Abstract

Background

   miR-182-5p (miR-182) is an oncogenic microRNA (miRNA) found in
   different tumor types and one of the most up-regulated miRNA in
   colorectal cancer (CRC). Although this microRNA is expressed in the
   early steps of tumor development, its role in driving tumorigenesis is
   unclear.

Methods

   The effects of miR-182 silencing on transcriptomic profile were
   investigated using two CRC cell lines characterized by different in
   vivo biological behavior, the MICOL-14^h-tert cell line (dormant upon
   transfer into immunodeficient hosts) and its tumorigenic variant,
   MICOL-14^tum. Apoptosis was studied by annexin/PI staining and cleaved
   Caspase-3/PARP analysis. The effect of miR-182 silencing on the
   tumorigenic potential was addressed in a xenogeneic model of
   MICOL-14^tum transplant.

Results

   Endogenous miR-182 expression was higher in MICOL-14^tum than in
   MICOL-14^h-tert cells. Interestingly, miR-182 silencing had a strong
   impact on gene expression profile, and the positive regulation of
   apoptotic process was one of the most affected pathways. Accordingly,
   annexin/PI staining and caspase-3/PARP activation demonstrated that
   miR-182 treatment significantly increased apoptosis, with a prominent
   effect in MICOL-14^tum cells. Moreover, a significant modulation of the
   cell cycle profile was exerted by anti-miR-182 treatment only in
   MICOL-14^tum cells, where a significant increase in the fraction of
   cells in G0/G1 phases was observed. Accordingly, a significant growth
   reduction and a less aggressive histological aspect were observed in
   tumor masses generated by in vivo transfer of anti-miR-182-treated
   MICOL-14^tum cells into immunodeficient hosts.

Conclusions

   Altogether, these data indicate that increased miR-182 expression may
   promote cell proliferation, suppress the apoptotic pathway and
   ultimately confer aggressive traits on CRC cells.

Electronic supplementary material

   The online version of this article (10.1186/s12885-019-5982-9) contains
   supplementary material, which is available to authorized users.

   Keywords: Colorectal cancer, microRNA, Apoptosis, Cell proliferation,
   Tumorigenesis

Background

   MicroRNAs (miRNAs) regulate fundamental cellular processes, such as
   proliferation, differentiation, migration, angiogenesis and apoptosis,
   by repressing translation or inducing cleavage of their targets. MiRNAs
   are also involved in cancer development and progression, where they act
   as oncogenes or tumor suppressors [[47]1]. A large variety of miRNAs
   have been shown to be involved, either as single elements or in
   combination [[48]2], in the regulation of multiple tumorigenic
   processes and neoplastic phenotypes. In colorectal cancer (CRC),
   specific miRNA expression patterns were associated with tumor stage and
   other clinical parameters [[49]3]. For instance, increased miR-21
   expression in tumor tissue has been linked to decreased disease-free
   survival [[50]4], and high miR-21 levels in plasma may be considered as
   a potential biomarker for the diagnosis of CRC [[51]5]. Furthermore,
   up-regulation of miR-185, miR-221, miR-182, miR-17-3p, miR-34a,
   miR-106a, and down-regulation of miR-133b, miR-150, miR-378 (and
   combinations thereof), have been associated with cancer progression,
   recurrence and poor survival [[52]6–[53]12]. Moreover, miR-10b,
   miR-885-5p, miR-210, and miR-155 may provide predictive biomarkers of
   metastasis and recurrence [[54]13, [55]14]. Differential response to
   chemotherapy has also been linked to miR-21, miR-320a, miR-150 and
   miR-129 expression levels [[56]15–[57]18].

   In reference to CRC development, we identified miR-182-5p (miR-182) as
   one of the most up-regulated miRNAs in primary tumors compared to
   normal colon mucosa, thus suggesting its potential impact on target
   genes de-regulated in CRC [[58]19]. A significant miR-182 increase is
   observed in the early phases of tumor development and is maintained in
   the metastatic process [[59]20, [60]21]. Plasma miR-182 concentrations
   were higher in CRC patients at stage IV than in controls, and
   significantly decreased 1 month after radical hepatic metastasectomy,
   indicating that evaluation of circulating miR-182 may integrate the
   array of non-invasive blood-based monitoring and screening biomarkers
   [[61]20].

   miR-182 has been described as an oncogenic miRNA implicated in the
   development of various malignant histotypes by several studies
   (reviewed in [[62]22]). In CRC, available evidence collectively
   indicates that miR-182 is one of the major players involved in the
   acquisition of malignant properties and it is associated with
   pro-proliferative signaling pathways and tumor invasion
   [[63]23–[64]25]. Nevertheless, the mechanisms underlying the ability of
   miR-182 to promote the tumorigenic process are not yet clarified. To
   fill this gap, we investigated the impact of miR-182 silencing in two
   human CRC cell lines endowed with different tumorigenic potential.
   Analysis of transcriptomic and in vitro readouts of miR-182 silencing
   indicated that this miRNA counteracts apoptosis and affects cell
   proliferation. In addition, the in vivo results showed that miR-182
   sustains tumor growth by altering tumor cell cycle dynamics and
   morphology.

Methods

Cell lines and patients

   HT-29, Caco2 and LoVo cells were obtained from the American Type
   Culture Collection (ATCC HTB-38, ATCC HTB-37, ATCC CCL-229). The
   CG-705, MICOL-S and MICOL-14^h-tert cell lines have been previously
   described [[65]26] and were kindly provided by Dr. P. Dalerba (Columbia
   University, NY). Briefly, the CG-705 cell line was derived from a
   primary tumor of the right colon; MICOL-S cell line was derived from
   the hepatic metastasis of a primary right colon cancer; the
   MICOL-14^h-tert cell line was derived from a lymph-node metastasis of a
   patient with rectal cancer. MICOL-S and MICOL-14^h-tert cell lines have
   similar in vitro morphology and express the same differentiation
   markers, but they were derived from individuals with different primary
   cancer locations, as reported in Table 1 of the above quoted paper
   [[66]26]. Both cell lines were unstable in vitro (i.e. they undergo
   growth arrest after a few in vitro passages) and were immortalized by
   h-TERT cDNA gene transfer. The MICOL-14^h-tert cell line behaves as
   non-tumorigenic in immunodeficient mice [[67]27]. However, we
   demonstrated that the subcutaneous (s.c.) injection of MICOL-14^h-tert
   cell line into non-obese diabetic severe combined immunodeficient
   (NOD/SCID) mice in combination with angiogenic factors translated into
   the acquisition of an in vivo tumorigenic phenotype [[68]27, [69]28].
   This property was consistently maintained thereafter, and in vivo
   tumorigenesis experiments confirmed that MICOL-14^h-tert cells behaved
   as dormant, whereas NOD/SCID mice injected with the tumorigenic variant
   MICOL-14^tum developed aggressive tumors within 6 weeks (not shown).
   Authentication of specific genetic fingerprint by short tandem repeat
   (STR) DNA profile analysis showed that the two cell lines presented
   exactly the same loci number profile, and confirmed their genetic
   identity (data not shown); moreover, these cell lines were tested and
   scored negative for mycoplasma contamination when experiments were
   performed. All cell lines were grown in RPMI-1640 medium (Invitrogen,
   Milan, Italy) supplemented with 10% fetal bovine serum (FBS; Gibco,
   Invitrogen), L-glutamine, Pen/Strep and HEPES, and used within 6 months
   of thawing and resuscitation. The cells were harvested with
   trypsin-EDTA in their exponentially growing phase, and maintained in a
   humidified incubator at 37 °C with 5% CO[2] in air. For this study, 5
   patients with sporadic stage IV CRC were also selected [[70]19], and
   their tumor tissue and normal mucosa samples were analyzed by qRT-PCR.
   The Ethics Committee of the University Hospital of Padova approved the
   study, and all patients provided written informed consent.

RNA extraction, reverse transcription and quantitative RT-PCR analysis

   RNA was extracted from cells 24, 48 and 72 h after their transfection
   using Trizol reagent (Thermo Fisher Scientific, MA), according to
   manufacturer’s instructions. RNA concentration and purity were measured
   with Nanodrop (Bio-Tek Instruments, Winooski, VT) and Agilent (Agilent
   Technologies, Santa Clara, CA). Reverse transcription and qRT-PCR
   experiments were conducted as previously described [[71]19] using
   Taqman Gene Expression Assay (Applied Biosystem by Thermo Fisher
   Scientific). Expression data were normalized using as a reference RNU44
   for miRNAs, and HPRT1 for transcripts.

miRNA silencing by transient in vitro transfection

   Cells were seeded in 6- or 24-well plates in complete RPMI medium for
   24 h. The medium was then replaced with Opti-MEM® I Reduced Serum
   Medium (Thermo Fisher Scientific) and specific hsa-miR-182 mirVana™
   miRNA inhibitor (Ambion by Thermo Fisher Scientific) was added to a
   total of 150 pmol/well; to allow cell transfection, Lipofectamine
   RNAiMAX transfection reagent (Invitrogen) was mixed with the miRNA
   inhibitor, according to protocol instructions. The mixture was
   incubated in the dark for 5 min at room temperature and then added to
   each well. In parallel, an equal number of cells were treated with an
   anti-miR-NC (mirVana™ miRNA inhibitor Negative Control #1; Ambion), as
   a control for data normalization of anti-mir-182-independent
   transfection effects. Cells plated in the medium used for the
   transfection, but without treatment, provided an additional control.

   Moreover, to monitor inhibitor uptake efficiency by flow cytometry
   analysis, the same number of cells were transfected with a
   carboxyfluorescein-labeled RNA oligonucleotide (FAM™-labeled Anti-miR™
   Negative Control; Ambion). After overnight incubation, the Opti-MEM
   medium supplemented with miRNA inhibitor or control was replaced with
   complete RPMI, and miRNA silencing was evaluated by qRT-PCR at
   different time points. At each time point, cells were also harvested to
   perform the experiments for miRNA function investigation. In all
   silencing experiments, transfection efficiency consistently exceeded
   80%, and miRNA expression levels were decreased > 70% in transfected
   cells compared to controls.

Apoptosis and cell cycle assay

   To detect cell death, the Annexin-V-FLUOS staining kit (Roche,
   Mannheim, Germany) was used according to manufacturer’s instructions.
   For cell cycle analysis, cells were fixed with cold ethanol, stained
   with anti-human Ki67 (BD Biosciences, Franklin Lakes, NJ, USA) and then
   incubated for 1 h in a DAPI/RNAse solution. Cytofluorimetric analysis
   was performed on a FACS Calibur flow cytometer (Becton-Dickinson
   Immunocytometry Systems, NJ; excitation/emission wavelengths of 488/525
   and 488/675 nm for Annexin-V and PI, respectively).

Western blot analysis

   Cell lysates were obtained in RIPA buffer containing protease
   inhibitor, and protein contents were quantified using Quantum Micro
   Protein Assay Kit (Euroclone, Milan, Italy). Experiments were conducted
   as previously described [[72]29] using the following primary
   antibodies: rabbit anti-Cleaved Caspase-3 (1:1000; Cell Signaling
   Technology, MA), rabbit anti-PARP (1:1000; Cell Signaling Technology)
   and mouse anti-β-actin (1:1000; Santa Cruz Biotechnologies, CA). The
   following secondary antibodies were used: goat anti-rabbit (1∶5000;
   Bioss Antibodies, MA) or goat anti-mouse (1∶5000; Calbiochem
   MerckMillipore, Darmstadt, Germany) conjugated to horseradish
   peroxidase and visualized using Supersignal West Pico Chemiluminescent
   Substrate Kit (Thermo Fisher Scientific) with the Chemidoc XRS System
   and Quantity One 4.6.9 software (both from Bio-Rad, Hercules, CA).
   Densitometric analysis was performed with the ImageJ software (NIH).

In vivo tumorigenesis assay

   Non obese diabetic/severe combined immune deficiency (NOD/SCID) mice
   were bred in our SPF animal facility. All procedures involving animals
   and their care conformed to institutional guidelines that comply with
   national and international laws and policies (EEC Council Directive
   86/609, OJ L 358, 12 December 1987). Before in vivo transfer, the
   tumorigenic MICOL-14^tum cells were treated with miR-182 inhibitor or
   anti-miR-NC as a control. For tumor establishment, 7 to 9-week-old mice
   were s.c. injected into both dorsolateral flanks with exponentially
   growing untreated or miR-182 silenced MICOL-14^tum cells (0.5 × 10^6
   cells in a 100 μl volume containing Matrigel). After 1 week, mirVana™
   miR-182 inhibitor in vivo ready (Life Technologies by Thermo Fisher
   Scientific) or negative control were combined with Invivofectamine 2.0
   Reagent (Life Technologies) and used for intratumoral injection to
   maintain in vivo miRNA silencing. The resulting tumor masses were
   inspected and measured as previously described [[73]28]. In all
   experiments, the mice survived until the experimental endpoint, when
   they were sacrificed by cervical dislocation. Tumors were harvested by
   dissection, and either snap-frozen or fixed in formalin and embedded in
   paraffin for further analysis. Isofluran anaesthetic was used prior to
   injecting mice with tumor cells and before sacrifice.

CRC grading and mitotic index evaluation

   The tumor sections were evaluated by Hematoxylin and Eosin (H&E)
   staining for CRC grading and mitotic index evaluation. The 2010 WHO
   scoring for CRC Grading, based upon the percentage of gland formation
   (> 75%; 35–75% and < 35%, respectively), is as follows: G1 well
   differentiated cancer, G2 moderately differentiated cancer and G3
   poorly differentiated cancer, and is. Main growth patterns were from
   less to more aggressive: glandular, trabecular and solid. The mitotic
   index, mirroring the ratio between the number of cells in a population
   undergoing and not undergoing mitosis, was calculated by counting the
   number of mitosis in 10 fields at 40X magnification.

Gene expression analysis

   Expression data were generated using the Affymetrix GeneChip PrimeView
   Human Gene Expression Array (Affymetrix by Thermo Fisher Scientific)
   using total RNA isolated from MICOL-14^h-tert and MICOL-14^tum cells
   transfected with either anti-miR-182 or anti-miR-NC. Raw data quality
   control was performed using the R package ‘affyQCreport’ [[74]30].
   Expression matrix reconstruction was obtained by ‘affy’ package
   [[75]31] using RMA for data summarization and normalization.
   Transcript-level annotation of probesets, based on Ensembl (release
   88), was obtained with R package ‘primeviewcdf’. Differential
   expression tests were conducted using Limma package [[76]32], setting
   significance threshold to 0.05 for p-value, adjusted using FDR method
   for multiple testing correction.

   Pathway enrichment analysis of differentially expressed genes was
   conducted using DAVID (Database for Annotation, Visualization and
   Integration Discovery, release 6.8) [[77]33]. Significant GO terms, PIR
   keywords, and KEGG and Reactome pathways were selected considering
   adjusted p-values (Benjamini-Hochberg) at most 0.05. Experimentally
   validated and predicted miR-182 target transcripts were downloaded from
   MirTarBase (release 6.0) [[78]34] and from TargetScanHuman (release
   7.1) [[79]35], respectively.

Statistical analysis

   Results were expressed as mean values ± SD. Two-tail Student’s t-test
   was performed on parametric groups. Values were considered significant
   at *p ≤ 0.05 and **p ≤ 0.01. All analyses were performed with SigmaPlot
   (Systat Software Inc. San Jose, CA).

Results

miR-182 is up-regulated in CRC cell lines and can be efficiently silenced in
tumorigenic and non-tumorigenic cell lines

   miR-182 expression levels were evaluated by qRT-PCR in normal colon
   mucosa samples as a reference, and in a panel of seven CRC cell lines.
   Significant miR-182 up-regulation was observed in all the analyzed
   cancer cell lines (Fig. [80]1A), strengthening the evidence that
   increased miR-182 expression is a shared feature of CRC [[81]19].
   Highest miR-182 expression levels were measured in MICOL-14^tum cells
   followed by parental MICOL-14^h-tert cells. Based on these results, we
   focused subsequent experiments of miR-182 silencing in MICOL-14^tum and
   MICOL-14^h-tert cells, as a model of two cell lines which share the STR
   DNA profile but differ in key phenotypic properties such as the ability
   to generate tumors in immunodeficient recipients.

Fig. 1.

   Fig. 1
   [82]Open in a new tab

   Expression of miR-182 in healthy colon mucosa and a panel of CRC cell
   lines. a The CRC cell lines were investigated by qRT-PCR for miR-182
   expression levels compared to a pool of normal colon mucosa samples.
   All cell lines showed high levels of miR-182, and in particular in the
   tumorigenic variant MICOL-14^tum compared to MICOL14^h-tert. Colon N,
   pool of normal colon mucosa. nRQ, normalized Relative Quantity. Mean
   values ± SD of 3 consecutive experiments are shown. *p < 0.01. b
   mir-182 inhibition in MICOL-14^h-tert and MICOL-14^tum cells. The
   evaluation of miR-182 expression levels was performed by qRT-PCR at 24,
   48, and 72 h after the treatment. Data analysis was performed by ΔΔCt
   method, and the control groups (NT and anti-miR-NC treated cells) were
   used as a sample reference at each time point. Data were expressed as
   mean value ± SD of 3 independent experiments. nRQ, normalized Relative
   Quantity. *p < 0.01

   Treatment with anti-miR-182 effectively inhibited miR-182 expression in
   both cell lines. In particular, 24 h after treatment, the miR-182
   expression resulted significantly repressed by a factor of 0.55
   (p = 0.0034) and 0.17 (p = 0.0008) in MICOL-14^h-tert and MICOL-14^tum,
   respectively. Silencing was maintained at all the time points
   considered and lasted for over 72 h in both cell lines (Fig. [83]1b).

miR-182 silencing strongly increases apoptosis and affects cell cycle

   We next wondered whether miR-182 silencing could affect some key
   properties of MICOL-14^h-tert and MICOL-14^tum cells lines, such as
   apoptosis and cell cycle dynamics. Judging from annexin/PI staining,
   miR-182 inhibition was associated with a significant increase in
   apoptosis in both cell lines, compared to untreated cells (NT) and
   control anti-miR-NC treated cells (Fig. [84]2a). At 24 h
   post-treatment, the increase in apoptosis was comparable in
   MICOL-14^h-tert and MICOL-14^tum cells, whereas at later time points
   (48 and 72 h), apoptosis levels were significantly increased in the
   tumorigenic cell line compared to the dormant counterpart.

Fig. 2.

   [85]Fig. 2
   [86]Open in a new tab

   Effect of anti-miR-182 treatment on apoptosis and cell cycle
   progression of MICOL-14^h-tert and MICOL-14^tum cell lines. a miR-182
   silencing was associated with increased sensitivity of cells to
   apoptosis in both MICOL-14^h-tert and MICOL-14^tum cell lines, as
   determined by Annexin V/PI staining at different time points following
   treatment. The results of three independent experiments in triplicate
   were expressed as mean fold change ± SD over the baseline apoptosis. b
   Western blot analysis (left panel) of cleaved Caspase-3 and PARP in
   MICOL-14^h-tert and MICOL-14^tum cell lines non-transfected (NT) and
   transfected with anti-miR-182 or control vector (miR-NC). The right
   panel shows the densitometric analysis of the ratio between cleaved and
   total PARP. β-actin was used as a loading control. The WB image is
   representative of three independent experiments; mean values ± SD of 3
   consecutive experiments are shown in the right panel. c The cell cycle
   analysis was performed in MICOL-14^h-tert and MICOL-14^tum cell lines
   48 h after treatment using Ki67 and DAPI staining. The control
   populations (NT and anti-miR-NC cells) were used as references at each