Background
Colorectal cancer (CRC), a malignant tumor of the colon or rectum, is one of the main reasons of cancer-related mortality in the world [
1‐
3]. Although in recent years, a considerable progress has been made in the diagnosis and therapy of CRC, the therapeutic efficacy of CRC patients is still poor [
4,
5]. Thus, elucidation of the mechanism underlying CRC progression is of great significance to find a novel therapeutic strategy for CRC.
Circular RNAs (circRNAs) are a new subtype of endogenous non-coding RNAs, which is characterized by a covalently closed continuous loop [
6‐
9]. CircRNAs have been proposed to participate in regulating a wide range of gene expression. Abnormal expression of circRNAs was reported to be linked to the development of human diseases, which make them potential biomarkers and possible therapeutic targets for human diseases [
10]. Therefore, circRNA research has become the hotspot of the study of various human diseases. Previous studies have showed that circRNAs were closely related to the occurrence and development of human cancers, such as breast cancer [
11], gastric cancer [
12], and CRC [
13]. hsa_circ_0137008, a newly identified circRNA, has been found to be reduced in CRC tissues [
14]. However, we still lack a clear understanding of the functional role of hsa_circ_0137008 in CRC.
MicroRNAs (miRNAs), a class of small non-coding RNAs with approximately 20 nucleotides, able to directly bind to the 3ʹ untranslated region of mRNAs, thereby regulating mRNA degradation and translational inhibition [
15,
16]. MiRNAs have been reported to be related to various physiological and pathological processes [
17]. In recent years, an increasing number of studies suggests that miRNAs serve as the oncogenic or tumor-suppressive factors in the progression of human cancers [
18]. MiR-338-5p is a member of the miR-338 family and has been documented to be involved in the development of multiple human cancers, such as melanoma [
19], hepatocellular carcinoma [
20], and gastric cancer [
21]. MiR-338-5p has been reported to be differentially expressed in CRC tissues, which make it a classifier for CRC detection [
22]. Previous researches have suggested that circRNAs acted as miRNA sponges to participate in the progression of human cancers [
23]. However, whether hsa_circ_0137008 regulates CRC progression through sponging miR-338-5p remains largely elusive till now.
In the current study, we investigated the function and molecular mechanism of hsa_circ_0137008 in CRC cells. We found that hsa_circ_0137008 was significantly downregulated in CRC tissues and cell lines. Mechanistically, hsa_circ_0137008 acted as miR-338-5p sponge to regulate CRC progression, indicating that hsa_circ_0137008 may be a potential therapeutic strategy for CRC patients.
Materials and methods
Patient tissue samples
Human CRC tissues (N = 30) and adjacent normal tissue samples (N = 30) were collected from CRC patients at The Second Affiliated Hospital of Guangzhou University of Chinese Medicine. The patients had not received any treatment before surgery. After excision, the tissues were immediately plunged into liquid nitrogen and stored at − 80 °C. This study was approved by The Second Affiliated Hospital of Guangzhou University of Chinese Medicine and in accordance with the ethical standards formulated in the Helsinki Declaration, and written informed consents were obtained from all the participants.
Cell culture
Human normal colon epithelial cell line (FHC) and CRC cell lines (HT29, HCT116, HCT8, LOVO, SW480, and SW620) were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Roswell Park Memorial Institute-1640 medium (RPMI-1640; HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37 °C in a humidified atmosphere with 5% (v/v) CO2.
Cell transfection
Empty pcDNA3.1 vector (Vector) and hsa_circ_0137008-overexpressing plasmid (circ_0137008) were synthesized by GeneCopoecia (Guangzhou, China). While miR-338-5p mimic and miRNA negative control (miR-NC) were obtained from RiboBio (Shanghai, China). SW480 and HCT116 cells were transfected with these molecules by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. At 48 h later, the cells were collected for the following experiments.
CircRNA plasmid stable transfection
Transfection was performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. The transfected cells were selected by screening of G418 (Sigma-Aldrich, St Louis, MO, USA) for 6 weeks. Surviving cells were subjected to identification of overexpression efficiency. The cells stably overexpressing hsa_circ_0137008 could be used for further experiments.
Quantitative real-time PCR (qRT-PCR) assay
Total RNA was isolated from tissues and cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The quality of isolated RNA was determined using NanoDrop ND-1000 spectrophotometer. Then the RNA was reverse transcribed into cDNA using PrimeScript RT Reagent Kit (Takara, Dalian, China), following by qRT-PCR analysis on Bio-Rad CFX96 system (Bio-Rad, Hercules, CA, USA). TaqMan miRNA assay was performed to examine the expression of miR-338-5p. The relative expression levels of hsa_circ_0137008 and miR-338-5p were calculated by the 2−ΔΔCt method, using β-actin and U6 as internal reference genes, respectively.
Western blot
After transfection, SW480 and HCT116 cells were collected, followed by protein extraction using RIPA lysis buffer. After 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis, isolated proteins were electro-transferred to polyvinylidene fluoride membranes and then blocked for 60 min with 5% skim milk. Subsequently, the membranes were incubated with primary antibodies from Boster (Wuhan, China) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary goat antibody (catalog number: BA1056) from Boster for 1 h at room temperature. The primary antibodies used in this experiment were list as follows: mouse anti-Ki-67 antibody (catalog number: M00254-1, Boster), mouse anti-proliferating cell nuclear antigen antibody (PCNA, catalog number: BM0104, Boster), rabbit anti-β-actin antibody (catalog number: BM3873, Boster), rabbit anti-E-cadherin antibody (catalog number: M00063-3, Boster), rabbit anti-Vimentin antibody (catalog number: BM4029, Boster), mouse anti-N-cadherin antibody (catalog number: BM1573, Boster).
Cell proliferation assay
The proliferation of SW480 and HCT116 cells was tested by using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Laboratories, Japan). After transfection, SW480 and HCT116 cells were seeded in 96-well plates (1 × 104 cells/well) and cultured for indicated times at 37 °C with 5% (v/v) CO2. Then, the cells were treated with 10 µl CCK-8 solution at 37 °C for 2 h. Subsequently, the absorbance of each well at 450 nm was assessed by mean of a spectrophotometer.
Colony formation assay was used to evaluate the proliferation of SW480 and HCT116 cells. After transfection, SW480 and HCT116 cells were plated into 6-well plates. The cells were incubated at 37 °C in a humidified incubator with 5% (v/v) CO2 for 14 days. Afterward, the cells were fixed with glutaraldehyde and stained with crystal violet. Colonies were pictured and counted under a stereomicroscope. Image J software was used to calculate the number of colonies (more than 50 cells).
Migration assay
The migration of SW480 and HCT116 cells was measured by wound-healing assay. After transfection, SW480 and HCT116 cells (1 × 105 cells/well) were seeded in 6-well plates and incubated for 24 h. A straight scratch was introduced by dragging the tip of the sterile pipette across the monolayer of SW480 and HCT116 cells. After washing with sterilized phosphate buffer saline (PBS), the cells were incubated in serum-free medium. The width of wound was pictured at 0 h and 48 h under a microscope.
Invasion assay
Transwell invasion assay was performed to evaluate the invasion of SW480 and HCT116 cells. After transfection, SW480 and HCT116 cells (5 × 104 cells/well) were suspended in serum-free medium and plated into the upper chamber pre-coated with Matrigel (BD Biosciences, San Jose, CA, USA). The lower chamber was filled with RPMI-1640 medium plus 10% FBS. After 48 h of incubation, a cotton swab was used to remove the cells on the upper surface of the chamber. The invaded cells on the lower surface of the chamber were fixed with 4% paraformaldehyde, and then stained with 0.1% crystal violet. The stained cells were counted with an inverted microscope (Thermo Fisher Scientific, Waltham, MA, USA).
Subcellular fraction assay
The nuclear and cytoplasmic RNA from SW480 and HCT116 cells were isolated using the PARIS Kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions, and then tested for hsa_circ_0137008 expression by qRT-PCR.
Luciferase reporter assay
The online database CircInteractome (
https://circinteractome.nia.nih.gov) was used to predict the potential miRNA downstream of hsa_circ_0137008. The wild type (WT) and mutated (MUT) sequence of hsa_circ_0137008 were cloned into pcDNA3.1 vector. Then, SW480 and HCT116 cells were plated into 24-well plates and co-transfected with circ_0137008-WT or circ_0137008-MUT and miR-NC or miR-338-5p using Lipofectamine 2000 (Invitrogen). At 48 h after transfection, dual luciferase reporter assay kit (Promega, Madison, WI, USA) was used to measure the luciferase activity, according to the manufacturer’s manual.
RNA pull-down assay
SW480 and HCT116 cells were transfected with biotinylated miR-NC (Bio-NC) or biotinylated miR-338-WT (Bio-miR-338-WT) or biotinylated miR-338-MUT (Bio-miR-338-MUT), which were commercially synthesized by RiboBio (Guangzhou, China). At 48 h post-transfection, SW480 and HCT116 cells were collected and lysed in lysis buffer containing 20 mM pH 7.5TRIS-HCl, 5 mM MgCl2, 100 mM NaCl, and 0.05% Igepal, protease inhibitors, 1 mM DTT, and 60 U/ml Superase-In. The cell lysates were cultured with M-280 streptaviden magnetic beads (Sigma) for 3 h at 4 °C and washed five times by ice-cold lysis buffer. The bound RNAs were subjected to qRT-PCR analysis.
Mouse xenograft model
The effect of hsa_circ_0137008 on the growth of CRC in vivo was measured by the xenograft experiments. Five-week-old BALB/c nude mice (N = 10) were acquired from Vital River Laboratories (Beijing, China) and handled in strict with the procedures approved by the Ethics Committee of The Second Affiliated Hospital of Guangzhou University of Chinese Medicine. SW480 cells stably expressing circ_0137008 were inoculated into nude mice. Tumor size was monitored every 7 days and the volume of xenograft tumors was calculated according to the following formula: Volume = 0.52 × length × width2. At the 35th days after inoculation, mice were killed and the tumors were resected, photographed and weighed.
Statistical analyses
Statistical analysis was performed by the Statistical Program for Social Sciences (SPSS) 20.0 (IBM, NY, USA) software. The data represent the mean ± standard deviation (SD) of at least three independent experiments. The difference between groups was analyzed by using one-way ANOVA or Student’s t test. Spearman tests were used to analyze the correlation between hsa_circ_0137008 with miR-338-5p. A P-value of less than 0.05 was considered as statistically significant.
Discussion
Recently, circRNAs have been widely studied because of their characteristics, including stable, abundant, conserved, and diverse [
24‐
27]. Deregulation of circRNA has been proposed to be implicated in the development of human cancers, including CRC. For instance, circITGA7 sequestered miR-370-3p away from neurofibromin 1 and then inhibit the activation of Ras signaling, thereby resulting in the suppression of the proliferation and metastasis of CRC cells [
28]. circCCDC66 has been reported to be upregulated in CRC and its downregulation repressed the proliferation, migration, invasion and anchorage-independent growth of CRC cells [
29]. These literatures implied that the development of CRC is often accompanied by abnormal expression of circRNAs, and circRNAs play a critical role in regulation of various biological processes of cancer cells, which is tightly linked to the initiation and progression of CRC. As a newly discovered circRNA, however, the contribution of hsa_circ_0137008 to CRC carcinogenesis has never been defined. Herein, our findings suggested that the expression levels of hsa_circ_0137008 were remarkably reduced in CRC tissues and cell lines. Overexpression of hsa_circ_0137008 could inhibit the proliferation, migration and invasion, of CRC cells, as evidenced by reduced expression of Ki67, PCNA, vimentin and N-cadherin and elevated expression of E-cadherin. Moreover, upregulation of hsa_circ_0137008 inhibited the growth of CRC in vivo. These results strongly supported the idea that hsa_circ_0137008 functioned as a tumor suppressor in CRC carcinogenesis. These findings suggested that hsa_circ_0137008 may be a potential biomarker for diagnosis and prediction of prognosis of patients with CRC.
Up to now, accumulating evidence suggested that circRNAs exert their biological functions via serving as competing endogenous RNA during the tumorigenesis of human cancers [
6,
30,
31]. As an example, overexpression of hsa_circ_0000673 suppressed the proliferation and invasion of gastric cancer cells by sponging miR-532-5p [
32]. Moreover, circRNA_LARP4 has been demonstrated to function as a sponge of miR-424 to regulate the progression of gastric cancer [
33]. In bladder cancer, circRNA-MYLK served as miR-29a sponge and then activated VEGFA/VEGFR2 and downstream Ras/ERK signaling pathway, thereby promoting the proliferation, metastasis and epithelial-mesenchymal transition of bladder cancer cells [
34]. In hepatocellular carcinoma, circADAMTS13 was downregulated in hepatocellular carcinoma tissues and its expression was negatively correlated with tumor size. circADAMTS13 interacted with miR-484 and then suppressed the proliferation of hepatocellular carcinoma cells [
35]. These studies demonstrated that circRNAs can act as miRNA “sponges”, thus restraining their ability to target mRNAs. Similar to the above researches, we identified the “sponge” role of hsa_circ_0137008 using luciferase reporter assay and RNA pull-down assay. Functionally, gain-of-function experiments demonstrated that hsa_circ_0137008 the cell proliferation, migration and invasion abilities in two CRC cell lines.
Several researches have proved that miR-338-5p exerts distinct functional roles in different types of cancers. For instance, in esophageal squamous cell carcinoma, miR-338-5p repressed the proliferation, migration, and invasion of CE-81T cells, as well as sensitized CE-81T cells to cisplatin through inhibiting the expression of fermitin family homolog 2, suggesting the anti-oncogenic role of miR-338-5p [
36]. In glioma, upregulation of miR-338-5p repressed the proliferation, metastasis and stemness abilities, and promoted the apoptosis and senescence of glioma cells [
37]. On the contrary, another study showed that miR-338-5p is associated with tumor staging, distant metastasis and poor prognosis in CRC patients. Upregulation of miR-338-5p enhanced the metastasis of CRC cells through inhibiting phosphatidylinositol 3-kinase, catalytic subunit type 3-mediated autophagy pathway [
38]. Consistent with this previous study in CRC, our results of gain-of-function experiments showed that miR-338-5p is upregulated in CRC and functions as an oncogene in vitro. To be specific, upregulation of miR-338-5p promoted the proliferation, migration and invasion of SW480 and HCT116 cells, as indicated by increased expression of Ki67, PCNA, vimentin and N-cadherin and decreased expression of E-cadherin. Importantly, upregulation of hsa_circ_0137008 attenuated the promoting effects of miR-338-5p on CRC cell proliferation, migration and invasion, indicating that hsa_circ_0137008 modulates the progression of CRC by sponging miR-338-5p. Our findings elucidated the upstream regulatory mechanism of ectopic expresson of miR-338-5p in CRC. Beyond miRNA “sponge”, other potential functions of the differentially expressed hsa_circ_0137008 need further investigation in CRC.
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