Background
Colon cancer is a common malignant tumor of digestive tract in clinic, and its incidence and mortality are high [
1]. With the adjustment of lifestyle and diet, the incidence of colon cancer is increasing year by year and becoming younger in China [
2]. Like most malignant tumors, the pathogenesis of colon cancer is not entirely clear. At present, colon cancer is considered to be the combined effect of environmental factors and genetic factors. Studies have shown that the main factors affecting the incidence of colon cancer include environment, intestinal homeostasis, diet, alcohol and tobacco addiction and physical exercise [
3]. Colon cancer treatment still is primarily surgical, chemotherapy and radiotherapy are supplementary. For therapeutic effect, there are significant individual differences among patients with colon cancer. In the patients with advanced colon cancer, the defects of the above therapy are obvious resulting in a poor prognosis. Postoperative metastasis for colon cancer chiefly includes hematological metastasis, peritoneal metastasis and distant lymph node metastasis, which are frequently accompanied by local recurrence [
4]. Hematogenous metastasis is the dominating cause of failure in the treatment of colon cancer. The survival rate of colon cancer is overtly relevant to clinical stage, and the 5-year survival rates of patients with no metastasis, local metastasis and distant metastasis are 90, 70 and 10%, respectively [
5]. Therefore, to find the markers of early diagnosis and explore the key molecules involved in the growth and metastasis of colon cancer is the focus of current research.
Long-stranded non-coding RNA is a class of RNA molecules whose transcriptional length exceeds that of 200 nt and can’t carry out coding proteins [
6]. LncRNA usually is located in cytoplasm or nucleus. The number of lncRNA in the human genome is astonishingly large [
7]. LncRNA participates in the regulatory processes of chromatin modification, transcriptional interference, transcriptional activation, nuclear transport, selective splicing and regulation of proto-oncogene activation, so as to regulate gene expression at epigenetic, transcriptional or post-transcriptional levels [
8,
9]. Abnormal expression and functions of lncRNA are involved in the occurrence and development of many diseases, especially malignant tumors. It is reported in colon cancer that LINC01082 and lncRNA THOR can regulate cell proliferation, migration and invasion [
10,
11] . LncRNA can not only directly participate in the post-transcriptional regulation of mRNA, including variable splicing, RNA editing, protein translation and transport, but also affect the expression of target genes by controlling microRNA [
12]. In some tumor cells, lncRNA carries the seed sequence of miRNA to prevent miRNA from binding to its target mRNA. The functions of lncRNA AWPPH in proliferation of colon cancer cells were regulated by targeting GLUT-1 [
13]; LncRNA CCAT1 promotes autophagy of liver cancer cells via regulating ATG7 by sponging miR-181 [
12]; LncRNA HOTAIR promotes colon cancer progress by targeting miR-34a [
14]. It is reported that the high expression of LINC00662 in lung cancer, gastric cancer and oral cancer promotes the occurrence and development of cancer [
15,
16]. This suggests that LINC00662 is signally connected with cancer development. However, its role and related mechanisms in the initiation and progression of colon are unknown.
ERK, a serine/threonine protein kinase, is a signal transduction protein that transmits mitogen signals and is located in the cytoplasm. The activated ERK was transferred to the nucleus to regulate the activity of transcription factors and produce cellular effect. It is known that there are five subfamilies in the ERK family, including ERK1~ERK5. ERK1 and ERK2 are involved in regulating a series of physiological processes in different cells, including meiosis and mitosis. A variety of stimulators such as growth factors, cytokines, viruses, ligands of G protein-coupled receptors and oncogenes can activate ERK1 and ERK2 [
17]. ERK signaling pathway is involved in the regulation of cell proliferation, differentiation and apoptosis. Activated ERK signaling pathway promotes the occurrence and development of a variety of cancers [
18,
19]. mRNAs, miRNAs, lncRNAs and circRNAs affect the occurrence and development of cancer by regulating the activation of ERK signaling pathway. It is reported that lncRNA HOXD-AS1 influences the proliferation and invasion of hepatocellular carcinoma cells by regulating the activation of ERK signaling pathway [
20]; miR-98 inhibits cell growth and invasion in retinoblastoma by targeting ERK signaling Pathway [
21]; cicrRNA_006528 promotes the occurrence and development of breast cancer by activating ERK signaling pathway [
22]; ectonucleoside triphosphate phosphohydrolase-7 (ENTPD7) inhibit the proliferation of lung cancer by inhibiting the activation of ERK signaling pathway [
19]. Our previous studies have shown that CLDN8 promotes the proliferation and metastasis of colon cancer cells by activating MAPK/ERK signaling pathway [
23]. Therefore, the role of LINC00662 in ERK signal pathway needs to be further studied.
Methods
Clinical samples
Cancer tissue and adjacent normal tissue in from 72 patients with colon cancer that resected in surgical procedures were collected from the First Affiliated Hospital of Zhengzhou University from July 2015 to July 2018. The patients’ clinical information is listed in Table
1. Liquid nitrogen was used for tissue store at − 80 °C. Each participant provided written informed consent. The use of human clinical tissues was approved by the Institutional Human Experiment and Ethics Committee of the First Affiliated Hospital of Zhengzhou University. All experiments were conducted under the rule of the Declaration of Helsinki.
Table 1Relationship between LINC00662 expression and clinical parameters
Age | | | | 0.001 | 0.974 |
< 60 | 41 | 20 | 21 | | |
> =60 | 31 | 15 | 16 | | |
Gender | | | | 0.259 | 0.611 |
male | 31 | 14 | 17 | | |
female | 41 | 21 | 20 | | |
Location | | | | 0.859 | 0.354 |
left | 39 | 17 | 22 | | |
right | 33 | 18 | 15 | | |
Histology | | | | 0.845 | 0.358 |
adenocarcinoma | 41 | 18 | 23 | | |
mucinous adenocarcinoma | 31 | 17 | 14 | | |
Stage | | | | 37.605 | 0.000 |
I + II | 33 | 29 | 4 | | |
III + IV | 39 | 6 | 33 | | |
T stage | | | | 8.354 | 0.004 |
Tis | 41 | 26 | 15 | | |
T1-T3 | 31 | 9 | 22 | | |
N stage | | | | 16.068 | 0.000 |
N0 | 36 | 26 | 10 | | |
N1 + N2 | 36 | 9 | 27 | | |
M stage | | | | 22.363 | 0.000 |
M0 | 31 | 25 | 6 | | |
M1 | 41 | 6 | 31 | | |
Status | | | | | |
death | 12 | 2 | 10 | 5.882 | 0.015 |
alive | 60 | 33 | 27 | | |
Cell line culture
Human colon epithelial cells (NCM460) and colon cancer cell lines (HCT116, HCT8, HCT29, LOVO, SW480, CT26 and LS174T) and were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Modified RPMI-1640 medium (ThermoFisher) which is supplemented with 10% FBS including 100 μg/L penicillin and 100 μg/L streptomycin was applied to maintain all cells at 5% CO2 and 37 °C.
Cell transfection
lncRNA LINC00662 pcDNA3.1 expression vector (5′-TCTACTTATATTTTATTCAAAAATTTA-3′), CLDN8 pcDNA3.1 expression vector (5′-ATGGCAACCCATGCCTTAGAAATCGCTG-3′), IL22 pcDNA3.1 expression vector (5′-ATGGCCGCCCTGCAGAAATCTGTGAGCTCTTT-3′) and empty vectors (pcDNA3.1-vector; 5′-ATCGCGCGTGTGCCGTGCAAAACTGCTACCAGT-3′) were designed and constructed by Sangon Biotech Co., Ltd. LINC00662 small interfering RNAs (siRNA; 5′- TAAATTTTGTAATAAAATATAAGTAGA-3′) and negative control (NC) siRNA were purchased form Thermo Fisher Scientific, Inc. miR-340-5p mimics (5′-TTATAAAGCAATGAGACTGATT-3′), miR-340-5p inhibitors (5′-AATCAGTCTCATTGCTTTATAA-3′), NC mimics (5′-TACTACGCATTATCCCATGCA-3′) and NC inhibitors (5′-TTAAACGTGTGTCGTACTGAA-3′) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Cell transfections were performed using Lipofectamine® 2000 reagent (Thermo Fisher Scientific, Inc.) at 37 °C with 10 nM of vectors, 40 nM of siRNAs and 40 nM of miRNAs. Cells were incubated with the transfection mixtures for 6 h. Cells treated with Lipofectamine® 2000 reagent only were used as untreated control cells. Cells transfected with empty vectors, NC siRNA, or NC miRNA were used as transfection controls. Cells transfected with pcDNA3.1-LINC00662, siRNA-LINC00662, siRNA-LINC00662 and CLDN8, siRNA-LINC00662 + IL22, miR-340-5p mimics and miR-340-5p inhibitors were collected 12 h after transfection prior to subsequent experimentation. Transfection efficiency was detected by RT-qPCR and western blot assays.
Cell counting kit-8 (CCK8) assay
Cells transfected with pcDNA3.1-LINC00662, siRNA-LINC00662, siRNA-LINC00662 and CLDN8, siRNA-LINC00662 + IL22, miR-340-5p mimics and miR-340-5p inhibitors (2 × 104 cells/mL) were incubated at 5% CO2 and 37 °C on 96-well plates (100 μL/well) for 24 h. CCK8 solution (Beyotime, Shanghai, China) was then added to each well after 24, 48, 72 and 96 h. Cell viability was estimated by a microplate reader which measure the absorbance values at a wavelength of 450 nm.
Cells transfected with pcDNA3.1-LINC00662, siRNA-LINC00662, siRNA-LINC00662 and CLDN8, siRNA-LINC00662 + IL22, miR-340-5p mimics and miR-340-5p inhibitors were seeded into 12-well plate and incubated with complete medium at 37 °C for 14–21 days. Then, the cells were fixed with 4% paraformaldehyde and stained with 2% crystal violet. The images were obtained using an inverted microscope.
Transwell assay
Cells (5 × 104) were suspended in serum-free DMEM and added to chambers (8 mm, BD Biosciences) coated with BD BioCoat Matrigel. After incubation, the cells on the upper membrane surface were removed with a cotton tip. Then, crystal violet was used to stain them and then 5 representative microscopic fields were selected to count cells under an Olympus fluorescence microscope (Tokyo, Japan) to measure the rate of invasion. Experiments were conducted 3 times.
Wound healing assay
In this study, cells in each group were implanted into 6-well culture plates with the density of 1.0 × 106cells/well. After the cells had fused, a scratch was scraped with a pipette tip on the cell monolayer, and PBS (Beyotime, Wuhan, China) was subsequently applied to wash cells for 3 times, and FBS-free medium was used to seed cells. At 0 and 48 h incubation, the colon cancer cell lines were photographed using the inverted microscope (Olympus, Japan) and the scratch area was assessed using Image J software (National Institutes of Health, Bethesda, MD, USA). migration rate = migration distance/original distance.
Flow cytometry
Cells grown normally without treatment were used as normal controls. Cell apoptosis was detected using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Sigma) by flow cytometry. Cells (2 × 105) were seeded into 6-well plates for 48 h. Subsequently, cells were washed with PBS and resuspended in binding buffer, followed by staining with 10 μL Annexin V-FITC for 10 min and 5 μL PI for 10 min in the dark according to the manufacturer’s instructions. The apoptotic (FITC positive and PI positive or negative) cells were analyzed by using a flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Target prediction and luciferase reporter assay
The putative targets of LINC00662 were predicted by the starbase v2.0. The reporter vector pmiRGLO- LINC00662-wild-type (LINC00662 WT) or miRGLO- LINC00662-mutant (LINC00662 MUT) containing the predicted miR-340-5p binding sites were purchased from GenePharma (Shanghai, China). LINC00662 WT or LINC00662 MUT was co-transfected with miR-340-5p mimics/inhibitors or NC mimics/inhibitors using Lipofectamine 2000 (invitrogen, USA). The putative targets of miR-340-5p were predicted by the TargetScan and miRDB. The reporter vector pmiRGLO- CLDN8-wild-type (CLDN8 WT) or miRGLO-CLDN8-mutant (CLDN8 MUT) and the reporter vector pmiRGLO-IL22-wild-type (IL22 WT) or miRGLO-IL22-mutant (IL22 MUT) containing the predicted miR-340-5p binding sites were purchased from GenePharma (Shanghai, China). CLDN8/IL22 WT or CLDN8/IL22 MUT was co-transfected with miR-340-5p mimics/inhibitors or NC mimics/inhibitors using Lipofectamine 2000 (invitrogen, USA). After 48 h, firefly and renilla luciferase activities were measured with the Dual Luciferase Reporter assay system (Promega, USA). The luciferase activities were normalized with the renilla luciferase activity.
The 5’UTR, coding sequences (CDS) and 3′UTR of IL22 were in- serted into PMIR-Reporter vector, denoted as Luc-IL22–5’UTR, Luc- IL22-CDS and Luc- IL22–3′UTR, respectively. The promoter sequences of IL22 were cloned into pGL3 vector, named as pGL3- IL22. For confirming the interaction between LINC00662 and IL22, Luc- IL22–5’UTR or Luc-S IL22-CDS or Luc- IL22–3′UTR or pGL3- IL22 was co-transfected with pcDNA3.1-LINC00662 infection in colon cancer cells (HCT29, LS174T, LOVO and CT26 cells). 72 h later, cells were lysed with Reporter lysis buffer (cat. no, E397 A; Promega Corporation, Madison, WI, USA) and luciferase activity was measured with VivoGlo Luciferin kit (cat. no., P1041; Promega Corporation) using a lumin- ometer (Thermo Fisher Scientific, Inc.) and normalized to β-gal activity.
Biotin RNA pull-down assay
This assay was conducted as previously reported. Biotin-labeled sense or antisense oligos of LINC00662 were incubated with HCT29, LS174T, LOVO and CT26 cell lysate for 1 h. The complex was pull down by streptavidin-coupled agarose beads (Invitrogen). Sense probes included 5′-(biotin-) TGTGGAGATGGCTGGTACCAGT-GCAAGACG-3′, 5′-(biotin-) GGTACAGGACGCAACCAGA-GACGGGAGGTA-3′ and 5′-(biotin-)AGGTAGGAGTGCGG-TACAGGTACGGGCACC-3′. Antisense probes comprised 5′- (biotin-)CCTGAACCCTTGCCAGTATCCTGACCACGT-3′, 5′- (biotin-)ACCTCCTGTCCTAGGTCCTGCGTCCTTTCG-3′ and 5′-(biotin-) CCTACTGCGCTAGCCGGGTCCACCACTTCT-3′. The isolated RNA was transcribed into cDNA and then the amounts of LINC00662 and miR-340-5p were measured by RT-PCR as described in the method of RT-PCR.
Functional enrichment analysis
The gene targets of miR-340-5p were identified using TargetScan and miRDB database. The Venn tool (Venn v2.0.2) was used to filter miRNA target genes into all three programs. Gene ontology (GO) categories (Biological Process, Cellular Components, and Molecular Processes) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Arraystar Inc., Rockville, USA) were used to perform the functional analysis for predicted miRNA target genes.
Immunohistochemistry
Immunohistochemistry was performed as described previously [
24]. Briefly, the antibody against VEGF and MMP-2 (Proteintech) was tested on sections from a tumor tissue array. To quantify the status of VEGF and MMP-2 protein expression in those groups, the intensity of the VEGF and MMP-2 immunoreaction was scored as follows: 0, none; 1, weak; 2, moderate; and 3, intense.
Xenograft tumor model
For tumor growth assay, HCT29 cells transfected with pcDNA3.1-vector or pcDNA3.1-LINC00662 and CT26 cells transfected with NC-siRNA or LINC00662-siRNA were trypsinized and washed and resuspended in DMEM without FBS. 20 male athymic nude mice (SLAC laboratory animal Center, Shanghai, China) were randomly divided into 4 groups (5 mice/group), and 2 × 106 HCT29 or CT26 cells were subcutaneously injected into the right armpit of mice. The tumor size was determined every 3–4 days after tumor formed (around 1–2 weeks). At 30 days after injection, the mice were euthanized and the excised tumor tissues were formalin-fixed, paraffin-embedded, sectioned and then analyzed with VEGF and MMP-2 immunohistochemistry, and the tumors weight were weighted by a digital balance and tumor volume were measured by a ruler.
Co-immunoprecipitation (co-IP)
Co-immunoprecipitation was performed as described previously [
25]. Both the input and IP samples were analyzed by Western blot using various antibodies at the following dilutions: IL22 antibody (1:1000), CLDN8 antibody (1:1000), Flag-tag antibody (1:1000), HA-tag antibody (1:1000) and normal rabbit/mouse IgG (CST).
Western-blot analysis
Total protein was extracted from cells. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime, Jiangsu, China) and then transferred onto PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed at room temperature for 2 h. Anti-CLDN8, anti-IL22, anti-p-ERK, anti-ERK, anti-cleaved caspase-3, anti-bax, anti-bcl-2, anti-XIAP, anti-VEGF, anti-MMP-2, anti-E-cadherin, anti-N-cadherin and anti-GAPDH (1:800, abcam) were added overnight at 4 °C. The membranes were subsequently incubated with goat anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (1:5000, abcam) at room temperature for 2 h. Finally, proteins were visualized using a WestrenBright ECL Kit (Advansta, USA).
RNA extraction and real-time PCR
The total RNA was extracted by using a TRIzol reagent. The first-strand cDNA was synthesized from 1 μg of total RNA using the Reverse Transcription System Bestar qPCR RT Kit according to the manufacturer instruction. Real-time PCR was carried out with an ABI 7500 Real-Time PCR System (Applied Biosystems, Lincoln Centre Drive, Foster City, CA 94404, USA). Each assay was performed in triplicate, and β-actin was used as the endogenous control gene. The relative amount of LINC00662, miR-340-5p, CLDN8 and IL22 were calculated using with a 2−ΔΔCt method and normalized using GAPDH as an internal control. The primers used in this study were shown below: for miR-340-5p, 5′-CCGTTAGTTACGATTCGAAG-3′ (forward), 5′- AGGCCGCGCGTAGTGATGCAACA-3′ (reverse); for U6: 5′- AACCTTATATCGGGCGGGA-3′ (forward), 5′-TTACGGCGATGCATAAT-3′ (reverse); for LINC00662: 5′-CGGGCGATTATCGACGATC-3′ (forward), 5′- TCGGGATCGACTACCCTAGGTAC-3′ (reverse); for IL22: 5′- AATGGCGGGCTAGGGGCCCTT-3′(forward), 5′- CCTAGCTACGAATCCTAGGAGA-3′ (reverse); for CLDN8: 5′- ATATACGTGTGCGTACGT-3′(forward), 5′-CGGCGTAGCTGAACCCTGGTA-3′ (reverse); for GAPDH: 5′- CCTAGGTAAACTAGACGA-3′(forward), 5′- ATTATCTGTGTCTGCATGGC-3′ (reverse).
Statistical analysis
All statistical analyses were conducted using statistical analysis software (IBM SPSS Software, Version 18.0). The relationship between the expression of miR-340-5p and LINC0062 was measured by Spearman rank correlation. In this study, we defined the relative LINC0062 expression > 4 as high expression and 4 is derived from the mean values of all samples. Survival analysis were compared using the univariate Cox proportional hazard model among different LINC0062 mRNA expression levels in colon cancer tissue. The correlations between LINC0062 expression and clinicopathological characteristics was examined by χ2 test. The results presented as mean ± SD were analyzed with a two-sided Student’s t-test for two groups and one-way ANOVA test for three or more groups. P < 0 .05 was considered as statistically significant. Survival curves were constructed using the Kaplan-Meier method and analyzed by the log-rank test.
Discussion
LncRNA plays an important role in the occurrence and development of cancer [
6,
8,
11,
15]. In this study, high expression of LINC00662 was detected in colon cancer tissues and cell lines, and the survival rate of colon cancer patients with high expression of LINC00662 was lower than that of patients with low expression of LINC00662. Cox model analysis further showed that the expression of LINC00662 was significantly correlated with OS. This suggests that LINC00662 may play a role in the occurrence and development of colon cancer. The expression of LINC00662 was relatively low in HCT29 and LS174T cells, while LINC00662 was relatively high in LOVO and CT26 cells. Therefore, HCT29 and LS174T cells were used in cell transfection of LINC00662 overexpression, LOVO and CT26 cells were used in cell transfection of LINC00662 knockdown (Fig.
1). Cell proliferation, apoptosis, migration and invasion are the basic biological functions of tumor cells for tumor growth and metastasis [
8]. The following results showed that in HCT29 and LS174T cells, high expression of LINC00662 prominently elevated cell vitality, clone formation ability, cell migration and invasion, and memorably declined apoptosis. On the contrary, LINC00662 knockout distinctly depressed cell activity. Clone forming ability, cell migration and invasion, overtly motivated cell apoptosis (Fig.
2). These results manifest that LINC00662 can visibly regulate the biological function of colon cancer cells in vitro. As a star molecule in the caspase family, Caspase-3 participates in the regulation of apoptosis, and its activity can be inhibited by XIAP [
26]. Bcl-2 and Bax belongs to the bcl-2 family, which is not only involved in regulating the activity of caspase-3, but also can be regarded as he substrate of caspase-3 to act directly on the downstream genes of caspase-3 [
26]. Therefore, caspase family and bcl-2 family are not only related to each other but also control each other in the process of apoptosis transmission and play a role in regulating apoptosis in a variety of cancer cells. Our results showed that high expression of LINC00662 notably reduced the expression of pro-apoptotic protein (caspase-3 and Bax) and promoted the expression of anti-apoptotic protein (bcl-2 and XIAP). On the contrary, LINC00662 knockout dramatically up-regulated the expression of pro-apoptotic protein (caspase-3 and Bax). The expression of anti-apoptotic protein (bcl-2 and XIAP) was descending. VEGF is a powerful cytokine that can produce a variety of biological effects. It can specifically act on vascular endothelial cells, induce vascular endothelial cell proliferation, and then promote tumor growth [
27]. Therefore, VEGF is considered to be a marker of cell proliferation. Matrix metalloproteinases (MMPs) can promote tumor metastasis by degradation of extracellular matrix and basement membrane [
28]. It is reported that MMP2 knockout can inhibit tumor metastasis [
29]. In this study, LINC00662 overexpression distinctly expedited the protein levels of VEGF and MMP2, and LINC00662 knockout significantly inhibited the protein levels of VEGF and MMP2 in colon cancer cells (Fig.
3). Tumor formation experiment in nude mice further confirmed that LINC00662 significantly regulated tumor growth and metastasis (Fig.
4). To sum up, LINC00662 affects the biological function of colon cancer cells by regulating the expression of proliferation and apoptosis-related proteins and the expression of migration and invasion-related proteins in vivo and in vitro.
It is reported that overexpressed miR-340-5p signally inhibit the proliferation and invasion of lung cancer cells 18. However, the role and mechanism of miR-340-5p in colon cancer is unknown. Based on the starbase v2.0 database, we predict that miR-340-5p contains LINC00662 binding sites. Co-transfection of LINC00662-WT and miR-340-5pmimcs markedly inhibited the relative luciferase activity, while co-transfection of LINC00662-WT and miR-340-5p inhibitors obviously increased the relative luciferase activity. In addition, after HCT29 and LS174T cells were transfected with LINC00662 overexpression, miR-340-5p expression in mRNA was significantly decreased. After LOVO and CT26 cells were transfected with siRNA-LINC00662, miR-340-5p expression in mRNA was significantly increased. Further results showed that miR-340-5p was significantly down-regulated in colon cancer tissues and cell lines. There was a negative correlation between miR-340-5pexpression and LINC00662expression in mRNA level. The results of functional experiments show that the functions of miR-340-5p regulating cell proliferation, apoptosis, invasion and migration were coincidence with that of LINC00662 overexpression (Fig.
5 and Fig.
6). miRNA affects cellular biological function by targeting its target genes. We predicted the target gene of miR-340-5p by miRDB and TargetScan database. GO and KEGG enrichment analysis were used to predict the biological functions and pathways of 1962 target genes from miRDB and TargetScan databases (Fig.
7). Our previous study found that CLDN8 was overtly up-regulation in colon cancer tissues and cell lines, promoting cell proliferation, migration and invasion by activating MAPK/ERK signaling pathway [
23]. Both IL22 and CLDN8 are target genes of miR-340-5p and are co-expressed in colon cancer cells. Through western blot assay, we found that the high expression of LINC00662 significantly increased the expression of CLDN8 and IL22 in protein level and activated the ERK signaling pathway in colon cancer cells. Combined with the previous results, the effects of LINC00662 overexpression and CLDN8 overexpression on the biological function of colon cancer cells were consistent. However, the high expression of LINC00662 up-regulated the protein levels of CLDN8 and IL22 in colon cancer cells and activated ERK signaling pathway were markedly reversed by miR-340-5p overexpression (Fig.
8). It was uncovered that LINC00662 regulated CLDN8/IL22 co-expression and the activation of ERK signaling pathway by competitively binding with miR-340-5p. The results of rescue experiment indicated that the functions of LINC00662 knockdown inhibiting cell proliferation, invasion and migration and promoting cell apoptosis were reversed by CLDN8 or IL22 overexpression (Fig.
9). Meanwhile the functions of LINC00662 knockdown inhibiting CLDN8, IL22 protein levels were reversed by CLDN8 or IL22 overexpression. The functions of LINC00662 inhibition inhibiting the activation of ERK signaling pathway was counteracted by CLDN8 or IL22 overexpression. It is reported that the activated ERK signaling pathway promotes the occurrence and development of cancer by up-regulating the expression of anti-apoptotic proteins, proliferation-related proteins and migration and invasion-related proteins. CLDN8 or IL22 overexpression reversed the effects of LINC00662 on the expression of Bax, Bcl-2, XIAP, VEGF, MMP-2, E-cadherin and N-cadherin in protein level (Fig.
10). Previous studies have shown that highly expressed LINC00662 activates ERK signaling pathway, and highly expressed CLDN8 can also activate ERK signaling pathway. miR-340-5p can target LINC00662 and CLDN8/IL22. Therefore, LINC00662 knockdown inhibited CLDN8/IL22 co-expression to inhibit the activation of ERK signaling pathway by competitively binding with miR-340-5p.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.