Reciprocal regulation of miR‐206 and IL‐6/STAT3 pathway mediates IL6‐induced gefitinib resistance in EGFR‐mutant lung cancer cells

Abstract Persistently activated IL‐6/STAT3 pathway promotes acquired resistance to targeted therapy with epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs) in non–small‐cell lung cancer (NSCLC) treatment. miR‐206 has been verified to be dysregulated and plays as a negative regulator in lung cancer. However, whether miR‐206 may overcome IL6‐induced gefitinib resistance in EGFR‐mutant lung cancer remains elusive. In this study, we investigated the role of miR‐206 in IL6‐induced gefitinib‐resistant EGFR‐mutated lung cancer cell lines. We showed that forced miR‐206 expression restored gefitinib sensitivity in IL6‐induced gefitinib‐resistant EGFR‐mutant lung cancer cells by inhibiting IL6/JAK1/STAT3 pathway. Specifically, mechanistic investigations revealed that miR‐206 blocked IL‐6/STAT3 signalling via directly targeting the 3'‐UTR of intracellular IL‐6 messenger RNA. Moreover, IL‐6 induced miR‐206 down‐regulation by reducing the cropping process of primary miR‐206 (pri‐miR‐206) into the Drosha/DGCR8 complex. Taken together, our findings reveal a direct role of miR‐206 in regulating IL‐6/STAT3 pathway and contrarily activated IL‐6/STAT3 signalling mediates the miR‐206 maturation process in gefitinib‐resistant EGFR‐mutant lung cancer cells.

addition to somatic mutation, activation of phenotypic or histological transformation, like epithelial-mesenchymal transition (EMT), and bypass signalling pathway which is driving survival of the bulk population are also the main mechanisms facilitating EGFR-TKI resistance. 5 STAT3 is a transcription factor that can promote oncogenesis, and it is commonly activated in cancer as well as in tumour-associated myeloid cells, which is frequently activated by inflammatory cytokine interleukin (IL)-6. 6 Experimental evidence suggested that deregulation of STAT3 activation by IL-6 correlates with pancreatic cancer, 7 colitis-associated cancer, 8 breast cancer 9 and hepatocellular carcinoma. 10 Functionally, over-activated IL-6/STAT3 signalling can induce drug resistance. [11][12][13] Previous reports have demonstrated that IL-6 promoted the EMT of lung cancer cell [14][15][16] and increased IL-6 activated signal transducer and activator of transcription 3 (STAT3) signalling pathway in lung adenocarcinoma. 17 Apparently, IL-6/STAT3 signalling pathway may be implicated in EGFR-TKI resistance. Yao et al 18 20 Specifically, evidence suggested that dysregulation of specific miRNAs may be involved in the acquisition of resistance to a number of cancer treatments, thereby modulating the sensitivity of cancer cells to such therapies. 21 In lung cancer, most miRNAs affect EGFR-TKI resistance by targeting c-Met, 22 PI3K/AKT, 23,24 apoptosis 25 and EGFR expression, 26,27 although EGFR-TKI-treated lung cancer cells inherently engage a positive feedback activation of STAT3 upon EGFR inhibition. 28 Several differentially expressed miRNAs were identified and found to regulate IL-6/STAT3 signalling pathway repression of multiple cancers. [29][30][31] However, it has not been determined whether certain miRNA is also involved in IL-6/STAT3 signalling to regulate EGFR-TKI sensitivity in lung cancer.
MiR-206 was first discovered as a central regulator in the regulation of myogenesis, muscle development and muscle remodelling. 32 Further study has revealed that deregulation of miR-206 occurs in lung adenocarcinoma correlating with poor prognosis and survival. 33 Chen et al have found miR-206 regulates cisplatin resistance and EMT in human lung adenocarcinoma cells partly by targeting Met. 34 In addition, miR-206 plays a role, involved in the invasion and metastasis of lung cancer, 35 in HGF-induced gefitinib-resistant human lung cancer cells through inhibition of c-Met signalling and EMT. 36 These promising studies point to the importance of further understanding the role of miR-206 in drug resistance in lung cancer. Here, we investigated the role of miR-206 in regulating IL-6/STAT3 pathway and gefitinib resistance in lung cancer.

| Clinical tissues
Tissue and serum specimens were obtained from 37 patients (Table S1) and 14 healthy participants in Qilu Hospital between 2015 and 2018.
The patients were diagnosed with NSCLC based on histopathological evaluation and treated with gefitinib (Iressa, Tocris Bioscience) for at least 6 months. No local or systemic treatment was conducted in these patients before surgery. All collected tissue and serum samples were immediately snap-frozen in liquid nitrogen and stored at −80°C until use. The study was approved by the Research Ethics Committee of Qilu Hospital, China. Written informed consent was obtained from all patients.

| Cell line culture
PC-9 and HCC827 cells were purchased from the Committee on Type Culture Collection of Chinese Academy of Sciences PC-9 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) and supplemented with 10% foetal bovine serum (FBS) and antibiotics and cultured at 37°C in humidified air with 5% CO 2 .
HCC827 cells were cultured in RPMI 1640 (Gibco) containing 10% FBS and antibiotics and cultured at 37°C in humidified air with 5% CO 2 . Recombinant human IL-6 was bought from R&D Systems.

| Measurement of IL-6
IL-6 levels in patients' and healthy participants' plasma samples were determined using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems). For intracellular IL-6 assessment, cells were lysed in Trizol reagent (Invitrogen) and treated with DNase I (Invitrogen) for qRT-PCR assay.

| Cell viability assays
Cells were seeded at a density of 5 × 10 3 cells per well in 96-well culture plates. The next day, cells were treated with miRNA mimics at indicated gefitinib concentration in a final volume of 100 μL for 72 hours. 10 µL of CCK-8 solution (Cell counting KIT-8, Solarbio) was added into each well, and the absorbance at 450 nm was measured after incubation for 30 minutes at 37°C to reflect the number of viable cells. All experiments were conducted by Multiskan Sky (Thermo Fisher) in triplicate.

| Cell apoptosis analysis
Cell apoptosis analyses were performed using Annexin V-FITC/PI Apoptosis Detection Kit (DOJINDO). Cells were seeded in 6-well plates at 1 × 10 6 per well. The next day, cells were treated with 0.1 μmol/L gefitinib and/or miRNA mimics. 24 hours after transfection, cells were harvested and resuspended at 1 × 10 6 in 100 μL volume. Cells were labelled with 5 μL Annexin V and 5 μL PI for 15 minutes in dark place. 400 μL of 1 Annexin V Binding Solution was added, and the samples were detected by flow cytometry within 1 hour.

| Colony formation assays
PC-9 and HCC827 were plated in 6-well culture dishes at a density of 500 cells/well. Cells were treated with 0.1 μmol/L gefitinib and/ or 20 nmol/L miR-206 mimics for 7 days. Cells were stained with crystal violet on the plates. Cell colonies were photographed under an inverted microscope.

| Co-immunoprecipitation and Western blot
For co-immunoprecipitation, cells were lysed using RIPA protein extraction reagent (Beyotime) supplemented with a protease inhibitor cocktail (Roche) and PMSF (Roche). The protein concentration was measured using the Bio-Rad protein assay kit. The supernatants were collected and incubated with anti-pStat3, Drosha at 4°C for 12 hours. Protein A Sepharose CL-4B beads (GE) were incubated with the mixture at 4°C for 2 hours. Then, the beads were washed three times with RIPA buffer. The bound proteins were eluted with SDS-PAGE loading buffer and used for Western blot. For Western blot assay, approximately 40 μg of protein extract was electrophoresed on a 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto 0.22-μm nitrocellulose membrane (Sigma) and incubated with specific antibodies. The ECL chromogenic substrate was used to visualize the bands. The intensity of the protein bands was quantified by Quantity One software (Bio-Rad ChemiDoc XRS). GAPDH was used as a control. Antibodies for Jak1, p-Jak1, Stat3, p-Stat3, Ago, IL-6, Drosha, DGCR8 and GAPDH were purchased from Abcam.

| Cell transfection
miRNA mimics or siRNA transfection was conducted with PEIpro (Polyplus) according to the manufacturer's instructions. Briefly, the cells were transfected with 20 nmol/L miRNA mimics or 50 nmol/L siRNA for 6 hours with PEIpro reagent and replaced with fresh growth medium. The next day, cells were treated with gefitinib for Western blot, qRT-PCR or MTT assay. siRNA for IL-6 was synthesized by Sangon Biotech, and the sequences were shown in Table S2.

| Luciferase reporter assay
The 3′-UTR of wild or mutant IL-6 was synthesized by Sangon Biotech and inserted into pMiR-Report firefly luciferase vector (GenePharma). pRL-Tk Renilla luciferase reporter was used for luciferase assay normalization.
PC-9 cells were transfected with 50 ng wild-type or mutant luciferase reporters and miR-206 mimics or control, along with 10 ng Renilla luciferase vector with the PEIpro (Polyplus) reagent. After 48 hours, luciferase activity was detected by the Dual-Luciferase Reporter Assay System (Promega) and relative luciferase activity was normalized to Renilla luciferase activity.

| RNA-ChIP assay
The cells were crosslinked and processed according to the RNA ChIP-IT ® Magnetic Chromatin Immunoprecipitation Kit (Active Motif, 53024) protocol. Antibodies to Ago control IgG (Abcam) were used at 4 μg per 10 μg sheared chromatin. 20 μL sonicated but pre-immunoprecipitated RNA from each sample was used as input control.
ChIP results were analysed by qRT-PCR. The primers locating the flank of pri-miR-197 stem-loop were designed and shown in Table   S2.

| Statistical analysis
All data were presented as mean ± SD values or min to max values. Student's t test (two-tailed) was performed to analyse the data from the experiments performed in triplicate, *P < .05, **P < .01, ***P < .001. For analysing the association of miR-206 expression and serum IL-6 levels, Spearman's correlation in Prism 7 was used with the P values indicated.

| miR-206 is dramatically down-regulated and negatively correlated with IL-6 in gefitinib-resistant EGFR-mutant lung carcinoma
To determine whether miR-206 is involved in IL-6/STAT3 signalling to regulate gefitinib sensitivity in lung cancer, we evaluated the expression of miR-206 and IL-6 in 37 NSCLC patients harbouring EGFR mutations and 14 healthy participants as IL-6 secreted by tumour cells was postulated as a potential mechanism for the primary resistance or low sensitivity to EGFR-TKIs. 37 The patients' backgrounds and clinical characteristics are listed in Table S1. The expression levels of miR-206 were dramatically reduced in tumour tissues compared to healthy participants' normal lung tissues ( Figure 1A), whereas the levels of serum IL-6 were significantly increased in NSCLC patients ( Figure 1B). Spearman's rank test showed a negative correlation between the expression of miR-206 and that of IL-6 (r = −.7762, P < .001, Figure 1C). In parallel, we adapted two EGFRmutant and TKI-sensitive lung cancer cell lines, PC-9 and HCC827, to IL-6 and cultured for 72 hours to simulate the in vivo microenvironment. In accordance with prior study, 38 activation of IL-6 could induce resistance to EGFR inhibitor ( Figure 1D). Surprisingly, we also found the reciprocal regulation of miR-206 and IL-6 in the gefitinib setting ( Figure 1E,F). These data suggested that miR-206 may be relevant to IL-6 downstream signalling pathway in EGFR-mutant lung cancer cells.

| miR-206 inactivates IL-6/JAK1/STAT3 pathway in IL6-induced gefitinib-resistant EGFR-mutant lung cancer cells
The significantly suppressive effect of miR-206 on IL6-induced gefitinib-resistant EGFR-mutant lung cancer cells prompted us to investigate its downstream signalling pathway. Previous reports have confirmed that IL-6/JAK1/STAT3 pathway is the basic mechanism to promote gefitinib resistance lung cancer. 38,39 In comply with these we examined whether STAT3 directly participated in miR-206-mediated gefitinib sensitivity by activating STAT3. We used colivelin, a STAT3 activator ( Figure S1), which suppresses neuronal death by activating STAT3. 40 The viability assay showed that addition of 50 nmol/L colivelin significantly abrogated miR-206 increased gefitinib sensitivity ( Figure 3D). Taken together, these results showed that miR-206 mediated IL6-induced gefitinib sensitivity by targeting JAK1/STAT3 pathway.

| Intracellular IL-6 is the direct target of miR-206
The F I G U R E 4 Intracellular IL-6 was the direct target of miR-206. A, Western blot was used to detect the Ago2-RISC complex using the Ago antibody in PC-9 cells with miR-206 mimics or control; IgG was used as a negative control. B, RNA-ChIP analysis was conducted to detect levels of mRNAs that bound with the Ago2-RISC complex from PC-9 cells transfecting with miR-206 mimics or control for 4 h, as measured by qRT-PCR; C, Western blot or D, qRT-PCR was used to detect the protein or mRNA levels of intracellular IL-6 in PC-9 and HCC827 cells; PC-9 and HCC827 cells were cultured for 72 h with 10 ng/mL rhIL-6 prior to miR-206 mimics or control treatment, and the cells collected for WB were washed five times to remove remnant membrane-bounded rhIL-6 with PBS before lysis. E, bioinformatics predicted and mutated miR-206 binding sites with IL-6. F, luciferase activity of the reporter construct containing the wild-type or mutant 3' UTR of IL-6 was measured after cotransfection with different amounts of miR-206 mimics in PC9 cells. G, knockdown of intracellular IL-6 abolished IL-6-induced p-Stat3 in PC-9 cell; PC-9 cells were cultured for 72 h with 10 ng/mL rhIL-6 prior to siRNA transfection. H, knockdown of intracellular IL-6 decreased gefitinib resistance in PC-9 cells. The mean ± SD values are shown. *P < .05, **P < .01, ***P < .001 in a dose-dependent manner in PC-9 cells, whereas that with mutated 3' UTR was not altered significantly ( Figure 4F). Moreover, knockdown of intracellular IL-6 mRNA by siRNA reduced the activation of STAT3 ( Figure S2 and Figure 4G) and decreased the IC 50 of gefitinib in PC-9 cells ( Figure 4H). In sum, these data support that the intracellular IL-6 serves as a direct target of miR-206 in EGFR-mutant lung cancer cells.

| IL-6 induces down-regulation of miR-206 by decreasing incorporation of pri-miR-206 into the Drosha/DGCR8 complex
In Figure 1E, IL-6 could induce down-regulation of miR-206, while the relative mechanism has not been elucidated. We first speculated that the biogenesis of miR-206 may be the most direct way to modulate its expression by IL-6. Hence, we measured the expression changes of pri-miR-206, pre-miR-206 and mature miR-206 from PC-9 cells. As shown in Figure Figure 5C). The above data implied that p-STAT3 may directly be associated with pri-miR-206, not Drosha/DGCR8 complex, to reduce its maturation process. To confirm this speculation, RNA-ChIP analysis was performed on PC-9 cells supplemented with IL-6 or not, and an enhanced association of p-STAT3 with pri-miR-206 was detected ( Figure 5D). Further, the association between pri-miR-206 and Drosha was significantly reduced in PC-9 cells cultured with IL-6 ( Figure 5E). Collectively, these data prove that p-STAT3 specifically binds with pri-miR-206 to inhibit its recruitment into the Drosha/ DGCR8 complex.

| D ISCUSS I ON
Alterations in the EGFR itself, somatic mutation or ALK rearrangement, and activation of alternative signalling pathways have been shown to induce acquired TKI resistance in EGFR-mutant NSCLC. 42 In several studies, EGFR inhibition with TKI concurrently activated STAT3 signalling in EGFR-mutant lung cancer cells, 28 In most cancers, STAT3 is typically active, but its activation can also occur through the influences of the microenvironment and in particular IL-6. 48 IL-6 is a multi-functional chronic inflammation cytokine, and high systemic IL-6 expression level is associated with worse prognosis in patients with NSCLC. 49 Except for influencing by microenvironment, it has been reported that STAT3dependent drug resistance could be mediated via autocrine IL-6 production. 38,50 Hence, the autocrine IL-6 production could exacerbate the intrinsic feedback activation of STAT3 and accelerate TKI resistance process.
STAT3 has emerged as an important regulator of gene expression, including miRNA, and in turn, several miRNAs can regulate the activity state of STAT3 in tumours. 51 For example, STAT3 interacted directly with the promoter of miR-21 in myeloma cells 52 and mediated reduction of the let-7 family via up-regulation of Lin-28 in breast cancer. 53 Conversely, up-regulation of miR-19a/b targeted SOCS-1, which is a negative regulator of IL-6R/STAT3 pathway. 54 59 In this study, we also found a post-transcriptional regulation of pri-miR-206 by binding of p-STAT3, which resulted in reduced expression of mature miR-206 and gefitinib resistance in EGFR-mutant lung cancer cells.
In summary, our findings reveal a reciprocal regulation of miR-206 and IL-6/STAT3 pathway that mediates IL-6-induced gefitinib resistance in EGFR-mutant lung cancer cells. We demonstrated F I G U R E 5 IL-6-induced p-Stat3 interacted with Drosha/DGCR8 complexes and decreased incorporation of pri-miR-206 into the complex to hinder its processing. A, relative expression of pri-miR-206, pre-miR-206 and mature miR-206 after 10 ng/mL rhIL-6 treatment in PC-9 cell. B, co-immunoprecipitation of pStat3 was performed in PC-9 cells by determination of protein levels of the associated Drosha and DGCR8 through Western blot. C, inhibition of pStat3 restored miR-206 maturation pathway; the qRT-PCR was performed 4 h after 5 nmol/L P6 or DMSO treatment in 10 ng/mL rhIL-6 treated PC-9 cell. D-E, RNA-ChIP was used to detect association between pri-miR-206 and p-Stat3 or Drosha. The mean ± SD values are shown. **P < .01, ***P < .001

ACK N OWLED G EM ENT
This work was supported by the Qingdao Science and Technology Bureau (NO. 13-1-4-129-jch).

CO N FLI C T S O F I NTE R E S T
The authors confirm that there is no conflict of interest. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available because of privacy or ethical restrictions.