miR-532 promoted gastric cancer migration and invasion by targeting NKD1

Accepted Manuscript miR-532 promoted gastric cancer migration and invasion by targeting NKD1

Shaobo Hu, Qichang Zheng, Heshui Wu, Chunyou Wang, Tao Liu, Wei Zhou PII: DOI: Reference:

S0024-3205(17)30110-8 doi: 10.1016/j.lfs.2017.03.019 LFS 15166

To appear in:

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Received date: Revised date: Accepted date:

6 December 2016 24 March 2017 25 March 2017

Please cite this article as: Shaobo Hu, Qichang Zheng, Heshui Wu, Chunyou Wang, Tao Liu, Wei Zhou , miR-532 promoted gastric cancer migration and invasion by targeting NKD1. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi: 10.1016/j.lfs.2017.03.019

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ACCEPTED MANUSCRIPT miR-532 promoted gastric cancer migration and invasion by targeting NKD1 Shaobo Hu1, Qichang Zheng1, Heshui Wu2, Chunyou Wang2, Tao Liu2, Wei Zhou2# 1

Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College,

Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P.R. China Department of Pancreatic Surgery, Union Hospital, Tongji Medical College,

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2

Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P.R. China

Corresponding author: Wei Zhou, Department of Pancreatic Surgery, Union Hospital,

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#

Jiefang

Avenue,

Wuhan,

Hubei

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Tongji Medical College, Huazhong University of Science and Technology, 1277 Province,

P.R.

China.

E-mail:

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[email protected].

430022.

Abstract Gastric cancer is one of the most common human malignant neoplasms,

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especially in China, its regulatory mechanism is important to develop new therapy approaches. miRNAs have been demonstrated to regulate gastric cancer progression. We found miR-532 was overexpressed in gastric cancer tissues and cells, Wound

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healing and transwell assay revealed that its overexpression promoted gastric cancer

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cell migration and invasion, its knockdown inhibited gastric cancer cell migration and invasion. Wnt/β-catenin antagonist naked cuticle homolog 1 (NKD1) was the target of miR-532, miR-532 inhibited NKD1 expression. TOP/FOP luciferase activity analysis

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suggested miR-532 also increased Wnt/β-catenin pathway activity. Overexpression miR-532 and NKD1 inhibited gastric cancer cell migration and invasion, consistent

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with miR-532 knockdown. These findings revealed miR-532 promoted gastric cancer cell migration and invasion through inhibiting NKD1 and activated Wnt/β-catenin pathway. We provide a potential target for gastric cancer therapy. Keywords: miR-532; gastric cancer, NKD1; migration; invasion

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ACCEPTED MANUSCRIPT Introduction Gastric cancer which is a malignant epithelial neoplasm. Dietary patterns, food storage, the availability of fresh produce and Helicobacter pylori are the main factors for gastric tumorigenesis (Torre et al. , 2015). The well-known regulatory mechanisms are essential for developing new therapy approaches. Since miRNAs have been discovered in 1993, the role of miRNAs in tumor

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progression has been studied widely. miRNAs are short (about 19-24nt) non-coding single-stranded RNAs, they could regulate approximately one-third of mammalian expressing genes at the post-transcriptional level, they inhibit translation and/or

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degrade targeted mRNA by binding to the 3’UTR of targeted mRNA (Esteller, 2011).

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Many miRNAs were reported to regulate gastric cancer development, such as miR-137, miR-421, miR-337-3p and miR-1(Du et al. , 2016, Ge et al. , 2016, Han et

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al. , 2015, Zheng et al. , 2016).

miR-532 is a potential biomarker in serum of deep vein thrombosis (Qin et al. , 2015), it’s also one of three-miRNA signature (miR-532, miR-628-3p and

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miR-425-3p) diagnostic marker for early-stage lung adenocarcinoma (Wang et al. , 2016). But the role and regulatory mechanism of miR-532 in gastric cancer progression hasn’t been researched. In this study, we examined the role of miR-532 in

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gastric cancer cell migration and invasion. We first compared miR-532 expression in

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gastric cancer cells and tissues to normal gastric cells and tissues, and then determined the role of miR-532 in gastric cancer migration and invasion. Finally, we studied the target of miR-532 to reveal the regulatory mechanism of miR-532. We found miR-532

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promoted gastric cancer cell migration and invasion by targeting NKD1.

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Materials and methods

Cell culture and specimens Human normal gastric epithelial cell (HEGC) and gastric cancer cells MGC-803, AGS, MKN-28, MKN-45 and SGC7901 were cultured in RPMI-1640 medium (Hyclone) supplemented with 10% fetal calf serum (FCS, GIBCO) and L-glutamine (GIBCO). Normal gastric tissues and gastric tumor tissues were obtained from Tongji Medical College, Huazhong University of Science and Technology, the specimens were obtained from patients undergoing gastric cancer and collected at surgery, and immediately frozen in liquid nitrogen until used. This study was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and 2

ACCEPTED MANUSCRIPT Technology, Informed consent was obtained from every patient. The detailed information was shown in Supplemental Table 1. Quantitative real-time PCR (qRT-PCR) Total RNA was isolated using Trizol Reagent (TaKaRa), and reversely transcribed into cDNA using PrimeScript™ RT reagent Kit (TaKaRa) and specific primer for the

miR-532,

sequence

was

5’

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GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACGGTC3’. The levels of miR-532 were determined using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa) on a CFX96 Touch Real-time PCR Detection system The

primers

were

shown

as

follows:

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(Bio-Rad).

forward,

5’

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CGCATGCCTTGAGTGTAGGAC3’, reverse, 5’GTGCAGGGTCCGAGGT3’. U6 was used as the internal control.

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Western blot

Total protein was isolated using RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS and protease inhibitors). Anti-NKD1 (#2201, Cell Signaling Technology),

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β-Catenin (#8480, Cell Signaling Technology), TCF4 (#2569, Cell Signaling Technology), LEF1 (#2286, Cell Signaling Technology) antibodies were used. Anti-EF1α antibody (#3586, Cell Signaling Technology) was used as the loading

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control for nuclear protein. Anti-GAPDH antibody (G9295, Sigma) was used as the

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loading control for total protein.

Vector, oligonucleotides and transfection The stem loop of miR-532 with 200bp flanking sequence was cloned into

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pMSCV-pur vector (Clontech), and transfected into gastric cancer cell AGS using Lipofectamine 3000 (Life Technologies), puromycin was used to screen stable cell

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lines. Cells transfected with empty vector were used as the negative control. Synthetic miR-532 mimic, miR-532 inhibitor, mutational miR-532 mimic and the negative control were purchased from Guangzhou RiboBio Co., Ltd. These oligonucleotides were transfected into gastric cancer cells using Lipofectamine 3000. To overexpress NKD1, the CDS of NKD1 was cloned into pBABE-neo vector, G418 was used to construct stable cell lines. Wound healing assay Cells were seeded at six-well plates, and grew to confluence, a pipette tip was used to create streaks in the monolayer. Progression of migration was observed and photographed at 24h after wounding. 3

ACCEPTED MANUSCRIPT Transwell assay Transwell assay was performed according previous method (Li et al. , 2016b). Cells were seeded at the upper compartment of chamber with coated Matrigel, and cultured using serum-free medium, the lower chamber contained medium supplemented with 10% FCS. 24h after, cells which passed through the Matrigel membrane were fixed and stained with crystal violet and counted in 5 random microscopic fields. Luciferase reporter assay

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To analyze β-catenin transcriptional activity, TOP-flash luciferase (plasmid 16558, Addgene) and FOP luciferase (plasmid 16559, Addgene) were transfected into gastric

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cancer cells using Lipofectamine 3000 (Shih et al. , 2000). The sequence of 3’UTR of

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NKD1 was cloned into psiCHECK2 vector, and cotransfected with different amount of miR-532 mimic, miR-532 inhibitor or mutational miR-532 mimic into gastric cancer cells. 48h after transfection, luciferase activity was measured using Dual

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Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s protocol. The independent experiments were performed in triplicate.

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Statistical analysis

The data are presented as mean ± SD from three independent experiments. Two-tailed Student’s t test was used to compare the difference groups. miRNASeq

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and RNASeqV2 date for gastric cancer samples were downloaded from TCGA dataset.

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Gene-set enrichment analysis (GSEA) analysis was performed using the Java desktop software (http://software.broadinstitute.org/gsea/index.jsp). A p value < 0.05 was

Results

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considered significant. All analyses were performed using SPSS 20.0.

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miR-532 was upregulated in gastric cancer tissues and cells To examine the role of miR-532 in gastric cancer progression, we downloaded miRNA expression profiles of normal stomach tissues and gastric cancer tissues from TCGA

dataset

(https://gdc-portal.nci.nih.gov/projects/TCGA-STAD),

statistical

analysis suggested miR-532 was significantly upregulated in gastric cancer tissues compared to the normal stomach tissues, we also found that miR-532 was also significantly upregulated in gastric cancer tissues compared to the adjacent normal stomach tissues (ANT, Figure 1A). To further examine the miR-532’s role in gastric cancer progression, GSEA analysis suggested miR-532 was upregulated in early gastric cancer (Figure 1B). To confirm above results, we used specimens collected 4

ACCEPTED MANUSCRIPT from hospital, and found miR-532 was also significantly upregulated in gastric cancer tissues compared to normal gastric tissues (Figure 1C). We also determined miR-532’s expression in gastric cancer cells, and revealed miR-532 was also significantly upregulated gastric cancer cells (Figure 1D). These findings suggested miR-532 was upregulated in gastric cancer tissues and cells. To determine the role of miR-532 in gastric cancer progression, we overexpressed miR-532 in AGS cell with

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low miR-532 level, and downregulated in MKN-45 cell with high miR-532 level (Figure 1E).

miR-532 promoted gastric cancer cell migration and invasion

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Tumor metastasis is the main reason for tumor death and relapse, we examined

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whether miR-532 could regulate tumor metastasis. GSEA analysis suggested patients with high miR-532 had high metastasis rate, patients with low miR-532 had low

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metastasis rate (Figure 2A), suggesting miR-532 was associated with gastric cancer metastasis. Wound healing assay suggested miR-532 overexpression promoted gastric cancer cell migration, miR-532 knockdown inhibited gastric cancer cell migration

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(Figure 2B). Transwell assay suggested miR-532 overexpression promoted gastric cancer cell invasion, miR-532 knockdown inhibited gastric cancer cell invasion (Figure 2C). These results suggested miR-532 promoted gastric cancer cell migration

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and invasion.

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miR-532 promoted gastric cancer progression through targeting NKD1 miRNA regulates biological functions through regulating target gene expression. TargetScan, Pictar and miRANDA were used to predict the target genes of miR-532,

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suggesting NKD1 was the target gene of miR-532 (Figure 3A), NKD1 is an antagonist of the canonical Wnt/β-catenin pathway (Yan et al. , 2001). miR-532

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overexpression inhibited NKD1 expression, while miR-532 knockdown promoted NKD1 expression (Figure 3B), suggesting the expression of miR-532 and NKD1 was opposite. Luciferase reporter assay suggested miR-532 overexpression decreased luciferase activity in a dose-dependent manner, miR-532 knockdown increased luciferase activity in a dose-dependent manner. Mutational miR-532 overexpression didn’t change the luciferase activity (Figure 3C), suggesting miR-532 directly bound to the 3’UTR of NKD1. NKD1 is an antagonist of Wnt/β-catenin pathway, TOP/FOP luciferase ratio suggested miR-532 overexpression increased Wnt/β-catenin activity, while miR-532 knockdown inhibited Wnt/β-catenin activity (Figure 3D), suggesting miR-532 regulated Wnt/β-catenin pathway. We further confirmed that whether 5

ACCEPTED MANUSCRIPT miR-532 regulates Wnt/β-catenin activity, western blot assay found miR-532 overexpression enhanced β-catenin nuclear translocation and, consequently promote the transcriptional activity of TCF/LEF, suggesting miR-532 overexpression increased Wnt/β-catenin activity, miR-532 knockdown reduced these effects, and inhibited Wnt/β-catenin activity (Supplemental Figure 1). We performed rescue experiment to determine whether miR-532 promoted gastric

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cancer cell migration and invasion by targeting NKD1, NKD1 overexpressed in gastric cell overexpressing miR-532 inhibited cell migration and invasion determined by wound healing assay and transwell assay (Figure 3E and 3F). These findings

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suggested miR-532 promoted gastric cancer cell migration and invasion by targeting

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NKD1.

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Discussion

In present study, we found miR-532 was upregulated in gastric cancer tissues and cells, it promoted gastric cancer cell migration and invasion by targeting NKD1, and

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inhibiting Wnt/β-catenin pathway. In the absence of Wnt stimulation, the destruction complex including proteins adenomatous polyposis coli (APC), GSK3β and AXIN phosphorylates β-catenin and mediates β-Catenin ubiquitylation and proteasomal

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degradation. Wnt/β-catenin pathway is activated by 19 Wnt ligands, once ligands bind

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to the Frizzeled family of atypical heptahelical G-protein-coupled receptors, the destruction complex activity is inhibited, β-catenin is accumulated and enters the nucleus, acting as a co-activator for TCF/LEF-mediated transcription to promote Wnt

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target genes expression (Anastas and Moon, 2013). TOP/FOP luciferase activity analysis could be used to determine Wnt pathway activity (Veeman et al. , 2003),

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previous study reveals that Wnt/β-catenin pathway promotes gastric tumorigenesis and self-renewal of gastric cancer stem cells (Chiurillo, 2015). We found miR-532 overexpression increased Wnt/β-catenin pathway activity, miR-532 knockdown inhibited

Wnt/β-catenin

pathway

activity,

suggesting

miR-532

regulated

Wnt/β-catenin pathway, confirming miR-532 promoted gastric cancer progression. We found NKD1 was the target of miR-532, NKD1 is a tumor suppressor. For example, NKD1 inhibits non-small-cell-lung cancer invasion, and is a favorable prognostic factor (Zhang et al. , 2011). NKD1 suppresses hepatocellular carcinoma metastasis by inhibiting Rac1 expression and activity (Koch et al. , 2005, Li et al. , 2016a). NKD1 is a favor prognostic factor for patients with breast invasive ductal 6

ACCEPTED MANUSCRIPT carcinoma (Lv et al. , 2015). The role of NKD1 in gastric cancer progression also be demonstrated, Guo group finds that HOXA11 inhibits gastric cancer cells proliferation, migration, invasion and induces apoptosis by upregulating NKD1 which inhibits Wnt/β-Catenin pathway (Cui et al. , 2015). Our rescue experiment suggested NKD1 inhibited the effect of miR-532 overexpression, suggesting NKD1 inhibited gastric cancer cell migration and invasion. Although we found miR-532 promoted

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gastric cancer cell migration and invasion, and miR-532 levels was positively correlated with tumor metastasis, but the in vivo metastasis assay also is needed to perform further.

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In summary, we found miR-532 was upregulated in gastric cancer cells and tissues,

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and promoted gastric cancer cell migration and invasion by targeting NKD1.

The authors declare no conflict of interest.

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Acknowledgements

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Conflict of interest

This work was supported by The Natural Science Foundation of China (No. 81302115

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and No. 81272658).

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ACCEPTED MANUSCRIPT Figure legends Figure 1. miR-532 is overexpressed in gastric cancer tissues and cells. A. miR-532 was upregulated in gastric tumor tissues compared to the normal stomach tissues, it was also upregulated in gastric cancer tissues (Tumor) compared to the adjacent normal gastric tissues (ANT). Date was downloaded from TCGA dataset. B. GSEA assay showed miR-532 was upregulated in early gastric cancer. C. qRT-PCR assay

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suggested miR-532 was upregulated in gastric cancer tissues compared to the normal gastric tissues. D. qRT-PCR assay suggested miR-532 was upregulated in gastric cancer cells compared to normal gastric epithelial cell (HEGC). E. qRT-PCR assay

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confirmed the effect of miR-532 overexpression in AGS or knockdown in MKN-45.

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Error bars indicate the SD. *p< 0.05.

Figure 2. miR-532 promotes gastric cancer cell migration and invasion. A. GSEA

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assay showed miR-532 was associated with gastric cancer metastasis. B. Wound healing assay suggested miR-532 overexpression promoted AGS cell migration, miR-532 knockdown inhibited MKN-45 cell migration. C. Transwell assay suggested

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miR-532 overexpression promoted AGS cell invasion, miR-532 knockdown inhibited MKN-45 cell invasion.

Figure 3. miR-532 promotes gastric cancer cell migration and invasion by

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inhibiting NKD1. A. miR-532 bound to the 3’UTR of NKD1 among many species

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predicted using TargetScan, Pictar and miRANDA. B. Western blot assay suggested there was an opposite level between NKD1 protein expression and miR-532 expression, GAPDH was used as the loading control. C. Luciferase activity assay

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suggested miR-532 directly bound to the 3’UTR of NKD1, miR-532 mimic reduced luciferase activity in a dose-independent manner, miR-532 inhibitor increased

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luciferase activity in a dose-independent manner. Mutated miR-532 couldn’t regulate luciferase activity. D. TOP/FOP luciferase activity analysis suggested miR-532 overexpression activated β-catenin activity, miR-532 knockdown inactivated β-catenin activity. E. Wound healing assay suggested miR-532 and NKD1 overexpression inhibited gastric cancer cell migration. F. Transwell assay suggested miR-532 and NKD1 overexpression inhibited gastric cancer cell invasion. Error bars indicate the SD. *p< 0.05. Supplemental Figure 1. Western blot analyzed β-Catenin expression in the nucleus after miR-532 overexpression or knockdown, EF-1α was used as the loading control. Western blot analyzed TCF4 and LEF1 expression, after miR-532 overexpression or 10

ACCEPTED MANUSCRIPT knockdown, GAPDH was used as the loading control.

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Supplemental Table 1. The detailed clinicopathologic information of patients.

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