Downregulation of KIF21B by miR-132-3p suppresses cellular functions in gastric cancer via regulating Wnt/β-catenin signaling pathway

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 mechanisms underlying gastric cancer (GC) pathogenesis remain largely uncovered. Here, we rst found that the expression of KIF21B was upregulated in GC tissues compared with adjunct normal tissues by analysis of Oncomine microarray gene expression datasets and clinical specimens. Knockdown of KIF21B signicantly suppressed the proliferation, migration and invasion in GC cell lines using CCK-8 assay and transwell assay. By luciferase reporter assay, KIF21B was conrmed as the target of miR-132-3p in GC cells and suppressed after miR-132-3p overexpression. 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, western blot analysis manifested that 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. In summary, our ndings provide the rst evidence that down-regulation of KIF21B by miR-132-3p suppresses cellular functions in gastric cancer via regulating Wnt/β-catenin signaling.


Introduction
Gastric cancer (GC) is considered as the fth most frequently occurred malignancy and third leading cause of tumor-associated death worldwide, remaining a global health problem [1,2]. Despite advances in comprehensive therapies, including surgery, adjuvant chemotherapy and radiation interventions, the prognosis of GC patients remains poor primarily ascribed to the diagnosis of GC at an advanced stage and uncontrolled metastasis [3,4]. Therefore, having a better understanding the molecular mechanisms associated with GC pathogenesis will be great importance to improve the clinical outcomes of GC patients.
Kinesin superfamily proteins (KIFs), as microtubule-dependent molecular motors, perform a series of cellular processes, including mitosis, motility and organelle transportation [5][6][7]. KIFs have been recently found to be aberrantly expressed and participated in tumorigenesis by modulating chromosomal and spindle movement [8][9][10]. As a member of the KIFs, kinesin family member 21B (KIF21B), located at 1q32.1, contains a motor domain, a stalk and a tail domain binding to microtubules, which is essential for neuronal morphology [11]. Interestingly, accumulating evidence has demonstrated that KIF21B was involved in several diseases, especially tumorigenesis. For instance, abundant KIF21B expression is associated with severe multiple sclerosis and Alzheimer's disease pathology with accelerated neurodegeneration [12,13]. The in uence of KIF21B may be relevant to mechanism underlying autism spectrum disorders etiology [14]. Zhao et al [15] showed that KIF21B was identi ed as an independent risk factor for overall survival and disease-free survival in patients with hepatocellular carcinoma. Sun et al [16] not only manifested that high expression of KIF21B predicted a poor prognosis of patients with non-small cell lung cancer (NSCLC), but also promoted the proliferation, migration and invasion of A549 and H1299 cells. However, the expression pattern and functional role of KIF21B in GC have not yet been investigated until now.
MicroRNAs (miRNAs/miRs), a type 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 the migration and invasion of breast carcinoma cells through lysosomal-associated protein transmembrane 4 beta (LAPTM4B) by mediating epithelial-mesenchymal transition signals [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, we performed loss-of-function assays 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 (Shandong Province, China).

Reverse transcription quantitative PCR analysis
Total RNA sample was extracted from tissue specimens or cell lines using TRIzol reagent (Invitrogen).

Cell counting kit-8 (CCK-8) assay
Transfected cells at a density of 4 × 10 3 cells per well were seeded into 96-well plates and cultured at 37°C .

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.

Results
The expression of KIF21B was upregulated in GC tissues To investigate the expression pattern of KIF21B in GC, we determined the differential mRNA expression of KIF21B between GC tissues and normal gastric tissues by analysis of the Oncomine microarray gene expression datasets. As shown in Fig. 1A, the expression of KIF21B 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. The results showed that the expression of KIF21B mRNA was signi cantly upregulated in GC tissues compared with adjacent tissues (Fig. 1B). Moreover, the results from IHC analysis (Fig. 1C) indicated that representative photomicrographs of different degrees of KIF21B expression intensity were observed in the cytoplasm. Expression of the KIF21B protein was detected in 78.6% of GC-derived tissues and in 18.5% of noncancerous tissues, which suggested that KIF21B was highly expressed in GC tissues.

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 ( Fig. 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 (Fig. 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 (Fig. 2C). Similarly, knockdown of KIF21B by si-KIF21B#2 transfection signi cantly reduced the number of invasive cells in AGS and SNU-5 cells (Fig. 2D).

Kif21b Was A Direct Target Of Mir-132-3p
The miRNAs that target the expression of KIF21B were predicted using TargetScan 7.1 database, which led to the identi cation of miR-132-3p as a direct target gene. As depicted in Fig. 3A, miR-132-3p exhibited identical seed regions, with complementary binding to the 3′-UTR of KIF21B. Then, luciferase reporter assay was conducted to examine whether miR-132-3p directly targets KIF21B. The results showed that miR-132-3p mimics signi cantly suppressed the luciferase activity of AGS (Fig. 3B) and SNU-5 (Fig. 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, overexpression of miR-132-3p signi cantly downregulated the expression of KIF21B mRNA (Fig. 3D) and protein (Fig. 3E) 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 Fig. 3F, the expression of miR-132-3p was signi cantly down-regulated in GC tissues compared with adjacent tissues. Furthermore, the expression of miR-132-3p was inversely correlated with KIF21B expression in 30 cases of GC tissues (Fig. 3G, r = -0.4132, p = 0.0232).
Overexpression of KIF21B reversed the suppressive effects of miR-132-3p on GC cell proliferation, migration and invasion 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 (Fig. 4A). Then, AGS and SNU-5 cells were transfected with miR-NC, miR-132-3p mimics or miR-132-3p mimics + KIF21B, respectively. As shown in Fig. 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. The in vitro experiments indicated that ectopic KIF21B expression effectively reversed the effects of miR-132-3p overexpression on cell proliferation (Fig. 4C), migration (Fig. 4D) and invasion (Fig. 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, migration and invasion. Thus, we next investigated whether Wnt/β-catenin signaling pathway was involved in the regulation of GC cells induced by miR-132-3p targeting KIF21B. As shown in Fig. 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 (Fig. 5B). These results indicated that KIF21B was a direct target of miR-132-3p involved in Wnt/β-catenin pathway in GC cells.

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 inhibition signi cantly reduced cell viability and colony formation, inhibited 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.
By bioinformatic analysis and luciferase reporter assay, 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 overexpression of KIF21B signi cantly alleviated the suppressive effects of miR-132-3p on cell proliferation, migration and invasion in AGS and SNU-5 cells. 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-offunction 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, and ectopic miR-338-3p expression mimicked the inhibitory functions of KIFC1 silencing on renal cell carcinoma cells [45]. MiR-30a could speci cally interact with KIF11 and suppress its expression in breast cancer [46]. Additionally, miR-206 directly suppresses KIF2A to inhibit ovarian cancer cell proliferation, migration, and invasion [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.
It has been widely held that the Wnt/β-catenin signaling pathway is 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 is closely associated with EMT process and plays a critical role in metastasis [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 (E-cadherin 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 EMT-correlated 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 Ncadherin 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 the migration and invasion of GC cells by targeting KIF21B-mediated Wnt/β-catenin pathway based on the correlation of EMT process and Wnt/β-catenin pathway.

Conclusion
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.  . All data are expressed as the means ± SD. **p < 0.01, ***p < 0.001, compared with si-NC group MiR-132-3p negatively regulated the Wnt/β-catenin pathway by directly targeting KIF21B. AGS and SNU-5 cells were transfected with miR-NC, miR-132-3p mimics or miR-132-3p mimics plus KIF21B, respectively.
Western blot assay was used to compare the protein levels of Wnt1, c-Myc, β-catenin, PCNA, E-cadherin and N-cadherin in transfected AGS (A) and SNU-5 (B) cells. GAPDH was used as an internal control.