Deregulation of the MiR-193b-KRAS Axis Contributes to Impaired Cell Growth in Pancreatic Cancer

Modulation of KRAS activity by upstream signals has revealed a promising new approach for pancreatic cancer therapy; however, it is not clear whether microRNA-associated KRAS axis is involved in the carcinogenesis of pancreatic cancer. Here, we identified miR-193b as a tumor-suppressive miRNA in pancreatic ductal adenocarcinoma (PDAC). Expression analyses revealed that miR-193b was downregulated in (10/11) PDAC specimens and cell lines. Moreover, we found that miR-193b functioned as a cell-cycle brake in PDAC cells by inducing G1-phase arrest and reducing the fraction of cells in S phase, thereby leading to dampened cell proliferation. miR-193b also modulated the malignant transformation phenotype of PDAC cells by suppressing anchorage-independent growth. Mechanistically, KRAS was verified as a direct effector of miR-193b, through which the AKT and ERK pathways were modulated and cell growth of PDAC cells was suppressed. Taken together, our findings indicate that miR-193b-mediated deregulation of the KRAS axis is involved in pancreatic carcinogenesis, and suggest that miR-193b could be a potentially effective target for PDAC therapy.


Introduction
MicroRNAs (miRNAs) are a class of very small, non-coding RNAs that are evolutionarily conserved in many organisms. miRNAs suppress the expression of protein-coding genes in metazoans by binding to the 3 0 untranslated region (3 0 -UTR) or even the coding region of their corresponding mRNA [1][2][3]. In this process, specific miRNAs pair-bond with target genes, leading to translational repression or/and mRNA destabilization [4]. Recent bioinformatic analyses have indicated that a great number of mRNAs are conserved target transcripts of miR-NAs in mammals [5,6]. miRNAs regulate a variety of cellular and developmental processes, including cell proliferation, survival, differentiation, animal development and disease [7][8][9]. Importantly, miRNAs display aberrant expression patterns in tumors and have emerged as surgery, the cancer status of PDAC and matched adjacent pancreas was reconfirmed in frozen sections of each specimen prior to RNA extraction. Patient characteristics, including sex, age, pathological diagnosis, differentiation and lymph node involvement, are presented in S1 Table. RNA quantitation and in situ hybridization Total RNA was extracted from frozen tissue samples or cultured cell lines using TRIzol reagent (Life Technologies). RNA concentration was determined on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). miRNA was converted to cDNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems); reverse transcriptase-minus and no-template controls were included. Mature hsa-miR-193b expression levels were assessed by real-time polymerase chain reaction (PCR) using TaqMan MicroRNA assays (Applied Biosystems). Reactions were performed in triplicate on an IQ5 instrument (Bio-Rad) using the 2 -ΔΔCt method; U6 snRNA was used as an endogenous control.

Apoptosis and colony-formation assays
Early-stage apoptosis was detected in MIA PaCa-2 and PANC-1 pancreatic cancer cells by first rinsing cells briefly with 0.125% trypsin (0.5×; GIBCO, Life Technologies) and then staining using a FITC Annexin V apoptosis Detection Kit I (BD Biosciences). Stained cells were analyzed on an Accuri C6 Flow Cytometer (BD Biosciences). Colony formation by cells treated with miRNA mimics (GenePharma Co.) was assayed as previously described [25].

DNA constructs and luciferase reporter assay
A fragment of wild-type KRAS 3 0 -UTR was amplified by PCR from PANC-1 genomic DNA using TopTaq DNA Polymerase (QIAGEN) with the primers, 5 0 -GCT CTA GAA AGG CCA TTT CCT TTT CAC A-3 0 and 5 0 -GCT CTA GAT GCA TGA CAA CAC TGG ATG A-3 0 (underlined bases correspond to an XbaI restriction site). Antarctic phosphatase (New England Biolabs Inc.) was used to prevent self-cyclization. A luciferase reporter construct was prepared by digesting the PCR product and inserting it into the pGL3-Control Vector (Promega) at a site immediately downstream of the firefly luciferase gene. Reporter constructs containing mutated KRAS 3 0 -UTR, synthesized by Sangon Biotech, were also prepared. The miR-193b complementary sequence, prepared by annealing the two primers 5 0 -CTA GAA GCG GGA CTT TGA GGG CCA GTT T-3 0 and 5 0 -CTA GAA ACT GGC CCT CAA AGT CCC GCT T-3 0 (underlined bases correspond to an XbaI restriction site), was used to verify the transfection efficiency.

Statistical analysis
Experiments were repeated at least three times. The results were expressed as means ± SD. Differences were assessed with a two-tailed Student's t test or Wilcoxon rank-sum test, and a pvalue < 0.05 was considered statistically significant.

miR-193b expression is downregulated in PDAC tissues and cell lines
To explore the expression pattern of miR-193b in PDAC, we determined miR-193b levels in 11 surgically resected PDAC specimens with matched adjacent benign tissues. In addition to primary selection for PDAC based on paraffin-embedded sections, we reconfirmed the PDAC status by histological analysis of frozen sections of each specimen before RNA extraction (S1 Fig). Real-time PCR, used to analyze the expression levels of miR-193b, revealed that miR-193b expression was significantly decreased in 10 out of 11 PDAC samples compared to matched, adjacent benign tissues (Fig 1A and 1B). Overall, the expression levels of miR-193b in bulk PDAC samples were decreased 2.1-to 8.6-fold compared with adjacent normal tissues.
Since tumor tissues are a mix of many tumor cells and stroma cells, and benign tissues are predominantly made up of acinar cells. We further performed LNA in situ hybridization of paraffin-embedded PDAC specimens (n = 22). The expression of miR-193b was lower in PDAC samples than matched adjacent tissues (Fig 2; Table 1). In benign adjacent tissues, miR-193b was strongly (3+) positive in cytoplasm of acinar cells (20/22). Ductal cells that made up a small portion of benign tissues showed various staining levels from negative to moderately (2+) positive (Fig 2C and 2D). In PDAC tissues, no strong miR-193b expression was observed in tumor cells. 2+ miR-193b staining levels were detected in tumor cells, in 14 out of 22 cases (Fig 2E and 2F). 1+ miR-193b levels were seen in 8 out of 22 cases (Fig 2G and 2H).
We next examined endogenous miR-193b expression levels in different pancreatic cancer cell lines. Compared with non-tumorigenic pancreatic duct hTERT-HPNE cells, miR-193b expression was significantly decreased in four different pancreatic cancer cell lines ( Fig 1C).
Taken together, these data suggest that a decrease in miR-193b expression is a common feature of pancreatic cancer both in vitro and in vivo.  The viability of cells was examined on days 1 to 4 using CCK-8 assays. Delivery of miR-193b mimics into cells significantly reduced the viability of both MIA PaCa-2 and PANC-1 cells compared with transfection of scrambled control miRNA (Fig 3A).
In order to study the mechanism underlying the miR-193b-induced decrease in cell viability, we further examined proliferation using BrdU-incorporation assays and evaluated cells for cell-cycle distribution. miR-193b overexpression suppressed the proliferative rate in both cell lines, reducing the percentage of BrdU-positive MIA PaCa-2 and PANC-1 cells by 16.8% and 26.4%, respectively, compared to scrambled controls ( Fig 3B). Moreover, a cell-cycle analysis revealed that miR-193b overexpression induced a 12.5% increase in G1 phase and a 7.0% decrease in S phase cells in the MIA PaCa-2 cell line compared to scrambled controls; a similar increase in G1-phase cells (19.5%) and decrease in S-phase cells (17.3%) was observed in the PANC-1 cell line ( Fig 3C). These results indicate that elevated expression of miR-193b in pancreatic cancer cells inhibits cell growth and cell-cycle progression by promoting G1-phase arrest and a subsequent reduction in the S-phase population.

miR-193b overexpression is associated with apoptosis in MIA PaCa-2 and inhibits clonogenic potential
To determine whether miR-193b acts via an apoptotic mechanism, we treated MIA PaCa-2 cells with miR-193b mimic or scrambled oligonucleotide and tested for the presence of Annexin V-stained cells by flow cytometry. As expected, treatment of MIA PaCa-2 cells with the miR-193b mimic induced an increase in Annexin V-positive, early-apoptotic cells (Fig 4A). We also confirmed this result by determining the levels of the apoptosis-associated proteins, c-PARP and c-caspase-3 using Western blot analysis ( Fig 4B). Interestingly, these assays showed no evidence of apoptosis induction in PANC-1 cells (Fig 4), suggesting a difference in the underlying signaling mechanisms in these two cell lines.
To investigate the effects of miR-193b on the anchorage-independent growth of pancreatic cancer cell lines, we transfected MIA PaCa-2 and PANC-1 with miR-193b mimic or scrambled oligonucleotide and performed colony-formation assays. After 2 weeks, there were fewer and smaller colonies in the miR-193b-overexpression group compared with the scrambled miRNA group in both cell lines (Fig 5).

miR-193b downregulates KRAS expression by targeting its 3 0 -UTR
To investigate the molecular mechanism of miR-193b, we first combined the computational approach, TargetScan (http://www.targetscan.org), with a consideration of oncogenes crucial in pancreatic carcinogenesis to identify putative target mRNAs. As shown in Fig 6A and 6B, computational analyses showed evolutionarily conserved miR-193b binding sites in the 3 0 -UTR of KRAS, which is known to be an important oncogene in pancreatic cancer.
We next assessed the functional interaction of miR-193b with KRAS using a luciferase reporter strategy. Constructs containing a segment of wild-type or mutant KRAS 3 0 -UTR inserted into the pGL-3 vector immediately downstream of the luciferase coding region were cotransfected with miR-193b mimic or scrambled miRNA into 293A cells; co-transfected pRL-TK plasmid served as an endogenous control. Whole-cell extracts were then obtained and assessed for luciferase activity using Dual-Luciferase Reporter Assays. These assays showed that transfection with miR-193b significantly decreased luciferase activity in cells cotransfected with wild-type KRAS 3 0 -UTR constructs compared with those co-transfected with mutant 3 0 -UTR controls (Fig 6C), verifying that KRAS is a direct target gene of miR-193b. Consistent with this, Western blot analyses showed that KRAS protein level was downregulated by miR-193b overexpression ( Fig 6D); CCND1, a known target of miR-193b [18], was used as a positive control. Collectively, these data suggest that miR-193b suppresses KRAS expression by directly binding to its 3 0 -UTR. Finally, we explored the downstream mechanisms underlying the antitumor effects of the miR-193b-KRAS axis. We found that restoration of miR-193b in PANC-1 cells triggered the downregulation of p-ERK and p-Akt (Fig 6E).

Discussion
Pancreatic cancer-often diagnosed at late stages when a patient is beyond cure-is characterized by its lethal nature. It is therefore important to understand its cellular origin, which is still a matter of debate. It has been reported that acinar-ductal metaplasia (ADM) is associated with pancreatic intraepithelial neoplasia (PanIN) formation [26] and pancreatic carcinoma [27]. Recent reports have found that PDAC might originate from acinar cells and that KRAS induced acinar-to-ductal reprogramming plays a key role in PDAC initiation [28]. It has also been extensively reported that pancreatic cancer exhibits aberrant patterns of miRNA expression. In the present study, deregulated miR-193b was observed in PDAC samples, which prompted us to further investigate the role of miR-193b in the development of this disease.
Here, we found that miR-193b was downregulated in most (10/11) of the tested bulk specimens from PDAC patients compared to matched adjacent tissues. As tumor tissues comprise a mix of many different cell types, we further used LNA in situ hybridization (n = 22 samples) to visualize the expression pattern of miR-193b in detail. In general, the expression level of miR-193b was high in "normal" acinar cells (3+), but comparably lower in PDAC cells (1+-2+). To examine the potential involvement of miR-193b in the early stage of PDAC development, we assessed the expression of miR-193b in chronic pancreatitis (CP). In peritumoral CP-like CP is a noted risk factor for PDAC [29], and ADM or tissues in which acinar cells have dedifferentiated to ductal-like structures are prone to transformation [30]. Thus, our results implicate decreased miR-193b is involved in the early stage of PDAC carcinogenesis. This raises the question: What are the functional roles of miR-193b in chronic inflammation versus PDAC? There are two possible explanations. First, previous reports showed that elevated levels of KRAS signaling in acinar cells is a direct linker between CP and PDAC: high KRAS activity induces inflammation and fibrosis, which then progress to PDAC [31,32]. In CP, KRAS may be upregulated when its inhibitor (miR-193b) is downregulated, and the higher KRAS activity could induce a series of inflammatory programs that lead to cancer. Second, accumulating evidence suggests that PDAC originates from acinar cells, and that the precursor lesion of PDAC (PanIN) occurs through a duct-like state in acinar cells [28,30,33]. Chronic irritation or KRAS mutation can cause ADM in the pancreas [34]. Furthermore, previous studies indicated that KRAS and MAPK are required for the development of ADM and PanIN in mice [35,36], and MAPK was found to be upregulated in pancreatitis [37]. Here, we show that miR-193b is downregulated in duct-like structures and PDAC. KRAS is directly targeted by miR-193b, MiR-193b in Pancreatic Cancer leading to regulation of the KRAS effector, MAPK. Thus, miR-193b might be involved in sustaining inflammation-induced ADM, which is prone to tumorigenesis. Further studies are needed to clarify the underlying mechanism. However, we speculate that miR-193b is downregulated during the formation of PDAC from acinar cells via ADM, and that this alteration, along with changes in other factors, contributes to acinar cell differentiation and finally PDAC carcinogenesis.
Many previous studies found that KRAS oncogene is mutated in more than 95% of PDAC tissues. Our present study observed that miR-193b was frequently decreased in PDAC samples. In PDAC cell lines, we found that miR-193b expression is higher in BxPC-3 cells bearing wildtype KRAS compared to the AsPC-1, MIA PaCa-2 and PANC-1 cells, which harbor mutant KRAS (mutation statuses were obtained from www.atcc.org and www.sanger.ac.uk). Based on these findings, we speculate that the KRAS mutation status may be similar in patient materials and pancreatic cancer cell lines. This would further suggest that the downregulation of miR-193b may be associated with the mutation status of KRAS. But how do oncogenic KRAS mutations affect the function of miR-193b in PDAC development?
A group searching for MAPK-regulated microRNAs in pancreatic cancer cells found that MAPK activation negatively regulated miR-193b [21]. It is also reported that MAPK activity is sustained in KRAS mutant mice, but transient in wild-type mice [35]. Our data showed that miR-193b directly targets KRAS and thereby modulates ERK signaling. Taken together, these findings suggest that a KRAS-MAPK-miR-193b positive feedback loop may contribute to upregulating KRAS during pancreatic tumorigenesis in the presence of mutant KRAS. Furthermore, given that KRAS mutation is the initiating event in PDAC, mutant KRAS probably attenuates miR-193b expression, thereby contributing to KRAS-driven PDAC tumorigenesis. Similarly, KRAS mutation inhibits the expression of miR-143/145 through RREB1, thereby increases the level of KRAS (miR-143/145 target), forming a positive feedback loop affecting KRAS pathway [38]. The presence of inflammatory irritants plus KRAS mutation can induce the positive feedback circuit mediated by NF-κB signaling, triggering a sustained and pathological up-regulation of KRAS and result in inflammation and PanIN [39]. These kinds of positive feedback mechanisms might be universal in sustaining high levels of oncogenic KRAS activity, which then drive the initiation and development of PDAC [40]. Further investigations are needed to address the impact of mutant versus wild-type KRAS on miR-193b activity in PDAC cells, and to clarify the mechanisms underlying the interaction of KRAS with miR-193b.
Restoration of miR-193b in pancreatic cancer cells induced a remarkable array of biological processes, including inhibition of proliferation, cell-cycle arrest, and suppression of colony formation. We also found that the beginnings of reduced miR-193b levels compared with normal acinar cells were detected in duct-like changes around tumor. These data suggest that miR-193b downregulation and the miR-193b-KRAS axis might be involved in PDAC initiation. Using luciferase reporter assays, we verified that KRAS is indeed a direct target of miR-193b. Western blot analyses further showed that overexpression of miR-193b decreased KRAS levels, consistent with an effector role of KRAS in PDAC cells/tissues with downregulated miR-193b expression.
In 2007, two groups used microarray analysis to reveal differentially expressed miRNAs in pancreatic adenocarcinoma, chronic pancreatitis, and normal pancreas [41,42]. They found that several miRNAs were decreased in pancreatic cancers compared to chronic pancreatitis and normal pancreas. Greither et al. identified four miRNAs that correlated with poorer survival of pancreatic cancer patients [43]. It has been shown that miR-193b is downregulated in other cancers in addition to pancreatic cancer, such as melanoma [18], non-small-cell lung carcinoma [44], hepatic cell carcinoma [19], and endometrial adenocarcinoma [20]. Thus, miR-193b appears to exhibit reduced expression levels in diverse cancers compared to matching benign tissues, suggesting a universal effect of this miRNA in cancer development. In addition, miR-193a and miR-193b shared the same seed sequence and targets such as PLAU and KRAS [45]. However, they differ in chromosomal locations and non-seed nucleotide sequences; potential non-redundancy among miRNA families is still unclear.
Interestingly, miR-193b was found to significantly induce apoptosis in MIA PaCa-2 cells but not in PANC-1 cells. This result, which was confirmed by different methods (Western blot detection of the apoptosis markers c-PARP and c-caspase-3, flow cytometry analysis of the Annexin V staining), suggests different underlying apoptotic mechanisms in these two cancer cell lines. Our interpretation of these findings is that miR-193b dampens pancreatic cell proliferation, mainly by acting as a brake on the cell cycle.
Downstream effectors of KRAS signaling in pancreatic cancer were widely explored. Among them, Ras/Raf/ERK and Ras/PI3K/AKT signals are major effector pathways in PDAC tumorigenesis [46], especially through cell cycle regulation [47]. Our colleagues have previously demonstrated that other miRNAs (miR-96, miR-217 and miR-27a) function in pancreatic cancer by directly or indirectly affecting KRAS activity, a process that involves PI3K/AKT or RAF-MEK-ERK signaling cascades [25,48,49]. To explore the downstream mechanisms underlying the antitumor effects of the miR-193b-KRAS axis, we found that both p-ERK and p-AKT were downregulated in miR-193b-treated PDAC cells. Recent findings showed that Ras/ Raf/ERK could function with AKT synergistically in genesis and maintenance of PDAC [50]. Thus, key effectors in KRAS signaling may be tightly regulated by miR-193b in pancreas, and miR-193b restoration could target Ras/Raf/ERK and Ras/PI3K/AKT to suppress PDAC growth.
In our work, Cyclin D1 showed decreased level after miR-193b treatment in PDAC cells. Ras-dependent Cyclin D1 expression relies on sustained RAF/MEK/ERK activation, and correspondingly affects G1 to S phase transition [51]. miR-193b directly targeted to KRAS and subsequently caused ERK pathway deregulation, may contribute to Cyclin D1 downmodulation, resulting in dampened cell cycle progression. Interestingly, previous reports showed that miR-193b repressed melanoma cell proliferation by directly targeting Cyclin D1 [18]. Actually, a group of targets concurrently modulated by a single miRNA make up the biological network, resulting in miRNA-associated functions [52]. Therefore, other unknown functional molecules targeted by miR-193b, maybe together contribute to the tumor-inhibiting effect, which might arouse interesting further exploration in revealing the comprehensive network of miR-193b-KRAS axis in PDAC.
Collectively, our findings demonstrate that miR-193b is frequently downregulated in PDAC samples and has potential tumor-suppressor activity. Dysregulation of the miR-193b-KRAS axis appears to be involved in pancreatic carcinogenesis, indicating that miR-193b is a potentially new therapeutic target in PDAC.