
Cells were lysed and centrifuged, and the precipitated material was transferred to a centrifuge tube and centrifuged at 1,000 rpm for another 5 min, after which the supernatant was discarded, and TRIzol (Invitrogen, Waltham, USA; 15596026) reagent was added to the pellet. The solution was then gently mixed several times to rupture the cells and shear the DNA. The Reverse Transcription Kit (Roche, Penzberg, Germany; 11483188001) was then used to generate complementary DNA (cDNA). The PCR system with FastStart Universal SYBR Green aster (Roche; FSSGMMRO) and a C1000 thermal cycler were used to perform quantitative real-time polymerase chain reaction (qPCR). Internal controls were GAPDH and U6, and the data were evaluated using the 2–ΔΔCt technique.14 All primer sequences are listed in Supplementary Table 1.
Cell transfection
To perform cell transfection, 125 uL of serum-free Dulbecco’s modified Eagle’s medium (DMEM) was added to each Eppendorf (EP) tube and labeled accordingly. Next, 5 uL of Lipofectamine 3000 (Invitrogen; V518472) was added along with 5 uL of diluted siRNA to each of the 2 EP tubes. The contents of the EP tubes were mixed thoroughly by gentle pipetting to homogenize and then allowed to stand at room temperature for 15 min. When the cells reached 40–60% confluency, the supernatant was discarded, and the cells were washed twice with sterile phosphate-buffered saline (PBS). Next, the serum-free DMEM medium was added to the cells, and then the mixture from the EP tubes was put into the corresponding well of the 6-well plate. The contents were mixed well, and the plate was placed in the incubator for further cultivation. After 6–8 h, the medium was replaced with complete medium. To detect the RNA content, transient transfection was performed for 48–72 h, and for protein content and functional experiments, it was performed for 72 h.
Ribonuclease R (RNase R) and actinomycin D treatment
The loop structure of circ_0000673 was confirmed via RNase R treatment. A total of 5 μg of RNA, extracted from DLD-1 and RKO cells, was treated with 2 U/μg of RNase R (Geneseed, Guangzhou, China; R0301) at 37°C for 30 min. Subsequently, the enzyme was inactivated at 70°C for 5 min. DEPC-treated water (MilliporeSigma, St. Louis, USA) served as a control. Actinomycin D (2 μg/mL) or dimethyl sulfoxide (DMSO; MilliporeSigma; D2650-100ML) were introduced to media containing DLD-1 or RKO cells for 0, 4, 8, and 12 h to analyze actinomycin D disposal. Then, we used qPCR to detect the expression of circ_0000673 and corresponding linear RNA.
Cell Counting Kit-8 assay
Colorectal cancer cells post-transfection were grown in 96-well plates with a density of 1×105 cells per well. At each designated time point (0, 24, 48, and 72 h), 10 μL of Cell Counting Kit-8 (CCK-8) reagent (Beyotime Biotechnology, Shanghai, China; C0037) was incorporated per well and then incubated for an additional 2 h at 37°C within a humidified incubator. Finally, the absorbance at 450 nm was read using a microplate reader (imark; Bio-Rad, Hercules, USA).
5-ethynyl-2’-deoxyuridine assay
The target cell line in logarithmic growth was selected, and a cell suspension was prepared. The cells were inoculated into a 96-well plate, and 100 µL of diluted 5-ethynyl-2’-deoxyuridine (EdU) in complete medium (50 µM) (Beyotime Biotechnology; C0071S) was added and incubated for 2 h. In the dark, cells were subjected to flow incubation, fixed with 4% paraformaldehyde, and successively treated with permeabilizing solution, Click-Interaction mixture and fluorescent nuclear stains. After each treatment, cells were washed with an immunostaining blocking buffer. Finally, images of the cells were captured using a fluorescent microscope (Leica DMI4000 B; Leica Camera AG, Wetzlar, Germany) and the cells were counted with different fluorescent colors.
Transwell assay
The cells to be treated were initially resuspended in serum-free medium. Subsequently, cell counting was performed, and the cells were appropriately diluted to obtain a concentration of 1×105/mL. Then, 200 uL of the cell suspension was aspirated and added dropwise to the upper chamber of the transwell. Following this, complete medium (0.7 mL) was added to the lower chamber. For invasion experiments, a diluted layer of Matrigel (BD Biosciences, San Jose, USA; 353095) was applied to the surface of the chambers. After a 24-h incubation period, the underlying cells were fixed for 30 min with 4% paraformaldehyde and stained for 1 h with 0.5% crystal violet. Finally, images were acquired using an inverted optical microscope (Leica DMI4000 B) and the quantity of migrating or invading labeled cells was counted using ImageJ software (National Institutes of Health (NIH), Bethesda, USA).
Wound healing assay
DLD-1 and RKO cells were seeded at a density of 6×105 per well in 6-well plates and allowed to grow for 48 h. Once 90% confluence was achieved, a linear scratch was made in each well using a 200 μL pipette tip. Then, cells were washed in PBS and incubated in complete medium. After 24 h of incubation, the width of the wound was analyzed.
Spheroid formation assay
Spheroid formation assay was performed in serum-free medium supplemented with ×1 B27, 20 ng/mL each of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF, Invitrogen; 17504-044, 266775, 266475), along with 100 U/mL penicillin and 0.1 mg/mL streptomycin (Beyotime Biotechnology; c0222) and required special ultra-low attachment plates (Corning Company, Corning, USA; 7007). The quantity and size of spheroids were measured using an inverted optical microscope (Leica DMI4000 B) after 10 days. The efficiency of spheroid formation was calculated using the formula as the number of colonies divided by the number of seeded cells.
Dual-luciferase reporter assay
The binding sequence of circ_0000673 or CPSF6 to miR-548b-3p was predicted, and genomic DNA from CRC cells was extracted and amplified. The double-enzymatic luciferase reporter gene empty plasmid used was pGL3 (Promega, Madison, USA). The luciferase reporter vector generated (pmirGLO-circ_0000673-WT, pmirGLO-circ_0000673-MUT, pmirGLO-CPSF6-WT, pmirGLO-CPSF6-MUT) and miR-NC or miR-548b-3p mimics were co-transfected into DLD-1 and RKO cells. Luciferase activity was detected using the dual-luciferase reporter detection system kit (Promega; E1910) after transfection for 48 h.
Western blot assay
After lysing cells or tissues with RIPA Lysis Buffer (Beyotime Biotechnology; P0013B) and 1% phenylmethylsulfonyl fluoride (PMSF) (Seven, Beijing, China; SW106) to collect proteins, the protein concentration was determined using the BCA Protein Concentration Assay Kit (Beyotime Biotechnology; P0009), and the proteins subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins on polyacrylamide gels were subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA) using electrophoretic techniques. The PVDF membranes were sealed with a sealing solution at room temperature for a period adjusted for the different antibodies, followed by overnight incubation at 4°C in a cold room immersed in primary antibody. The next day the membranes were washed 3 times with phosphate-buffered saline with Tween (PBST) and then incubated with the secondary antibody for 1.5 h at room temperature and washed again in PBST. The expression level of the target protein was assessed using a BeyoECL kit (Beyotime Biotechnology; P0018S), with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serving as an internal reference. All antibodies were sourced from Immunoway (Texas-Plano, USA; NESTIN: YN2050, SOX2: YM0594) or Abcam (Cambridge, USA; CPSF: ab175237, OCT4: ab200834). The antibody was diluted in accordance with the manufacturer’s instructions.
Xenograft assay
We purchased nude BALB/c mice from Vital River (Beijing, China) and housed them in a barrier system at 45–50% humidity and 25–27°C. A CRC xenograft model was established by subcutaneously injecting 5×106/150 μL of transfected CRC tumor cells into the anterolateral groin of the left hind limb and 5×106/150 μL of sh-NC transfected tumor cells into the anterolateral groin of the right hind limb of each mouse. Tumor volume was measured once a week. After 4 weeks, the mice were euthanized by cervical dislocation, and the tumors were dissected and weighed, and the expression of genes under study was also assessed. The Ethics Committee of The Second Affiliated Hospital of Harbin Medical University approved the study (approval No. SYDW2021-052).
Statistical analyses
The data were analyzed using IBM SPSS v. 27.0 (IBM Corp., Armonk, USA). Data visualization was performed using GraphPad Prism v. 9 (GraphPad Software, San Diego, USA). Data were expressed as mean ± standard deviation (±SD) and underwent analysis based on the grouping or pairing conditions, employing distinct Mann–Whitney test, Kruskal–Wallis test and post hoc multiple comparisons. The clinicopathologic data in Table 1 were examined using Fisher’s exact test. Due to the limited sample size, non-parametric tests were used, leading to marginal significance in the reported results, and the statistical methods and detailed parameters utilized for all results were documented in Supplementary Table 2.
Results
Circ_0000673 was upregulated in CRC tissues and cells
We first identified the basic characteristics of circ_0000673, which is 251 nucleotides in length produced by exons 4 and 5 of RSL1D1 mRNA (Figure 1A). To explore the relevance of circ_0000673 to CRC progression, we examined its expression in CRC cells. Quantitative real-time polymerase chain reaction assays pointed to higher circ_0000673 enrichment being detected in CRC cell lines (DLD-1, RKO and HT-29), compared to NCM460 cells (Figure 1B). After RNase R treatment, we found that circ_0000673 was resistant to RNase R digestion when compared to RSL1D1 (Figure 1C,D). After total RNA synthesis was inhibited by actinomycin D, the expression of circ_0000673 and RSL1D1 mRNA were detected at different time points, and again, the stability of circRNA was found to be much stronger than that of linear mRNA (Figure 1E,F).
The relationship between circ_0000673 and clinicopathologic data of CRC patients
Considering the elevated expression of circ_0000673 in CRC tissues compared to normal tissues (Figure 2A), we investigated the relationship between circ_0000673 expression and the pathological advancement of CRC. Initially, we categorized the patients based on the presence of lymph node metastasis or TNM (tumor-node-metastasis) stage and subsequently performed quantitative comparisons of circ_0000673 expression levels within each group using qPCR (Figure 2B,C). However, to explore whether circ_0000673 could play a role in the clinical setting, we also analyzed its correlation with pathological data after dividing all cases into circ_0000673 high- and low-expression groups (Table 1). Survival correlation, as analyzed using a Kaplan–Meier estimation, revealed that patients exhibiting high circ_0000673 expression experienced poorer overall survival (OS) compared to their counterparts with low circ_0000673 expression (log-rank p = 0.043; Figure 2D). To evaluate the prognostic efficacy of circ_0000673, we plotted receiver operating characteristic (ROC) curves, and the results confirmed that circ_0000673 had clinical prognostic significance (p < 0.001; Figure 2E).
Circ_0000673 downregulation restricted the proliferation, invasion and migration of cancer cells
Once the transfection efficacy of si-circ_0000673 was assessed (Figure 3A), CCK-8 and EdU assays revealed a significant reduction in the vitality of DLD-1 and RKO cells following si-circ_0000673 transfection compared to the si-NC group (Figure 3B–D). Building upon the findings presented above, the downregulation of circ_0000673 exhibits the capacity to curb the proliferation of CRC cells. We extended our investigation to assess the influence of circ_0000673 on the invasive and migratory capabilities of CRC cells, employing both wound healing and transwell assays. The results indicated that in cells transfected with si-circ_0000673, the migration distance of tumor cells and the number of migrating cells were notably lower compared to the control group (Figure 4A,B). Moreover, the transwell assay showed a reduction in the number of invasive cells in the experimental group following the removal of the stromal gel layer (Figure 4C). The number of invasive cells in the transwell layer was also reduced (Figure 4D). A subsequent attenuation of tumor cell stemness after knockdown of circ_0000673 can be seen in the spheroid formation assay, and some stemness markers like NESTIN, OCT4 and SOX2 were also downregulated at the protein expression level (Figure 5A,B).
Targeted regulation of miR-548b-3p expression by circ_0000673 in colorectal cancer cells
Circinteractome, starBase and circBANK predicted that miR-548b-3p may act downstream of circ_0000673 in regulating CRC (Figure 6A,B). To validate this hypothesis, we conducted a dual-luciferase reporter assay. The experimental results confirmed our hypothesis, showing that the luciferase activity was significantly reduced in DLD-1 and RKO cells co-transfected with miR-548b-3p mimics and the recombinant plasmid circ_0000673-WT (Figure 6C). Using qPCR assay, miR-548b-3p expression level was significantly increased after transfection with si-circ_0000673 compared to that with small interfering RNA negative control (si-NC) group (Figure 6D), and miR-548b-3p expression was found to be downregulated in CRC cells and tissues (Figure 6E). It is worth noting that the expression of circ_0000673 and miR-548b-3p in CRC tissues presented a negative relationship (Figure 6F).
CPSF6 severed as a downstream effector of miR-548b-3p in CRC
Bioinformatics analysis disclosed that miR-548b-3p could target CPSF6 (Figure 7A). The results obtained from the experiment indicate that luciferase activity was considerably weaker in the CPSF6-WT cells transfected with miR-548b-3p compared with the control group, while the luciferase activity was not significantly changed in the CPSF6-MUT group cells transfected with miR-548b-3p mimics (Figure 7B,C). In CRC tissues, we observed that both CPSF6 mRNA and protein were increased and CPSF6 expression was negatively correlated with miR-548b-3p expression (Figure 7D–F). Similarly, the CPSF6 nucleic acid and protein expression levels were elevated in CRC cells (DLD-1 and RKO) (Figure 8A,B). The addition of anti-miR-548b-3p significantly increased CPSF6 abundance, but miR-548b-3p overexpression decreased CPSF6 expression (Figure 8C).
Circ_0000673 regulated CRC malignant biological behaviors via miR-548b-3p/CPSF6
We performed rescue experiments to further characterize the regulatory relationship between circ_0000673 and CPSF6. It was found that circ_0000673 knockdown suppressed CPSF6 expression at both the nucleic acid and protein levels, and then rescued CPSF6 repression by silencing miR-548b-3p in part (Figure 8D). The findings suggest that circ_0000673 could function as a ceRNA, potentially binding to miR-548b-3p to elevate CPSF6 in CRC cells. Subsequent rescue experiments regarding cellular phenotype were also conducted. Cell Counting Kit-8 and EdU rescue assays provided further confirmation that the reduced cell proliferation due to circ_0000673 knockdown could be restored by suppressing miR-548b-3p (Figure 9A–C). Additionally, transwell assays showed that the circ_0000673 knockdown-induced inhibition of migration and invasion capabilities could be partially reversed by inhibiting miR-548b-3p. Moreover, the downregulation of cell proliferation, migration and invasion by miR-548b-3p was partially mitigated upon transfection with si-CPSF6 (Figure 10A–C). Finally, overexpression of CPSF6 significantly amplified the proliferation, migration and invasion of tumor cells, while the knockdown of circ_0000673 partly countered these effects (Figure 11A–C). In conclusion, circ_0000673/miR-548b-3p/CPSF6 axis is involved in the tumorigenic process of CRC.
Circ_0000673 silencing restrains xenograft tumor growth in vivo
A CRC xenograft model was established to confirm the effects of circ_0000673 on CRC tumorigenicity. RKO cells at the logarithmic growth stage were inoculated into nude mice after transfection with sh-circ_0000673 or short hairpin RNA negative control (sh-NC) and monitored for 4 weeks. Both tumor volume and weight of the sh-circ_0000673 transfected group were decreased (Figure 12A,B), indicating that circ_0000673 silencing was able to inhibit the growth of xenograft tumors in vivo. Quantitative real-time polymerase chain reaction and western blot on tumor tissues showed circ_0000673 expression was severely decreased in sh-circ_0000673 group tumors (Figure 12C). Conversely, the expression of miR-548b-3p was upregulated due to the silenced circ_0000673 (Figure 12D). In addition, mRNA and protein expression of CPSF6 were significantly decreased in sh-circ_0000673 tumor tissues compared to sh-NC group (Figure 12E,F).
Discussion
The incidence of CRC has remained consistently high over time.15 Emerging evidence highlights the significant roles that circRNAs play in various human cancers. For instance, circ_0120175 has been shown to promote the proliferation, migration and invasion of lung squamous cell carcinoma (LSCC) cells through the miR-330-3p/SLC7A11 axis, while concurrently inhibiting apoptosis.16 Similarly, circ-ZNF609 has been demonstrated to selectively bind to miR-432-5p, leading to increased LRRC1 expression and facilitating cholangiocarcinoma (CCA).17, 18 In this study, we have confirmed the elevated expression of circ_0000673 in CRC tissues and cells. Moreover, we observed that high circ_0000673 expression was associated with shorter OS among CRC patients, and ROC curve analysis suggested that circ_0000673 might hold potential as a diagnostic biomarker. Notably, the inhibition of circ_0000673 effectively curtailed the proliferation, migration and invasion of CRC cells while diminishing their stemness. Finally, circ_0000673 knockdown resulted in the suppression of tumor growth in in vivo experiments. Collectively, these findings underscore the regulatory role of circ_0000673 as a pro-cancer factor in CRC.
In recent years, accumulating evidence has underscored the pivotal role of circRNAs in gene expression regulation, often achieved through their capacity to sponge miRNAs.19 In our study, we unveiled that circ_0000673 functions as a miR-548b-3p sponge in CRC cells. Previous research has highlighted the tumor-suppressive effects of elevated miR-548b-3p in breast cancer, achieved by inhibiting MDM2 expression.20 In the context of lung cancer, Wang et al. demonstrated that miR-548b-3p acts as an oncogenic suppressor by regulating the PI3K/AKT signaling pathway,21 and miR-548b-3p suppresses the malignancy of tumor cells by inhibiting the expression of CIP2A and SP1.22 Our study has provided conclusive evidence that miR-548b-3p is downregulated in CRC tissues and cells. Notably, anti-miR-548b-3p partially counteracted the effects of circ_0000673 silencing on the malignant behaviors of CRC cells. These data collectively suggest that miR-548b-3p plays a tumor-suppressive role in CRC, whereas circ_0000673 functions as an accelerator of CRC progression by sponging miR-548b-3p.
Our study uncovers the role of CPSF6 as a target gene of miR-548b-3p in CRC cells. In gastric cancer, CPSF6 has been shown to negatively regulate VHL expression through alternative polyadenylation (APA), and the VHL short 3’UTR heterodimer induced apoptosis and hindered the growth of gastric cancer cells.23 Similarly, CPSF6 has been implicated in the progression of hepatocellular carcinoma (HCC) by upregulating NQO1 expression through APA.24 In a study by Liu et al., CPSF6 was found to inhibit BTG2 expression, promote glycolysis and suppress apoptosis in HCC cells.25 In our investigation, we observed an upregulation of CPSF6 in CRC tissues and cells, and the increased CPSF6 levels counteracted the inhibitory effects of miR-548b-3p overexpression and si-circ_0000673 on the malignant behavior of CRC cells. These findings collectively suggest that CPSF6 plays an oncogenic role in CRC. Therefore, we conclude that circ_0000673 regulates the progression of CRC by sponging miR-548b-3p to modulate the expression of CPSF6.
Limitations
Our study revealed the function and mechanism of circ_0000673 in CRC development. However, we did not investigate further the upstream regulatory mechanisms of circ_0000673, such as whether there is a strongly associated transcription factor causing abnormal expression of circ_0000673 in CRC. Furthermore, studies related to the clinical application of circ_0000673 have not been performed, including those examining its relationship with tumor drug resistance capacity.
Conclusions
To sum up, circ_0000673 assumes a tumor-promoting role within the context of CRC, actively driving its progression by orchestrating the miR-548b-3p/CPSF6 axis. These findings propose that circ_0000673 holds potential as a promising target for the diagnosis and treatment of CRC in clinical practice.
Supplementary data
The Supplementary materials are available at https://doi.org/10.5281/zenodo.10867861. The package includes the following files:
Supplementary Table 1. Primers used in this study.
Supplementary Table 2. Statistical methods and test results used.
Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Consent for publication
Not applicable.
