Kinesin family member 3C (KIF3C) is a novel non-small cell lung cancer (NSCLC) oncogene whose expression is modulated by microRNA-150-5p (miR-150-5p) and microRNA-186-3p (miR-186-3p)

ABSTRACT This study is aimed at investigating the biological function of kinesin family member 3 C (KIF3C) in non-small cell lung cancer (NSCLC) progression and its upstream regulatory mechanism. Quantitative real-time PCR, Western blot and immunohistochemistry were adopted to examine microRNA-150-5p (miR-150-5p), microRNA-186-3p (miR-186-3p) and kinesin family member 3 C (KIF3C) expression levels. NSCLC cell proliferation, migration, and invasion were detected through cell counting kit-8 (CCK-8) assay, EdU assay, and Transwell assay. The metastasis of NSCLC cells was evaluated utilizing a pulmonary metastasis model in nude mice in vivo. The targeted relationship among KIF3C 3ʹUTR, miR-186-3p, and miR-150-5p were verified by dual-luciferase reporter gene assays. It was confirmed that in NSCLC tissues and cells, KIF3C expression level was increased and KIF3C overexpression promoted NSCLC cell proliferation and metastasis. Additionally, miR-150-5p and miR-186-3p directly targeted KIF3C to repress its expression. Our data suggest that KIF3C, which is negatively regulated by miR-150-5p and miR-186-3p, is an oncogenic factor in NSCLC progression.


Introduction
Non-small cell lung cancer (NSCLC) cases take up about 85% of all lung cancer (LC) cases, and globally, NSCLC is the leading cause of cancerrelated death in both men and women [1]. The therapies for NSCLC mainly include surgery, radiotherapy, chemotherapy, and immunotherapy [2]. Despite dramatic advances in these therapies in recent years, due to the strong metastasis ability of NSCLC cells, NSCLC patients' five-year survival rate remains less than 15% [2]. It is urgent to delve deeper into NSCLC pathogenesis and offer a theoretical basis for exploring more effective treatment for the patients.
Kinesin family member 3 C (KIF3C) is a member of the KIF3 subfamily of kinesin superfamily proteins (KIFs), and it features prominently in regulating many biological processes [3]. KIF3 complex is associated with neuronal development and differentiation [4]. Furthermore, it regulates tumor cells' growth, metastasis and chemoresistance [5,6]. Specifically, the up-regulation of KIF3C is accompanied by docetaxel resistance of breast cancer cells [5]. Another study shows that KIF3C expression in the low-grade glioma tissue is higher in comparison to the high-grade glioma tissue, and patients with high KIF3C expression have a longer survival time; functionally, KIF3C inhibits glioma cells' growth through modulating the PI3K/Akt/mTOR pathway [6]. The above studies indicate that KIF3C can exert tumorpromoting effect or tumor-suppressive effect in different cancers. Through bioinformatics analysis, the current study revealed that KIF3C was upregulated in NSCLC tissues, and it was linked to the poor prognosis of NSCLC. Thus it was hypothesized that KIF3C could probably play a cancer-promoting role in NSCLC.
In this study, we hypothesized that KIF3C had the potential to be the diagnostic biomarker and therapeutic target for NSCLC. We investigated the biological function of KIF3C on the malignant biological behaviors of NSCLC cells, and the regulatory function of miR-186-3p and miR-150-5p on KIF3C. Herein, we report that, KIF3C, which is negatively regulated by miR-150-5p and miR-186-3p, is an oncogenic factor in NSCLC progression.

Ethics statement and tissue samples
From February 2018 to May 2020, normal and cancer tissue samples from 42 NSCLC patients diagnosed in the Affiliated Hospital of Chengde Medical College were collected. All patients were diagnosed with NSCLC through clinical, imaging, and pathological examinations, and they did not suffer from other malignancies. The informed consent was signed by all subjects. This study was endorsed by the Affiliated Hospital of Chengde Medical College Research Ethics Committee. Before the surgery, none of the subjects had undergone radiotherapy, chemotherapy, or targeted therapy. The tissue samples were frozen in liquid nitrogen after surgical removal and reserved for follow-up research.

Transwell assays
Transwell assays were performed to detect cell migration and invasion. Cell migration assay: the cell suspension was prepared with serum-free medium. The lower chamber of Transwell system (Costar, Cambridge, MA, USA) was added with 500 μL of serum-containing culture solution, and 200 μL of cell suspension (containing about 5 × 10 4 cells) was added to the top compartment. Then the cells were put in the incubator. After 24 h of incubation, the chambers were taken out, and the liquid in the upper chamber was removed. Cotton balls were utilized to gently wipe away the cells in the upper chamber. Next, the cells which were on the below surface of the membrane, were stained with crystal violet staining solution at room temperature for 15 min. After the cells were washed by PBS and air-dried, the cells in 5 visual fields were counted and photographed under the inverted microscope (×200). Cell invasion assay: a layer of Matrigel was added onto the membrane of the Transwell chamber in advance, and the other procedures were the same with the migration assay.

EdU assay
An EdU kit (Beyotime Biotechnology, Shanghai, China) was utilized to perform EdU assay following the manufacturer's instructions. Transfected H226 and A549 cells in logarithmic growth phase were incubated in 96-well plates with 50 μmol/L EdU medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). After incubation for 2 h, the cells were cleaned with PBS. Subsequently, paraformaldehyde was used to fix the cells. The addition of 200 μL of 2 mg/ml glycine and 5 min of incubation were followed by cell washing with PBS for 5 min on a shaker. Next, the cells were added with 0.5% TritonX-100 for decolorized incubation on a shaker for 10 min. Next, PBS was used to wash the cells twice, 5 min for each time. Next, the cells were stained with Apollo in darkness at room temperature for 30 min, and then stained with DAPI staining solution in darkness at room temperature for 20 min. After PBS washing, a fluorescence microscope was employed to photograph the cells.

Immunohistochemistry (IHC)
The NSCLC tissue samples and adjacent normal tissue samples were fixed in 10% formaldehyde and embedded in paraffin. Next, the tissues blocks were sliced, the tissues sections were deparaffinized and hydrated. The deparaffinized sections were incubated at room temperature with 1% H 2 O 2 for 10 min to block endogenous peroxidase. After PBS washing 3 times and blocking for 1 h with immunostaining blocking solution, the sections were incubated with anti-KIF3C antibody (ab236748, 1:100) in a wet box overnight at 4°C. PBS was utilized to rinse the sections, which were then incubated with biotinlinked antiserum at room temperature for 1 h. Next, the sections were rinsed again and stained for 1 min with 3,3-diaminobenzidine hydrochloride. Ultimately, double distilled water was utilized to wash the sections, which were subsequently stained for 1 min with hematoxylin, and then the sections were observed under the microscope.

Western blot assay
RIPA lysis buffer (Pierce, Rockford, IL, USA) was used to lyse NSCLC cells, and after highspeed centrifugation, the supernatant was collected. The BCA reagent (Pierce, Rockford, IL, USA) was applied for determining the total protein concentration, and the supernatant was heated for 10 min in a 100°C water bath to denature the proteins. Protein separation was performed by SDS-PAGE, and then the proteins were electro-transferred to the PVDF membrane (Millipore, Bedford, MA, USA). After being blocked with 5% skim milk, the membranes were incubated overnight with primary antibodies at 4°C: anti-GAPDH antibody (ab8245, 1:500, Abcam, Cambridge, UK) and anti-KIF3C antibody (ab236748, 1:500, Abcam, Cambridge, UK). After being rinsed with TBST, the membranes and the secondary antibody (goat anti-rabbit IgG coupled with HRP, ab205718, 1:1000, Abcam, Cambridge, UK) were incubated at room temperature for 1 h. After the membranes were washed with TBST again, the protein bans were developed with a hypersensitive ECL kit (Millipore, Bedford, MA, USA). An ImageJ software (NIH, Bethesda, MD, USA) was adopted to quantify the protein bands.

Lung metastasis model in vivo
From Hebei Medical University Animal Center (Shijizhuang, China), BALB/c nude mice (male, 4-5 weeks old) were obtained. The mice were randomly separated into two groups (KIF3C overexpression group and control group; 5 mice in each group). H226 cells (1 × 10 7 cells/mouse) were injected via the caudal vein into each mouse. The mice were euthanized after 3 weeks, and their lungs were fixed in formalin and embedded in paraffin. Next, the tissues were sliced, and the lung tissues were stained with hematoxylin/eosin. Subsequently, the metastatic nodules in the lung tissues of the mice were observed under a microscope.

Statistical analysis
All experiments were conducted in triplicate. The data analysis tool was SPSS 23.0 software (SPSS Inc., Chicago, IL, USA). 'Mean ± standard deviation' was the expression form of the data. The differences between the two groups were evaluated by Student's t-test. One-way analysis of variance was utilized to conduct the comparison among multiple groups. P < 0.05 signified that a difference was statistically significant.

Results
We hypothesized that KIF3C could promote the progression of NSCLC. Loss-of-function and gainof-function models were established, and we demonstrated that KIF3C could regulate the malignant biological behaviors of NSCLC cells in vitro and in vivo. Additionally, we demonstrated that miR-150-5p and miR-186-3p directly targeted KIF3C, and the up-regulation of miR-150-5p and miR-186-3p weakened the promoting effects of KIF3C on proliferation, migration, and invasion of NSCLC cells.

KIF3C is significantly high-expressed in NSCLC tissues and cells
Firstly, GEPIA database (http://gepia.cancer-pku.cn/) was applied to analyze KIF3C expression. The data showed that KIF3C expression level in NSCLC tissues (lung adenocarcinoma: LUAD; lung squamous carcinoma: LUSC) was markedly higher compared with normal tissues (Figure 1a). Then, qRT-PCR was conducted to evaluate KIF3C expression in 42 pairs of NSCLC and para-cancerous tissues, and it was shown that KIF3C mRNA expression in NSCLC tissues was markedly up-regulated (Figure 1b). Compared with HBE, KIF3C mRNA was also significantly highly expressed in NSCLC cell lines (Figure 1c). IHC was used to further examine KIF3C protein expression in NSCLC, and it was observed that KIF3C was highexpressed in NSCLC tissues (chi-square = 7.2446, p = 0.026, Figure 1d). Through the analysis of the correlation between KIF3C expression and the patients' clinicopathological features, it was suggested that high KIF3C expression was significantly associated with higher TNM stage (Table 1). Moreover, the KM-plotter database (https://kmplot.com/analy sis/) indicated that high KIF3C expression was strongly correlated with the shorter overall survival time (OS), faster first progression (FP), and shorter post-progression survival time (PPS) of NSCLC patients (Figure 1e-g).

KIF3C overexpression promotes NSCLC cell proliferation and metastasis
The above results showed that among NSCLC cells, KIF3C expression was the highest in A549 cells, whereas KIF3C expression was the lowest in H226 cells (Figure 1c). Therefore, KIF3C overexpression plasmids were transfected into H226 cells and si-KIF3C was transfected into A549 cells, and the transfection was verified by Western blotting to be successful (Figure 2a). To explore KIF3C's biological role in regulating the malignant biological behaviors of NSCLC cells, we conducted CCK-8, EdU, and Transwell assays, and it showed that compared with the control group, KIF3C overexpression remarkably promoted cell proliferation, migration, and invasion, while KIF3C knockdown repressed cell proliferation, migration, and invasion (Figure 2b-e). Besides, the metastasis of NSCLC cells in vivo was evaluated using a lung metastasis model in nude mice, and it was unveiled that KIF3C overexpression facilitated the lung metastasis of H226 cells in vivo (Supplementary Figure 1a). The above findings suggest that KIF3C facilitates NSCLC cells' proliferation, migration, and invasion.

MiR-150-5p targets and represses KIF3C to inhibit NSCLC progression
To further confirm whether miR-150-5p participated in NSCLC progression via modulating KIF3C, H226 cells were co-transfected with pcDNA-KIF3C and miR-150-5p mimics, and A549 cells were co-transfected with si-KIF3C and miR-150-5p inhibitors, and Western blotting showed that the transfection was a success (Figure 4a). Subsequently, cell proliferation, migration, and invasion were detected through CCK-8, EdU, and Transwell assays, and as shown, overexpression of KIF3C remarkably promoted H226 cell proliferation, migration, and invasion, while the transfection of miR-150-5p mimics weakened this effect (Figure 4b-d); on the other hand, knocking down KIF3C inhibited A549 cell proliferation, migration, and invasion, while the transfection of miR-150-5p inhibitors reversed this effect (Figure 4b-d).

Discussion
KIF3C belongs to the kinesin superfamily protein (KIF) family. Known as a type of conserved proteins which regulate microtubule-dependent movement, KIFs feature prominently in modulating many biological processes, for instance, cell morphology, intracellular macromolecule transport, cytoskeletal dynamics, cell division, and cell migration [13][14][15]. Reportedly, KIF3C is highly enriched in nervous tissues such as the brain, spinal cord, and retina, and KIF3C is involved in regulating axon regeneration at growth cones after neuronal injury [16][17][18]. Importantly, accumulating evidence supports the regulatory function of KIF3C in cancer biology. For instance, KIF3C expression level is increased in breast cancer, and high KIF3C expression is associated with lymph node metastasis and tumor recurrence; additionally, KIF3C knockdown inhibits the EMT process via suppressing the TGF-β signal pathway and inhibits BC cell proliferation by inducing G2/M phase arrest, which suggests that KIF3C can serve as a biomarker and target for the diagnosis and therapy of breast cancer [13]. Two recently published studies report that, in glioma, up-regulation of KIF3C is associated with patients' unfavorable prognosis; functionally, KIF3C can regulate the PI3K/AKT/mTOR signal pathway to inhibit glioma cell growth [6,19]. To our best knowledge, our study was the first to confirm that in NSCLC tissues, KIF3C was highly expressed, and its high expression was significantly associated with the patient's adverse prognosis; furthermore, KIF3C overexpression dramatically promoted NSCLC cell proliferation, migration, and invasion, and its depletion repressed these malignant phenotypes of NSCLC cells. The findings indicate that KIF3C serves as an oncogene in NSCLC progression. It has been reported that miRNAs play a vital role in regulating the progression of multiple cancers, and miRNAs can participate in regulating cell proliferation, differentiation, apoptosis, metabolism, inflammation, angiogenesis, and many other biological processes by modulating the expression of target genes [20]. Importantly, a lot of studies have confirmed that miRNAs serve as tumor suppressors or promoters in NSCLC [21,22]. For example, in NSCLC tissues and cells, miR-148a expression level is remarkably reduced, and low miR-148a expression is significantly related to the poor prognosis of the patients; functionally, miR-148a represses tumor cell migration and invasion via targeting Wnt1 [21]. Another study reports that miR-199 is a tumor-suppressing miRNA, and its expression is reduced in NSCLC, and miR-199 suppresses the malignant progression of NSCLC via targeting RGS17 [22]. The role of miR-150-5p in NSCLC is controversial. It is reported that miR-150-5p can repress LKB1 to promote NSCLC progression [23]. However, other studies report that miR-150-5p inhibits Wnt-β-catenin signaling via targeting GSKIP and HMGA2, or suppresses MMP14, or GLUT1 to exert its tumor-suppressive properties in NSCLC [24][25][26]. In the present work, our data showed that miR-150-5p was lowly expressed in NSCLC tissues, and it counteracted the oncogenic effects of KIF3C, supporting it is a tumor suppressor. Previously, the biological function of miR-186-3p in NSCLC was obscure. In cervical cancer, miR-186-3p blocks tumorigenesis via suppressing MCM2 [27]. In breast cancer, miR-186-3p overexpression markedly restrains EREG expression, thus inhibiting the drug resistance and glycolysis of cancer cells [12]. Our data suggested that miR-186-3p was down-regulated in NSCLC tissues, and its overexpression counteracted the effects of KIF3C on Figure 4. Effects of miR-150-5p / KIF3C axis on NSCLC cell proliferation, migration, and invasion. a H226 cells were co-transfected with pcDNA-KIF3C and miR-150-5p mimics, and A549 cells were co-transfected with si-KIF3C and miR-150-5p inhibitors. After transfection, KIF3C expression was detected by Western blot. B&C. CCK-8 and EdU assays were used to detect the changes in cell proliferation. D&E. Transwell assays were used to detect the changes in cell migration and invasion. * P < 0.05, ** P < 0.01 and *** P < 0.001. NSCLC cells. These results suggest that miR-186-3p is a tumor suppressor in NSCLC, which is similar with its role in cervical cancer and breast cancer [12,27]. Importantly, this study also revealed that miR-150-5p and miR-186-3p directly targeted KIF3C and negatively regulated its expression, and our demonstrations partly explain the mechanism of KIF3C overexpression in NSCLC.

Conclusion
Collectively, our study reports that high expression of KIF3C implies unfavorable prognosis of NSCLC patients. Also, with in vitro and in vivo experiments, this study also confirms that KIF3C promotes the malignant biological behaviors of NSCLC cells. Additionally, as a downstream target of miR-186-3p and miR-150-5p, KIF3C is modulated by miR-186-3p and miR-150-5p in NSCLC cells. This study offers a novel understanding into the pathogenesis of NSCLC. However, more clinical samples are needed to further evaluate the potential of KIF3C as a prognostic biomarker for NSCLC.

Highlights
KIF3C expression was up-regulated in NSCLC, and it was associated with poor prognosis.
Overexpression of KIF3C promoted NSCLC cell proliferation and metastasis.