MiR-345-5p functions as a tumor suppressor in pancreatic cancer by directly targeting CCL8

MiR-345-5p functions as a tumor suppressor in pancreatic cancer by directly targeting CCL8

Biomedicine & Pharmacotherapy 111 (2019) 891–900 Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsev...

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Biomedicine & Pharmacotherapy 111 (2019) 891–900

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

MiR-345-5p functions as a tumor suppressor in pancreatic cancer by directly targeting CCL8 Tinggang Moua, Fei Xiea, Pingyong Zhonga, Hao Huaa, Liang Laia, Qin Yangb, Jie Wanga, a b

T



Department of Hepatic-Biliary-Pancreatic Surgery, the First people's Hospital of Neijiang, Neijiang, Sichuan, China Department of Gastroenterology, the First people's Hospital of Neijiang, Neijiang, Sichuan, China

A R T I C LE I N FO

A B S T R A C T

Keywords: PDAC miR-345-5p CCL8 Proliferation Migration Invasion

Background: Increasing evidence has demonstrated that microRNAs (miRNAs) are key regulators of human diseases and can serve as prognostic markers for several cancers, such as pancreatic ductal adenocarcinoma (PDAC). Previous studies have revealed various functions for miR-345-5p in several cancers. However, the role and potential mechanism of miR-345-5p in PDAC have not been resolved. Methods: Quantitative RT-PCR was performed to investigate the expression levels of miR-345-5p in pancreatic cancer tissues and cell lines, and the effect of miR-345-5p on the proliferation and invasiveness of pancreatic cancer was examined in Transwell assays with miR-345-5p overexpression. We used Western blot assay to explore the underlying mechanisms. Immunofluorescence staining was performed to examine changes in the cytoskeleton of PANC-1 cells in response to miR-345-5p. Luciferase assays were used to clarify the target and regulation mechanism of miR-345-5p. Results: miR-345-5p expression was downregulated in PDAC cells and tissues. Upregulated miR-345-5p expression inhibited the proliferation and metastasis of PDAC cells. We identified CCL8 as a direct target of miR345-5p and found CCL8 expression was inversely correlated with miR-345-5p expression in PDAC samples. CCL8 could activate the NF-κB signaling pathway to promote the proliferation and invasiveness of PDAC cells. These results suggested that miR-345-5p inhibited PDAC progression by inactivating NF-κB signaling. Conclusions: Here we demonstrated that miR-345-5p was a tumor-suppressive miRNA in pancreatic cancer progression by targeting CCL8. Our results suggest miR-345-5p may be a potential therapeutic biomarker for pancreatic cancer treatment.

1. Introduction Pancreatic duct adenocarcinoma (PDAC) is highly malignant and is characterized by progressive growth, early-stage metastasis, and adverse reactions to radiotherapy and chemotherapy [1]. Pancreatic cancer is the fourth leading cause of cancer-related death in the United States, with a 5-year survival rate of less than 5% [2]. Although diagnostic and surgical treatment methods have been developed over the past decades, the morbidity and postoperative mortality rates of pancreatic cancer remain high and these treatments do not improve survival. Thus, there is an urgent need for identifying targets that can enable earlier diagnosis and individualized treatment for treating patients at high risk of PDAC [3]. MicroRNAs (miRNAs) are small non-coding RNAs (18–22 nucleotides) that play crucial roles in the post-transcriptional regulation of genes and protein expression. MiRNAs interact with their target coding



mRNAs, resulting in either mRNA degradation or inhibition of mRNA translation [4]. Multiple studies have demonstrated that miRNAs participate in various biological processes, including cancer development [5]. Multiple studies have also demonstrated that miRNAs can function as oncogenes or tumor suppressors in cancers. For example, a recent report showed that miR-345-5p expression was notably upregulated in prostate cancer patient serum compared with the serum of non-tumor patients, and miR-345-5p promoted growth and metastasis of prostate cancer cells [6]. In contrast, another report showed that miR-345-5p expression was downregulated in gastric cancer and miR-345-5p could inhibit epithelial-mesenchymal transition (EMT) to prevent the metastasis of gastric cancer cells [7]. In addition, miR-345 is downregulated and associated with patient prognosis in non-small cell lung cancer [8]. These data indicate that miR-345 plays different functional roles in human cancers. However, whether miR-345-5p functions in the pathogenesis of PDAC remains unknown.

Corresponding author at: No. 31, Tuozhong lane, Jiaotong road, Neijiang, Sichuan, 641000, China. E-mail address: [email protected] (J. Wang).

https://doi.org/10.1016/j.biopha.2018.12.121 Received 10 October 2018; Received in revised form 29 December 2018; Accepted 30 December 2018 0753-3322/ © 2019 Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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Fig. 1. miR-345-5p is downregulated in pancreatic cancer. (a) RT-PCR was used to quantify miR-345 mRNA expression in 10 pairs of PDAC tissue and adjacent, normal pancreas tissues. A decreased expression of miR-345-5p is observed in cancer tissues as compared with the normal pancreas. (b) A differential expression pattern of miR-345-5p is observed in PDAC cell lines. (c–f) PANC-1 and SW1990 cells were transfected with lentivirus overexpressing miR-345-5p (defined as lv-miR345-U) or lentivirus with short hairpin RNA targeting miR-345-5p (defined as lv-miR-345-KD). Cells transfected with empty lentiviral vectors served as a negative control (lv-NC). The miR-345-5p expression was analyzed by RT-qPCR after transfection; All experiments were performed three times and data were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

in PDAC. Bioinformatics analysis identified CCL8 as a target of miR345-5p. Our findings indicate that miR-345-5p may be a valuable target for PDAC treatment.

Chemokine (C-C motif) ligand 8 (CCL8, also known as MCP2) is a small cytokine member of the CC chemokine family that interacts with multiple cellular receptors to attract and activate human leukocytes. CCL8 also functions as a cancer-promoting factor in tumorigenesis, tumor progression, translation, and invasion processes [9,10]. In previous reports, CCL8 was shown to promote the invasion and migration of esophageal squamous cell carcinoma cells by the activating NF-κB signaling pathway [10]. In our study, we examined the function and potential mechanism of miR-345-5p in PDAC. We demonstrated that miR-345-5p is downregulated in PDAC tissues and cells and exhibits tumor suppressor roles

2. Materials and methods 2.1. Cell culture and generation of stable cell lines The human pancreatic duct epithelial (HPDE) cell line and human PDAC cell lines (BxPC-3, AsPC-1, PANC-1, SW1990 and MIA-PaCa-2) were purchased from ATCC and authenticated though STR typing. 892

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Fig. 2. Upregulated expression of miR-345-5p suppresses the proliferation and migration of PDAC cells in vitro. (a, b) Transwell invasion assay demonstrates the suppressed invasive ability of lv-miR-345-U control compared to the negative control in PANC-1 and SW1990 cell lines (100×). (c, d) The effect of miR-345-5p expression on migration of PDAC cells using wound scratch healing assay (100×). (e) CCK-8 assays were performed to compare proliferation of miR-345-5p overexpressing (lv-miR-345-U) with negative control (lv-NC) in PANC-1 and SW1990 cells. (f, g) Colony formation assay were performed to explore the cell proliferation in lv-miR-345-U and lv-NC. **p < 0.01, ***p < 0.001. 893

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Fig. 3. Upregulated expression of miR-345-5p suppresses the proliferation of PDAC cells in vivo. (a) Images of the subcutaneous xenograft of the miR-345-5p overexpression (lv-miR-345-U) group and negative control (lv-NC) group in PANC-1 cell line, n = 7. (b) Tumor volume of the subcutaneous xenografts were shown. (c) Weight change curve. (d) IHC staining for miR-345-5p and representative images of one pair of subcutaneous xenograft tissue (100×).(bar: 50 μm) (e) The relative expression of Ki67 in tumor tissue. *p < 0.05, **p < 0.01, ***p < 0.001.

PDAC patients included four women and six men with an average age of 58.8 years (range, 42–68 years). Histopathological diagnosis classified all 10 tumor samples as adenocarcinomas. The study was approved by the Human Research Ethics Committees of the First People’s Hospital of Neijiang, Neijiang, Sichuan.

SW1990, AsPC-1, BxPC-3, and HPDE cells were cultured with 1640 medium (Gibco, USA), while PANC-1 and MIA-PaCa-2 cells were grown with DMEM medium (Gibco). All cells were supplemented with 10% fetal bovine serum (10% FBS, Gibco), 100 U/mL penicillin G, and 100 μg/ml streptomycin. Human miR-345-5p overexpressed control (lvmiR-345-U), knockdown control (lv-miR-345-KD) and negative control (NC) lentivirus were obtained from Genechem (Shanghai, China). Infections were carried out according to the manufacturer’s instructions.

2.3. Cell counting kit (CCK-8) assay Cells were plated in a 96-well plate and the cells were cultured at 37 °C and 5% CO2 in the incubator. Next, 10 μl of CCK-8 (Dojindo, Tokyo, Japan) were added into each well. The cells were cultured for 1–4 h and the absorbance at 450 nm was determined with an enzyme marker.

2.2. Human tissue samples Paired human tissue samples were collected from 10 PDAC patients who underwent surgical resection from April 2017 to May 2018. The 10 894

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Fig. 4. Upregulated expression of miR-345-5p inhibited the invision of PDAC cells in vivo. (a) Images of the liver metastasis model of the miR-345-5p overexpression (lv-miR-345-U) group and negative control (lv-NC) group in PANC-1 and SW1990 cell lines, n = 5. (b) Liver micrometastases were counted. (c) Serial sections of whole liver were H&E stained.(bar: 50 μm) ***p < 0.001.

2.4. Colony-forming assays

2.6. Quantitative real-time PCR

Cells (1 × 103) were plated in a 6-well plate and cultured for 1 week. The cells were rinsed with PBS three times, fixed with 4% polyformaldehyde for 30 min and then stained with 0.1% crystal violet for 30 min. The number of clones was counted under the microscope.

RNA was isolated from tissues or cell lines using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA was reverse-transcribed to cDNA using the ReverTra Ace qPCR RT Kit (TransGen, China). Quantitative real-time PCR was performed using the SYBR Green Realtime PCR Master Mix (TransGen), according to the manufacturer’s instructions. The primers for miR-345-5p, U6, CCL8 and GAPDH were obtained from Shenggong (Shanghai, China). The primer sequences are as follows: GAPDH forward 5′-GGAGCGAGATCCCTCCA AAAT-3′, reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′; and CCL8 forward 5′-TCTACGCAGTGCTTCTTTGCC-3′, reverse: 5′-AAGGGGGAT CTTCAGCTTTAGTA-3′. U6 was the normalization control for miR-3455p while GAPDH was the normalization control for CCL8.

2.5. Immunofluorescence Coverslips were placed in cell culture plate. Cells were fixed with polyformaldehyde and sealed by sealed liquid. Cells were incubated with primary antibodies F-actin (Abcam, 1:150) and β-tubulin (Abcam, 1:150) at 4 °C for 20 h. Cells were then stained with secondary antibodies, CY3-conjugated goat anti-rabbit antibody and FITC-conjugated goat anti-mouse antibody, at 37 °C for 1 h. Slides were then counterstained with DAPI to visualize the cell nuclei. All immunofluorescence staining procedures were performed according to the manufacturers’ protocols. Finally, a fluorescence quenching agent was added to slides, and the cells were photographed under a laser scanning confocal microscope.

2.7. Transwell assays Cells were plated in 24-well transwell plates. The Matrigel was mixed with medium at a 1:8 ratio and placed on the upper surface of each insert. Chambers were held in an incubator for 6 h. PDAC cells were seeded into the upper chamber (1 × 104 per well) and the lower chambers contained 600 μl DMEM or 1640 with 20% FBS. Migration ability was detected at 12 h and invasion ability at 24 h. Cells on the 895

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Fig. 5. miR-345-5p inhibits the activity of NF-κB signaling through CCL8 in PDAC cells. (a) The wild-type and mutant of putative miR-345-5p target sequences of CCL-8 3′UTR. (b, c) After 24 h, luciferase reporter assays were performed in PANC-1 (b) and SW1990 cells (c). (d, e) Western blot determined the expression of CCL8, NF-κB and the phosporylation level of NF-κB in PANC-1 (d) and SW1990 cells (e). qRT-PCR determined the relative expression of CCL8 level in PANC-1 (f) and SW1990 (g). **p < 0.01.

2.9. Western blot analysis

lower surface were stained with crystal violet. A microscope was used to count the number of migrated or invaded cells.

Western blot analysis was performed as previously described [11]. The following antibodies were used: rabbit anti-CCL8 (Abcam, USA; 1:500), mouse anti-GAPDH (Boster, China; 1:5000), goat anti-rabbit IgG secondary antibody (CST; 1:2000), rabbit anti-NF-κB, and anti-pNF-κB (both CST, USA; 1:500).

2.8. Wound healing assay Cells were plated in 6-well plates. After 24 h, cells were covered with the bottom of culture plates and mitomycin C (10 μg/ml) was added to the cells for 2 h. A scratch was made in the cell layer by a sterile plastic tool. The dissociated cells were immediately removed with PBS, and the rest of the cells were cultured with medium containing 1% FBS for another 24 h. Finally, cell layers were photographed under an Olympus BX51 microscope.

2.10. In vivo metastasis and proliferation assays PANC-1 cell line was used for the xenograft histological analysis. For the orthotopic implantation model, we used 8-week-old wild-type BALB/c nude mice. For subcutaneous transplantation, we used 6-week896

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Fig. 6. miR-345-5p changes the cytoskeleton of PANC-1 cell and overexpression of CCL8 in miR-345-5p-overexpressing cells blocked the inhibitory effects of upregulated miR-345 on proliferation and invasion. (a) Immunofluorescence staining shows that F-actin and β-tubulin polymerized in negative control cells, while the upregulated expression of miR-345-5p cells resulted in F-actin and β-tubulin depolymerization in PANC-1 cells. Upregulation of CCL8 in miR-345-5p-overexpressing cells restored F-actin polymerization. (bar: 10 μm) (b, c) A CCK-8 assay was performed to compare proliferation of overexpressed CCL8 (CCL8-U) in miR345-5p-overexpressing cells with negative control PDAC cells. (d–g) Transwell assays were performed to compare overexpressed CCL8 in miR-345-5p-overexpressing cells and negative control cells from the PANC-1 and SW1990 cell lines. *p < 0.05, **p < 0.01, ***p < 0.001.

evaluated macroscopically and microscopically [12].

old BALB/c nude mice. All mice used in experiments were female. For orthotopic implantation, hair was removed from the abdomen under pentobarbital sodium anesthesia. An abdominal longitudinal incision was made to expose the pancreas, and 20 μl of cell suspension (approximately 1 × 106 cells) was injected directly into the pancreas. The incision was closed with sutures. For subcutaneous transplantation, a total of 2 × 106 transfected cells were subcutaneously injected into the right armpit of mice under pentobarbital sodium anesthesia. The weight of each mouse and the tumor diameter were measured every week. All mice were killed after initiation of treatment for 8 weeks. Tumors were

2.11. Luciferase assay The 3′ untranslated region (UTR) of CCL8 containing the binding sites of miR-345-5p was cloned into the dual luciferase reporter vector pGL3 (pGL3-CCL8-3′UTR). Cells were plated in triplicate in 48-well plates at 2 × 104 cells/well. After 24 h, the cells were co-transfected with the reporter vector containing either wild-type or mutant 3′UTR of CCL8 along with miR-345-5p mimic using Lipofectamine 2000 897

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model, we also found that upregulated expression of miR-345-5p inhibited tumor cell metastasis in vivo (Fig. 4a). Hematoxylin and eosin staining of serial whole liver sections showed reduced numbers of micrometastases in the miR-345-5p overexpression group compared with the control group (Fig. 4b, c). Together, these results showed that upregulated expression of miR-345-5p suppressed the growth and migration of PDAC cells in vivo.

(Invitrogen) according to the manufacturer’s instructions. After 48 h, luciferase and Renilla activities were measured using the Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s instructions. 2.12. Statistical analysis For continuous variables, all data are presented as mean ± SD. The statistical analysis was performed using a two-tailed unpaired t-test (between two groups) or a one-way analysis of variance (ANOVA) (among three or more groups). Statistical analyses were performed using IBM SPSS Statistics software version 20.0 (Chicago, IL, USA). P < 0.05 was considered statistically significant.

3.4. MiR-345-5p directly targets and inhibits CCL8 and inhibits NF-κB activation To identify the molecular mechanism by which miR-345-5p suppresses the proliferation and metastasis of PDAC cells, we searched for potential targets of miR-345-5p using bioinformatics prediction tools, such as TargetScan, miRanda, and miRWalk. Among the identified candidates (Fig. 5a), we selected CCL8 for subsequent analyses, as previous studies have demonstrated its involvement in many human malignancies. To examine whether miR-345-5p expression affects the expression of CCL8, we used Western blot assay, PCR and immunohistochemistry. We found that upregulated expression of miR345-5p in PANC-1 and SW1990 cell lines significantly decreased CCL8 protein expression, while downregulation of miR-345-5p resulted in high CCL8 levels compared with negative controls (Fig. 5d, e). We further observed that upregulated miR-345-5p led to a decrease in CCL8 mRNA expression (Fig. 5f, g). In addition, immunostaining of tumor samples from BALB/c nude mice revealed that upregulated miR-345-5p was result in a lower staining degree compared with negative control (Supplemental Figure). To more closely examine the mechanism by which CCL8 is regulated by miR-345-5p, we examined the 3′UTR of CCL8 and identified miR345-5p binding sites. We generated a luciferase vector containing the wild-type 3′UTR of CCL8 or a mutated construct in which the putative miR-345-5p-binding sequences were mutated. Co-transfection of miR345-5p mimics into PANC-1 and SW1990 cell lines resulted in decreased luciferase activity with the wild-type reporter construct compared with the control cells (Fig. 5b, c). Furthermore, miR-345-5pmediated repression of luciferase reporter activity was abolished with the mutated reporter construct. Previous studies showed that CCL8 regulated migration and invasion through activation of NF-κB. We thus next examined the potential effects of miR-345-5p on the NF-κB signaling pathway. Western blot analysis showed that overexpression of miR-345-5p reduced the protein levels of NF-κB in the nucleus and the total protein levels of phosphorylated NF-κB, with no impact on total protein levels of NF-κB compared with controls (Fig. 5d, e).

3. Results 3.1. MiR-345-5p expression is greatly decreased in PDAC cells and tissues To examine whether miR-345-5p was connected with pancreatic cancer, we used RT-PCR to determine the relative expression of miR345-5p in 10 pancreatic cancer tissues and paired adjacent normal tissues. We also evaluated the expression of miR-345-5p in various PDAC lines and HPDE cells by RT-PCR. The results showed that miR345-5p was significantly downregulated in PDAC tissues compared with the matched adjacent non-tumor tissues (Fig. 1a). Furthermore, miR345-5p was greatly decreased in PDAC lines, such as PANC-1, BxPC-3, SW1990, MIA PaCa-2 and AsPC-1, compared with the normal pancreatic line HPDE (Fig. 1b). These observations indicated that miR-3455p might function as a tumor suppressor in PDAC. 3.2. Upregulated expression of miR-345-5p suppresses the proliferation and migration of PDAC cells in vitro To investigate whether miR-345-5p functions as a tumor suppressor in PDAC, we used lentivirus to upregulate miR-345-5p in PANC-1 and SW1990 cell lines. Our results confirmed that miR-345-5p was significantly overexpressed 5 days after lentivirus infection in PANC-1 and SW1990 cell lines compared with cells infected with negative control virus (Fig. 1c–f). We next performed transwell assays to determine whether miR-3455p affects SW1990 and PANC-1 cell metastasis. The results showed that overexpression of miR-345-5p inhibited SW1990 and PANC-1 cell metastasis (Fig. 2a, b). MiR-345-5p overexpression also dramatically suppressed the migration of SW1990 and PANC-1 cells in wound healing assays (Fig. 2c, d). In addition, upregulated expression of miR-345-5p in SW1990 and PANC-1 cells inhibited the growth of PDAC cells in colony formation and CCK-8 assays (Fig. 2e–g). Notably, while control group cells showed polymerization of F-actin and β-tubulin, cells with upregulated expression of miR-345-5p showed F-actin and β-tubulin depolymerization, which suggested overexpression of miR-345-5p with the lower migration ability (Fig. 6a). Together these data demonstrated that miR-345-5p inhibits proliferation and migration of PDAC cells in vitro.

3.5. Effect of CCL8 on proliferation and migration of PDAC cells To examine the effect of CCL8 on migration and proliferation of PDAC cells, we knocked down CCL8 using siRNA (si-CCL8) compared with negative control siRNA (NC) in PANC-1 and SW1990 cell lines. Transwell assay and CCK-8 assay were performed to evaluate the migration and proliferation of the si-CCL8 cells and NC groups. The results showed that knockdown of CCL8 inhibited the migration and proliferation of PDAC cells (Supplemental Figure). We next evaluated whether overexpression of CCL8 by plasmid transfection in miR-345-5p overexpressing cells would restore miR-3455p-mediated inhibition of invasion and proliferation. The results showed that overexpression of CCL8 in miR-345-5p-overexpressing cells blocked the inhibitory effects of upregulated miR-345 on proliferation and invasion (Fig. 6b–f). Furthermore, upregulation of CCL8 in miR-345-5p-overexpressing cells restored F-actin polymerization (Fig. 6a).

3.3. Upregulated expression of miR-345-5p suppresses the proliferation and migration of PDAC cells in vivo To further examine whether miR-345-5p affects the proliferation and migration of PDAC cell in vivo, we established a subcutaneous xenograft model and a liver metastasis model of human PDAC cells in BALB/c nude mice. In the subcutaneous xenograft model, mice with miR-345-5p overexpression had a slower increase in tumor diameter and volume and a slower reduction in weight compared with controls (Fig. 3a–c). Moreover, immumohistochemical staining showed that Ki67 was reduced in tumors from the overexpression group compared with those from the control group (Fig. 3d, e). In the liver metastasis 898

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4. Discussion

PDAC development and progression [29]. Our studies showed that high expression of miR-345-5p led to low expression of CCL8, which reduced nuclear protein levels of NF-κB, as well as levels of phosphorylated NFκB. Inactivation of the NF-κB pathway would lead to the inhibited proliferation and migration of PDAC cells. In summary, our results demonstrate that miR-345-5p plays a crucial role in suppressing cell proliferation and metastasis of PDAC through targeting CCL8. Further exploration of the potential mechanism between miR-345-5p and the progression of pancreatic cancer may provide a new target for anti-pancreatic cancer treatment.

This study demonstrated that the expression of miR-345-5p is significantly decreased in PDAC tissues and cells, suggesting that miR-3455p might suppress PDAC progression. We further demonstrated that overexpression of miR-345-5p inhibits the proliferation and metastasis of SW1990 and PANC-1 cell lines in vitro. Subcutaneous and orthotopic transplantation models confirmed that overexpression of miR-345-5p could inhibit the growth of subcutaneous tumors and resulted in reduced metastases in the liver of nude mice. Furthermore, we demonstrated that miR-345-5p suppressed PDAC progression by directly suppressing CCL8, which might inhibit the progression of pancreatic cancer through inactivating NF-κB signaling. Together, our findings suggest that miR-345-5p may function as a tumor suppressor in PDAC by targeting CCL8. Previous studies have demonstrated an involvement for miR-345-5p in the initiation, proliferation, invasion, and metastasis of numerous types of human cancers [6,7]. However, the exact biological function of miR-345-5p in tumor cells has been controversial. For example, one study reported that miR-345-5p was significantly overexpressed in prostate cancer and that overexpressed miR-345-5p promoted the proliferation and migration of prostate cancer cells in prostate cancer by inhibiting CDKN1A [13]. However, other reports suggested that miR-345-5p was downregulated and functions as a suppressor in other tumors [7,14]. In the study by Srivastava et al., miR-345 suppressed pancreatic cancer cell growth by directly suppressing the expression of BCL2, which plays a key role in inhibiting apoptosis [15]. However, our results show that miR-345-5p suppressed the expression of CCL8, which may lead to inactivation of NF-κB to inhibit proliferation and metastasis. CCL8 is a sub-family of the CC chemokines, which is a class of structurally related chemotactic cytokines [16,17]. Together with their cognate receptors, chemokines can regulate a variety of cellular functions, including immunodeficiency virus type I infection, cancer metastasis, arthritis, asthma, and neurodegenerative diseases [16]. CCL8 is an agonist of C-C chemokine receptor type 2 (CCR2) and CCR5, playing a pivotal role in the control of leukocyte chemotaxis, HIV entry, and other inflammatory diseases [18–21]. In previous studies, CCL8 was shown to induce EMT to promote metastasis in esophageal squamous cell carcinoma cells, and EMT was reported to correlate with activation of NF-κB signaling pathways [10,22]. CCL8 also activated the NF-κB pathway, as shown by use of BAY11-708, a specific inhibitor of the NFκB signaling pathway [10]. In breast cancer, CCL8 not only promoted intravasation in the primary sites of tumor growth but also extravasation and establishment of secondary growth by enhancing cancer cell seeding [9,23]. Furthermore, Barbai et al. demonstrated that CCL8 expression in dermal fibroblasts was increased by soluble factors, produced by the tumor itself, and CCL8 in return enhanced the migration activity of tumor cells as a chemoattractant [24]. In addition, the expression of CCL8 enhances the migration of inflammatory cells and suppresses carcinogenic progression [25,26]. Hiwatashi et al. showed that CCL8 could attract tumor-associated macrophages, which are key elements in tumor-promoting inflammatory reactions, and that it also had the potential to suppress tumor activity [27]. The authors also found that STAT3, as an effective inhibitor of NF-κB, might directly promote the transcription of MCP2/CCL8, which directly inhibited melanoma cell proliferation or the engraftment of melanoma cells into the tissues [27]. Together, these data suggest that CCL8 exhibits different effects in tumor progression. Our results showed that upregulated expression of miR-345-5p suppressed the expression of CCL8 in pancreatic cancer cells and reduced expression of CCL8 led to reduced metastasis. NF-κB plays critical roles in inflammation, cell proliferation and differentiation, immune response, and cancer [28]. NF-κB transcriptional factors are constitutively activated in the majority of PDACs, and NF-κB pathways are involved in the regulation of numerous aspects of

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