Background
Colorectal cancer (CRC), a common malignant tumor, is one of the main causes of cancer-related deaths [
1]. The rapid development of diagnostic methods, multidisciplinary diagnosis and treatment models, small-molecular targeted drugs, and immunotherapy drugs in recent years has significantly improved the diagnosis and treatment of CRC [
2‐
5]. However, the prognosis of CRC patients needs to be improved further due to the unclear pathogenesis of CRC and lack of effective therapeutic drugs. Moreover, CRC treatment also levies a heavy economic burden on the patients [
6]. There is still a lot to be done to ensure effective and inexpensive drugs for CRC.
Sodium butyrate (NaB) is a short-chain fatty acid produced by intestinal microflora during the anaerobic fermentation of dietary fiber [
7]. It is readily available and inexpensive. As a component of intestinal microecological environment, its short-term effects on the human body are not obvious [
8]. NaB can maintain the stability of the gut environment by regulating various cellular functions, including proliferation, differentiation, apoptosis, and intestinal epithelial permeability [
7,
9,
10]. By regulating immune cells, including regulatory T cells, dendritic cells, natural killer cells, and macrophages, NaB inhibits the expression of HDAC [
11], increases the acetylation of regulatory proteins, and regulates various molecular signaling pathways [
12], thereby inhibiting the proliferation of malignant tumor cells and reducing the risk of cancer [
13].
We confirmed the inhibitory effect of NaB on CRC cells in the present study. The molecular mechanism of NaB on CRC cells is complex, and there are inconclusive and inconsistent reports regarding this. Compiling the latest gene database information and by analyzing the genomic data of CRC cell lines after NaB intervention, a molecular network of the effect of NaB on CRC cells was constructed. In addition, the analysis of differentially expressed RNA related to CRC prognosis will provide support for further clinical application of NaB in CRC.
Methods
Cell culture
Human CRC cell lines SW480, LOVO, HCT116, and HCT8 were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc.) was used as the cell culture medium. These CRC cell lines were incubated in a humidified incubator (Sanyo XD-101; Sanyo Electric Co., Ltd., Osaka, Japan) with 5% CO2 at 37 °C.
CCK-8 assay
We seeded the exponentially growing CRC cell lines (5000 cells/100 μl) into 96-well plates, pre-incubated them for 24 h, added NaB at concentrations ranging from 0 to 32 mmol/L (10 μl), and then incubated them for 24, 48 and 72 h. Following this, we added 10 μl of CCK-8 solution (KGA317, Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) into the 96-well plates and incubated them for 3 h. A microplate reader (ELx800; BioTek Instruments, Inc., Winooski, VT, USA) was used to measure the absorbance at 450 nm. Physiological saline was used as blank, and untreated CRC cells were used as the control. The inhibition rate (IR) was calculated as follows: IR = [1 - (A450 values of the experimental group – A450 values of the blank group) /(A450 values of the control group – A450 values of the blank group)] × 100%.
Analysis of cell apoptosis
CRC cells (5 × 105 cells/well) were treated with NaB at a concentration of 0, 2.5, 5, 10 mmol/L for 48 h at 37 °C and seeded in 6-well plates. Following this, they were washed twice with 0.1 mmol/L phosphate-buffered saline (PBS), mixed with 5 μl annexin V fluorescein isothiocyanate (FITC; KGA105; KeyGen Biotech Co., Ltd.) and propidium iodide (PI; KGA511, KeyGen Biotech Co., Ltd), incubated for 15 min at room temperature in the dark, and then analyzed using FACS flow cytometer (Becton.Dickinson, Franklin Lakes, NJ, USA) with an argon laser (488 nm). The cell number in each quadrant was calculated by the internal software system.
Cell cycle analysis
We cultured CRC cells along with HCT116 cell lines and HCT8 cell lines in 6-well plates (3.0 × 105 cells/well) with medium for 24 h, treated them with NaB (5 mmol/L) for 48 h, then washed, collected, and fixed the cells using 70% ethanol and stored them at 4 °C overnight. Following this, Tris-HCl buffer (pH 7.4) containing 1% RNase A (cat. no KGA511; KeyGen Biotech Co., Ltd) was added to the cells and they were stained using PI (5 mg/ml) in each well. Flow cytometry (FACSCalibur; Becton-Dickinson) and cell cycle analysis software (FlowJo, version 7.6.5; KeyGen Biotech Co., Ltd.) were used to analyze the data. The experiment was repeated three times for each sample.
Scratch-wound assay
CRC cells were cultured in 12-well plates, which then formed mono-layer cells; after this, a line was scratched using a sterile 20-ul pipette tip, and the cultured monolayer cells were washed three times and then treated with NaB (5 mmol/L) for 24 h. The width of the scratch area was used to estimate the migration capacity.
Transwell invasion assay
CRC cells were collected and cultured with 200 μL serum-free medium in the upper chambers of Matrigel-coated transwell plates (2 × 104 cells per well) with a pore size of 8 μm; cell culture medium was added to the lower chamber. After incubation for 48 h at 37 °C and 5% CO2, the cells were fixed with a 4% formaldehyde solution for 30 min and then stained with a 0.4% crystal violet solution for 10 min at room temperature. Cells that invaded into the bottom of the transwell membranes in each chamber were randomly selected and photographed with a microscope. The invasion capacity of untreated HCT8 and HCT116 cells and those treated with NaB (5 mmol/L) was assessed by counting the number of cells in 5 microscope fields (200 folds).
Statistical analysis
SPSS software version 18.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analyses. Differences between measurement date for the groups were assessed using independent sample T tests. Cell counting data were assessed using the chi-square test. P < 0.05 was considered to indicate a statistically significant difference.
Molecular network construction
Data processing and re-annotation
The data set No. GSE54127 (species:
Homo sapiens) from the NCBI GEO [
14] (Gene Expression Omnibus, GEO,
http://www.ncbi.nlm.nih.gov/geo/) database includes human genome information from 6 HCT116 cell samples and 6 HCT116 cells treated with NaB. Data were obtained using Agilent-026652 Whole Human Genome Microarray 4x44K v2 (Probe Name version) platform. The original chip probe signals were processed using data standardization, including background correction, normalization, and the determination of expression using the RMA (robust multi - array business) method in the R [
15] affy package (Version 1.52.0,
http://www.bioconductor.org/packages/2.9/bioc/html/affy.html). The human reference genome (GRCh38) from the GENCODE database [
16] (
https://www.gencodegenes.org/releases/current.html) was compared to the above probe sequences by using seqmap software [
17]. Probes with unique maps were retained, and the corresponding genes for each probe were identified based on chromosome position and positive- and negative-chain information according to the human gene annotation file (Release 25) provided by GENCODE. Annotation information for the corresponding probe “protein_coding” probe was as mRNA, while that for the corresponding “antisense”, “sense_intronic”, “lincRNA”, “sense_overlapping” or “processed_transcript” probes was as lncRNA. Finally, probes that did not match gene symbols were removed. The mean values for different probes were taken as the final mRNA/lncRNA expression values.
Differential RNA analysis
The classical Bayesian method provided via the limma package [
18] (Version 3.10.3,
http://www.bioconductor.org/packages/2.9/bioc/html/limma.html) was used to analyze the differential mRNA and lncRNA between the experimental group (treated with NaB) and control group (HCT116). The Benjamini & Hochberg method [
19] was used to perform multiple inspection and correction for the corresponding
p value and logFC value. The
p value < 0.05 after the correction and |logFC| > 2 (4-fold change) was as the difference expression threshold.
Functional and pathway enrichment analysis
The R package clusterProfiler [
20] (version: 3.8.1,
http://bioconductor.org/packages/release/bioc/html/clusterProfiler.html) was used to perform the Gene Ontology BP (biological process) [
21] and KEGG [
22] pathway enrichment analysis with regard to differentially expressed mRNA. Pearson correlation coefficients were obtained for differentially expressed mRNAs and lncRNAs. Relationships with r > 0.9 and p value< 0.05 were screened for subsequent ceRNA network construction. The mRNA was as potential target gene of lncRNA. The KEGG pathway enrichment analysis for the mRNA corresponding to the lncRNA was carried out using the clusterProfiler package in R, and lncRNA function was indirectly predicted via the enrichment analysis results. An adjusted
P value < 0.05 was considered to indicate significant enrichment after “BH” correction.
Protein interaction (PPI) network construction
The interaction between the proteins encoded by the differentially expressed genes obtained from the above analysis was analyzed based on the STRING database (Version:10.0,
http://www.string-db.org/) [
23]. The species was set to
Homo sapiens and the PPI score was set to 0.9 (highest confidence). The Cytoscape software [
24] was used to construct the network basing on the PPI pairs. The CytoNCA plug-in [
25] (Version 2.1.6,
http://apps.cytoscape.org/apps/cytonca) was used to analyze the topological properties of the node network. The parameter was set to without weight and the results included Degree Centrality (DC), Betweenness Centrality (BC), and Closeness Centrality (CC). The important nodes (hub proteins) involved in protein interaction in the PPI network were determined by ranking the topological properties of each node.
MiRNA prediction and ceRNA network construction
The lnCeDB [
26] database was used to predict the targeted miRNA corresponding to the differentially expressed lncRNA based on the lncRNA-mRNA pairs identified I the above analyses. Six databases—miRWalk, miRanda, miRDB, PITA, RNA22, and Targetscan—and miRWalk2.0 software [
27] (
http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/) were used to predict the targeted miRNA corresponding to the differentially expressed mRNAs. The lncRNA-miRNA-mRNA pairs were obtained by screening the mRNA-miRNA pairs and lncRNA-miRNA pairs regulated by the same miRNA. The Cytoscape software was used to construct the ceRNA network via analysis of the correlation of the lncRNA-miRNA-mRNA pairs (Correlation coefficient > 0.9). CytoNCA was used to analyze the degree of connectivity of each node in the ceRNA network.
Survival analysis of differential mRNA and lncRNA
Clinical information and exon-seq data of colorectal cancer (COADREAD) from the Broad Institute’s GDAC Firehose platform (
http://gdac.broadinstitute.org/) were used for the survival analysis of differential mRNAs and lncRNAs. Chromosome position annotation data for lncRNA and protein-coding RNA in Gencode database were compared with the expression profile data of exon-seq. The starting and ending positions of an exon contained lncRNA or mRNA in the annotated database, and was consistency with the positive and negative chains. Finally, 369 cases of cancer with survival information were obtained by comparison of samples,
Clinical information, including overall survival (OS) and OS status, was included. The lncRNAs and mRNAs identified in the above ceRNA network were selected as candidate genes. The expression value of these candidate genes was screened using TCGA data, and divided into high expression and low expression groups based on the median expression value. The log-rank statistical test, with a threshold of p < 0.05, was conducted. The relationship between candidate genes and patient prognosis was analyzed, and the K-M survival curve was plotted.
qPCR analysis
Human colorectal cancer cell lines HCT116 and HCT8 from the Chinese Academy of Sciences were selected. HCT116 and HCT8 cells were treated with 5 mmol/L NaB for 48 h as the experimental group, and not treated as the control group. Our research group cultured cell lines in RPMI-1640 (Gibco; Thermo Fisher Scientifc, Inc., Waltham, MA, USA), which mixed with 10%FBS and 1%P/S, and incubated them in a humidified incubator (Sanyo XD-101; Sanyo Electric Co., Ltd., Osaka, Japan) at 37 °C and 5% CO
2.Using TRIzol reagent (Invitrogen; Yi Sheng Biotechnology Scientifc, China) extracted total RNA from those cells according to its instruction to reverse transcription and amplify PCR. Using the FTC-3000 real-time fluorescence quantitative PCR system (Funglyn Biotech, Inc., Ontario, Canada) performed PCR reactions. The reaction conditions are as follows. First, performe for 3 min at 50 °C, and then Increase temperature to 95 °C for 3 min. After 10s, cool to 60 °C for 30 s. The reaction is repeated over 40 cycles. The experiment was repeated three times. Researcher measured the relative expression of HMGA2, LOXL2 and ST7 by the 2
-△△Ct method. The GAPDH was regarded as a reference gene. Information on primers was shown in Table
1.
Table 1
Primers of genes used for qRT-PCR
GAPDH-hF | GAAGGTGAAGGTCGGAGTC |
GAPDH-hR | GAAGGTGAAGGTCGGAGTC |
HMGA2-F | CGAAAGGTGCTGGGCAGCTCCGG |
HMGA2-R | CCATTTCCTAGGTCTGCCTCTTG |
LOXL2-F | GGGTGGAGGTGTACTATGATGG |
LOXL2-R | CTTGCCGTAGGAGGAGCTG |
ST7-F | CGCGGATCCCCTCTGTGTGTGTGTGTGTAAC |
ST7-R | CCGGAATTCGCATTCCTGGGCAGGTCGGT |
EdU assay
The EdU assay was performed to detect cell proliferation by using the KeyFluor488 Click-it EdU imaging kit (Jiangsu Kaiji Biotechnology Co., LTD., China, KGA331–100). The SW480 cells treated with 5 mmol/L NaB for 48 h, which were the experimental group, oppositely untreated cells were the control group. After cell digestion and counting, cell suspension with a concentration of 1 × 104 /mL was prepared. After adding 200 μL cell suspension (2 × 103 cells/well) to each well of 96-well cell culture plates, the plates were placed in an incubator at 37 °C with 5% CO2 for 24 h.After repeated cleaning, incubation, and decolorization shaker incubation, a high-content cell imaging system (Top Biotek Co, Ltd., USA, ImagExpress Micro HCS) was used for detection.
Discussion
NaB is the final product of dietary fiber decomposition by intestinal microorganisms, such as
Butyricicoccus pullicaecorum, Bifidobacterium longum, Butyrivibrio fibrisolvens, Ruminococcus bromii, and
Lachnospiraceae, in the colorectum [
28]. Although its concentration is low in the colorectum, NaB has important biological activities in intestinal mucosal cells [
29]. NaB is also involved in the occurrence and development of CRC [
30]; NaB levels have been reported to be decreased in stool samples of CRC patients [
31,
32]. In the present study, we investigated the biological effects of NaB on CRC and explored its underlying molecular mechanism(s) of action. We found that in vitro, NaB induced apoptosis and inhibited CRC cell proliferation, invasion, and metastasis by modulating complex molecular networks. NaB also induced G1 phase block in CRC cells. The RNA prediction and molecular network construction may provide novel directions for future research.
Although some molecular mechanistic studies have been conducted to elucidate how NaB inhibits CRC [
33,
34], the molecular mechanism remains obscure because of the complexity of intracellular molecular signaling pathways. To address this, we constructed a PPI network and a ceRNA network using multiple databases. The ceRNA (competing endogenous RNA) hypothesis shows the interaction mechanisms between RNA [
35]. The competition between lncRNA and microRNA leads to the change of downstream molecular expression and thus altered cell function [
36,
37]. Increasing evidence in recent years has implicated that ceRNAs play important role in the development of many cancers, such as breast cancer [
38], gastric cancer [
39], cholangiocarcinoma [
40], CRC [
41] and so on. We constructed a PPI network and a ceRNA network basing on multiple databases. Our results may provide guidance and direction for further research on the molecular mechanism of NaB inhibition CRC.
The relationship between CRC prognosis and differentially expressed RNA was analyzed basing on the gene expression value and clinical survival information of CRC from TCGA. Three differentially expressed mRNAs including HMGA2, LOXL2, and ST7 were significantly correlated with the prognosis of CRC. A high expression of HMGA2 indicated a poor prognosis of CRC, and HMGA2 was significantly downregulated after NaB treatment. Low LOXL2 expression predicted the poor prognosis of CRC, and LOXL2 was significantly upregulated after NaB treatment. These two genes may be potential molecular targets of NaB in inhibiting CRC. The low ST7 expression predicted the poor prognosis of CRC. However, the ST7 gene in CRC cells is significantly downregulated after NaB treatment. It may be related to the side effects of NaB.
We found that HMGA2, LOXL2, and ST7 were significantly correlated with the prognosis of CRC. The prognostic factors are not only related to the biological function of tumor cells, but also related to recurrence and metastasis, drug sensitivity, PS score of patients, nutritional status and other factors. We will try to study the prognostic differential genes, such as HMGA2, LOXL2 and ST7 in the further studies.
There are some limitations in the present study. Although we built the network based on database analysis and RNA prediction, we did not further verify its molecular network. The critical nodes of the PPI network and ceRNA network may provide some direction for further molecular validation. In addition, although some studies have indicated that NaB is reduced in the stools from CRC patients, the interaction of numerous microorganisms and various metabolites in stools leads to a complex intestinal microecosystem. The clinical application of NaB is limited by the complexity, variability and individuality of intestinal microecosystem.
Conclusions
We investigated the effects of NaB on CRC cells. In addition, we explored its molecular pathways basing on database analysis and RNA prediction. NaB induces the apoptosis and inhibition of CRC cell proliferation, invasion, and metastasis by modulating complex molecular networks. This will provide a theoretical basis for NaB as an anticancer agent.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.