Background
Colon cancer is a frequently occurring gastrointestinal tumor, which is responsible for over 1 million newly diagnosed cases across the world per year [
1]. Colon cancer has been regarded as the fourth most fatal cancer in the world, with a mortality rate of about 50% [
2]. Colon cancer is characterized by symptoms like obstruction, perforation as well as bleeding in the colon [
3]. The possible etiology of colon cancer includes the conversion of cholesterol and δ5–7-dehydrocholesterol, dietary fat changes, and etc. [
4]. At present, the first-line therapy for colon cancer is the combined application of surgical resection and adjuvant chemotherapy [
5]. It is noteworthy that colon cancer is comprised of a small number of cancer stem cells (CSCs) that aid in tumor maintenance and confer resistance to cancer therapies, which is likely to allow for tumor recurrence upon the stopping of the treatment [
6]. Interestingly, microRNAs (miRs) have been reported to be crucial regulators on CSCs and regarded to serve as a promising therapeutic target for colon cancer treatment [
7].
It has been noted in a previous study that the inhibitory role of miR-195-5p in the stem-like ability of colorectal cancer cells [
8]. Moreover, miR-497 could serve as an anti-tumor gene in diverse cancer, including colorectal cancer [
9]. Intriguingly, an existing study has reported that miR-497/195 could be inhibited in myoblasts, as well as skeletal muscle tissues by nuclear factor κB (NF-κB) [
10], a transcription factor which is identified as a type of transcription factor dimer composed of p50/NFKB1, p52/NFKB2, c-Rel, p65/RelA as well as RelB [
11]. The activation of NF-κB has been demonstrated to encounter multiple solid as well as hematological tumors [
12]. It has also been reported that NF-κB was capable of promoting stem-like properties of colon cancer stem cells (CCSCs) [
13,
14].
More importantly, the binding site between microRNA (miR)-195-5p/497–5p and minichromosome maintenance marker 2 (MCM2) has been identified based on the prediction results on the starBase website. MCM2 is a component of the replicative helicase machinery that is capable of interacting with histones H3 and H4 via the N-terminal domain in the process of replication [
15]. MCM2 can increase the sensitivity of ovarian cancer cells to carboplatin through p53-dependent apoptotic response, thereby improving the therapeutic application of carboplatin in ovarian cancer patients [
16]. Besides, extent of HMGA1 phosphorylation has been found to be differentially expressed in response to MCM2 perturbation and has a significant role to play in modulating cell behaviors of lung cancer cells [
17]. MCM2 also has wide clinical application value in breast cancer diagnosis and prognosis [
18]. Of note, MCM2 has been proved to be closely related to stem cells. For instance, decreased MCM2 expression has been reported to cause serious deficiency in stem cells [
19]. Moreover, portions of retinoblastoma cells have been detected to display immunoreactivity to MCM2, as one of the stem cell markers [
20]. Although the relation between miR-195-5p/497–5p and MCM2 in colon cancer has rarely been studied before, MCM2 has been reported to be targeted by miR-31 in nasopharyngeal carcinoma and prostate cancer [
21,
22]. To the best of our knowledge, this is the first study reporting the binding relation between miR-195-5p/497–5p and MCM2 in colon cancer. In this study, we hypothesized that NF-κB and miR-497-5p/195-5p may participate in the regulation of CCSCs with the involvement of MCM2, and thus this study was performed to verify this hypothesis.
Materials and methods
Ethics statement
The study was approved by the Medical Ethics Committee of Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences and carried out in strict accordance with the Helsinki Declaration. All participating patients have signed the written informed consent. All animal experiments were performed with approval of the Animal Ethics Committee of Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences and in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
Study subjects
In this study, we collected colon cancer tissues and adjacent tissues from 35 patients with colon cancer (including 23 males and 12 females, aged 47–69 years) who underwent surgery in Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences from January 2018 to March 2019. All specimens were confirmed as primary colorectal cancer by pathological examination, and none of the patients had received radiotherapy or chemotherapy prior to the surgery. Five colon cancer cell lines (LoVo, SW620, SW1116, SW480, HCT-116) and one immortalized normal colon epithelial cell line (NCM460) (American Type Culture Collection (ATCC), VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or Roswell Park Memorial Institute (RPMI)-1640 (Gibco Company, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco Company, Grand Island, NY, USA) in an incubator at 37 °C with 5% CO2.
Selection and characterization of CCSCs
SW620 and LoVo cells were seeded into an ultra-low attachment cell culture plate (Corning Glass Works, Corning, N.Y., USA), and cultured in the medium prepared as previously reported [
23]. The cultured SW620 and LoVo cells were separately labeled with anti-AC133 microbeads conjugated antibody (1: 10) and anti-EpCAM microbeads conjugated antibody (1: 10) following the manufacturer’s instructions of the kit (Miltenyi Biotec, Bergisch-Gladbach, Germany) to isolate AC133+ SW620 cells and EpCAM+ LoVo cells on the FACS Calibur Flow Cytometer (Becton Dickinson, San Jose, Canada). AC133 is a type of antibody that is usually applied to isolate CSCs by testing a glycosylated epitope of CD133 on the cells [
24].
Cell treatment
CCSCs were transfected with 100 nM miR-195-5p/497–5p mimic or negative control (NC), 70 nM si-MCM2/p65 or NC, 100 nM pcDNA-MCM2/p65 (Guangzhou Ribobio, Guangzhou, China) according to the manufacturer’s instructions of Lipofectamine 2000 reagent. Cultured CCSCs were then assigned into the following groups: (1) to detect the relationship between NF-κB and miR-195-5p/497–5p: i. the si-NC group; ii. the si-p65 group; iii, the pcDNA-3.1 + miR-NC group; iv. the pcDNA-p65 + miR-NC group; v. the pcDNA-p65 + miR-497-5p group; vi. the pcDNA-p65 + miR-195-5p; (2) to detect the relationship between miR-195-5p/497–5p and MCM2: i. the si-NC group; ii. the si-MCM2 group; iii. The pcDNA-3.1 + miR-NC group; iv. The pcDNA-MCM2 + miR-NC group; v. the pcDNA-MCM2 + miR-497-5p group; vi. the cDNA-MCM2 + miR-195-5p group.
Dual-luciferase reporter gene assay
The artificially synthesized MCM2 3’UTR gene fragment was introduced into the psiCHECK-2 vector (Promega Corporation, Madison, WI, USA). The complementary sequence mutation sites of seed sequences were designed based on the wild type (WT) of MCM2. Specifically, the first binding site (− 604 ~ 594 bp) was mutated as WTΔ1, the second biding site (− 377 ~ 367 bp) as WTΔ2, and the third binding site (+ 106 ~ 116 bp) as WTΔ3. Meanwhile, WT binding sites were set as WT. All the above-mentioned binding sites were inserted into the psiCHECK-2 vector reporter plasmid. The correctly sequenced luciferase reporter plasmids MCM2 3’UTR-WT (100 ng) and MCM2 3’UTR-mutant type (MUT; 100 ng) were co-transfected into HEK-293 T cells (CRL-1415, Xin Yu Biotechnology, Shanghai, China) with miR-195-5p/497–5p mimic/NC-mimic (2 nM, Dharmacon, Lafayette, CO, USA), respectively. Following 48 h of transfection, the cells were collected and lysed. In addition, the fragments containing predicted p65 binding sites were amplified through P195 and then inserted into the PGL3 vector (Promega Corporation, Madison, WI, USA) by restriction endonucleases MluMlu I (Fermentas, ME, USA) and Nhe I (Fermentas, ME, USA). pNF-κB-TA-luc (Beyotime, Shanghai, China) reflects the changes in the activity of NF-κB. Luciferase activity was detected on a Glomax 20/20 luminometer fluorescence detector (Promega Corporation, Madison, WI, USA) using a luciferase detection kit (RG005, Beyotime, Shanghai, China).
Cell counting kit-8 (CCK-8) assay
A CCK-8 detection kit (Dojindo Laboratories, Kumamoto, Japan) was used to detect cell viability. In brief, CCSCs (4 × 103 cells/well) were seeded into a 96-well plate. Subsequently, 10 μL of CCK-8 reagent was added into each well and incubated for 2 h, followed by measurement of the optical density at 450 nm.
Flow cytometry
Annexin V and propidium iodide (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) was applied for the detection of cell apoptosis. In brief, the CCSCs were collected and rinsed with phosphate buffer saline (PBS), followed by rinsing with binding buffer. Next, the CCSCs were incubated with 5 μL of Annexin V for 15 min in the dark. Following another rinse, binding buffer and 5 μL of propidium iodide were sequentially added to the CCSCs. After on-ice incubation at 2 °C–8 °C, the cell mixture was subsequently analyzed using flow cytometry with the aid of the FACSAria II Special Order System (BD Biosciences, Franklin Lakes, NJ, USA).
The transfected CCSCs were treated by trypsin and prepared into cell suspension with CCSCs medium. The cell suspension (1 × 102 cells/well) was seeded into a 96-well ultra-low adherence culture plate (Corning Glass Works, Corning, N.Y., USA) and cultured in a 37 °C incubator for 5 days. After the incubation, the number of the formed microspheres in each well was observed and photographed under an inverted microscope (IX53, OLYMPUS, Tokyo, Japan).
A 6-well plate was coated with 2 mL of 0.7% low-melting-point agarose and supplemented with the cell-agarose mixture (0.35% agarose) at a cell density of 1 × 104 cells for every 100 cm2. Cells were replaced once every 2 to 3 days during the culture, which was terminated after 1 month. The culture dishes were taken out and the cells were counted under an inverted microscope (IX53, OLYMPUS, Tokyo, Japan). The cell mass with more than 50 cells was regarded as one cell colony, which was then photographed and counted.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from tissues or cells using a TRIzol kit (15596–018, Solarbio, Beijing, China) in strict accordance with the manufacturer’s instructions, followed by the determination of the RNA concentration. The primers were synthesized by Takara (Dalian, China) (Table
1). The reverse transcription was carried out according to the manufacturer’s instructions provided by the one-step miRNA reverse transcription kit (D1801, Haigene, Harbin, China), as well as the complementary (cDNA) reverse transcription kit (K1622, Yaanda Biotechnology Co., Ltd., Beijing, China). Using 2 μg total cDNA as the template, as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 serving as internal references, the fold changes in gene expression were calculated via relative quantification (2
-ΔΔCt method) with the use of a fluorescent qPCR (ViiA 7, DAAN Gene Co., Ltd. Of Sun Yat-sen University, Guangzhou, China).
Table 1
Primer sequences for RT-qPCR
miR-195-5p | Forward: ACACTCCAGCTGGGTAGCAGCACAGAAAT |
Reverse: TGGTGTCGTGGAGTCG |
miR-497-5p | Forward: CAGCAGCACTGTGGTTTGT |
Reverse: CGACAGCAGCACACTGTGGTT |
MCM2 | Forward: CCTCTGTGCTTTATGGACAC |
Reverse: GGAGGCTCACGAAACAGAGG |
U6 | Forward: CTCGCTTCGGCAGCACA |
Reverse: AACGCTTCACGAATTTGCTTC |
GAPDH | Forward: TCAAGGCTGAGAACGGGAAG |
Reverse: TGGACTCCACGACGTACTCA-3 |
Western blot analysis
High-efficiency radio-immunoprecipitation assay (RIPA) lysate (R0010, Solarbio, Beijing, China) was employed to extract the total protein from tissues or cells, in strict accordance with the manufacturer’s instructions. After protein separation through polyacrylamide gel electrophoresis, the protein was electrotransferred onto a polyvinylidene fluoride membrane (Merck Millipore, Billerica, MA, USA) using the wet transfer method. The membrane was then probed with the following diluted anti-rabbit primary antibodies (all purchased from Cell Signaling Technologies (CST), Beverly, MA,USA) against p65 (#8242, 1: 1000), p-p65 (#3039 s, 1: 1000), MCM2 (#3619, 1: 1000), CD133 (#64326, 1: 1000), epithelial cell adhesion molecule (EpCAM; #2626, 1: 1000), B-cell leukemia/lymphoma 2 (Bcl-2; #4223, 1: 1000), Bcl-2 associated X protein (Bax; #5023, 1: 1000), Nanog (#4903S, 1: 500), Oct-4 (#2890S, 1: 500), and Sox2 (#3579S, 1: 500), then subsequently re-probed with goat anti-rabbit immunoglobulin G (IgG; #7074, 1: 2000) diluent labeled with horseradish peroxidase and incubated for 1 h at room temperature. The ImageJ 1.48u software (National Institutes of Health, Bethesda, Maryland, USA) was utilized for the protein quantitative analysis. The ratio of gray value of the target protein band to that of the GAPDH internal reference band was regarded as the relative protein expression.
Human colon cancer xenografts in nude mice
CCSCs were inoculated into ultra-low adhesion culture plates and the transfected cells were assigned into the following groups: (1) to validate the effect of NF-κB on the tumorigenesis by regulating miR-195-5p/497–5p: I. the miR-NC + PBS group; ii. The miR-497-5p/195-5p agomir + PBS group; iv. the miR-497-5p/195-5p agomir + LPS group; (2) to validate the effect of miR-195-5p/497–5p on tumorigenesis by targeting MCM2: i. the miR-NC group; ii. the miR-497-5p/195-5p agomir group; iii. The miR-497-5p/195-5p agomir + pcDNA-3.1 group; iv. The miR-497-5p/195-5p agomir + pcDNA-MCM2 group. Cell microspheres were collected in a 10 mL centrifuge tube 7 days after culture, followed by centrifugation with the supernatant discarded. After treatment with 0.25% trypsin, a single-cell suspension was prepared using CCSCs medium suspension. Cell count was carried out using an amount of 10 μL single-cell suspension. Cell suspension (1 × 105 cells) was prepared, re-suspended in 50 mL saline and then sufficiently mixed with 50 mL Matrigel Matrix (1: 1). Finally, the suspension mixture was subcutaneously injected into the BALB/c-nu nude mice (5–6 weeks, 19–24 g, n = 6 in each group, Hunan Slac Laboratory Animals Co., Ltd., Changsha, Hunan, China).
Statistical analysis
The SPSS 21.0 (IBM Corp., Armonk, NY, USA) was applied for statistical data analysis. All data were presented as mean ± standard deviation (s.d.). Paired t-test was applied to compare data of the colon cancer tissues and adjacent tissues that conformed to normal distribution and homogeneity of variance. Unpaired t-test was utilized to analyze the data conforming to normal distribution and homogeneity of variance between two groups. Comparisons among multiple groups were analyzed using the one-way analysis of variance, and a Tukey’s test was performed for post-hoc test. Repeated measures analysis of variance was used for comparing data among multiple groups at different time points, followed by Bonferroni post-hoc test. A value of p < 0.05 indicates a statistically significant difference.
Discussion
Colon cancer is one of the common malignancies that occur in the human digestive system with a high mortality rate worldwide [
25]. MicroRNAs (miRs) have been reported to overcome chemoresistance in CSCs in colorectal cancer [
26]. In the present study, the major objective was to explore the role of NF-κB and miR-195-5p/497–5p in the stem-like properties of CCSCs, with the involvement of MCM2. The obtained findings from the present study demonstrated that NF-κB was capable of downregulating miR-195-5p/497–5p expression, thereby upregulating the expression of MCM2, which resulted in the enhancement of stem-like properties of CCSCs.
Initially, the current study found that miR-195-5p and miR-497-5p were poorly expressed in CCSCs, while MCM2 was highly expressed in primary colon cancer tissues. Consistent with our findings, a previous study demonstrated that miR-195-5p could regulate NOTCH2-mediated EMT of tumor cells in colorectal cancer tissues using integrated analysis [
27]. Downregulation of miR-497 was also found in colorectal cells, which was closely associated with amplified insulin-like growth factor 1 receptor-involved DNA copy number reduction [
28]. Intriguingly, as reported by another previous study, the expression of both miR-497 and miR-195 displayed a significant decline in colorectal cancer cells [
29]. Moreover, MCM2 showed a higher mRNA expression in patients with colonic adenomas with high-grade dysplasia, suggesting that MCM2 could be a potential biomarker for early diagnosis of colorectal cancer [
30]. In addition, similar to our findings, high expression of MCM2 was also found in CSCs marker-positive breast cancer cells [
31].
Another important finding obtained in the present study was that NF-κB could negatively regulate miR-195-5p/497–5p expression, thus promoting stem-like properties of CCSCs, as well as facilitating tumorigenesis and stem-like properties of CCSCs in vivo. This finding was validated not only by the decreased in protein expression of Bax and increased in protein expression of CD133, EpCAM, and Bcl-2, but also by the promoted cell viability, volume of microspheres, cell invasion and migration, and colony formation ability, as well as decreased cell apoptosis. In line with our finding, Moreover, a previous study has reported that miR-195-5p could downregulate YAP1 in a mouse colorectal cancer xenograft model, thereby notably decreases the tumor development in vivo [
32]. Besides, increased miR-497-5p has been reported to able to suppress proliferation as well as invasion of colorectal cancer cells by targeting PTPN3 [
33]. In addition, NF-κB-mediated signaling pathways displayed direct participation in the maintenance of properties of CSCs which closely related to tumor development, including colon cancer [
13]. Moreover, compound 19-inactivated NF-κB pathway was found to aid in the suppressive role of compound 19 in the progression of colorectal CSCs, which resulted in promoted cell apoptosis [
34]. Besides, it has been revealed that a novel signaling pathway, NF-κB/miR-497/SALL4 axis, is involved with inflammation and stemness properties in hepatocellular carcinoma cells [
35]. All the aforementioned results support the functions of overexpression of miR-195-5p/497–5p and that of NF-κB in colon cancer or CSCs, as demonstrated in the present study. Furthermore, results from RT-qPCR demonstrated that the overexpression of p65, a subunit of NF-κB, could significantly reduce the expression of miR-497-5p and miR-195-5p, indicating the negative regulation of miR-195-5p/497–5p by NF-κB in CCSCs, which was consistent with some existing reports. For instance, NF-κB inhibition by oxytocin could induce the up-regulation of miR-195 which promotes apoptosis and inhibits proliferation of breast cancer cells [
36]. In addition, miR-497 has been identified as a regulatory miR by NF-κB in a previous study [
37].
Furthermore, our results revealed that miR-195-5p/497–5p could target and downregulate the expression of MCM2, thereby contributing to the enhancement in stem-like properties of CCSCs both in vitro and in vivo. Consistent with our findings, the downregulation of MCM2 by siRNA has led to cell cycle arrest and apoptosis in colon cancer cells [
38]. Moreover, inhibition of MCM2 was also found to be able to reduce the foci forming of RAD51 in colon cancer cells [
39]. It was previously pointed out and demonstrated that MCM2 was presented in stem/progenitor cells of the subventricular zone within the brain and MCM2 could enhance green fluorescent protein expression which was specific to stem/progenitor cells [
40]. Additionally, cells that were positive in regard to MCM2, which serves as neural stem marker, showed a higher percentage in the retinoblastoma tumors that were invasive [
41]. The above-mentioned reports support the stimulatory role of MCM2 in CCSCs properties. In the current study, based on the starBase database, MCM2 was found to be a downstream target gene of miR-497-5p and miR-195-5p, and there were specific binding sites existed between miR-195-5p/497–5p and MCM2. This targeting relationship was further verified by dual-luciferase reporter gene assay. Moreover, the results from RT-qPCR demonstrated that the overexpressed miR-195-5p/497–5p could significantly decrease the expression of MCM2. A negative correlation was also detected between the expression of miR-497-5p/miR-195-5p and the expression of MCM2 in colon cancer tissues. Therefore, it can be concluded that miR-195-5p/497–5p could affect stem-like properties of CCSCs through the negative regulation of MCM2.
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