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GKN1 expression in gastric cancer cells is negatively regulated by miR-544a
Biochimie 167 (2019) 42e48
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Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
GKN1 expression in gastric cancer cells is negatively regulated by miR544a Chiara Stella di Stadio a, 1, Raffaella Faraonio a, 1, Antonella Federico a, 1, Filomena Altieri a, Emilia Rippa a, **, Paolo Arcari a, b, * a b
Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy CEINGE, Advanced Biotechnology Scarl, Via Gaetano Salvatore 486, I-80145, Naples, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 April 2019 Accepted 6 September 2019 Available online 8 September 2019
Gastrokine1 (GKN1), important for maintaining the physiological function of the gastric mucosa, is highly expressed in the stomach of healthy individuals but is down-regulated or absent in gastric tumor tissues and derived cell lines. The mechanisms underlying GKN1 gene inactivation are still unknown. We previously showed that GKN1 downregulation in gastric tumors is likely associated with an epigenetic transcriptional complex that negatively regulates GKN1 expression. In addition, TSA-mediated inhibition of HDACs leads to GKN1 restoration at the transcriptional level, but no at the translational level. These findings led to hypothesize the activation of a second regulatory mechanism microRNAs-mediated, thus resulting in translational repression and gene silencing. Bioinformatic analyses performed with 5 different algorithms highlighted that 4 miRNAs contained a seed sequence for the 30 UTR of GKN1 mRNA. Among these, only two miRNAs, hsa-miR-544a and miR-1245b-3p directly target the GKN1-30 UTR as evaluated by luciferase reporter assays. TaqMan miRNA assay performed on gastric cancer cell lines after TSA treatment showed a stronger increase of miR-544a expression than that of miR-1245b-3p. Finally, co-transfection of AGS cells with GKN1-30 UTR and premiR-544a showed compared to controls, a strong reduction of GKN1 expression both at translational and transcriptional levels. The up-regulation of miR544a could be crucially involved in the GKN1 translational repression, thus suggesting its potential role as a biomarker and therapeutic target in GC patients. These findings indicate that epigenetic mechanisms leading to the inactivation of GKN1 play a key role in the multi-step process of gastric carcinogenesis and would provide an essential starting point for the development of new therapeutic strategies based on epigenetic targets for alternatives gene. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Keywords: Gastrokine 1 Gastric cancer Epigenetics Translation regulation miRNA
1. Introduction Gastrokine 1 (GKN1) is a secretory protein that is localized within the granules just under the apical plasma membrane [1e3]. Its expression is confined to the gastric epithelium, except for trace levels in the placenta [3]. The protein is highly expressed in the gastric mucosa of healthy individuals, but markedly downregulated in samples derived from Helicobacter pylori infected
* Corresponding author. Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, I-8031, Naples, Italy. ** Corresponding author. Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, I-8031, Naples, Italy. E-mail addresses: [email protected] (E. Rippa), [email protected] (P. Arcari). 1 CSDS, RF and AF equally contributed to the work.
patients [4]. Furthermore, GKN1 is down-regulated or completely absent in GC tissues [4,5]. Although the biological function of this protein is currently unknown, a role in cell proliferation and differentiation has been hypothesized, as well as the potential involvement in the replenishment of the surface lumen epithelial cell layer and the maintaining of mucosal integrity [6e8]. GKN1 can also induce Fas-mediated apoptosis and senescence [9] and to reduce the proliferation of AGS cells compared to non-gastric cancer cells (H1355) [10]. Recent evidence has demonstrated that GKN1 is involved in gastric mucosal inflammation by regulating the production of inflammatory mediators [11] and that is involved also in the epithelial-mesenchymal transition. In particular, the recovery of GKN1 expression suppresses cell migration and invasiveness abrogating the expression of PI3K/akt pathway proteins, concomitant with the re-expression of E-cadherin [12]. These data suggest
https://doi.org/10.1016/j.biochi.2019.09.005 0300-9084/© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
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2. Materials and methods Abbreviations 2.1. Materials DMSO dimethyl sulfoxide; GC gastric cancer GKN1 gastrokine 1 G6PD glucose-6-phosphate dehydrogenase HAT histone acetyltransferases HDAC histone deacetylase H3K9triMe trimethylation of Histone 3 at lysine 9 RNU6 U6 Small nucleolar RNA TSA trichostatin A SUV39H1 Histone-lysine N-methyltransferase 30 UTR 30 -untranslated regions
that GKN1 plays a role as a gastric specific tumor suppressor and that be considered a biomarker for GC because individuals with a lower expression of the protein have an increased risk of developing gastric diseases [13]. The mechanism by which GKN1 gene is inactivated in GC remains still unknown. Therefore, recently we have investigated the possible causes of GKN1 gene silencing to determine if epigenetic mechanisms could play an important role in its down-regulation. To these aim, chromatin immunoprecipitation (ChIP) assays for the repressive trimethylation of histone 3 at lysine 9 (H3K9triMe) and its specific histone-lysine N-methyltransferase (SUV39H1) were performed on biopsies of normal and tumor human gastric tissues. GKN1 down-regulation showed to be associated in gastric cancer tissues with high levels of H3K9triMe and with the recruitment of SUV39H1 on GKN1 promoter. This finding suggested the presence of an epigenetic transcriptional complex that negatively regulates GKN1 expression in gastric tumor [14]. It was also investigated whether underacetylation might contribute to GKN1 transcriptional inhibition using TSA to increase general histone acetylation. Inhibition of HDACs led to an increase of GKN1 mRNA of about 160 fold compared to untreated AGS gastric cancer cells. However, this large increase was not associated with the restoration of GKN1 expression at the translational level [15]. These results suggested the presence of a second regulatory block occurring at the translational level through mechanisms mediated by microRNAs (miRNAs), resulting in translational repression and gene silencing. Recently, microRNA has emerged as molecular regulators that can have key roles in pathogenesis and progression of different malignancies, including gastric cancer. miRNAs are a class of small non-coding RNAs (19e25 nucleotides) that act as important epigenetic players in many cellular processes, such as differentiation, proliferation, and apoptosis, exerting great influence in cancer pathogenesis [16,17]. The mature miRNAs act as posttranscriptional regulators interacting with the 30 -untranslated regions (30 UTR) of the target transcripts and resulting in either mRNA degradation or inhibition of translation. Changes in miRNA expression profiles have been observed in a variety of human tumors, including gastric cancer [18]. Therefore, the possible involvement of miRNAs in this process was investigated. The results obtained showed that GKN1-30 UTR was a direct target of hsa-miR-544a and miR-1245b-3p and showed an increase of miR-544a expression in the gastric cancer cell lines after TSA treatment. The up-regulation of miR-544a could be the cause of the GKN1 translational repression since co-transfection in a gastric cancer cell of GKN1 cDNA containing the 30 UTR and premiR-544a showed, compared to control, a strong reduction of GKN1 expression level.
Dulbecco's modified Eagle's medium (DMEM-F12) and fetal bovine serum (FBS) were purchased from Cambrex (Rutherford, NJ, USA). Mouse GKN1 monoclonal antibody (M01), clone 2E5, was purchased from Abnova (Taipei, Taiwan). Trichostatin A (TSA) was from Sigma (Milan, Italy). a-Tubulin was from Abcam (Cambridge, UK). 2.2. Cell cultures, transfection, human tissues, and Western blotting Human gastric adenocarcinoma cell lines (AGS, MKN28, KATO III) and human embryonic kidney 293 (HEK293) cells were grown in DMEM-F12 and DMEM, respectively, supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin and 1% L-glutamine at 37 C in a 5% CO2 atmosphere. AGS were transfected with 4 mg of vector pcDNA 3.1, pcDNA3.1flGKN1(His)6 encoding the full-length GKN1 (flGKN1, containing the first 20 amino acids leader peptide and His6-Tag sequence at the Cterminal) as already described [9]. The efficiency of transfection of gastric cancer cells with flGKN1 was always evaluated by a parallel transfection using EGFP vector as control. In general, after transfection, the average value of the ratio between the number of green fluorescent cells/total number of cells was 0.5 ± 0.1. Proteins from cell extracts (about 20 mg) were analyzed by Western blotting using mouse anti-GKN1 at 1:500. Detection was performed using the enhanced chemiluminescence detection kit (SuperSignal West Pico) following the manufacturer's instructions. Western blot band intensity was measured with ImageJ 1.46r software. 2.3. RNA isolation and real-time qPCR Total RNA was extracted from AGS, MKN28, and KATO III gastric cancer cell lines using TRIzol reagent (Invitrogen) and quantified by Nanodrop (Thermo Scientific, Wilmington, DE). cDNA was synthesized using the reverse transcription kit from Roche Molecular Systems (Roche, Penzberg, Germany) according to the manufacturer's protocol. GKN1 transcript levels were quantified using SYBR Green PCR MasterMix (Applied Biosystems) IQ SYBR green Supermix (Bio-Rad) on a CFX96 Real-Time System instrument (Bio-Rad) using the following primers pairs: 50 -ctttctagctcctgccctagc-3’; 50 -GTTGCAGCAAAGCCATTTCC-3’. Real-Time qPCRs were performed in triplicate under the following conditions: 10 min at 95 C, followed by 40 cycles (15 s at 95 C and 1 min at 60 C). The relative fold changes were calculated using the 2-DDCt method by the formula: 2- (sample DCt e control DCt), where DCt is the difference between the amplification fluorescent thresholds of the gene of interest and the internal reference gene (G6PD) used for normalization [19]. TaqMan miRNA Assay kit (Applied Biosystems, Foster City, CA, USA) was used to detect the expression miRNAs (miR-544a, miR1245b-3p) in DMSO or TSA treated cells lines. Briefly, 100 ng of total RNA was reversely transcribed (RT) at 16 C for 30 min, 42 C for 30 min and 85 C for 5 min in 15 ml reaction volume. Two ml of RT product was used for PCR reaction in a final volume of 20 ml. The PCR reaction started with an initial denaturation step at 90 C for 10 min, followed by 40 cycles of 95 C for 15 s and 60 C for 1 min. Small nucleolar RNA RNU6 (Applied Biosystems, Foster City, CA) was used for normalization. PCR reactions were performed in triplicate and fold changes were calculated using the 2eDDCT method, where DCt is the difference between the amplification
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fluorescent thresholds of the miRNA of interest and the RNA of RNU6. 2.4. Treatment of gastric cancer cell lines with TSA
independently, followed by combination of them. Since these platforms use different parameters and/or databases, putative miRNAs predicted by at least three algorithms have been selected for further studies [26e30].
AGS, MKN28 and KATO III cells were plated in 10 cm culture dishes and grown for 24 h before drug treatment. The next day, about 8.8 106 cells were incubated in fresh culture medium containing a TSA/DMSO solution up to a final concentration of 300 nM. Control cells were treated with an equivalent volume of DMSO. After 24 or 48 h, cells were harvested and used either to evaluate the GKN1 mRNA or miR expression by qRT-PCR.
2.7. Statistical analysis
2.5. Plasmid construction and luciferase reporter assay
3. Results
The 30 UTR of GKN1 (157bp) was amplified by PCR using AGS DNA with primer pairs containing XbaI site. Oligonucleotide sequences were as follows: 50 -ATCATTCTAGAGGATCCACAATTTTTTAAAGCCACTATGG-3'; 50 -ATCATTCTAGATGAATTCAATGCTAAATGATTTTAT-3'. The PCR products were cloned into the pGL3-control vector, downstream from the Firefly luciferase 30 UTR (Promega, Madison, WI, USA). The orientation of the inserted fragments was established by digestion and confirmed by sequencing. The pGL3 constructs with the reverse orientation were used as a negative control. The full-length GKN1 cDNA containing also the 30 UTR (GKN10 3 UTR) was synthesized by TwinHelix (Milan, Italy) and cloned in the pcDNA 3.1 (þ) expression vector in the BamHI/XbaI cloning sites. The construct was then controlled by digestion and sequencing. flGKN1(His)6 was as already described [9]. Luciferase assays were performed as previously described [20]. Briefly, human HEK-293 cells were co-transfected with pGL3 constructs (200 ng) in presence of 100 nM pre-miRs (Ambion) and with the Renilla luciferase reporter plasmid (10 ng) as an internal control, using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured at 24 h after transfection using a dual-luciferase reporter assay (Promega) according to manufacturer's instructions and performed on a 20/20n Luminometer (Turner BioSystems, Sunnyvale, CA, USA). Relative luciferase activity was calculated by normalizing the firefly luminescence to the Renilla luminescence. All transfection experiments were done in triplicate and each experiment was repeated three times.
3.1. Bioinformatic analysis for miRNAs targeting GKN1-30 UTR
2.6. Bioinformatic analyses For the identification of putative miRNAs which can target GKN1-30 UTR, 5 different software algorithms were used, namely TargetScan7.0 (http://www.targetscan.org) [21], an improved tool that predicts effective miRNAs targets by searching for seed-region matches (6e8 mer sites) in the 30 UTRs of transcripts, complemented with estimation of miRNA targeting efficacy and occurrence of evolutionary motif conservation; miRanda (http:// www.microrna.org) [22], an algoritm that selects strict seed matches of miRNA with transcript 30 UTRs and then filteres the mRNA:miRNA duplex for free energy criterion and conserved binding sites from Drosophila to humans; miRDB (http://mirdb. org) [23], a database that recovers functional interactions by combining miRNA target predictions using MirTarget as in silico tool, with expression data of the putative targets; PITA (http:// genie.weizmann.ac.il) [24], that focuses principally on target site accessibility of the mRNAs which is strictly connected to the secondary structure of the transcript analyzed and DIANA-microT-CDS (v5.0) (http://www.microrna.gr/microT-CDS) [25], an updated version of DIANA-microT, that is designed to examine miRNArecognition elements (MREs) in both 30 UTRs and coding regions
Statistical analysis was performed by two-tailed paired Student's t-test using KaleidaGraph 4.1.1 software. Western blot band intensity was evaluated with ImageJ 2.0.0 software. Data were reported as means ± standard deviation (SD). The significance was accepted at the level of p < 0.05.
In previous work, we showed that a possible epigenetic mechanism could underline the GKN1 gene silencing in gastric carcinogenesis. The model proposed involved the activity of a transcription factor that might regulate the GKN1 promoter function in association with the enzymes SUV39H1 (histone methylation) and HDACs (histone deacetylases). Treatment of MKN28, AGS, and KATO III GC cell lines with trichostatin A (TSA), an inhibitor of HDACs, led by qRT-PCR to an increase of GKN1 mRNA levels of about 50-, 160- and 110-fold, respectively [14]. However, the up-regulation of the GKN1 mRNA levels were not associated with the re-expression of the protein evaluated by western blotting. This finding led to hypothesize the occurrence of a post-translation regulation of GKN1 expression mediated by miRNAs. To search for putative miRNAs that could target the 30 UTR of GKN1 mRNA, we first combined putative miRNAs targeting GKN1 30 UTR from the most popular tools: TargetScan7.0 and PITA, that focus on different features like seed matching/conservation of sites and secondary structure of transcript, respectively. Subsequently, because the predicted interactions were poorly conserved, we explored three other computational methods with different performances to implement the specificity of our analyses: miRanda tool, that analyzes free energy and non-conserved sites, DIANAmicroT-CDS (v5.0), that examines miRNA-recognition elements (MREs) also comprised in coding region, and miRDB that recovers functional annotations (Fig. 1A). The cut-off used for our bioinformatic analyses was driven by published studies [28e30] and the putative miRNAs were selected following a level of stringency based on the intersections of 3 different algorithms [28e30] (Fig. 1B). These computational predictions highlighted 4 putative miRNAs: hsa-miR-1245b-3p (30 -AUAUCCGGAAAUCUAGUAGACU50 , 22 nts), hsa-miR-186e5p (30 -UCGGGUUUUCCUCUUAAGAAAC50 , 22 nts), hsa-miR-325 (30 -UGUGAAUGACCUGUGGAUGAUCC-50 , 23 nts), hsa-miR-544a (30 -CUUGAACGAUUUUUACGUCUUA-50 , 22 nts); the underlined sequence represents the miRNA seed region. Fig. 2 shows the nucleotide sequence of GKN1-30 UTR and the corresponding segments where the identified miRNAs form a base pairing. 3.2. GKN1-30 UTR is a direct target of hsa-miR-544a and miR1245b-3p The functional activity of the identified miRNAs was performed using constructs containing the GKN1-30 UTR (WT) and GKN1 reverse 30 UTR (MUT) as negative control cloned downstream of the luciferase genes. These report plasmids were co-transfected in presence of premiR negative control (miR-NEG) or the 4 premiRs in HEK-293 cells and the luciferase activity was assessed. As shown in Fig. 3, over-expression of the selected miRNAs resulted in a
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Fig. 1. Bioinformatic analysis used to highlight putative miRNAs targeting GKN1-30 UTR. Flow chart (panel A) and Venn diagram (panel B) showing the strategy used for the prediction of miRNAs targeting the 30 UTR of GKN1 mRNA to be tested subsequently in a luciferase assay. miRNAs population of each algorithm is given in parenthesis.
3.4. GKN1 expression in gastric cancer cells is down-regulated by miR- 544a
Fig. 2. Annealing regions of predicted miRNAs in the GKN1-30 UTR. The nucleotide sequence of the 30 UTR of GKN1 mRNA showing the corresponding miRNAs target sequences (boxed).
significant inhibition of the activity of the reporter constructs bearing the wild-type (WT) 30 UTRs of GKN1 gene when compared with miR-NEG-transfected cells only for miR-544a and miR-1245b3p (Fig. 3C and D) whereas for miRNA-186e5p and miR-325, no effect was instead observed (Fig. 3A and B). The transfection of the constructs containing the inverted 30 UTR regions (MUT) abolished the effect of miR-544a and miR-1245b-3p. These results indicated that GKN1-30 UTR is a direct target of miR544a and miR-1245b-3p.
3.3. TSA-induced up-regulation of GKN1 mRNA is accompanied in GC cell lines by up-regulation of miR-544a Because TSA-inhibition of HDACs led to an increase of GKN1 mRNA of about 160 fold compared with untreated AGS gastric cancer cells [14], we tried to assess by TaqMan MiRNA Assay the expression level of miR-544a and miR-1245b-3p in MKN28, AGS and KATO III cell lines after treatment of the cells with TSA. The results showed an increase of miR-544a expression of about 20-, 140- and 60-fold in MKN28, AGS, and KATO III cells, respectively, compared with untreated cells (Fig. 4A); the expression of miR1245b-3p was detectable only in AGS cells and TSA treatment induced an increase of about 7-fold (Fig. 4B), whereas no effect was observed in MKN28 and KATO III cells after TSA treatment. These results suggested that the up-regulation of miR-544a could represent one important factor leading to GKN1 translational repression.
We then tried to assay the potential effect of miR-544a on the expression of GKN1 in gastric cancer cells. To this purpose, a new pCDNA 3.1 vectors containing the full-length GKN1 cDNA plus the complete 30 UTR was constructed (GKN1-30 UTR). This vector was used to co-transfect MKN28 cells with premiR-544a and with a premiR negative control. As reported in Fig. 5, compared to GKN130 UTR alone (Fig. 5, lane 1), cells co-transfected with premiR-544a showed a marked reduction of GKN1 protein that was about 73% (Fig. 5, lane 3) whereas about 32% reduction was observed in cells co-transfected with the negative control (Fig. 5, lane 2). Control of transfection was made with flGKN1(His)6 owing to a higher molecular weight (Fig. 5, lane 4). The down-regulation of GKN1 was also observed at the transcription level. As reported in Fig. 6, qRTPCR performed on total RNA extracted from AGS cells transfected with GKN1-30 UTR plus negative control or plus premiR-544a showed a reduction of about 80% of the GKN1 transcript in cells treated with premiR-544a. 4. Discussion In previous work, we showed that epigenetic mechanisms leading to the inactivation of GKN1 gene seem to play a key role in the multi-step process of gastric carcinogenesis. Treatment of GC cell lines with TSA strongly increased GKN1 mRNA expression thus suggesting that histone deacetylation represents an important mediator of GKN1 repression associated with DNA methylation [14]. However, it should be considered that in the experimental condition used, the up-regulation of GKN1 mRNA level was still very low because the cycle numbers required for its amplification resulted of about 10-fold higher than those required for the amplification of housekeeping glucose-6-phosphate dehydrogenase (data not shown). Nevertheless, no GKN1 protein reexpression was detected under these conditions by Western blotting, also in AGS cells treated with the proteasome inhibitor (MG132). This finding suggested the presence of further regulation at the translational level, perhaps by mechanisms mediated by miRNAs, resulting in translational repression and gene silencing. For
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Fig. 3. Putative miRNAs are validated by luciferase assay in HEK293 cells. The luciferase constructs bearing WT or inverted (MUT) GKN1-30 UTR were co-transfected in presence of premiR negative control (miR-NEG) or the 4 premiRs in HEK-293 cells. Renilla luciferase was used as an internal control. Dual-luciferase assays were performed as described in Materials and Methods. Triplicate transfection ratio (±SD) firefly/Renilla luciferase activity of three independent experiments was averaged and expressed as a percentage of the corresponding transfections with miR-NEG control. *p < 0.05.
example, in recent work, it has been reported that miRNA-544 directly targets the 30 UTR of the newly-identified tumor suppressor gene IRX1, whose hypermethylation decreases expression of the protein in GC [31]. Dysregulation of miRNAs occurs in cancer cells and accumulating evidence indicates that miRNAs can behave either as oncogenes or tumor suppressors, with context-dependent functions. For example, miR-494 suppresses gastric cancer by targeting c-myc
[32], but in other cellular contexts can function as an oncogene and also influences cell response to therapy [33,34]. For the identification of putative miRNAs which can target GKN1-30 UTR, a bioinformatic approach was undertaken by analyzing 5 different software algorithms. The intersection of 3 programs resulted in a list of 4 potential miRNAs: hsa-miR-1245b3p, hsa-miR-186e5p, hsa-miR-325, hsa-miR-544a. To verify if the selected miRNAs were able to target the 30 UTR of GKN1 mRNA, a
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Fig. 4. Expression levels of miR-544a, and miR-1245b-3p in gastric cancer cell lines TSA-treated. qRT-PCR analysis of miR-544a (A) and miR-1245b-3p (B) in MKN28, AGS and KATO III gastric cancer cell lines after TSA treatment of the cells for 48 h. RNU6 was used as an internal standard for normalization The relative expression was evaluated using as control cells treated with DMSO. Data from a representative experiment are reported as mean values ± SD. *p < 0.05, **p < 0.001, ***p < 0.01.
Fig. 5. Effect of miR-544a on the expression level of GKN1 in MKN28 cells. MKN28 gastric cancer cells were treated as below reported and after 36 h from transfection, cell extracts were analyzed by Western blot using anti-GKN1 antibody. Lanes: 1, cells transfected with GKN1-30 UTR alone. 2, cells co-transfected with GKN1-30 UTR plus negative control. 3, cells co-transfected with GKN1-30 UTR plus premiR-544a. 4, cells transfected with flGKN1(His)6. 5, cells transfected with GFP. 6, untransfected MKN28 cells. Densitometric analysis of the Western blot was performed only for the samples of lanes 1, 2, and 3, since the samples of lanes 4, 5, and 6 were used as transfection controls.
luciferase reporter assay was performed. The results of these experiments showed that GKN1-30 UTR was mainly a direct target of hsa-miR-544a and to a lower extent of miR-1245b-3p (Fig. 3). On the bases of these results, it was tried to highlight the possible involvement of these miRNAs in the post-translation regulation of GKN1 expression. To this purpose, it was evaluated the expression level of miR-544a and miR-1245b-3p in MKN28, AGS and KATO III gastric cancer cell lines after TSA treatment in which we observed a strong increase of GKN1 mRNA expression that was not accompanied by the expression of the protein. Compared with untreated cells, the results showed an evident upregulation of miR-544a expression of about 20-, 140- and 60-fold in MKN28, AGS, and KATO III cells, respectively. The expression of miR-1245b-3p was instead detectable only in AGS with an increase
Fig. 6. PremiR-544a induces down-regulation of GKN1 mRNA in a gastric cancer cell line. qRT-PCR analysis of GKN1 mRNA in AGS gastric cancer cell line after transfection of the cells with GKN1-30 UTR plus premiR negative control (miR-NEG) or plus premiR544a. The experiment was performed in triplicates and data are reported as mean values ± SD. *p < 0.0001.
of about 7-fold only, whereas no effect was observed in MKN28 and KATO III cells. Therefore, one might postulate that the upregulation of miR-544a could be the cause of GKN1 translational repression. In fact, by co-transfecting in AGS cells GKN1-30 UTR and premiR-544a, it was observed, compared to untreated cells, a strong reduction of GKN1 expression both at protein and mRNA levels. The negative regulation of GKN1 observed (Figs. 5 and 6) regarded most likely the transfected gene since the GKN1 protein is not expressed in gastric cancer cells whereas the GKN1 mRNA level
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is almost undetectable [14]. These results suggested a potential role of GKN1 as a biomarker and as a therapeutic target in GC patients. 5. Conclusions In conclusion, the data here presented are hopeful since miR544a has been shown to play an important role in gastric cancer by inducing epithelial-mesenchymal transition (EMT) [35]. Our findings suggest that epigenetic mechanisms leading to the inactivation of GKN1 play a key role in the multi-step process of gastric carcinogenesis and would provide an essential starting point for the development of new therapeutic strategies based on epigenetic targets for alternatives gene. Conflicts of interest The authors declare no conflict of interest. Authors' contributions CSDS and AF were involved in all experiments, FA was involved in tissue culture and transfection, RF was involved in bioinformatic analyses and manuscript revision, ER take care of cell tissue culture, cell growth and transfection, ER and PA were involved in the preparation of a draft manuscript and in the reading and approval of the final manuscript. Author agreement All the authors that participated to the present work declare that there are no conflict of interest and that the Material submitted is original. All authors are in agreement to have the article published. Acknowledgments This work was supported by funds from Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale (2008BKRFBH_003), from FIRB (N_RBNE08NKH7_003) and PON Ricerca e Competitivit a 2007e2013 (PON01_02782). References [1] T.E. Martin, C.T. Powell, Z. Wang, S. Bhattacharyya, M.M. Walsh- Reitz, K. Agarwal, F.G. Toback, A novel mitogenic protein that is highly expressed in cells of the gastric antrum mucosa, Am. J. Physiol. Gastrointest. Liver Physiol. 285 (2003) G332eG343. [2] E. Rippa, F. Altieri, C.S. Di Stadio, G. Miselli, A. Lamberti, A. Federico, V. Quagliariello, F. Papale, G. Guerra, P. Arcari, Ectopic expression of gastrokine 1 in gastric cancer cells up-regulates tight and adherens junction proteins network, Pathol. Res. Pract. 211 (8) (2015) 577e583. [3] F.B. Fahlbusch, M. Ruebner, H. Huebner, G. Volkert, C. Zuern, F. Thiel, M. Koch, C. Menendez-Castro, D.L. Wachter, A. Hartner, W. Rascher, The tumor suppressor gastrokine-1 is expressed in placenta and contributes to the regulation of trophoblast migration, Placenta 34 (11) (2013) 1027e1035. [4] G. Nardone, E. Rippa, G. Martin, A. Rocco, R.A. Siciliano, A. Fiengo, G. Cacace, A. Malorni, G. Budillon, P. Arcari, Gastrokine 1 expression in patients with and without Helicobacter pylori infection, Dig. Liver Dis. 39 (2) (2007) 122e129. [5] G. Nardone, G. Martin, A. Rocco, E. Rippa, G. La Monica, F. Caruso, P. Arcari, Molecular expression of gastrokine 1 in normal mucosa and in Helicobacter pylori-related preneoplastic and neoplastic lesions, Cancer Biol. Ther. 7 (12) (2008) 1890e1895. [6] E.R. Lacy, G.P. Morris, M.M. Cohen, Rapid repair of the surface epithelium in human gastric mucosa after acute superficial injury, J. Clin. Gastroenterol. 17 (1993) S125eS135. [7] D.K. Podolsky, Healing the epithelium: solving the problem from two sides, J. Gastroenterol. 32 (1997) 122e126. [8] M.M. Walsh-Reitz, E.F. Huang, M.W. Musch, E.B. Chang, T.E. Martin, S. Kartha, F.G. Toback, AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins, Am. J. Physiol. Gastrointest. Liver Physiol. 289 (2005)
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Oncol. 137 (2011) 1697e1704. [13] S.F. Moss, J.W. Lee, E. Sabo, A.K. Rubin, J. Rommel, B.R. Westley, F.E. May, J. Gao, P.A. Meitner, R. Tavares, M.B. Resnick, Decreased expression of gastrokine 1 and the trefoil factor interacting protein TFIZ1/GKN2 in gastric cancer: influence of tumor histology and relationship to prognosis, Clin. Cancer Res. 14 (2008) 4161e4167. [14] C.S. Di Stadio, F. Altieri, A. Federico, G. Miselli, A. Niglio, M. De Palma, E. Rippa, P. Arcari, Down-regulation of GKN1 in gastric cancer is not associated with the RUNX3 expression, J. Biosci. Med. 5 (11) (2017) 1e11. [15] F. Altieri, C.S. Di Stadio, A. Federico, G. Miselli, M. De Palma, E. Rippa, P. Arcari, Epigenetic alterations of gastrokine 1 gene expression in gastric cancer, Oncotarget 810 (2017) 16899e16911. [16] M. Malumbres, miRNAs and cancer: an epigenetics view, Mol. Asp. Med. 34 (4) (2013) 863e874. [17] R. Kala, G.W. Peek, T.M. Hardy, T.O. Tollefsbol, MicroRNAs: an emerging science in cancer epigenetics, J. Clin. Bioinform. 3 (1) (2013) 6. [18] F. Tong, P. Cao, Y. Yin, S. Xia, R. Lai, S. Liu, MicroRNAs in gastric cancer: from benchtop to bedside, Dig. Dis. Sci. 59 (1) (2014) 24e30. [19] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods 25 (2001) 402e408. [20] R. Caggiano, F. Cattaneo, O. Moltedo, G. Esposito, C. Perrino, B. Trimarco, R. Ammendola, R. Faraonio, miR-128 is implicated in stress responses by targeting MAFG in skeletal muscle cells, Oxid. Med. Cell Longev. 2017 (2017) 9308310. [21] V. Agarwal, G.W. Bell, J. Nam, D.P. Bartel, Predicting effective microRNA target sites in mammalian mRNAs, Elife 4 (2015), e05005. [22] B. John, A.J. Enright, A. Aravin, T. Tuschl, C. Sander, D.S. Marks, Human MicroRNA targets, PLoS Biol. 2 (11) (2004) e363. [23] L. Weijun, W. Xiaowei, Prediction of functional microRNA targets by integrative modeling of microRNA binding and target expression data, Genome Biol. 20 (2019) 18. [24] M. Kertesz, N.N. Iovino, U. Unnerstall, U. Gaul, E. Segal, The role of site accessibility in microRNA target recognition, Nat. Genet. 39 (10) (2007) 1278e1284. [25] M.D. Paraskevopoulou, G. Georgakilas, N. Kostoulas, I.S. Vlachos, T. Vergoulis, M. Reczko, C. Filippidis, T. Dalamagas, A.G. Hatzigeorgiou, DIANA-microT web server v5.0: service integration into miRNA functional analysis workflows, Nucleic Acids Res. 41 (W1) (2013) W169eW173. [26] D.E. Kuhn, M.M. Martin, D.S. Feldman, A.V. Jr Terry, G.J. Nuovo, T.S. Elton, Experimental validation of miRNA targets, Methods 44 (1) (2008) 47e54. [27] H. Dweep, C. Sticht, N. Gretz, In-silico algorithms for the screening of possible microRNA binding sites and their interactions, Curr. Genom. 14 (2) (2013) 127e136. [28] A.C. Oliveira, L.A. Bovolenta, P.G. Nachtigall, M.E. Herkenhoff, N. 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Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
GKN1 expression in gastric cancer cells is negatively regulated by miR544a Chiara Stella di Stadio a, 1, Raffaella Faraonio a, 1, Antonella Federico a, 1, Filomena Altieri a, Emilia Rippa a, **, Paolo Arcari a, b, * a b
Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy CEINGE, Advanced Biotechnology Scarl, Via Gaetano Salvatore 486, I-80145, Naples, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 April 2019 Accepted 6 September 2019 Available online 8 September 2019
Gastrokine1 (GKN1), important for maintaining the physiological function of the gastric mucosa, is highly expressed in the stomach of healthy individuals but is down-regulated or absent in gastric tumor tissues and derived cell lines. The mechanisms underlying GKN1 gene inactivation are still unknown. We previously showed that GKN1 downregulation in gastric tumors is likely associated with an epigenetic transcriptional complex that negatively regulates GKN1 expression. In addition, TSA-mediated inhibition of HDACs leads to GKN1 restoration at the transcriptional level, but no at the translational level. These findings led to hypothesize the activation of a second regulatory mechanism microRNAs-mediated, thus resulting in translational repression and gene silencing. Bioinformatic analyses performed with 5 different algorithms highlighted that 4 miRNAs contained a seed sequence for the 30 UTR of GKN1 mRNA. Among these, only two miRNAs, hsa-miR-544a and miR-1245b-3p directly target the GKN1-30 UTR as evaluated by luciferase reporter assays. TaqMan miRNA assay performed on gastric cancer cell lines after TSA treatment showed a stronger increase of miR-544a expression than that of miR-1245b-3p. Finally, co-transfection of AGS cells with GKN1-30 UTR and premiR-544a showed compared to controls, a strong reduction of GKN1 expression both at translational and transcriptional levels. The up-regulation of miR544a could be crucially involved in the GKN1 translational repression, thus suggesting its potential role as a biomarker and therapeutic target in GC patients. These findings indicate that epigenetic mechanisms leading to the inactivation of GKN1 play a key role in the multi-step process of gastric carcinogenesis and would provide an essential starting point for the development of new therapeutic strategies based on epigenetic targets for alternatives gene. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Keywords: Gastrokine 1 Gastric cancer Epigenetics Translation regulation miRNA
1. Introduction Gastrokine 1 (GKN1) is a secretory protein that is localized within the granules just under the apical plasma membrane [1e3]. Its expression is confined to the gastric epithelium, except for trace levels in the placenta [3]. The protein is highly expressed in the gastric mucosa of healthy individuals, but markedly downregulated in samples derived from Helicobacter pylori infected
* Corresponding author. Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, I-8031, Naples, Italy. ** Corresponding author. Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, I-8031, Naples, Italy. E-mail addresses: [email protected] (E. Rippa), [email protected] (P. Arcari). 1 CSDS, RF and AF equally contributed to the work.
patients [4]. Furthermore, GKN1 is down-regulated or completely absent in GC tissues [4,5]. Although the biological function of this protein is currently unknown, a role in cell proliferation and differentiation has been hypothesized, as well as the potential involvement in the replenishment of the surface lumen epithelial cell layer and the maintaining of mucosal integrity [6e8]. GKN1 can also induce Fas-mediated apoptosis and senescence [9] and to reduce the proliferation of AGS cells compared to non-gastric cancer cells (H1355) [10]. Recent evidence has demonstrated that GKN1 is involved in gastric mucosal inflammation by regulating the production of inflammatory mediators [11] and that is involved also in the epithelial-mesenchymal transition. In particular, the recovery of GKN1 expression suppresses cell migration and invasiveness abrogating the expression of PI3K/akt pathway proteins, concomitant with the re-expression of E-cadherin [12]. These data suggest
https://doi.org/10.1016/j.biochi.2019.09.005 0300-9084/© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
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2. Materials and methods Abbreviations 2.1. Materials DMSO dimethyl sulfoxide; GC gastric cancer GKN1 gastrokine 1 G6PD glucose-6-phosphate dehydrogenase HAT histone acetyltransferases HDAC histone deacetylase H3K9triMe trimethylation of Histone 3 at lysine 9 RNU6 U6 Small nucleolar RNA TSA trichostatin A SUV39H1 Histone-lysine N-methyltransferase 30 UTR 30 -untranslated regions
that GKN1 plays a role as a gastric specific tumor suppressor and that be considered a biomarker for GC because individuals with a lower expression of the protein have an increased risk of developing gastric diseases [13]. The mechanism by which GKN1 gene is inactivated in GC remains still unknown. Therefore, recently we have investigated the possible causes of GKN1 gene silencing to determine if epigenetic mechanisms could play an important role in its down-regulation. To these aim, chromatin immunoprecipitation (ChIP) assays for the repressive trimethylation of histone 3 at lysine 9 (H3K9triMe) and its specific histone-lysine N-methyltransferase (SUV39H1) were performed on biopsies of normal and tumor human gastric tissues. GKN1 down-regulation showed to be associated in gastric cancer tissues with high levels of H3K9triMe and with the recruitment of SUV39H1 on GKN1 promoter. This finding suggested the presence of an epigenetic transcriptional complex that negatively regulates GKN1 expression in gastric tumor [14]. It was also investigated whether underacetylation might contribute to GKN1 transcriptional inhibition using TSA to increase general histone acetylation. Inhibition of HDACs led to an increase of GKN1 mRNA of about 160 fold compared to untreated AGS gastric cancer cells. However, this large increase was not associated with the restoration of GKN1 expression at the translational level [15]. These results suggested the presence of a second regulatory block occurring at the translational level through mechanisms mediated by microRNAs (miRNAs), resulting in translational repression and gene silencing. Recently, microRNA has emerged as molecular regulators that can have key roles in pathogenesis and progression of different malignancies, including gastric cancer. miRNAs are a class of small non-coding RNAs (19e25 nucleotides) that act as important epigenetic players in many cellular processes, such as differentiation, proliferation, and apoptosis, exerting great influence in cancer pathogenesis [16,17]. The mature miRNAs act as posttranscriptional regulators interacting with the 30 -untranslated regions (30 UTR) of the target transcripts and resulting in either mRNA degradation or inhibition of translation. Changes in miRNA expression profiles have been observed in a variety of human tumors, including gastric cancer [18]. Therefore, the possible involvement of miRNAs in this process was investigated. The results obtained showed that GKN1-30 UTR was a direct target of hsa-miR-544a and miR-1245b-3p and showed an increase of miR-544a expression in the gastric cancer cell lines after TSA treatment. The up-regulation of miR-544a could be the cause of the GKN1 translational repression since co-transfection in a gastric cancer cell of GKN1 cDNA containing the 30 UTR and premiR-544a showed, compared to control, a strong reduction of GKN1 expression level.
Dulbecco's modified Eagle's medium (DMEM-F12) and fetal bovine serum (FBS) were purchased from Cambrex (Rutherford, NJ, USA). Mouse GKN1 monoclonal antibody (M01), clone 2E5, was purchased from Abnova (Taipei, Taiwan). Trichostatin A (TSA) was from Sigma (Milan, Italy). a-Tubulin was from Abcam (Cambridge, UK). 2.2. Cell cultures, transfection, human tissues, and Western blotting Human gastric adenocarcinoma cell lines (AGS, MKN28, KATO III) and human embryonic kidney 293 (HEK293) cells were grown in DMEM-F12 and DMEM, respectively, supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin and 1% L-glutamine at 37 C in a 5% CO2 atmosphere. AGS were transfected with 4 mg of vector pcDNA 3.1, pcDNA3.1flGKN1(His)6 encoding the full-length GKN1 (flGKN1, containing the first 20 amino acids leader peptide and His6-Tag sequence at the Cterminal) as already described [9]. The efficiency of transfection of gastric cancer cells with flGKN1 was always evaluated by a parallel transfection using EGFP vector as control. In general, after transfection, the average value of the ratio between the number of green fluorescent cells/total number of cells was 0.5 ± 0.1. Proteins from cell extracts (about 20 mg) were analyzed by Western blotting using mouse anti-GKN1 at 1:500. Detection was performed using the enhanced chemiluminescence detection kit (SuperSignal West Pico) following the manufacturer's instructions. Western blot band intensity was measured with ImageJ 1.46r software. 2.3. RNA isolation and real-time qPCR Total RNA was extracted from AGS, MKN28, and KATO III gastric cancer cell lines using TRIzol reagent (Invitrogen) and quantified by Nanodrop (Thermo Scientific, Wilmington, DE). cDNA was synthesized using the reverse transcription kit from Roche Molecular Systems (Roche, Penzberg, Germany) according to the manufacturer's protocol. GKN1 transcript levels were quantified using SYBR Green PCR MasterMix (Applied Biosystems) IQ SYBR green Supermix (Bio-Rad) on a CFX96 Real-Time System instrument (Bio-Rad) using the following primers pairs: 50 -ctttctagctcctgccctagc-3’; 50 -GTTGCAGCAAAGCCATTTCC-3’. Real-Time qPCRs were performed in triplicate under the following conditions: 10 min at 95 C, followed by 40 cycles (15 s at 95 C and 1 min at 60 C). The relative fold changes were calculated using the 2-DDCt method by the formula: 2- (sample DCt e control DCt), where DCt is the difference between the amplification fluorescent thresholds of the gene of interest and the internal reference gene (G6PD) used for normalization [19]. TaqMan miRNA Assay kit (Applied Biosystems, Foster City, CA, USA) was used to detect the expression miRNAs (miR-544a, miR1245b-3p) in DMSO or TSA treated cells lines. Briefly, 100 ng of total RNA was reversely transcribed (RT) at 16 C for 30 min, 42 C for 30 min and 85 C for 5 min in 15 ml reaction volume. Two ml of RT product was used for PCR reaction in a final volume of 20 ml. The PCR reaction started with an initial denaturation step at 90 C for 10 min, followed by 40 cycles of 95 C for 15 s and 60 C for 1 min. Small nucleolar RNA RNU6 (Applied Biosystems, Foster City, CA) was used for normalization. PCR reactions were performed in triplicate and fold changes were calculated using the 2eDDCT method, where DCt is the difference between the amplification
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fluorescent thresholds of the miRNA of interest and the RNA of RNU6. 2.4. Treatment of gastric cancer cell lines with TSA
independently, followed by combination of them. Since these platforms use different parameters and/or databases, putative miRNAs predicted by at least three algorithms have been selected for further studies [26e30].
AGS, MKN28 and KATO III cells were plated in 10 cm culture dishes and grown for 24 h before drug treatment. The next day, about 8.8 106 cells were incubated in fresh culture medium containing a TSA/DMSO solution up to a final concentration of 300 nM. Control cells were treated with an equivalent volume of DMSO. After 24 or 48 h, cells were harvested and used either to evaluate the GKN1 mRNA or miR expression by qRT-PCR.
2.7. Statistical analysis
2.5. Plasmid construction and luciferase reporter assay
3. Results
The 30 UTR of GKN1 (157bp) was amplified by PCR using AGS DNA with primer pairs containing XbaI site. Oligonucleotide sequences were as follows: 50 -ATCATTCTAGAGGATCCACAATTTTTTAAAGCCACTATGG-3'; 50 -ATCATTCTAGATGAATTCAATGCTAAATGATTTTAT-3'. The PCR products were cloned into the pGL3-control vector, downstream from the Firefly luciferase 30 UTR (Promega, Madison, WI, USA). The orientation of the inserted fragments was established by digestion and confirmed by sequencing. The pGL3 constructs with the reverse orientation were used as a negative control. The full-length GKN1 cDNA containing also the 30 UTR (GKN10 3 UTR) was synthesized by TwinHelix (Milan, Italy) and cloned in the pcDNA 3.1 (þ) expression vector in the BamHI/XbaI cloning sites. The construct was then controlled by digestion and sequencing. flGKN1(His)6 was as already described [9]. Luciferase assays were performed as previously described [20]. Briefly, human HEK-293 cells were co-transfected with pGL3 constructs (200 ng) in presence of 100 nM pre-miRs (Ambion) and with the Renilla luciferase reporter plasmid (10 ng) as an internal control, using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured at 24 h after transfection using a dual-luciferase reporter assay (Promega) according to manufacturer's instructions and performed on a 20/20n Luminometer (Turner BioSystems, Sunnyvale, CA, USA). Relative luciferase activity was calculated by normalizing the firefly luminescence to the Renilla luminescence. All transfection experiments were done in triplicate and each experiment was repeated three times.
3.1. Bioinformatic analysis for miRNAs targeting GKN1-30 UTR
2.6. Bioinformatic analyses For the identification of putative miRNAs which can target GKN1-30 UTR, 5 different software algorithms were used, namely TargetScan7.0 (http://www.targetscan.org) [21], an improved tool that predicts effective miRNAs targets by searching for seed-region matches (6e8 mer sites) in the 30 UTRs of transcripts, complemented with estimation of miRNA targeting efficacy and occurrence of evolutionary motif conservation; miRanda (http:// www.microrna.org) [22], an algoritm that selects strict seed matches of miRNA with transcript 30 UTRs and then filteres the mRNA:miRNA duplex for free energy criterion and conserved binding sites from Drosophila to humans; miRDB (http://mirdb. org) [23], a database that recovers functional interactions by combining miRNA target predictions using MirTarget as in silico tool, with expression data of the putative targets; PITA (http:// genie.weizmann.ac.il) [24], that focuses principally on target site accessibility of the mRNAs which is strictly connected to the secondary structure of the transcript analyzed and DIANA-microT-CDS (v5.0) (http://www.microrna.gr/microT-CDS) [25], an updated version of DIANA-microT, that is designed to examine miRNArecognition elements (MREs) in both 30 UTRs and coding regions
Statistical analysis was performed by two-tailed paired Student's t-test using KaleidaGraph 4.1.1 software. Western blot band intensity was evaluated with ImageJ 2.0.0 software. Data were reported as means ± standard deviation (SD). The significance was accepted at the level of p < 0.05.
In previous work, we showed that a possible epigenetic mechanism could underline the GKN1 gene silencing in gastric carcinogenesis. The model proposed involved the activity of a transcription factor that might regulate the GKN1 promoter function in association with the enzymes SUV39H1 (histone methylation) and HDACs (histone deacetylases). Treatment of MKN28, AGS, and KATO III GC cell lines with trichostatin A (TSA), an inhibitor of HDACs, led by qRT-PCR to an increase of GKN1 mRNA levels of about 50-, 160- and 110-fold, respectively [14]. However, the up-regulation of the GKN1 mRNA levels were not associated with the re-expression of the protein evaluated by western blotting. This finding led to hypothesize the occurrence of a post-translation regulation of GKN1 expression mediated by miRNAs. To search for putative miRNAs that could target the 30 UTR of GKN1 mRNA, we first combined putative miRNAs targeting GKN1 30 UTR from the most popular tools: TargetScan7.0 and PITA, that focus on different features like seed matching/conservation of sites and secondary structure of transcript, respectively. Subsequently, because the predicted interactions were poorly conserved, we explored three other computational methods with different performances to implement the specificity of our analyses: miRanda tool, that analyzes free energy and non-conserved sites, DIANAmicroT-CDS (v5.0), that examines miRNA-recognition elements (MREs) also comprised in coding region, and miRDB that recovers functional annotations (Fig. 1A). The cut-off used for our bioinformatic analyses was driven by published studies [28e30] and the putative miRNAs were selected following a level of stringency based on the intersections of 3 different algorithms [28e30] (Fig. 1B). These computational predictions highlighted 4 putative miRNAs: hsa-miR-1245b-3p (30 -AUAUCCGGAAAUCUAGUAGACU50 , 22 nts), hsa-miR-186e5p (30 -UCGGGUUUUCCUCUUAAGAAAC50 , 22 nts), hsa-miR-325 (30 -UGUGAAUGACCUGUGGAUGAUCC-50 , 23 nts), hsa-miR-544a (30 -CUUGAACGAUUUUUACGUCUUA-50 , 22 nts); the underlined sequence represents the miRNA seed region. Fig. 2 shows the nucleotide sequence of GKN1-30 UTR and the corresponding segments where the identified miRNAs form a base pairing. 3.2. GKN1-30 UTR is a direct target of hsa-miR-544a and miR1245b-3p The functional activity of the identified miRNAs was performed using constructs containing the GKN1-30 UTR (WT) and GKN1 reverse 30 UTR (MUT) as negative control cloned downstream of the luciferase genes. These report plasmids were co-transfected in presence of premiR negative control (miR-NEG) or the 4 premiRs in HEK-293 cells and the luciferase activity was assessed. As shown in Fig. 3, over-expression of the selected miRNAs resulted in a
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Fig. 1. Bioinformatic analysis used to highlight putative miRNAs targeting GKN1-30 UTR. Flow chart (panel A) and Venn diagram (panel B) showing the strategy used for the prediction of miRNAs targeting the 30 UTR of GKN1 mRNA to be tested subsequently in a luciferase assay. miRNAs population of each algorithm is given in parenthesis.
3.4. GKN1 expression in gastric cancer cells is down-regulated by miR- 544a
Fig. 2. Annealing regions of predicted miRNAs in the GKN1-30 UTR. The nucleotide sequence of the 30 UTR of GKN1 mRNA showing the corresponding miRNAs target sequences (boxed).
significant inhibition of the activity of the reporter constructs bearing the wild-type (WT) 30 UTRs of GKN1 gene when compared with miR-NEG-transfected cells only for miR-544a and miR-1245b3p (Fig. 3C and D) whereas for miRNA-186e5p and miR-325, no effect was instead observed (Fig. 3A and B). The transfection of the constructs containing the inverted 30 UTR regions (MUT) abolished the effect of miR-544a and miR-1245b-3p. These results indicated that GKN1-30 UTR is a direct target of miR544a and miR-1245b-3p.
3.3. TSA-induced up-regulation of GKN1 mRNA is accompanied in GC cell lines by up-regulation of miR-544a Because TSA-inhibition of HDACs led to an increase of GKN1 mRNA of about 160 fold compared with untreated AGS gastric cancer cells [14], we tried to assess by TaqMan MiRNA Assay the expression level of miR-544a and miR-1245b-3p in MKN28, AGS and KATO III cell lines after treatment of the cells with TSA. The results showed an increase of miR-544a expression of about 20-, 140- and 60-fold in MKN28, AGS, and KATO III cells, respectively, compared with untreated cells (Fig. 4A); the expression of miR1245b-3p was detectable only in AGS cells and TSA treatment induced an increase of about 7-fold (Fig. 4B), whereas no effect was observed in MKN28 and KATO III cells after TSA treatment. These results suggested that the up-regulation of miR-544a could represent one important factor leading to GKN1 translational repression.
We then tried to assay the potential effect of miR-544a on the expression of GKN1 in gastric cancer cells. To this purpose, a new pCDNA 3.1 vectors containing the full-length GKN1 cDNA plus the complete 30 UTR was constructed (GKN1-30 UTR). This vector was used to co-transfect MKN28 cells with premiR-544a and with a premiR negative control. As reported in Fig. 5, compared to GKN130 UTR alone (Fig. 5, lane 1), cells co-transfected with premiR-544a showed a marked reduction of GKN1 protein that was about 73% (Fig. 5, lane 3) whereas about 32% reduction was observed in cells co-transfected with the negative control (Fig. 5, lane 2). Control of transfection was made with flGKN1(His)6 owing to a higher molecular weight (Fig. 5, lane 4). The down-regulation of GKN1 was also observed at the transcription level. As reported in Fig. 6, qRTPCR performed on total RNA extracted from AGS cells transfected with GKN1-30 UTR plus negative control or plus premiR-544a showed a reduction of about 80% of the GKN1 transcript in cells treated with premiR-544a. 4. Discussion In previous work, we showed that epigenetic mechanisms leading to the inactivation of GKN1 gene seem to play a key role in the multi-step process of gastric carcinogenesis. Treatment of GC cell lines with TSA strongly increased GKN1 mRNA expression thus suggesting that histone deacetylation represents an important mediator of GKN1 repression associated with DNA methylation [14]. However, it should be considered that in the experimental condition used, the up-regulation of GKN1 mRNA level was still very low because the cycle numbers required for its amplification resulted of about 10-fold higher than those required for the amplification of housekeeping glucose-6-phosphate dehydrogenase (data not shown). Nevertheless, no GKN1 protein reexpression was detected under these conditions by Western blotting, also in AGS cells treated with the proteasome inhibitor (MG132). This finding suggested the presence of further regulation at the translational level, perhaps by mechanisms mediated by miRNAs, resulting in translational repression and gene silencing. For
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Fig. 3. Putative miRNAs are validated by luciferase assay in HEK293 cells. The luciferase constructs bearing WT or inverted (MUT) GKN1-30 UTR were co-transfected in presence of premiR negative control (miR-NEG) or the 4 premiRs in HEK-293 cells. Renilla luciferase was used as an internal control. Dual-luciferase assays were performed as described in Materials and Methods. Triplicate transfection ratio (±SD) firefly/Renilla luciferase activity of three independent experiments was averaged and expressed as a percentage of the corresponding transfections with miR-NEG control. *p < 0.05.
example, in recent work, it has been reported that miRNA-544 directly targets the 30 UTR of the newly-identified tumor suppressor gene IRX1, whose hypermethylation decreases expression of the protein in GC [31]. Dysregulation of miRNAs occurs in cancer cells and accumulating evidence indicates that miRNAs can behave either as oncogenes or tumor suppressors, with context-dependent functions. For example, miR-494 suppresses gastric cancer by targeting c-myc
[32], but in other cellular contexts can function as an oncogene and also influences cell response to therapy [33,34]. For the identification of putative miRNAs which can target GKN1-30 UTR, a bioinformatic approach was undertaken by analyzing 5 different software algorithms. The intersection of 3 programs resulted in a list of 4 potential miRNAs: hsa-miR-1245b3p, hsa-miR-186e5p, hsa-miR-325, hsa-miR-544a. To verify if the selected miRNAs were able to target the 30 UTR of GKN1 mRNA, a
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Fig. 4. Expression levels of miR-544a, and miR-1245b-3p in gastric cancer cell lines TSA-treated. qRT-PCR analysis of miR-544a (A) and miR-1245b-3p (B) in MKN28, AGS and KATO III gastric cancer cell lines after TSA treatment of the cells for 48 h. RNU6 was used as an internal standard for normalization The relative expression was evaluated using as control cells treated with DMSO. Data from a representative experiment are reported as mean values ± SD. *p < 0.05, **p < 0.001, ***p < 0.01.
Fig. 5. Effect of miR-544a on the expression level of GKN1 in MKN28 cells. MKN28 gastric cancer cells were treated as below reported and after 36 h from transfection, cell extracts were analyzed by Western blot using anti-GKN1 antibody. Lanes: 1, cells transfected with GKN1-30 UTR alone. 2, cells co-transfected with GKN1-30 UTR plus negative control. 3, cells co-transfected with GKN1-30 UTR plus premiR-544a. 4, cells transfected with flGKN1(His)6. 5, cells transfected with GFP. 6, untransfected MKN28 cells. Densitometric analysis of the Western blot was performed only for the samples of lanes 1, 2, and 3, since the samples of lanes 4, 5, and 6 were used as transfection controls.
luciferase reporter assay was performed. The results of these experiments showed that GKN1-30 UTR was mainly a direct target of hsa-miR-544a and to a lower extent of miR-1245b-3p (Fig. 3). On the bases of these results, it was tried to highlight the possible involvement of these miRNAs in the post-translation regulation of GKN1 expression. To this purpose, it was evaluated the expression level of miR-544a and miR-1245b-3p in MKN28, AGS and KATO III gastric cancer cell lines after TSA treatment in which we observed a strong increase of GKN1 mRNA expression that was not accompanied by the expression of the protein. Compared with untreated cells, the results showed an evident upregulation of miR-544a expression of about 20-, 140- and 60-fold in MKN28, AGS, and KATO III cells, respectively. The expression of miR-1245b-3p was instead detectable only in AGS with an increase
Fig. 6. PremiR-544a induces down-regulation of GKN1 mRNA in a gastric cancer cell line. qRT-PCR analysis of GKN1 mRNA in AGS gastric cancer cell line after transfection of the cells with GKN1-30 UTR plus premiR negative control (miR-NEG) or plus premiR544a. The experiment was performed in triplicates and data are reported as mean values ± SD. *p < 0.0001.
of about 7-fold only, whereas no effect was observed in MKN28 and KATO III cells. Therefore, one might postulate that the upregulation of miR-544a could be the cause of GKN1 translational repression. In fact, by co-transfecting in AGS cells GKN1-30 UTR and premiR-544a, it was observed, compared to untreated cells, a strong reduction of GKN1 expression both at protein and mRNA levels. The negative regulation of GKN1 observed (Figs. 5 and 6) regarded most likely the transfected gene since the GKN1 protein is not expressed in gastric cancer cells whereas the GKN1 mRNA level
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is almost undetectable [14]. These results suggested a potential role of GKN1 as a biomarker and as a therapeutic target in GC patients. 5. Conclusions In conclusion, the data here presented are hopeful since miR544a has been shown to play an important role in gastric cancer by inducing epithelial-mesenchymal transition (EMT) [35]. Our findings suggest that epigenetic mechanisms leading to the inactivation of GKN1 play a key role in the multi-step process of gastric carcinogenesis and would provide an essential starting point for the development of new therapeutic strategies based on epigenetic targets for alternatives gene. Conflicts of interest The authors declare no conflict of interest. Authors' contributions CSDS and AF were involved in all experiments, FA was involved in tissue culture and transfection, RF was involved in bioinformatic analyses and manuscript revision, ER take care of cell tissue culture, cell growth and transfection, ER and PA were involved in the preparation of a draft manuscript and in the reading and approval of the final manuscript. Author agreement All the authors that participated to the present work declare that there are no conflict of interest and that the Material submitted is original. All authors are in agreement to have the article published. Acknowledgments This work was supported by funds from Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale (2008BKRFBH_003), from FIRB (N_RBNE08NKH7_003) and PON Ricerca e Competitivit a 2007e2013 (PON01_02782). References [1] T.E. Martin, C.T. Powell, Z. Wang, S. Bhattacharyya, M.M. Walsh- Reitz, K. Agarwal, F.G. Toback, A novel mitogenic protein that is highly expressed in cells of the gastric antrum mucosa, Am. J. Physiol. Gastrointest. Liver Physiol. 285 (2003) G332eG343. [2] E. Rippa, F. Altieri, C.S. Di Stadio, G. Miselli, A. Lamberti, A. Federico, V. Quagliariello, F. Papale, G. Guerra, P. Arcari, Ectopic expression of gastrokine 1 in gastric cancer cells up-regulates tight and adherens junction proteins network, Pathol. Res. Pract. 211 (8) (2015) 577e583. [3] F.B. Fahlbusch, M. Ruebner, H. Huebner, G. Volkert, C. Zuern, F. Thiel, M. Koch, C. Menendez-Castro, D.L. Wachter, A. Hartner, W. Rascher, The tumor suppressor gastrokine-1 is expressed in placenta and contributes to the regulation of trophoblast migration, Placenta 34 (11) (2013) 1027e1035. [4] G. Nardone, E. Rippa, G. Martin, A. Rocco, R.A. Siciliano, A. Fiengo, G. Cacace, A. Malorni, G. Budillon, P. Arcari, Gastrokine 1 expression in patients with and without Helicobacter pylori infection, Dig. Liver Dis. 39 (2) (2007) 122e129. [5] G. Nardone, G. Martin, A. Rocco, E. Rippa, G. La Monica, F. Caruso, P. Arcari, Molecular expression of gastrokine 1 in normal mucosa and in Helicobacter pylori-related preneoplastic and neoplastic lesions, Cancer Biol. Ther. 7 (12) (2008) 1890e1895. [6] E.R. Lacy, G.P. Morris, M.M. Cohen, Rapid repair of the surface epithelium in human gastric mucosa after acute superficial injury, J. Clin. Gastroenterol. 17 (1993) S125eS135. [7] D.K. Podolsky, Healing the epithelium: solving the problem from two sides, J. Gastroenterol. 32 (1997) 122e126. [8] M.M. Walsh-Reitz, E.F. Huang, M.W. Musch, E.B. Chang, T.E. Martin, S. Kartha, F.G. Toback, AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins, Am. J. Physiol. Gastrointest. Liver Physiol. 289 (2005)
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