Background
Colorectal cancer (CRC) is one of the most prevalent malignant tumors, with high morbidity and mortality worldwide. In the USA, CRC is currently the third most common cancer type and the third leading cause of cancer-related death [
1]. Although advances in screening and treatment have improved the life expectancy of CRC patients in recent decades [
2], CRC remains a major health problem all over the world. Much more attention should be given to the exact mechanisms contributing to the initiation and development of CRC.
Although there are many risk factors for CRC (including obesity, smoking, dietary patterns, physical inactivity, and genetic and epigenetic factors) [
3‐
5], understanding the molecular basis of individual susceptibility to colorectal cancer and determining the factors that initiate the development of the tumor, drive its progression and determine its responsiveness or resistance to antitumor agents are the most important tasks in the study of this disease [
2]. Among the myriad CRC-related molecular factors, oncogene activation (e.g., KRAS and IGF1R) and tumor suppressor gene silencing (e.g., APC and PDCD4) play vital roles during CRC tumorigenesis [
6‐
9]. T-cell intracellular antigen 1 (TIA1) is an RNA binding protein and is linked to multiple biologic processes associated with RNA metabolism, both in the nucleus and in the cytoplasm [
10]. TIA1 is thought to be a new member of the tumor suppressor family [
11], as TIA1 regulates, modulates and/or interacts with many types of mRNA involved in cancer cell proliferation, apoptosis, angiogenesis, invasiveness and metastasis as well as in immune evasion [
12‐
16]. For example, it has been reported that knockdown of TIA1 triggers cell proliferation and invasion as well as tumor growth [
14]. Furthermore, TIA1 has been found to regulate many oncogenes (e.g., RAB40B) to inhibit cell proliferation [
12]. Moreover, TIA1 can promote cell apoptosis by regulating Fas alternative splicing [
17]. In CRC, TIA1 is also closely connected to tumorigenesis. For example, TIA1 has been found to regulate VEGF isoform expression, angiogenesis, tumor growth and bevacizumab resistance in CRC [
15]. Moreover, TIA1 can be used to supplement prognostic information related to TNM stage and adjuvant therapy in mismatch repair-proficient colorectal cancer patients [
16]. Because of the myriad of tumor suppressor functions of TIA1, it is imperative that we elucidate the mechanisms underlying how TIA1 is regulated during tumorigenesis, especially in CRC.
MicroRNAs (miRNAs) are small (19–23 nucleotides) non-coding RNA molecules [
18] that act as endogenous suppressors of gene expression by binding to the 3’-untranslated region (3’-UTR) of target mRNAs to induce translational repression or mRNA cleavage. Occasionally, miRNAs may bind directly to coding sequence of mRNAs or even function as activators of gene expression by binding to the 5’-UTR of target mRNAs [
19‐
21]. As vital post-transcriptional regulators, miRNAs are involved in numerous physiological and pathological processes, such as developmental timing [
22], hematopoietic cell differentiation [
23], cell proliferation [
24], organ development [
25] and tumorigenesis in particular [
26,
27]. Many miRNAs are directly or indirectly correlated with cancer genes and can function as either tumor suppressor miRNAs or oncomiRs [
27]. During CRC initiation and progression, some miRNAs show a significant alteration in their expression patterns and influence CRC cell proliferation, invasion and apoptosis. Among these miRNAs, miR-19a is one of the most important. miR-19a belongs to a well-known and important miRNA family named mir-17-92 (also known as oncomir-1) and is a miRNA polycistron with pleiotropic functions in cell survival, proliferation, differentiation and angiogenesis [
28‐
31]. miR-19a has been reported to be significantly overexpressed in CRC [
32]. Moreover, miR-19a has been found to be induced by PRL-3 to promote the proliferation and metastasis of CRC cells [
33]. miR-19a can also enhance the invasion and metastasis of CRC cells by targeting TG2 [
34]. More importantly, miR-19a is associated with lymph metastasis and mediates TNF-α-induced EMT in CRC [
32]. These studies have revealed an important oncogenic role of miR-19a in CRC. However, the precise molecular mechanism through which miR-19a influences CRC progression remains unknown.
In this study, we identified TIA1 as a direct target gene of miR-19a in CRC. We also detected an inverse correlation between miR-19a and TIA1 protein levels in CRC tissues. Moreover, we provided evidence that miR-19a can promote CRC cell proliferation and migration in vitro and accelerate tumor growth in vivo by targeting TIA1.
Methods
Tissue samples
CRC tissue and paired normal adjacent tissue samples were acquired from patients undergoing a surgical procedure at the Affiliated Drum Tower Hospital of Nanjing University Medical School (Nanjing, China). Both the tumor and normal tissues were sent for histological analysis and diagnostic confirmation. Written consent was obtained from all patients, and all protocols concerning the use of patient samples in this study were approved by the Ethics Committee of Nanjing University. Tissue samples were immediately frozen in liquid nitrogen at the time of surgery and stored at -80 °C. All experiments were performed in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) and approved guidelines of the Nanjing University. The clinical features of the patients are listed in Additional file
1: Table S1.
Cell culture
The three human CRC cell lines SW480, Caco2 and HT29 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). SW480 and HT29 cells were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) in a humidified incubator at 37 °C with 5% CO2. Caco2 cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) in a humidified incubator at 37 °C with 5% CO2.
Protein isolation and western blot
RIPA lysis buffer (Beyotime, Shanghai, China) with freshly added PMSF (Beyotime, Shanghai, China) was used to isolate protein from cells or tissues. Proteins were separated by 10% SDS-PAGE (Bio-Rad). Antibodies against TIA1, CMYC, PDCD4 and GAPDH were purchased from Santa Cruz Biotechnology (sc-365349, sc-40, sc-130545 and sc-25778, respectively; Santa Cruz, CA, USA).
We used an online database YM500 to analyze and compare the miRNA expression in colon cancer tissues and normal solid tissues. This database compared the miRNA expression profiles between 8 normal solid colon tissues and 429 primary solid colon tumors, and a total of 2578 miRNAs were analyzed. In total, 273 miRNAs were found to be significantly changed in the solid tumors compared to the normal tissues (128 were upregulated, and 145 were downregulated, Additional file
2: Table S2). Among the 128 upregulated miRNAs, 26 miRNAs were determined to be upregulated by an infinite fold change at an extremely low expression level in the normal group of these miRNAs. For these miRNAs, we set a threshold of 26.18 for the base mean of the primary solid tumors, which was the average value of the base means of these miRNAs. Above this threshold, we found 7 significantly upregulated miRNAs, and miR-19a ranked first among them. For the remainder of the 102 upregulated miRNAs, we set a threshold of 42.46 for the fold change, which was the average fold change value of these miRNAs. Above this threshold, we found another 22 significantly upregulated miRNAs, and miR-19b ranked first among these. In sum, we obtained 29 significantly upregulated miRNAs, and miR-19a/b was at the top of the list (Additional file
3: Table S3).
RNA isolation and quantitative RT-PCR
Total RNA was extracted from the cultured cells and tissues using Trizol Reagent (Invitrogen, CA, USA). To quantify mature miR-19a and 19b, TaqMan miRNA Assay Probes (Applied Biosystems, Foster City, CA) were used according to the manufacturer’s instructions. Quantitative real-time PCR was performed using a TaqMan PCR kit on an Applied Biosystems 7500 Sequence Detection System (Applied Biosystems). All of the reactions were run in triplicate. After the reactions were complete, the cycle threshold (CT) data were determined using fixed threshold settings, and the mean CT was determined from triplicate PCRs. A comparative CT method was used to compare each condition to the control reactions. U6 snRNA was used as an internal control, and the relative amount of miRNA normalized to U6 was calculated with the equation 2-ΔΔCT in which ΔΔCT = (CT
miR-19a − CT
U6)tumor − (CT
miR-19a − CT
U6)control.
To quantify TIA1 and GAPDH mRNA, oligo d(T)18 primers (TaKaRa) were used to reverse transcribe total RNA into cDNA. Then, a qRT-PCR was run by using SYBR Green dye (Invitrogen) and specific primers for TIA1 and GAPDH. The primer sequences were as follows: TIA1 (sense): TCCCGCTCCAAAGAGTACATATGAG; TIA1 (antisense): AAACAATTGCATGTGCTGCACTTTC; GAPDH (sense): CGAGCCACATCGCTCAGACA; and GAPDH (antisense): GTGGTGAAGACGCCAGTGGA. After the reactions were complete, the CT values were determined by setting a fixed threshold. The relative amounts of TIA1 mRNAs were normalized to GAPDH using a method similar to that described above.
miRNA overexpression and knockdown
miRNA overexpression and knockdown was achieved by transfecting CRC cells with miRNA mimics or inhibitors, respectively. Synthetic miRNA mimics and inhibitors and scrambled negative control RNAs (control mimic and inhibitor) were purchased from GenePharma (Shanghai, China). SW480, Caco2 and HT29 cells were seeded in 6-well plates and transfected using Lipofectamine 2000 (Invitrogen) on the following day when the cells were approximately 60–80% confluent. For miRNA overexpression and knockdown, 200 pmol of miRNA mimic or inhibitor and corresponding negative control were added to each well. At 6 h after transfection, the SW480 and HT29 cell medium was changed to RPMI-1640 supplemented with 2% FBS, and the Caco2 cell medium was changed to DMEM supplemented with 2% FBS. The cells were harvested 48 h after transfection for total RNA or protein isolation.
Plasmid construction and siRNA interference assay
Mammalian expression plasmids were purchased from Genescript (Nanjing, China). An empty plasmid served as a negative control (control plasmid). siRNAs designed to specifically silence TIA1 or c-MYC were purchased from GenePharma (Shanghai, China). A scrambled siRNA served as a control. The siRNA sequences were as follows: si-TIA1: TGCACAACAAATTGGCCAGTA and si-c-MYC: ACGGAACTCTTGTGCGTAA. The overexpression plasmids and siRNAs were transfected into CRC cells using Lipofectamine 2000 (Invitrogen). Total RNA and protein were isolated 48 h after transfection and were assessed by quantitative RT-PCR and western blot, respectively.
Pull-down assay
For miRNA pull-down, SW480 cells which were transfected with biotinylated miR-19a (miR-19a probe) or control probe (Genescript, Nanjing, China) were harvested in lysis buffer (20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5% NP-40 and 1 U/ul Recombinant RNAse inhibitor (TaKaRa)), and the total RNA was pretreated with DNaseI and then heated at 65 °C for 5 min, followed by an instant ice bath. Then the RNA was incubated with streptavidin-coated magnetic beads (New England BioLabs, S1420S) at 4 °C for 4 h, with constant rotation. After incubation, two washes with lysis buffer were performed and RNA was extracted with Trizol (Invitrogen, CA, USA).
For mRNA pull-down, two DNA probes complementary to TIA1 mRNA was synthesized with 3’ terminal biotin labels. A scrambled biotinylated probe was used as negative control (Genescript, Nanjing, China). The probes (8 pmol/ul) were then incubated with streptavidin-coated magnetic beads (New England BioLabs, S1420S) at 25 °C for 1 h to generate probe-coated magnetic beads. SW480 cells were harvested in lysis buffer as described above. Then the lysate was incubated with probe-coated beads at 37 °C for 3 h, with constant rotation. After incubation, two washes with lysis buffer were performed and RNA was extracted with Trizol (Invitrogen, CA, USA). The sequence of TIA1 probes were as follows: Probe 1: AAAATCTAAATTTTGCATATT; Probe 2: ATAAGAGAACAATGAGGGTCC.
The extracted RNAs were analyzed by qRT-PCR.
Luciferase reporter assay
An 800-bp fragment of the TIA1 3’-UTR containing the two conserved miR-19a binding sites was inserted into a luciferase reporter plasmid (Genescript, Nanjing, China). To test binding specificity, sequences that interacted with the miR-19a seed sequence were mutated from TTGCACA to AACGTGT, and the synthetic TIA1 3’-UTR mutant fragment was inserted into an equivalent reporter plasmid (Genescript, Nanjing, China). For the luciferase reporter assays, SW480 cells were cultured in 24-well plates, and each well was co-transfected with 0.2 μg of firefly luciferase reporter plasmid, 0.2 μg of β-galactosidase (β-gal) expression plasmid (Ambion), and equal amounts (50 pmol) of miR-19a mimic, miR-19a inhibitor or the scrambled negative control RNAs using Lipofectamine 2000 (Invitrogen). The β-gal plasmid was used as a transfection efficiency control. The cells were assayed using a luciferase assay kit 24 h post-transfection (Promega, Madison, WI, USA).
Cell proliferation assay
For CCK-8 assay, SW480 cells were plated at 2 × 104 cells per well in 96-well plates and incubated overnight in RPMI-1640 supplemented with 10% FBS. The cell proliferation index was measured using a Cell Counting Kit-8 (CK04-500, Dojindo, Japan) at 12, 24, 36, 48, and 60 h post-transfection according to the manufacturer’s instruction. Absorbance was measured at a wavelength of 450 nm.
For EdU assay, SW480 cells were seeded in 48-well plates (Corning). After transfection, when the confluency of SW480 cells reached 80%, an EdU assay kit (RiBoBio, Guangzhou, China) was used to determine the proliferation rate of the cells. The manufacturer’s instruction was followed except that the nucleus staining dye was changed from Hoechst 33342 (supplied with the kit) to DAPI (Beyotime). After staining, the cells were captured by photomicroscopy (BX51 Olympus, Japan).
Cell migration assay
Cell migration assays were performed using Millipore 24-well Millicell (Millipore) plates containing an 8-μm pore membrane. The bottom face of the membrane was coated with 10 μg/mL fibronectin (Gibco). Cells were harvested 24 h after transfection and suspended in FBS-free RPMI-1640 culture medium. The cells were then added to the upper chamber (2 × 104 cells/well), and 0.5 mL RPMI-1640 plus 20% FBS was added to the lower compartment. The Transwell-containing plates were incubated for 24 h in the incubator. After incubation, cells that had migrated to the lower surface of the filter membrane were fixed with 4% paraformaldehyde for 25 min at room temperature. The membrane was washed 3 times with PBS and stained with 0.1% crystal violet in methanol for 15 min at room temperature. Cells remaining on the upper surface of the filter membrane (non-migrating) were gently scraped off with a cotton swab. The lower surfaces (with migrating cells) were captured by photomicroscopy (BX51 Olympus, Japan), and the cells were counted blindly (five fields per chamber).
Immunofluorescence
SW480 cells were seeded on cell slide (Fisherbrand) in 24-well plates and then infected with miR-19a lentivirus or transfected with TIA1 vector. Two days later the cells were briefly washed with PBS, and then fixed in 4% paraformaldehyde for 30 min at room temperature (RT). After fixation, the cells were washed with PBS (3 × 5 min, RT), and then permeabilized and blocked using 5% BSA (Sigma) and 0.5% Triton X-100 in PBS for 1 h at RT. Next, the cells were incubated with primary antibody for Ki67 (Abcam, ab16667) in 5% BSA overnight at 4 °C, and then rinsed in PBS (3 × 5 min, RT). The cells were then incubated in secondary fluorescent antibody (Invitrogen, 594 nm) in 5% BSA in a light-proof environment for 1 h at RT. Next, the cells were stained with DAPI (Beyotime, Shanghai, China) a light-proof environment for 10 min at RT. Finally the cells were washed with PBS (3 × 5 min, RT) and visualized using a confocal microscope (C2+, Nikon).
SW480 cells that were infected with miR-19a lentivirus or transfected with TIA1 vector were suspended in RPMI-1640 with 0.3% Agarose L.M.P (Biocam) solution, and plated onto solidified 0.6% Agarose L.M.P in 60 mm dish (Corning) at a density of 3 × 106 cells/mL in triplicate. The seeded cells were maintained in culture by feeding with 0.5 ml fresh RPMI-1640 plus 10% FBS and Penicillin-Streptomycin antibiotics (Gibco) every 3 days, for a total time 18 days. Then the colonies were fixed with 4% paraformaldehyde, and stained with 0.05% crystal violet. Then rinse the dish softly to scour off the redundant crystal violet. The shapes of colonies were capture by photomicroscopy (BX51 Olympus, Japan), and the numbers were counted by visual inspection.
Establishment of tumor xenografts in mice
Four-week-old male SCID mice (
nu/
nu) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China) and maintained under specific pathogen-free conditions at Nanjing University. A lentiviral expression plasmid that can express miR-19a was purchased from GenePharma (Shanghai, China). Puromycin (Sigma-Aldrich, USA) was used to successfully obtain stably infected cells. SW480 cells were infected with a control lentivirus or a miR-19a lentivirus, transfected with a TIA1 overexpression plasmid, or co-transfected with a miR-19a overexpression lentivirus and a TIA1 overexpression plasmid. After infection and/or transfection, SW480 cells were subcutaneously injected into SCID mice (3 × 10
6 cells in 0.2 mL PBS per mouse, 5 mice per group). The needle was inserted into the left side of the armpit, midway down, 5 mm deep at a 45° angle. Mice were sacrificed 24 days after injection to remove the xenografted tumors, and the volumes and weights of the tumors were measured. The volume of tumors were calculated by the formula: Tumor volume = [length * (width)
2]/2 [
35]. A portion of the tissues was used for protein and total RNA extraction, and the remaining tissue was fixed in 4% paraformaldehyde for 24 h. The tissue was processed for hematoxylin and eosin (H&E) staining or immunohistochemical staining for TIA1 and Ki-67. All experiments were approved by the Institutional Review Board of Nanjing University (Nanjing, China) and performed in accordance with the U.K. Animals (Scientific Procedures) Act (1986) and the guidelines of the National Institutes of Health.
Statistical analysis
All of the images of the western blot assay, EdU assay, migration assay, immunofluorescence, colony formation or animal experiments were representative of at least three independent experiments or staining results. The quantitative RT-PCR assay, pull down assay, luciferase reporter assay, and proliferation assay were performed in triplicate, and each individual experiment was repeated several times. The results are presented as the means ± SE of at least three independent experiments. Observed differences were considered statistically significant at p < 0.05 by using Student’s t-test.
Discussion
In 2016, the American Cancer Society estimated that there would be more than 130,000 new cases of and more than 49,000 new deaths from CRC in the United States, making it the third leading cause of death from cancer [
1]. Although recent advances in medicine have improved the survival of CRC patients, CRC remains a major public health problem. The clinical behavior of colorectal cancer results from interactions at many levels [
2], and among these interactions, the genetic changes in oncogenes and/or tumor-suppressor genes are the most important driving force for tumorigenesis and cancer development. TIA1 is an important tumor suppressor in many cancers [
10], especially in CRC [
15,
16]. In this study, we demonstrated that TIA1 protein was significantly downregulated in CRC tissues and inhibited proliferation and migration of CRC cells and attenuated tumor growth in xenografted mice. Interestingly, we observed that TIA1 mRNA levels did not exhibit alterations consistent with the protein levels. This phenomenon inspired us to speculate that a post-transcriptional mechanism is involved in the repression of TIA1 expression in CRC. Therefore, we focused on a vital post-transcriptional regulator, miRNA, and provided the first evidence supporting the important role of miR-19a as an oncomiR in CRC targeting TIA1.
The discovery of miRNAs expanded the list of classical tumor suppressors and oncogenes. miRNA profiles undergo a wide array of alterations during the initiation and development of various types of cancer, including CRC [
47‐
49]. More importantly, new studies have established the potential usefulness of miRNAs as therapeutic molecules against cancer [
50‐
53]. For example, aberrantly dysregulated miRNAs can be restored by using antagomirs or miRNA mimics [
50,
53]. miR-19a belongs to a well-known oncomiR cluster, namely the miR-17-92 cluster. miR-19a has been widely studied and is thought to be a key oncogenic component of mir-17-92 [
54]. miR-19a is overexpressed and functions as an oncomiR in many cancer types, including bladder cancer [
55], cervical cancer [
56], gastric cancer [
57], pancreatic cancer [
58], renal cancer [
59], lung cancer [
60] and CRC [
32‐
34]. In this study, we performed a meta-analysis of the miRNA expression profile in CRC and found that some miRNAs (especially miR-19a) were dramatically upregulated. We then used 3 bioinformatic algorithms to predict miRNAs that could target TIA1 and identified miR-19a as an ideal candidate. Subsequently, we experimentally validated TIA1 as a genuine miR-19a target in three CRC cell lines. Furthermore, we revealed the important effects of miR-19a-driven suppression of TIA1 on the promotion of CRC cell proliferation and migration and on tumor growth in a xenografted nude mouse model. These results suggested that targeting miR-19a may be a practical method to control CRC development and ameliorate symptoms, as has been shown by other groups [
50,
51,
53]. More research should focus on characterizing the feasibility of targeting miR-19a in CRC therapy and developing simplified and cost-effective manipulation methods.
The c-Myc oncogene contributes to the genesis of many human cancers [
61,
62], including CRC [
63]. c-Myc functions as a transcriptional activator or inhibitor that modulates almost every aspect of tumor cell biology [
64]. In recent years, the ability of c-Myc to regulate miRNAs has received much attention [
65]. Many of the functions of c-Myc in cancers are executed through its downstream miRNAs [
66]. Among the miRNA targets of c-Myc, the miR-17-92 cluster is the most well-studied [
29,
44,
45]. c-Myc has been shown to upregulate the expression of the mir-17-92 cluster to accelerate tumor development in a mouse B-cell lymphoma model [
29]. Increased expression of the miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression is also associated with c-Myc expression [
45]. Interestingly, c-Myc influences its target miRNAs to indirectly regulate the target genes of the miRNAs. For example, the c-Myc-regulated miR-17-92 cluster can modulate E2F1 expression to promote cell cycle progression [
44]. In this study, we showed that c-Myc indirectly suppresses TIA1 expression through enhancing miR-19a expression in CRC. These findings provide new insights into understanding how c-Myc contributes to CRC development. In addition, another member of the TIA family, T-cell intracellular antigen 1 (TIA1)-related protein (TIAR), inhibits translation of c-Myc mRNA via AU-rich elements [
67]. This finding, combined with ours, reveals the complex relationship between c-Myc and TIA family.
As an important gene expression modulator, TIA1 regulates many downstream genes [
10]. Under conditions of rapid oxygen decline and extreme hypoxia, TIA1 co-aggregates with TIAR to suppress the HIF-1α pathway [
68]. TIA1 can also function as a translational silencer of COX-2 expression in neoplasia [
69]. Moreover, TIA1 even affects the expression of some miRNAs [
70]. It is easy to speculate that by suppressing TIA1, miR-19a may also influence the downstream genes of TIA1. In this study, we proved that miR-19a inhibits TIA1 to indirectly downregulate PDCD4, which is a TIA1 target [
46] as well as an important tumor suppressor [
9] in CRC. Some oncomiR functions of miR-19a may be indirectly executed by inhibiting its indirect target PDCD4. Thus, TIA1 functions as a bridge to link upstream regulators (such as miR-19a) and downstream effectors (such as PDCD4). We suppose that there are many other indirect regulation relationships similar to miR-19a and PDCD4, and these relationships greatly complicate the miR-19a—target regulatory network.
Acknowledgements
Not applicable.