MicroRNA‐188 inhibits biological activity of lung cancer stem cells through targeting MDK and mediating the Hippo pathway

New Findings What is the central question of this study? The aim was to investigate the function of microRNA‐188 in the biological characteristics of lung cancer stem cells and the molecular mechanisms involved. What is the main finding and its importance? This study highlights a new molecular mechanism involving microRNA‐188, MDK and the Hippo signalling pathway that plays a suppressive role in biological activity of lung cancer stem cells. This finding might offer new insights into gene‐based therapy for lung cancer. Abstract MicroRNAs (miRNAs) have been implicated in lung cancer and reported as new promising diagnostic and therapeutic tools for cancer control. Here, we investigated the action of microRNA‐188 (miR‐188) in lung cancer stem cells. We first tested miR‐188 expression in clinical samples of lung cancer patients, and a low expression profile of miR‐188 was found. Next, we analysed the role of miR‐188 in lung cancer stem cells with cell growth assays. To verify the in vitro results, we used a xenograft model to validate the capability of miR‐188 in tumorigenesis. Overexpression of miR‐188 reduced viability and metastasis of cancer stem cells. Similar results were reproduced in vivo, where overexpression of miR‐188 retarded tumour growth in mice. We also identified MDK as a target of miR‐188, and overexpression of MDK was found in lung cancer samples. Overexpressed MDK promoted the malignant behaviours of lung cancer stem cells. In addition, the Hippo pathway was found to be inactivated in lung cancer tissues, presenting as increased levels of YAP and TAZ. Suppression of the Hippo pathway also enhanced lung cancer stem cell activity and promoted the growth of xenograft tumours. To sum up, our results reveal that miR‐188 inhibits the malignant behaviours of lung cancer stem cells and the growth of xenograft tumours. This study might offer new insights into gene‐based therapies for cancer.

models, including prostate cancer, oral squamous cell carcinoma and hepatocellular carcinoma (Fang et al., 2015;Wang & Liu, 2016;Zhang et al., 2015). Also, it is reported that miR-188 is downregulated in lung cancer cells (Zhao et al., 2018). Midkine (MDK) is known as a heparinbinding growth factor that is upregulated in some malignant tumours, including lung cancers (Hao et al., 2013). The human MDK gene is positioned on chromosome 11p11.2, and there are four exons in the coding frames of the protein (Muramatsu, 2002). Interestingly, MDK is of significance in human tumour processes and in biological processes such as enhancement of fibrinolytic activity, induction of chemotaxis and angiogenesis and inhibition of apoptosis (Yuan et al., 2015). Overexpression of MDK has been revealed in a variety of cancers, including gastric cancer (Xu et al., 2012), breast cancer (Ibusuki et al., 2009) and lung cancer (Hao et al., 2013). The Hippo pathway is an important pathway for organ growth, whose aberrant expression has been linked to tumorigenesis. The core kinases MST1/2 and LATS1/2 are tumour inhibitors that suppress the activity of the oncogenic factors Yesassociated protein (YAP) and PDZ-binding motif (TAZ) (Park, Shin, & Park, 2018), and their correlation with tumorigenesis, control of organ size and stem cell renewal has been reported (Park et al., 2018;Tao et al., 2017). This pathway has also been found in lung development and tumorigenesis (Yeung, Yu, & Yang, 2016). An article by Teoh & Das (2017) highlighted the role of the core members, upstream modulators and downstream effectors in lung cancer development and suggested that YAP and TAZ might be promising targets for future drug delivery and treatment.
In this study, we explored the functions of miR-188 in the biological characteristics of lung cancer stem cells with the involvement of MDK and the Hippo pathway.

Ethical approval
All experimental procedures were performed in accordance with the guidelines by the Ethics Committee of the First Hospital of Jilin University (approval no. 2014-243) and were confirmed to meet the principles and regulations described by Grundy (2015). Signed

Clinical data collection
The First Hospital of Jilin University enrolled 75 patients with primary lung cancer from November 2014 to April 2015. The tumour tissues

New Findings
• What is the central question of this study?
The aim was to investigate the function of microRNA-188 in the biological characteristics of lung cancer stem cells and the molecular mechanisms involved.
• What is the main finding and its importance? cells were cultured in LHC-9 medium (Gibco). All cells were cultured in an incubator at 37 • C in air enriched with 5% CO 2 .

Immunomagnetic bead separation
A549 and H125 cells were detached in 0.25% trypsin to make singlecell suspension, centrifuged at 160 g for 2 min and resuspended in MACS Separation Buffer (Miltenyi Biotec, Auburn, CA, USA). Next, cells were mixed with 20 μl CD44 antibody magnetic bead marker (Miltenyi Biotec) and incubated at 4 • C for 15 min. CD44 + cells were collected using an Auto MACS instrument (Miltenyi Biotec) and counted, then labelled with 100 μl CD133 antibody magnetic bead marker (Miltenyi Biotec) and incubated at 4 • C for 45 min. The CD133 + /CD44 + cells were collected using the Auto MACS instrument, and the purity of CD133 + /CD44 + cells was detected using a flow cytometer (Attune NxT; Thermo Fisher Scientific Inc., Waltham, MA, USA).

Cell transfection
The

Flow cytometry
The cultured cells (1 × 10 6 ) were centrifuged, suspended in 0.3 ml PBS containing 10% calf serum and transferred into a 1.5 ml EP tube. The cells were fixed with 0.7 ml absolute ethanol at −20 • C for

Microarray analysis
The total miRNA molecules were randomly extracted from lung cancer tissues and adjacent tissues of three stage III patients using TRIzol was used to screen out miRNAs, P < 0.05, |log Fold change| > 2 was defined as differential miRNAs and plotted into a heatmap according to hierarchical clustering. Three independent experiments were performed.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
TRIzol reagent (Invitrogen) was implemented to extract total RNA from lung cancer tumour tissues and adjacent tissues. A PrimeScript Reagent Kit (TAKARA, Otsu, Shiga, Japan) was used for reverse transcription and to amplify complementary DNA. The qPCR was conducted with QuantStudio 3 and 5 Real-Time PCR Systems (Thermo-Fisher Scientific). Gene expression was quantified using a TaqMan Fast Advanced Master Mix Kit (ThermoFisher Scientific). All samples were subjected to three independent analyses. The analysis results were based on the −2 ΔΔCt method. The relevant primers are shown in Table 2. Three independent experiments were performed.

2.9
Cell counting kit-8 assay The cultured cells were detached with a mixture of 0.02% EDTA and 0.25% trypsin to prepare A549 and H125 cell suspensions. The cell suspension (100 μl) was added to each well in a 96-well plate and cultured in an incubator for 24 h. Next, 10 μl of cell counting kit-8 (CCK-8) reagent (TAKARA) was added to each well, and the cells were cultured for 2 h. The optical density (OD) value of each well was measured at 460 nm. Three independent experiments were performed.

Transwell assays
In aseptic conditions, the apical chambers were precoated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) for 30 min. Next, each chamber was loaded with 30 μl serum-free RPMI-1640 medium and placed in an incubator in air enriched with 5% CO 2 for later use.
The cells were trypsinized, centrifuged, resuspended in serum-free medium and diluted to a cell suspension of 5 × 10 5 cells ml −1 . Next, the basolateral chamber of the Transwell was supplemented with 500 μl RPMI-1640 medium containing 10% fetal bovine serum, and then 200 μl cell suspension was added to each apical chambe. The Transwell plates were incubated at 37 • C in air enriched with 5% CO 2 for 48 h.
After that, the cells in the inner membrane were washed using PBS. The remaining invaded cells were stained with Crystal Violet solution for 10 min, and photographs of the cells were captured under the microscope. Cell migration was measured in a similar manner without precoating with Matrigel in the apical chambers.

Xenograft transplantation model
Forty-eight female specific pathogen-free mice (4-6 weeks of age, weighing 20 ± 2 g) were purchased from Beijing Vital River Laboratory
A Lipofectamine 3000 transfection kit (Invitrogen) was used to cotransfect WT plasmid, MUT plasmid with miR-188 or mimic NC into cells. Cells were lysed after 24 h, and the intensity of luciferase activity was measured using a dual-luciferase reporter assay system (Promega Corporation, Madison, WI, USA; intensity = RLU1/RLU2, where RLU1 is the firefly luciferase reaction intensity and RLU2 the renilla luciferase reaction intensity) (Jiang et al., 2014).

Western blot analysis
The tumour tissue homogenate was treated with 1 ml of lysate

Statistical analysis
Statistical analysis was carried out with SPSS v.22.0 software (SPSS Inc., Chicago, IL, USA). Measurement data were depicted as the mean ± SD. Student's paired t test was used for comparisons between two groups. One-or two-way ANOVA was adopted for comparisons among multiple groups, after which Tukey's multiple comparisons test was applied for pairwise comparisons. The survival curve was calculated using the Kalpan-Meier method, and the post-statistical analysis was performed using the log rank test. A value of P < 0.05 was regarded as statistical significance.

MicroRNA-188, beneficial to the patient's prognosis, is downregulated in lung cancer tissues and cells
MicroRNA-188 was found to be reduced in lung cancer tissues by examining miRNA microarray differences between tumour tissues and adjacent normal tissues in lung cancer patients (Figure 1a). This result was validated using the RT-qPCR (Figure 1b). The survival of lung cancer patients was analysed and showed that miR-188 expression  Figure 1c). We also found that miR-188 was poorly expressed in lung cancer tumour cells relative to the BEAS-2B cells (Figure 1d). Next, the stem cells from A549 and H125 cell clusters were isolated by immunomagnetic beads for subsequent experiments (Figure 1e).

Overexpression of miR-188 inhibits lung cancer stem cell activity
The cancer stem cells were collected and treated with miR-188 mimic, and the transfection was successfully performed according to the RT-qPCR results (Figure 2a). The CCK-8 results demonstrated that the OD value at 460 nm was reduced after miR-188 overexpression (Figure 2b).

MicroRNA-188 targets MDK
We predicted MDK as a target mRNA of miR-188 on StarBase, and this binding relationship was validated using a dual luciferase assay.
The results suggested that co-transfection of pMIR-MDK-WT vector

Overexpressed MDK partly inhibits the biological effects of overexpressed miR-188
To validate the involvement of MDK in miR-188-mediated events, a rescue experiment was performed, in which cells transfected with miR-188 mimic were also transfected with lentiviral vectors over-  (Figure 5f).

MicroRNA-188 mediates Hippo pathway activity
The protein levels of key kinases of the Hippo pathway, YAP and TAZ, in cells were determined using western blot analysis. It was found that the F I G U R E 4 Overexpression of MDK promotes lung cancer stem cell activity. (a) Detection of MDK expression in cells with MDK by RT-qPCR ( * P < 0.05 according to two-way ANOVA). (b) Cell counting kit-8 assay was used to detect change in cell proliferation in cells with MDK ( * P < 0.05 according to two-way ANOVA). (c) Transwell assay was used to detect change in cell migration in cells with MDK ( * P < 0.05 according to two-way ANOVA). (d) Transwell assay was used to detect change in cell invasion in cells with MDK ( * P < 0.05 according to two-way ANOVA). (e) Flow cytometry was used to detect change in cell apoptosis in cells with MDK [with propidium iodide (PI) and annexin V as markers; * P < 0.05 according to two-way ANOVA]. (f) Changes in lung tumour volume and weight in mice in xenoplastic transplantation of MDK ( * P < 0.05 according to two-way ANOVA) F I G U R E 5 Overexpression of MDK partly inhibits the biological effects of overexpressed miR-188. (a) Detection of MDK expression in cells with miR-188 mimic + MDK by RT-qPCR ( * P < 0.05 according to two-way ANOVA). (b) Cell counting kit-8 assay was used to detect change in cell proliferation in cells with miR-188 mimic + MDK ( * P < 0.05 according to two-way ANOVA). (c) Transwell assay was used to detect change in cell migration in cells with miR-188 mimic + MDK ( * P < 0.05 according to two-way ANOVA). (d) Transwell assay was used to detect change in cell invasion in cells with miR-188 mimic + MDK ( * P < 0.05 according to two-way ANOVA). (e) Flow cytometry was used to detect change in cell apoptosis in cells with miR-188 mimic + MDK [with propidium iodide (PI) and annexin V as markers; * P < 0.05 according to two-way ANOVA]. (f) Changes in lung tumour volume and weight in mice in xenoplastic transplantation of miR-188 mimic + MDK ( * P < 0.05 according to two-way ANOVA)

F I G U R E 6
MicroRNA-188 (miR-188) mediates Hippo pathway activity. The protein levels in the Hippo pathway in cells with miR-188 mimic and miR-188 control, MDK and negative control (NC), miR-188 mimic + NC and miR-188 mimic + MDK were determined by western blot analysis levels of activated YAP and TAZ were increased in lung cancer tissues.
Overexpression of miR-188 was found to activate this signalling by suppressing the levels of YAP and TAZ, whereas the levels of these kinases were recovered by the further upregulation of MDK ( Figure 6).

Suppression of Hippo pathway promotes lung cancer stem cell activity
To validate the roles of the Hippo signalling pathway in cancer stem cell growth and tumorigenesis of lung cancer, a Hippo-specific inhibitor, XMU-MP-1, was administered into the stem cells ( Figure 7a).
Consequently, it was found that the malignant behaviours of the cells were promoted, presenting as enhanced cell viability (Figure 7b (Zhao et al., 2018). Another study revealed that miR-188 was downregulated in lung adenocarcinoma and that upregulation of miR-188 inhibited proliferation and resistance to apoptosis of lung adenocarcinoma cells (Lv et al., 2020). Furthermore, miR-188-5p enhanced the growth of gastric cancer cells in vitro and tumour metastasis in vivo (Li et al., 2019).
In addition, StarBase prediction and luciferase activity analysis suggested that miR-188 targeted MDK. Many miRNA targets have been characterized in different types of cancers. miR-188 was reduced in prostate cancer and retarded cancer cell growth via repression of LAPTM4B . Additionally, downregulation miR-188 was found in hepatocellular carcinoma, where it acted as a tumour suppressor through binding to FGF5 (Fang et al., 2015). It was also demonstrated that miR-188 inhibited oral squamous cell carcinoma by targeting SIX1, offering new insights into the detailed molecular mechanisms of oral squamous cell carcinoma (Wang & Liu, 2016). In the present study, we detected MDK expression in lung cancer tissues and cells, suggesting that MDK expression was increased in lung cancer, and overexpression of MDK

F I G U R E 8
The mechanistic diagram depicts that miR-188 targets the MDK-mediated Hippo pathway, inhibits the activation of YAP protein, weakens the proliferation, migration and invasion of lung cancer stem cells and enhances their apoptotic ability promoted lung cancer stem cell activity. MDK is able to activate some signalling pathways and results in various cellular process through interaction with its downstream proteins (Muramatsu, 2011;Weckbach, Muramatsu, & Walzog, 2011). MDK has also been found to be important in the process of human carcinogenesis, which might be a potential target for cancer therapy. MDK downregulation suppressed glioma cell proliferation and the tumour growth in nude mice (Luo et al., 2015). Furthermore, upregulation of MDK might lead to the stemness properties and cell survival of prostate cancer stem cells (Erdogan, Doganlar, Doganlar, Turkekul, & Serttas, 2017).
Next, the protein levels of YAP and TAZ in lung cancer cells were determined, and the findings suggested that Hippo pathway activity was suppressed in lung cancer tissues. Additionally, we also found that suppression of the Hippo pathway promoted lung cancer stem cell activity. As already mentioned, YAP and TAZ are crucial oncogenes that are activated once Hippo is suppressed.
On phosphorylation by LATS1/2, YAP is prevented from nuclear accumulation and consequently degraded in the cytoplasm, whereas non-phosphorylated YAP accumulates in the nucleus and promotes aberrant cell proliferation (Wennmann et al., 2014). Recent advances in understanding the components and functional implications of the Hippo pathway indicate its roles in tumorigenesis, tissue regeneration, organ development and stem cell self-renewal (Park et al., 2018). The Hippo signalling pathway has also been implicated in playing pivotal roles in lung cancer pathogenesis, including multidrug resistance and tumour development (Liu, Zuo, & Ou, 2018). Taking in vivo xenograft and in vitro cell culture approaches, Liu et al. (2016) showed that PDE/cGMP/PKG signalling maintains prostate cancer stem cell stemness by targeting to Hippo/TAZ pathway. Moreover, Bora-Singhal et al. (2015) showed that YAP was enhanced in NSCLC stem-like cells and resulted in their self-renewal and ability to form angiogenic tubules.
In summary, our study highlights a new molecular mechanism involving miR-188, MDK and the Hippo signalling pathway, which plays a suppressive role in biological activity of lung cancer stem cells ( Figure 8). Exploration of the function of miR-188 not only enhances our understanding of lung cancer carcinogenesis but also identifies miR-188 as a promising biomarker for lung cancer diagnosis and therapy.

COMPETING INTERESTS
None declared.

AUTHOR CONTRIBUTIONS
Study design: X.Y. Experimental work: X.Y., B.W. and W.C. Data analysis: X.Y., B.W. and W.C. Writing the manuscript: X.Y. Overall coordination and research governance: X.M. All authors read and approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.