MiR-132-3p suppresses cell proliferation and migration in gastric cancer by targeting KIF21B/Wnt/β-catenin signaling pathway

Background: Recently, kinesin family member 21B (KIF21B) has been reported to be an oncogene in non-small cell lung cancer and hepatocellular carcinoma. However, the functional role and related molecular mechanisms underlying gastric cancer (GC) pathogenesis remain largely uncovered. Methods: The expression of KIF21B was investigated by analysis of Oncomine microarray gene expression datasets and clinical specimens. The association between KIF21B and miR-132-3p was assessed by luciferase reporter assay. CCK-8 assay and transwell assay were performed to analyze the functional role of miR-132-3p/KIF21B in GC cells. Related protein expression levels were evaluated by immunohistochemistry and western blot analysis. Results: We rst found that the expression of KIF21B was upregulated in GC tissues compared with adjunct normal tissues. Knockdown of KIF21B signicantly suppressed the proliferation, migration and invasion in GC cell lines (AGS and SNU-5). KIF21B was conrmed as the target of miR-132-3p in GC cells. Moreover, miR-132-3p was down-regulated and inversely correlated with KIF21B expression in GC tissues. Further functional experiments demonstrated that overexpression of KIF21B remarkedly reversed the suppressive effects of miR-132-3p overexpression on GC cell proliferation, migration and invasion. Furthermore, miR-132-3p overexpression downregulated the protein levels of Wnt1, c-Myc, β-catenin, PCNA and N-cadherin, and upregulated E-cadherin expression in GC cells, which were all alleviated after KIF21B overexpression. Conclusions: In summary, our ndings provide the rst evidence that down-regulation of KIF21B by miR-132-3p suppresses cellular functions in GC via regulating Wnt/β-catenin signaling.

KIF21B may be relevant to mechanism underlying autism spectrum disorders etiology (14). Zhao et al (15) showed that KIF21B expression level was associated with overall survival and disease-free survival in hepatocellular carcinoma. Sun et al (16) not only manifested that increased KIF21B expression was correlated with worse survival prognosis, and uncontrolled cell proliferation and migration in nonsmall cell lung cancer (NSCLC). However, little is known about the functional role of KIF21B in GC.
MicroRNAs (miRNAs/miRs), a class of small non-coding RNAs, have been frequently demonstrated to function as pivotal regulators in tumorigenesis though modulating various biological processes, including proliferation, invasion and differentiation via binding to the 3′ untranslated region (3′-UTR) of target genes (17)(18)(19)(20). In recent years, miR-132-3p has been reported to be aberrantly expressed in tumor tissues and serve an important regulator in tumor cell functions. For example, miR-132-3p suppressed breast carcinoma cell migration and invasion by targeting lysosomal-associated protein transmembrane 4 beta (LAPTM4B) (21). Ectopic miR-132-3p aggravated cell apoptosis and inhibited cell proliferation in mantle cell lymphoma (22). In addition, miR-132-3p functions as a tumor suppressor in colorectal cancer (23,24), osteosarcoma (25,26) and bladder carcinoma (27). Here, our previous investigation revealed that KIF21B was a potential target gene of miR-132-3p. Moreover, Zhang et al (28) and Wang et al (29) consistently demonstrated that miR-132-3p expression was signi cantly altered between tumor tissues and normal tissues derived from patients with GC by microRNA pro ling.
Based on these facts, we speculated that miR-132-3p/KIF21B axis plays a critical role in regulating cell functions in GC cells.
To validate our hypothesis, we rst determined the expression pattern of KIF21B in GC tissues by searching Oncomine database and collecting clinical specimens. Then, loss-of-function assays were performed to analyze the functional role of KIF21B in GC cells. Furthermore, we further explored whether KIF21B was the downstream regulator involved in miR-132-3p regulating GC cell functions by modulating Wnt/β-catenin signaling. Our ndings may provide novel insights into the treatment of GC.

Oncomine gene expression analysis
We rst examined the expression of KIF21B gene in GC by searching microarray gene expression datasets derived from Oncomine database (www.oncomine.com). In brief, the Cancer Type was de ned as Gastric Cancer, Data Type was mRNA, and Analysis Type was Cancer versus Normal Analysis. Total three datasets, including Wang Gastric (30), Chen Gastric (31) and DErrico Gastric (32) were included in our analysis. The log-transformed, median-centered and normalized expression values of KIF21B were extracted, analyzed and read from the scatterplot accordingly.

Tissue specimens
Total thirty pairs of fresh tumor tissues and matched adjacent normal stomach mucosa tissues were collected from GC patients who received radical gastrectomy at the Second Hospital, Cheeloo College of Medicine, Shandong University (Shandong Province, China). All patients did not receive radiotherapy and chemotherapy before surgery. In addition, para n-embedded specimens of GC were also obtained to evaluate the KIF21B protein expression. Informed consent was obtained from all patients. This study was conducted in accordance with Helsinki Declaration and approved by the Medical Ethics Committee of the Second Hospital, Cheeloo College of Medicine, Shandong University (Approval number: CCM32-J2; 2018.6.3; Shandong, China).

Immunohistochemistry
Immunohistochemistry staining was performed to assess the protein expression of KIF21B in tissue samples using the EliVisionTMplus kit (Maixin, Fuzhou, China) according to the instructions provided.

Bioinformatic analysis and luciferase reporter assay
The potential miRNAs that target KIF21B were predicted by TargetScan (www.targetscan.org/vert_71/). From all these predictions, we selected miR-132-3p for further analysis. The mRNA 3′-UTR of KIF21B, carrying the predicted binding site or mutant binding site of miR-132-3p, was ampli ed by PCR and cloned into pmirGLO (Promega, Madison, Wisconsin), which were named as KIF21B wild-type (WT) and mutant (MUT) constructs, respectively. Next, cells were co-transfected with KIF21B WT or KIF21B MUT and miR-132-3p mimics or miR-NC using the transfection reagent Lipofectamine 2000. After 48 h incubation, cells were harvested and relative luciferase activity was measured via a Dual-luciferase reporter assay system (Promega).

Western blot analysis
Total protein sample was extracted with RIPA lysis buffer with protease inhibitor (Solarbio Science & Technology Co., Ltd., Beijing, China) and protein concentration was determined by a BCA kit (Beyotime, Shanghai, China). Then, equal amount of protein sample was separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocked with 5% non-fat milk, the membranes were incubated with primary antibodies against KIF21B, Wnt1, c-Myc, β-catenin, PCNA, Ecadherin, N-cadherin and GAPDH (All from Abcam, Cambridge, UK) at 4 °C overnight. Subsequently, the membranes were incubated with horseradish peroxidase-coupled secondary antibody for 2 h at room temperature, followed by detection of protein signals with an enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA, USA).

Statistical analysis
All quantitative data were presented as mean ± SD from three independent experiments. All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, California, USA). Differences between two groups were assessed by paired Student's t-test. Differences among three groups were evaluated by one-way analysis of variance (ANOVA), followed by Dunnett's test or Tukey's test. The correlation between miR-132-3p and KIF21B was evaluated using Pearson's correlation coe cient analysis. The p-value less than 0.05 was considered to be statistically signi cant.

KIF21B expression level was upregulated in GC tissues
Oncomine microarray gene expression datasets were rst searched to investigate the expression pattern of KIF21B in GC. As depicted in Figure 1A, KIF21B expression level was signi cantly increased in gastric cancer tissues (p = 7.73E-4) compared with the corresponding normal tissues in the Wang Gastric dataset, in diffuse gastric adenocarcinoma (p = 0.002) and gastric intestinal type adenocarcinoma (p = 0.005) compared with normal tissues in Chen Gastric dataset, as well as in gastric intestinal type adenocarcinoma (p = 0.010) and gastric mixed adenocarcinoma (p = 0.016) compared with normal tissues in DErrico Gastric dataset. To con rm the upregulation of KIF21B in GC, reverse transcription quantitative PCR was performed to analyze the expression of KIF21B mRNA in thirty pairs of fresh GC tissue and matched adjacent normal tissues. As shown in Figure 1B, the expression of KIF21B mRNA was signi cantly upregulated in GC tissues compared to that in adjacent tissues. Moreover, we observed the representative photomicrographs of different degrees of KIF21B expression intensity in the cytoplasm by IHC analysis (Figure 1C).

Knockdown of KIF21B inhibited the proliferation, migration and invasion of GC cells
Next, we explored the functional role of KIF21B in GC cells by performing loss-of-function assays in AGS and SNU-5 cells. At rst, the expression of KIF21B protein was suppressed by transfecting with two different siRNAs in AGS and SNU-5 cells, as demonstrated by western blot analysis (Figure 2A). CCK-8 assay showed that either si-KIF21B#1 or si-KIF21B#2 transfection signi cantly suppressed cell proliferation ability in AGS and SNU-5 cells (Figure 2A). Notably, si-KIF21B#2 generated more powerful suppressive effects on KIF21B expression and cell proliferation ability, in comparison with si-KIF21B#1, which was thus selected for the subsequent in vitro experiments. Subsequently, transwell assay was applied to analyze the effects of KIF21B knockdown on GC cell migration and invasion. Our data showed that the number of migratory cells was remarkedly decreased in si-KIF21B#2 group compared with si-NC group in both AGS and SNU-5 cells ( Figure 2C). Similarly, knockdown of KIF21B by si-KIF21B#2 transfection signi cantly reduced the number of invasive cells in AGS and SNU-5 cells ( Figure 2D).

KIF21B as a target gene of miR-132-3p
TargetScan 7.1 database was used to predict miRNAs that target KIF21B expression, which led to the identi cation of miR-132-3p as a direct target gene. As depicted in Figure 3A, miR-132-3p exhibited identical seed regions, with complementary binding to the 3′-UTR of KIF21B. Then, results from luciferase reporter assay showed that miR-132-3p mimics signi cantly suppressed the luciferase activity of AGS ( Figure 3B) and SNU-5 ( Figure 3C) cells transfected with KIF21B WT plasmid. However, the luciferase activity remained unchanged when the cells were transfected with KIF21B MUT without the miR-132-3p binding sites. What's more, miR-132-3p overexpression signi cantly downregulated KIF21B mRNA ( Figure  3D) and protein ( Figure 3E) expression levels in both AGS and SNU-5 cells. These ndings suggested that miR-132-3p could negatively regulate KIF21B expression through its 3′-UTR. In addition, we analyzed the expression of miR-132-3p in GC tissues. As shown in Figure 3F, miR-132-3p expression level was signi cantly down-regulated in GC tissues compared to that in adjacent tissues. Furthermore, miR-132-3p expression level was inversely correlated with KIF21B expression level in 30 cases of GC tissues ( Figure  3G, r = -0.4132, p = 0.0232).

KIF21B overexpression reversed the suppressive effects of miR-132-3p on GC cells
Since miR-132-3p was identi ed to target KIF21B and negative correlated with KIF21B expression in GC tissues, we thus explored whether miR-132-3p regulated GC cell functions by targeting KIF21B. At rst, the expression of miR-132-3p was overexpressed in AGS and SNU-5 cells after transfection with miR-132-3p mimics, as demonstrated by reverse transcription quantitative PCR ( Figure 4A). Then, AGS and SNU-5 cells were co-transfected with miR-132-3p mimics and KIF21B. As shown in Figure 4B, downregulated KIF21B protein level induced by miR-132-3p overexpression was attenuated after KIF21B overexpression plasmid transfection in both AGS and SNU-5 cells. Furthermore, ectopic KIF21B expression effectively reversed miR-132-3p overexpression-mediated suppressive effects on cell proliferation ( Figure 4C), migration ( Figure 4D) and invasion ( Figure 4E) abilities in AGS and SNU-5 cells.
MiR-132-3p suppressed the Wnt/β-catenin signaling pathway in GC cells via repressing KIF21B It has been reported that Wnt/β-catenin signaling pathway participates in regulating tumor cell proliferation and migration. Then, we investigated whether Wnt/β-catenin signaling pathway was involved in miR-132-5p-mediated suppressive effects on GC cells. As shown in Figure 5A, miR-132-3p overexpression obviously suppressed the expression of Wnt1, c-Myc and β-catenin, which was reversed after KIF21B overexpression in AGS cells. In addition, we found that miR-132-3p overexpression downregulated the expression of PCNA and N-cadherin, and upregulated E-cadherin expression in AGS cells, which was reversed after co-transfection with miR-132-3p mimics and KIF21B. Similarly, we observed that KIF21B overexpression abolished the effects of miR-132-3p overexpression on the protein levels of Wnt1, c-Myc, β-catenin, PCNA, E-cadherin and N-cadherin in SNU-5 cells ( Figure 5B).

Discussion
In the current study, we rst observed that KIF21B expression was upregulated in GC tissues compared with adjacent normal tissues by analysis of the Oncomine microarray gene expression datasets and clinical specimens. Next, we performed loss-of-function experiments to demonstrate that silencing of KIF21B suppressed the proliferation, migration and invasion of AGS and SNU-5 cells. Actually, KIFs play crucial roles in mitotic cell division, which make them involved in tumorigenesis (34,35). Similarly, upregulation of KIF14 could promote cancer metastasis in gastric cancer (36) and promote cell proliferation in colorectal cancer (37). KIF11 knockdown signi cantly inhibited proliferation, migration and invasion in breast cancer (38). Knockdown of KIF23 resulted in a marked inhibition of cell proliferation of GC in mice, with signi cant downregulation of Ki67 and PCNA expression (39). Consistent with our data, KIF21B was upregulated in hepatocellular carcinoma and NSCLC tissues, which promoted the corresponding tumor cell proliferation, migration and invasion (15,16). Herein, our data highlight the oncogenic role of KIF21B in GC.
We further demonstrated that KIF21B was a potential target of miR-132-3p in AGS and SNU-5 cells. What's more, decreased miR-132-3p expression was found to be inversely correlated with KIF21B mRNA levels in 30 cases of GC tissues. The rescue experiments manifested that KIF21B overexpression signi cantly alleviated the suppressive effects of miR-132-3p on GC cell proliferation, migration and invasion. These data suggested that miR-132-3p inhibited the cellular functions by directly targeting KIF21B in GC cells. In fact, miR-132-3p expression was demonstrated to be more frequently downregulated in human cancer, including colorectal cancer (40), pancreatic carcinoma (41) and glioma (42,43). Functionally, the suppressive effects of miR-132-3p have been reported on cell proliferation, migration and invasion in breast cancer (21), mantle cell lymphoma (22), colorectal cancer (23,24), osteosarcoma (25,26) and bladder carcinoma (27). Notably, He et al showed that inhibition of miR-132-3p enhanced GC cell proliferation and migration by targeting mucin 13 (MUC13) (44). Despite the suppressive role of miR-132-3p has been reported already, there are some major differences between it and our study as follows: 1) We performed gain-of-function assay to investigate the functional role of miR-132-3p, rather than loss-of-function assay in the above study from He et al. 2) Different target genes of miR-132-3p were identi ed. 3) Different molecular mechanisms underlying miR-132-3p regulating GC cell functions. 4) In addition to migration and invasion, we analyzed cell proliferation here. Until now, several KIFs have been reported to be directly regulated by miRNAs involved in the development of tumors. For instance, miR-338-3p could directly bind to the 3'-untranslated region (3'-UTR) of KIFC1 in renal cell carcinoma cells (45). MiR-30a could speci cally suppress KIF11 by targeting its 3'-UTR in breast cancer (46). Additionally, miR-206 inhibited the cell proliferation, migration, and invasion of ovarian cancer cells by directly targeting KIF2A (47). To our best knowledge, KIF21B has not been demonstrated to be a target of certain miRNA, which made the identi cation of KIF21B as the target of miR-132-3p provided a novel therapeutic target against the progression of GC. Wnt/β-catenin signaling pathway has been widely reported to be frequently over-activated in multiple types of tumors, which plays a fundamental role in regulation of cellular proliferation and invasion (48). What's more, Wnt/β-catenin pathway plays a critical role in metastasis and is closely associated with EMT process (49). Here, we found that miR-132-3p overexpression inhibited the activity of Wnt1, c-Myc and β-catenin, as key effector of Wnt/β-catenin signaling pathway and inactivated EMT markers (Ecadherin and N-cadherin), which were all reversed after KIF21B overexpression in GC cells. Similar with our data, miR-132-3p suppressed the migration and invasion of tumor cells by regulating the EMTcorrelated molecules in lung cancer (50,51) and cervical cancer (52). It has been con rmed that KIF26B regulates cell invasion in breast cancer through driving epithelial-mesenchymal transition (EMT) (53). KIF11 knockdown signi cantly upregulated E-cadherin expression, and downregulated the expression of N-cadherin and Vimentin in breast cancer cells (38). Although the association between miR-132-3p/KIF21B and Wnt/β-catenin pathway has not been reported yet, we thus speculated miR-132-3p suppressed GC cell proliferation, migration and invasion by targeting KIF21B-mediated Wnt/β-catenin pathway based on the correlation of EMT process and Wnt/β-catenin pathway.

Conclusions
In summary, we rst demonstrated that KIF21B knockdown suppressed the proliferation, migration and invasion in GC cells. Moreover, we con rmed that KIF21B was the downstream regulator underlying miR-132-3p exerted suppressive effects on GC cells by downregulating Wnt/β-catenin pathway. Thus, we ndings help us better understand targeting miR-132-3p/KIF21B axis as a promising therapeutic target for GC treatment.

Declarations
Ethics approval and consent to participate The present study was conducted in accordance with Helsinki Declaration and approved by the Ethics Committee of the Second Hospital, Cheeloo College of Medicine, Shandong University (Approval number: CCM32-J2; 2018.6.3; Shandong, China).

Authors' contributions
Supervision: Zhipeng Li; Interpretation of analysis of data: Bingtian Liu and Ling Qiang; Preparation of the manuscript: Bingxin Guan; Revision for important intellectual content: Ling Qiang. All authors read and approved the nal manuscript.

Availability of data and materials
All results and data generated or analyzed during the present study are included in this published article or are available from the corresponding author on reasonable request. This image is not available with this version.

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