Introduction
Colorectal cancer (CRC) is a malignancy that poses a significant challenge to the biotechnology field due to its high aggression and poor survival rates. Current data indicate that CRC has become leading cause of cancer deaths worldwide [
1]. Whereas, the multifactorial pathobiology of CRC is still indistinct, for which cure therapies and early diagnosis remain limited [
2,
3]. Therefore, further understanding in the occurrence and development of CRC diagnosis is urgent. Recent studies have shed light on the heterogeneous inner mechanisms of oncogenesis, revealing the role of specific genetic and epigenetic alterations in CRC development. In this study, we conducted an in-depth analysis of the driving factors involved in CRC cancerogenesis, with a particular focus on hsa_circRNA_102051. By exploring the genetic and epigenetic factors associated with CRC development, we hope to further our understanding of this complex disease and develop new biotechnological approaches for its diagnosis and treatment.
Previous reports have validated that circRNAs play a crucial role in various bioprocesses involved in malignancies by interacting with microRNAs and regulating transcription [
4,
5]. Hsa_circRNA_102051 was one of the circRNAs influencing tumor progression. In esophageal squamous cancer, upregulation of hsa_circRNA_102051 was found to influencing downstream microRNAs and mRNAs [
6]. Nevertheless, the expression and impacts of hsa_circRNA_102051 in CRC has never been reported. Here, this research discovered a novel pathway that hsa_circRNA_102051 could exert function by sponging miR-203a and subsequently modulate BPTF (Bromodomain PHD [plant homeodomain] Finger Transcription Factor) level in CRC cells and tissues. Our findings shed new light on the mechanisms involved in CRC development and provide a potential target for biotechnological interventions aimed at treating this deadly disease.
MiR-203a was proved to negatively affect multiple cancers, including CRC [
7]. And bioprocesses of malignancies suppressed by miR-203a covered cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) and so on [
8‐
10]. According to circBank, hsa_circRNA_102051 is derived from TADA2A gene, which has been previously reported to produce circTADA2As capable of blocking miR-203a expression and influencing downstream factors [
11,
12]. The correlation between hsa_circRNA_102051 and miR-203a was disclosed in this study, thereby highlighting the potential of circTADA2As as promising targets for biotechnological interventions in the cancer field.
In addition, BPTF was the downstream gene affected by hsa_circRNA_102051 and focused on in this paper. Being important in targeting the NURF (nucleosome remodeling factor) remodeling complex, BPTF was considered vital for the cell stemness [
13]. Numerous studies have reported that BPTF could enhance self-renewal capacity of tumor-initiating cells (TIC) and cancer stem cells (CSC) [
14,
15], thus promoting the progression and metastasis of malignancies, such as lung cancer and hepatocellular carcinoma (HCC) [
16,
17]. Recent research has shown that BPTF is highly expressed in CRC and activated cell proliferation [
18]. And another study we did showed that BPTF could be regulated by circRNAs in colon cancer, subsequently enhancing cell abilities [
19]. Our current study has identified a novel regulatory mechanism of BPTF in CRC, shedding light on its impacts on the disease.
Briefly, this paper discovered the regulatory function of hsa_circRNA_102051 on miR-203a/BPTF expression, as well as downstream biological processes, especially the Notch signaling pathway, which composed a complete axis influencing CRC progression and metastasis.
Method and material
Clinical sample
The tumor tissues and matched adjacent tissues of 20 CRC patients admitted to the Affiliated Hospital of Guizhou Medical University from January 2018 to December 2019 were collected. All cases were confirmed by histopathological examination, and none of them received chemotherapy or radiotherapy before surgery. The study followed the Declaration of Helsinki, and all patients signed informed consent. Twenty samples of intestinal cancer tissues and adjacent tissues (normal colorectal mucosa located more than 5 cm away from the tumor margin, without invasion and avoiding tissues with inflammatory reactions) were collected in strict accordance with the collection standards during the operation. Some samples were frozen at -80℃, and some samples were fixed with 4% paraformaldehyde, dehydrated by automatic dehydrator, and preserved by paraffin embedding.
GSE147597 chip was downloaded from the GEO database and then utilized to analyze circRNAs differentially-expressed in metastatic CRC, involving samples from 20 CRC tissues from patients with or without liver metastasis. Microarray analysis was performed using the software package “Limma” of R software (Ver. 3.6.3). The threshold was set to P value (corrected by Bonferroni - Holm) < 0.05, Fold Change > 2. Starbase and Circular RNA Interactome were respectively used to predict the miR-203a binding sites on hsa_circRNA_102051 and BPTF.
Cell maintenance and transfection
Human CRC Cell lines including SW480, HT-29, SW1463 and HCT116, as well as FHCs (fetal human cells), were obtained from ATCC. SW480 and SW1463 cells were seeded in ATCC-formulated Leibovitz’s L-15 medium. HT-29 and HCT116 cells were cultured in ATCC-formulated McCoy’s 5a medium modified. FHCs were maintained in DMEM:F12 medium. All the cell lines were incubated under 37 °C with 5% CO2 in air atmosphere, and routinely subcultured every 3 days.
Vector construction and cell transfection
Specific oligonucleotides and plasmids were designed to regulate the expression of hsa_circRNA_102051, miR- 203a, and BPTF. All the plasmids, siRNA, shRNA or mimics along with their corresponding negative controls, were designed and provided by GeneChem. The above oligonucleotides and plasmids were transfected into cell lines using a Lipofetamine 2000 transfection reagent according to the instructions. The expression efficiency was examined using quantitative PCR.
Quantitative real-time PCR (qRT-PCR)
Total RNAs were extracted from CRC tissues and cells using Trizol reagent (Invitgen), and cDNA was then synthesized using a reverse transcription kit (Takara). In the ABI7900 system, cDNA amplification was performed using the Power SYBR Green (Takara) reaction mixture. The expression levels of hsa_circRNA_102051, miR-203a and BPTF were calculated by 2
−ΔΔCt with U6 or GAPDH as internal reference. All primer sequences are listed in Supplementary Table
1.
Cell counting Kit-8 assay (CCK-8)
CCK-8 assay was performed to detect the viability of CRC cells (SW480 and HT-29 cell lines) with different transfections. Briefly, transfected cells were seeded in 96-well plates and supplemented with 10 µl CCK8 solution to each well at 0 h, 24 h, 48 h, and 72 h. After 2.5 h incubation, the cell viability was presented by the OD value at 450 nm under a microplate analyzer.
RNase R
RNA was extracted from CRC cell lines using Trizol reagent (Invitrogen, USA). After that, 100 µg extracted RNA was incubated with RNase R at 37 °C for 20 min, and then purified using an RNEasy Minelute Cleanup Kit (Tokyo, Qiagen, Japan). Finally, the expression of RNA was detected through PCR.
Immunohistochemistry (IHC) staining
All specimens were fixed in 4% formalin, embedded in paraffin and sectioned into 5 μm sections. Next, the sections were submerged in citrate buffer for antigen retrieval and incubated with 1% bovine serum albumin (BSA) to block nonspecific binding. Primary antibodies against Ki67 (1:500; ZSGB-BIO), BPTF (1: 500; ProMab) and appropriate secondary antibody (1:500, ZSGB-BIO) were utilized according to the manufacturer’s protocol. The sections were then incubated with DAB and hematoxylin and then scored independently by two observers. The score was based on both the proportion of positively stained tumor cells and the intensity of staining.
Fluorescence in situ hybridization (FISH)
The digoxin-labeled probes specific to hsa_circRNA_102051 and biotin-labeled probes against miR-203a were prepared by Servicebio. SW480 and HT-29 cells were maintained on coverslips and fixed with 4% paraformaldehyde in PBS for 15 min. The probes were diluted in hybridization solution in PCR tubes and heated at 95 °C for 2 min in a PCR block to denature the probe. The probe was immediately chilled on ice to prevent reannealing. The hybridization solution was drained, and 100 µL of diluted probe per section was added to cover the entire sample. The samples were covered with a coverslip to prevent evaporation and were incubated in the humidified hybridization chamber at 65 °C overnight. The signals were detected by Cy3-conjugated anti-digoxin and FITC-conjugated anti-biotin antibodies (Jackson ImmunoResearch Inc.). Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). The final images were obtained under a laser scanning confocal microscope (Nikon).
EdU cell proliferation assay
24-well plates were coated with laminin (300 µL/well; Sigma) and incubated at least 4 h at 37 °C, then washed with PBS prior to cell seeding. Then, CRC cells in suspension were seeded manually at 2500 cells/well (5 cells/µL). Afterward, EdU staining assays were conducted to evaluate cell proliferation. Briefly, 10 µM 5-ethynyl-2′- deoxyuridine (EdU; Sigma) solution was added to each well and incubated for 3 h. After being washed with PBS, the cells were fixed in 4% paraformaldehyde and stained with EdU staining (Riobio) based on the manufacturer’s instructions. A fluorescence microscope (Leica DMI6000B) was utilized for eventual visualization and measurement.
TUNEL fluorescence assay
According to the manufacture’s protocol, cells were firstly fixed with formaldehyde for 15 min on ice and then washed with PBS. After supplementation with ice-cold 70% ethanol and incubation for 30 min, cells were resuspended in wash buffer again. Staining solution was then added to the cells and maintained for 60 min at 37ºC. After another washing with rinse buffer, supernatant was discarded. The propidium iodide/RNAse A solution was applied for resuspending cells and 30 min incubation. And the eventual staining results were observed under a fluorescence microscope.
Xenograft mice model
4-weeks-old female BALB/c nude mice were obtained from the Model Animal Research Center of Guizhou Medical University. For the establishment of CRC xenograft models, SW480 cells (3 × 106) with different transfections were injected into the right flank of the nude mice. The tumor volumes were measured every 5 days using the formula: V = (length × width2)/2. After 25 days, mice were sacrificed, and tumors were collected, weighed and measured. All the animal experiments were performed under the guidelines of the Institution Animal Care and Use Committee.
Western blot
Cells were lysed on ice with RIPA buffer (protein lysis buffer containing protease inhibitor and phosphatase inhibitor) for 30 min. And then the protein extracted from the supernatant was quantified through a BCA kit (Thermo Fisher). Then, the cell lysates were separated on SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk, and then incubated with primary antibodies at 4˚C overnight and with the secondary antibodies at room temperature for 2 h. Afterward, the target proteins were visualized with an enhanced chemiluminescence detection system.
Dual-luciferase reporter assay
Dual luciferase reporter assays were performed to verify the binding sites between hsa_circRNA_102051 and miR-203a, as well as miR-203a and BPTF. Wild type or mutant sequences of above genetic factors were constructed using pmirGLO vectors (Promega). Cells were co-transfected with mimics or inhibitors, or their corresponding negative controls along with the luciferase reporter vector. After 48 h, luciferase activity of each system was measured using the dual luciferase reporter gene assay system (Promega) according to the manufacturer’s instructions.
RNA-binding protein immunoprecipitation (RIP)
RIP experiments were carried out utilizing a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) following the manufacturer’s instructions. Cell lysates from SW480 and HT-29 were incubated with protein A magnetic beads conjugated to either antiAgo2 or IgG antibody. The coprecipitated RNA was purified and reversely transcribed into cDNA using PrimeScript RT Master Mix (TaKaRa Bio). The enrichment of hsa_circRNA_102051 and miR-203a pulled down by Ago2 or IgG from endogenous complex was then measured through quantitative PCR.
A mixture culture medium was prepared including serum-free 1640 medium (Invitrogen), 2% B27 Supplement (Invitrogen), 20 ng/ml basal fibroblast growth factor (bFGF) (PeproTech), 20 ng/ml epidermal growth factor (EGF) (PeproTech), 0.4% BSA (Sigma-Aldrich), and 5 µg/ml insulin (Sigma-Aldrich). CRC cells were digested and resuspended at a density of 1000 cells/well into prepared medium. After being incubated at 37 °C and 5% CO2 with saturated humidity for 12–14 days, the tumor sphere was defined as > 2000 cells. The number of spheres divided by the original number of seeded cells was then counted and analyzed.
Statistical analysis
All experiments were conducted at least three times, with the data presented as mean ± standard deviation. Statistical analysis was performed using GraphPad 7.0 (La Jolla). Differences between two groups were compared using t-test, and differences between multiple groups were compared using one-way analysis of variance (ANOVA), followed by Tukey-Kramer post-hoc analysis. P value less than 0.05 was considered statistically significant.
Discussion
In this research, hsa_circRNA_102051 is screened out and identified with overexpression in metastatic CRC tissues. Upregulated hsa_circRNA_102051 is capable to suppress miR-203a and mediately trigger BPTF expression, which enhances the proliferation, migration, invasion and stemness of CRC cells, activating Notch signaling pathway and eventually promoting tumor growth and metastasis. Hsa_circRNA_102051/miR-203a/BPTF axis provided a novel angle of the modulation of CRC progression, and could be applied to clinic in future as predictive markers or therapeutic targets.
On the contrary to this study, previous researches on breast cancer reported that hsa_circRNA_102051 was downregulated in breast cancer patients, acting as a tumor suppressor and regulating miR-197-5p/CDH19 expression [
20,
21]. As described before, hsa_circRNA_102051 in esophageal squamous cancer was also significantly upregulated, which is corresponding with the present research [
6]. Therefore, the expression of hsa_circRNA_102051 could distinct within different categories of cancers. And hsa_circRNA_102051 could be capable to sponge various microRNAs besides miR-203a to exert other impacts, which deserves in-depth exploration.
As for miR-203a, abundant studies have its upregulation in CRC and several other cancers [
7,
10,
22]. The epigenetic value of miR-203a as a predictive, prognostic, and targeting factor has been well documented. This study further confirmed the downregulation of miR-203a in CRC and determined its inhibiting effects on tumor progression. In addition, BPTF is another gene that has been shown to promote malignancy in several studies. Several studies display that BPTF exerts positive impacts on tumor through the MYC pathway [
17,
23,
24], which is a promising signal for further monitoring.
In this study, stemness markers including BPTF, SOX9, OCT-4 and CD44 were all detected, indicating that overexpressed hsa_circRNA_102051 enhanced the stemness of CRC. Thereinto, SOX9 activation was proved to repress miR-203a transcription by binding to miR-203a promoter [
25], while BPTF has been identified to promote HCC growth and enhance cancer stem cell traits [
15]. Hence, investigating the underlying mechanisms of the interaction between stemness factors and miR-203a/BPTF axis could be a novel research direction in the future.
Conclusion
Overall, this study identified hsa_circRNA_102051 as a key regulator of CRC progression and metastasis. The study demonstrated that hsa_circRNA_102051 promoted the expression of stemness markers including BPTF, SOX9, OCT-4, and CD44 through its ability to suppress miR-203a. This, in turn, activated the Notch signaling pathway, which promoted CRC growth and metastasis. The study highlights the potential of hsa_circRNA_102051 as a predictive biomarker or therapeutic target for CRC treatment. However, there are still unanswered questions, such as the inner mechanisms of the interaction between stemness factors and miR-203a/BPTF, which may be explored in future research.
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