Background
Colorectal cancer (CRC) is the third most deadly cancer in the United States, which accounts for 8% of all cancer deaths in men and women [
1]. It has been estimated that there are nearly 140,250 new CRC cases and 50,630 deaths of CRC in the United States in 2018 [
1]. Surgery combined with adjuvant therapy resected cancerous lesions, inhibited CRC cell growth and decreased CRC metastasis{Acciuffi, 2018 #35}, thus reduced the occurance of CRC. However, 40–50% of CRC patients still had metastasis to lymph node, liver, lung, etc. [
2‐
4]. Increasing evidences show that epithelial-mesenchymal transition (EMT) plays an important role in the metastasis of CRC. Li et al. reported that inhibition of EMT induced the suppression of CRC cell migration and invasion [
5]. Vu and Datta reviewed that EMT led to the increase of invasiveness and metastasis in CRC [
6]. Besides, during EMT, epithelial marker E-cadherin is downregulated, and mesenchymal markers vimentin and N-cadherin are upregulated through regulating different EMT-related signaling pathways [
7].
KIAA1199 is a gene firstly reported in Deiters’ cells and considered as the cause of non-syndromic hearing loss in 2003. Studies have shown that KIAA1199 was upregulated in many human cancers and negatively related with the survival rate [
8,
9]. Researchers have shown that protein level of KIAA1199 was remarkably increased in colon cancer tissues and cells, and indicated markedly reduced survival [
10,
11]. KIAA1199, as a cell-migration inducing protein, is overexpressed in metastatic CRC tissue, and inhibition of KIAA1199 inhibited migration and invasion of CRC cells and suppressed CRC metastasis [
12]. However, the underlying mechanism of KIAA1199 in CRC is not fully revealed.
microRNAs, a class of small noncoding RNAs that modulate gene expression at post-transcriptional level, are involved in the development, progression and metastasis of CRC cancer [
13,
14]. miR-600 was first identified in breast cancer stem cells that regulated the balance between self-renewal and differentiation of breast cancer stem cells and influenced tumor progression [
15]. Later, studies showed that miR-600 was downregulated in cancers, such as acute myeloid leukemia, cervical cancer [
16,
17], which was associated with a positive prognosis of cancer. Recently, Zhang et al. found that miR-600 overexpression remarkably inhibited migration and invasion abilities of CRC cells [
18], however, the underlying mechanism of miR-600 in CRC metastasis is unclear. According to the bioinformatics software Targetscan, there were potential binding sites between miR-600 and KIAA1199. Therefore, we assumed miR-600 as a potential upstream molecular of KIAA1199, and might involve in modulating CRC metastasis.
Researchers have found long noncoding RNAs (lncRNAs) were abnormally expressed in CRC, which was necessary for the proliferation, apoptosis, migration and invasion. Our previous report found that lncRNA TUG1 was upregulated in CRC samples and cells and promoted metastasis by affecting EMT, indicating a poor prognosis for CRC [
19]. Bioinformatics software DIANA also predicted there were potential binding sites between TUG1 and miR-600. Thus, we assumed that lncRNA TUG1 promoted KIAA1199 expression via miR-600 to accelerate CRC metastasis and EMT.
Methods
Tissue collection
Seventy-six CRC tissues and matched adjacent normal tissues were collected from CRC patients who received surgical treatment at the department of Gastrointestinal Surgery, the First Affiliated Hospital of Zhengzhou University between March 2016 and June 2017. The patients were divided into two groups: miR-600 high expression group (n = 29) and miR-600 low expression group (n = 47) according to the mean relative expression level of miR-600 (mean fold 0.55 was used as the cut-off). No patients received radiotherapy, chemotherapy or targeted therapy before surgery. The study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University, and all patients signed informed consent. All specimens collected from surgery were immediately stored at − 80 °C.
Cell culture and transfection
Human CRC cell lines (HCT116, SW480, HT29, LOVO and SW620) and the normal human colon epithelial cell line NCM460 were purchased from the American Type Culture Collection (USA). The cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) at 37 °C with 5% CO2.
miR-600 mimic, miR-600 inhibitor, si-KIAA1199, pcDNA-KIAA1199, si-TUG1 and corresponding scrambled negative control (NC) vectors were synthesized by Invitrogen. Lentivirus miR-600 (lenti-miR-600) and lenti-vector were synthesized by GENECHEM (Shanghai, China). The vectors were transfected into CRC cells by Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions.
Quantitative real-time PCR
Total RNA was extracted from CRC tissues, matched adjacent normal tissues, CRC cells and the normal human colon epithelial cell using Trizol (Invitrogen, USA). cDNA was compounded using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan). Real-time PCR was performed to measure miR-600 and KIAA1199 expressions using One Step SYBR® PrimeScript™ PLUS RT-PCR Kit (Takara, Japan). Fold change expression of miR-600 and KIAA1199 was calculated using the 2−ΔΔCt method. Primers used in this study were synthesized by Invitrogen. miR-600 primer 5’-CCCGUCCCGACCGGACUCGUUCCGAGAACAGACAUUCAUUGAGGUGACGGACUGUCUGUUCUCGGAAGGAUACUUCGACCUCGGUGUGUCGUGCACACUGAA-3′; KIAA1199 primer 5’-AGGCGTGACACTGTCTCGGCTACAG-3′.
SW620, LOVO or HCT116 cells were plated in 6-well plates at 1 × 103 cells per well and maintained in DMEM containing 10% FBS for 2 weeks with the medium replaced every 4 days. After 2 weeks, the colonies were washed with PBS for 2 times, fixed with methanol and stained with crystal violet (Sinopharm Chemical Reagent, China). The number of colonies was counted under a microscope.
Wound healing assay
SW620, LOVO or HCT116 cells were transfected with miR-600 mimic, si-KIAA1199 pre-NC or si-NC. After 72 h, cells were collected and seeded into 6-well plates until 80% confluence. Then, a pipette tip was used to creat a wound. At 0 and 48 h, the spread extent of wound closure was observed by photograph.
Transwell assay
SW620 and LOVO cells were transfected with miR-600 mimic or NC, and HCT116 cells were transfected with miR-600 inhibitor. After 72 h, 3 × 104 cells were collected and placed in 100 μl serum-free DMEM medium in the upper chamber coated with Matrigel (BD, USA). DMEM medium supplemented with 10% FBS was added into the lower chamber. Twenty-four hours later, cells that migrated through Matrigel were fixed with methanol and stained with crystal violet (Sinopharm Chemical Reagent, China) for 15 min. To observe the invasion ability, cells were incubated for 48 h. Image J was used to quantify the number of migration and invasion cells.
Western blot
After transfection, CRC cells were lysed in Radio Immunoprecipitation Assay (RIPA) buffer (Thermo Scientific, USA), and protein concentration was measured by Pierce BCA Protein Assay Kit (Thermo Scientific, USA). Proteins were separated on a 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Invitrogen, USA), which were blocked with 5% skim milk for 2 h. Blots were incubated with primary antibodies against E-cadherin (Cell Signaling Technology, USA), N-cadherin (Cell Signaling Technology, USA), Vimentin (Cell Signaling Technology, USA), Fibronecin (Abcam, USA), KIAA1199 (Invitrogen, USA), β-actin (Sigma, USA) and horseradish peroxidase-conjugated secondary antibody (Abcam, USA). Protein bands were visualized using ChemiDoc MP imaging system (Bio-Rad, USA). β-actin acted as an internal control.
Luciferase reporter gene assay
pMIR-KIAA1199 3’UTR Wild Type (WT) (400 ng) or pMIR-KIAA1199 3’UTR mutant (MT) were transfected into HCT116 cells with 40 ng pRL-TK vectors (Promega, USA). miR-600 mimic or miR-600 inhibitor or NC was co-transfected with reporter plasmids for 48 h. Cells were collected to measure luciferase activity by dual Glo™ Luciferase Assay System (Promega).
Experimental mouse model
BALB/c nude mice (5-week-old, male) were purchased from The Animal Experimental Center of Zhengzhou University, and the animal study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. SW620 cells were transfected with lenti-miR-600 or lenti-vector and resuspended at a concentration of 1 × 106 cells//100 μl. A total of 100 μl SW620 cells were subcutaneously injected into each BALB/c nude mouse (n = 6). Seven days after inoculation, tumor volume was measured, which was calculated by the formula: 0.5 × length×width2. Nude mice were sacrificed after 25 days, and the weight of tumor tissues were measured. In addition, SW620 cells mentioned above were injected into the spleen subcapsular of each BALB/c nude mice at a concentration of 1 × 106 cells/100 μl (n = 6) to establish nude mice liver metastasis model of CRC. Nude mice were sacrificed after 5 weeks, and liver tissues were obtained to count tumor nodules.
RNA immunoprecipitation (RIP) assay
Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) was used for RIP experiment. Endogenous polycomb repressive complex2 (EZH2) (Invitrogen, USA) was used for RIP assay. Ago2 was assessed by IP-western, and TUG1 and miR-600 levels were measured by qRT-PCR.
Statistical analysis
SPSS software (version 18.0) was used for data analysis, and the data was expressed as mean ± standard error (SE). The overall survival was calculated using the Kaplan-Meier method. The data was analyzed by one-way ANOVA and t test, with P < 0.05 considered statistically significant.
Discussion
Numerous studies have shown that lncRNAs play critical role in modulating tumor processes, such as apoptosis, proliferation and metastasis [
20,
21]. Although our previous report has proved lncRNA TUG1 promoted the aggressiveness of CRC cells and facilitated EMT in CRC cells [
19], the exact molecular mechanism mediated by lncRNA TUG1 in CRC is still unclear. So, we further attempted to reveal the possible mechanism of lncRNA TUG1 mediated CRC metastasis in this study. It has been reported that lncRNAs could facilitate or suppress tumor processes via competitively binding with miRNAs, and bioinformatics software analysis is commonly used to predict the potential binding sites between miRNA and the interacted genes [
22]. In this study, bioinformatics software DIANA predicted there were potential binding sites between TUG1 and miR-600. We observed TUG1 knockdown increased miR-600 expression in CRC cells, whereas overexpression of TUG1 decreased miR-600 expression. Besides, TUG1 knockdown suppressed CRC cell migration and invasion and EMT, and miR-600 inhibitor reversed these inhibition effects, indicating lncRNA TUG1 could negatively regulated miR-600 to promote migration and invasion of CRC cells and EMT in CRC cells.
Evidence has shown that miRNAs play critical roles in modulating proliferation, metastasis and EMT of CRC, such as miR-590-5p, miR-198, miR-19b-3p, etc. [
23‐
25]. miR-600 is a newly identified miRNA and its role in proliferation, apoptosis, migration in cancer cells is largely unknown. Zeng et al. reported that miR-600 could be significantly associated with prolonged survival time in cervical cancer [
17]. Zhang et al. found that miR-600, as a negative regulator of p53, could suppress proliferation, migration and invasion in mutant p53-expressing human CRC cell [
18]. However, the expression of miR-600 in CRC cell lines or tissues has not been discussed, and its role in EMT and CRC metastasis has not been explored. In this study, we focused on the role of miR-600 in migration and invasion of CRC cell, EMT in CRC cells and CRC metastasis. We observed that miR-600 was downregulated in CRC tissues and cell lines and CRC metastatic tissue, and low miR-600 expression predicted a poor clinical prognosis for CRC patients. Besides, we found overexpression of miR-600 inhibited migration and invasion of CRC cells, which was consistent with previous report [
18]. Moreover, miR-600 overexpression inhibited EMT in CRC cells in vitro, suppressed tumor volume and weight in vivo, and reduced the number of liver metastasis in vivo, which brought new evidence to the role of miR-600 in CRC progression.
KIAA1199, also defined as cell migration inducing protein (CEMIP), has been reported to be overexpressed in many cancers and promoted cancer metastasis through different signaling pathways, such as Wnt signaling and MEK1/ERK1/2 signalling [
26,
27]. Evensen et al. found KIAA1199 was upregulated in breast cancer metastatic tissues, and resulted in EMT in breast cancer cells, inhibited cell migration in vitro and decreased metastasis in vivo [
8]. Recently, researchers showed that KIAA1199, one of the direct targets of miR-216a, was highly expressed in CRC metastatic tissues, and KIAA1199 downregulation inhibited CRC cell migration and invasion in vitro [
12]. However, there was no research studying the function of miR-600 on KIAA1199 in CRC metastasis. Our present study first proved that KIAA1199 was a direct target of miR-600, and was negatively regulated by miR-600 to modulate CRC cell migration and invasion, revealing new mechanism of miR-600 on CRC progression.
Limitations
our study had a relatively small sample size which may be an important factor affecting the extrapolation of our results. The statistical test for miR-600-related survival is of borderline significance. Thus, a larger study should be performed to clearly determine the relationship between miR-600 expression and CRC patients survival.
Conclusion
In this study, we discovered miR-600 was downregulated and KIAA1199 was upregulated in CRC tissues and cell lines, which predicted a poor clinical prognosis. Overexpression of miR-600 suppressed CRC cell migration/invasion and EMT-related proteins, inhibited tumor volume and weight, and decreased the number of CRC liver metastasis via KIAA1199. Inhibition of TUG1 suppressed CRC cell migration and invasion and EMT-related proteins via regulating miR-600. Our study first proved that TUG1/miR-600/KIAA1199 promoted CRC cell migration and EMT in vitro and metastasis in vivo.