miR‐1‐3p and miR‐206 sensitizes HGF‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT

Abstract Hepatocyte growth factor (HGF) overexpression is an important mechanism in acquired epidermal growth factor receptor (EGFR) kinase inhibitor gefitinib resistance in lung cancers with EGFR activating mutations. MiR‐1‐3p and miR‐206 act as suppressors in lung cancer proliferation and metastasis. However, whether miR‐1‐3p and miR‐206 can overcome HGF‐induced gefitinib resistance in EGFR mutant lung cancer is not clear. In this study, we showed that miR‐1‐3p and miR‐206 restored the sensitivities of lung cancer cells PC‐9 and HCC‐827 to gefitinib in present of HGF. For the mechanisms, we demonstrated that both miR‐1‐3p and miR‐206 directly target HGF receptor c‐Met in lung cancer. Knockdown of c‐Met mimicked the effects of miR‐1‐3p and miR‐206 transfections Meanwhile, c‐Met overexpression attenuated the effects of miR‐1‐3p and miR‐206 in HGF‐induced gefitinib resistance of lung cancers. Furthermore, we showed that miR‐1‐3p and miR‐206 inhibited c‐Met downstream Akt and Erk pathway and blocked HGF‐induced epithelial‐mesenchymal transition (EMT). Finally, we demonstrated that miR‐1‐3p and miR‐206 can increase gefitinib sensitivity in xenograft mouse models in vivo. Our study for the first time indicated the new function of miR‐1‐3p and miR‐206 in overcoming HGF‐induced gefitinib resistance in EGFR mutant lung cancer cell.

MicroRNAs (miRNAs) are endogenously expressed small noncoding RNA (about 22-25 nucleotides in length) acting as post-transcriptional regulators of gene expression. MiRNA can influence many biological processes, such as cell proliferation, invasion, metastasis, differentiation, metabolism and apoptosis. Multiple miR-NAs have been reported down-regulated in lung cancer and acted as suppressors in tumour development, including miR-145, 4 let 7, 5 miR-1, 6 miR-34b/c, 7 miR-449a, 8 and so on. miRNA-1 (mainly refer to miR-1-3p) and miR-206 are members of the muscle-specific miR-1 family of so-called myomiRs, 9 which play an important role in the myogenesis and development of cardiac and skeletal muscles. 10,11 A multitude of recent studies showed that deregulation of myomiR expression is associated with a variety of cancers. [12][13][14][15] In lung cancer, miR-1 and miR-206 have been found exhibited antitumour activities in multiple aspects, including repression of cell proliferation, migration, invasion and angiogenesis. For example, Korde et al 12 found that miR-1 levels were lower in NSCLC than cancerfree tissue and overexpression of miR-1 could reduce tumour growth and angiogenesis by targeting Mpl gene. Chiu et al 16 showed that ADAM9 down-regulated miR-1 expression, which in turn enhanced CDCP1 expression to promote lung cancer progression. Singh et al 17 reported that down-regulation of miRÀ1/À206 in NSCLC has major effects on carbon flux and the activity of metabolic pathways associated with cell proliferation and growth.
Furthermore, we and other researchers have found that low expression of miR-206 is related to lung cancer invasion and metastasis. 18,19 However, the role of miR-1-3p and miR-206 in HGFinduced gefitinib resistance of lung cancer is not clear.
This study was performed to investigate whether miR-1-3p and miR-206 increased the sensitivity of HGF-induced gefitinib resistance in EGFR mutant lung cancer cells. We assessed this issue using human lung cancer cells, PC9 and HCC-827, both harbouring deletions in exon19 of EGFR. The results showed that HGF-induced gefitinib resistance and miR-1-3p and miR-206 could circumvent the HGF-induced gefitinib resistance by targeting c-Met-Akt-Erk pathway and suppressing epithelial-mesenchymal transition (EMT).

| Cell cultures
The EGFR mutant (exon 19 deletion) human lung adenocarcinoma cell lines, PC-9, were provided by Affiliated Hangzhou Hospital of Nanjing Medical University. HCC827 cell line was purchased from the Cell Bank at the Chinese Academy of Sciences. Both the cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS and antibiotics.

| miRNA transfection
The miRNA mimics or negative control mimics were chemically synthesized by GenePharma Co., Ltd. (shanghai, China), and the transfection was conducted as we described previously. 20 All miRNA sequences are listed in Table S1. Transfection efficiency was confirmed by SYBR green (Takara) real-time PCR detection of miR-1-3p and miR-206 expression.

| c-Met knockdown and overexpression
For knockdown of c-Met, c-Met shRNA expression vector (PGPU6/ GFP/neo-shRNA-c-Met, designated as sh-Met) and the control vector PGPU6/GFP/neo-shControl (designated as sh-NC) were provided by GenePharma Inc (Shanghai, China). c-Met shRNA sequences are listed in Table S2

| Cell viability assay
The cell viability was detected by MTT method. Tumour cells (4 9 10 3 per well) were plated into 96-well plates and allowed to adhere overnight. Then, the cells were transfected with miRNA mimics or mimic NC. After 24 hours of incubation, different concentrations of gefitinib (0.001-1 lmol/L) and/or HGF were added, and incubation was continued for 48-72 hours. The cell survival rate was determined with MTT solution (5 mg/mL; Sigma). Each sample was plated in quintuplicate, and three independent experiments were performed.

| Migration and invasion assay
Wound healing experiment and transwell assays were used to determine the migration and invasion abilities of the cells, respectively.
The experiments were conducted as we described previously. 21

| Quantitative RT-PCR
Total RNA was isolated from tumour cells by Trizol reagent (Invitrogen) following manufacture's protocol. cDNA was synthesized by cDNA Synthesis Kit (Invitrogen), and RT-PCR was performed using SYBR green methods (Takara). U6 RNA was used as a miRNA JIAO ET AL.

| Western blot analysis
Cells were harvested and lysed on ice for 30 minutes in RIPA buffer (Beyotime) supplemented with protease inhibitor cocktail (Roche).
Lysates were subjected to Western blotting assay as described previously 21 and detected with antibodies against p-Met, c-Met, p-EGFR, EGFR, phospho-AKT, total AKT, phospho-ERK, total ERK, GAPDH, E-cadherin, Vimentin and Snail (Cell signalling technology).   Table S4; Cells were co-transfected with 25 ng of c-Met 3 0 -UTR reporter constructs and 20 nmol/L miR-1-3p or miR-206 mimics using Lipofectamine 2000 (Invitrogen) in 24-well plates. Luciferase assays were performed 24 hours after transfection using the Dual-Luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity. Gefitinib (25 mg/kg/d) was used by oral gavage. MiR-1-3p and miR-206 agomirs and their negative control (NC) (2 nmol; Genepharma, Shanghai, China) were given locally by direct injection into the xenografts every 3 days. In addition, we set 3 days of stop gefitinib (stop-GE) interval to confirm the effect of gefitinib and combined treatment. Tumour volumes were determined (in cubic millimetre) by measuring in two directions and were calculated as tumour volume = length 9 (width) 2 /2.

| Statistical analysis
All data were presented as the means AE standard deviation. Differences between samples were analysed using two-tailed Student's t test. P < .05 was considered statistically significant.

| RESULTS
3.1 | HGF induced gefitinib resistance and decreased expression of miR-1-3p and miR-206 in EGFR mutant lung cancer cells PC-9 and HCC827 cell lines (both harbouring EGFR exon 19 deletion) are highly sensitive to gefitinib. Our MTT results showed that HGF can induce gefitinib resistance in these two cells ( Figure 1A,B), which is consistent with previous report. 22 In addition, we found that HGF slightly promoted proliferation of PC-9 and HCC827 cells under our experimental conditions ( Figure S1A,B), but significantly enhanced the migration and invasion activities of two cells ( Figure S1C-F).
miR-1-3p and miR-206 have been identified as tumour suppressors in various human cancers, including lung cancer. 6,12,17,18,23,24 Exogenous expression of these two miRNAs could significantly reduce cell motility and invasiveness. 25 In this study, we evaluated miR-1-3p and miR-206 expression in HGF stimulation of PC-9 and HCC827 cells. Real-time PCR results showed that the expression levels of both miR-1-3p and miR-206 were dramatically decreased as compared with control cells ( Figure 1C,D). In addition, we found that gefitinib increased the expression of miR-1-3p and miR-206, whereas HGF attenuated this effect in both cells ( Figure S2A,B).
Then, we transfected miR-1-3p and miR-206 mimics to increase the expression of miR-1-3p and miR-206 in PC-9/HGF and HCC827/HGF cells, MTT assay was used to detect gefitinib sensitivity of these cells. The results showed that either miR-1-3p or miR-206 mimics could obviously reversed HGF-induced gefitinib resistance compared with mimics negative control (NC) ( Figure 2C).
These results suggested that miR-1-3p/miR-206 are potential suppressors of HGF-induced gefitinib resistance in PC-9 and HCC827 cells.   Figure S3). However, the combination of miR-1-3p or miR-206 mimics and gefitinib, significantly inhibited HGF-induced pc-Met expression. Moreover, the combination of miR-1-3p or miR-206 mimics and gefitinib also obviously inhibited phosphorylation of Akt and Erk1/2 in both cell lines. Interestingly, we also found that miR-1-3p or miR-206 mimic transfection could suppress the EGFR expression in PC-9 and HCC827 cells, which may increase the effect of two miRNAs on overcoming gefitinib resistance. Therefore, our results suggested that miR-1-3p/miR-206 is able to down-regulate c-Met and EGFR expression and inhibit downstream Akt and Erk pathways in HGF-induced gefitinib-resistant NSCLC cells.

| miR-1-3p/miR-206 inhibits EMT in HGFinduced gefitinib-resistant cells
Epithelial-mesenchymal transition (EMT) is extensively correlated with therapeutic resistance to EGFR-TKIs, 32,33 and we next examined whether miR-1-3p and miR-206 could reverse EMT in HGF-F I G U R E 2 miR-1-3p and miR-206 overcame HGF-induced gefitinib resistance in PC-9 and HCC827 cells. A, HGF overexpression lentivirus increased the production of HGF in PC-9 and HCC827 cells. The cells were incubated in medium contained lentivirus for 48 h, and culture supernatants were harvested. The level of HGF was determined by ELISA. B, HGF overexpressed PC-9 and HCC827cells increased gefitinib resistance. The PC-9/NC, HCC827/NC, PC-9/HGF, HCC827/HGF cells were incubated with increasing concentrations of gefitinib for 72 h. showed that PC-9/NC tumours regressed rapidly in response to gefitinib treatment. Surprisingly, when we stopped gefitinib for 3 days (day14-16), PC-9/NC tumour grew again. Finally, PC-9/NC tumours disappeared after 12 days of gefitinib treatment, whereas PC-9/HGF tumours were slightly suppressed following gefitinib treatment ( Figure 7A). Importantly, the combination of miR-1-3p (or miR-206) and gefitinib reduced the size of PC-9/HGF tumours (Figure 7A,B). Furthermore, MiR-206+GE is more effective than MiR-1-3p+GE in our mouse models, which is consistent with the results in vitro. These results suggest that HGF can induce resistance to gefitinib in vivo and that this resistance can be overcome by miR-1-3p and miR-206.

| DISCUSSION
Many miRNAs have been reported down-regulated in lung cancer, such as miR-34b/c, 7 miR-1, 25 miR-206, 17 miR-145 4 and let 7. 5 Among them, miR-1-3p and miR-206, which belong to muscle-specific miRNAs and play key roles in skeletal muscle differentiation, have found exhibited inhibitory function in lung cancer growth, migration and invasion in recent years. [16][17][18] However, other functions of these two miRNAs are not known. In this study, we F I G U R E 3 c-Met is a direct target of miR-1-3p and miR-206 in PC-9 and HCC827 cells. A, Alignment between the predicted binding motifs within the c-Met 3 0 UTRs and miR-1-3p/miR-206. Complementary sequences to the seed regions of miR-1-3p/miR-206 are also indicated. B, Target verification for miR-1-3p/miR-206 in PC-9 and HCC827 cells. PC-9 and HCC827 cells were co-transfected with miR-1-3p or miR-206, pmirGLO-c-Met-wt (3 0 UTR-wt) or pmirGLO-c-Met-mut (3 0 UTR-mut), along with a pRL-SV40 reporter plasmid. After 24 h, the luciferase activity was measured. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. *P < .05, **P < .01. C, c-Met expression after miR-1-3p or miR-206 mimics transfection was determined by Western blot analysis in PC-9 and HCC827 cells reported that miR-1-3p and miR-206 can overcome HGF-induced gefitinib resistance in EGFR mutant lung cancer cells in vitro and in vivo, and the mechanisms were related to inhibit Akt/Erk pathways and meanwhile suppress HGF-induced EMT (Figure 8). The study showed the novel anti-tumour function of miR-1-3p and miR-206 in lung cancer.
MiR-1-3p and miR-206 belong to the same miRNA cluster, which are frequently down-regulated in various types of cancers. F I G U R E 5 miR-1-3p/miR-206 suppresses c-Met/Akt and Erk pathway in HGF-mediated gefitinib-resistant cells. miR-1-3p and miR-206 inhibited Akt and Erk1/2 signalling, even in HGF treated PC-9 (A) and HCC827 (B) cells. PC-9 and HCC827 cells were transfected with miR-1-3p or miR-206 mimics and then treated with gefitinib (1 lmol/L) in the presence/ absence of HGF (50 ng/mL) for 1 h, and cell extracts were prepared and immunoblotted with the indicated antibodies. GE: gefitinib DNA methylation and acetylation of histones are two major epigenetic regulation mechanisms in miR-1-3p and miR-206 expression; meanwhile, many nuclear transcription factors, such as NRF2, 17 nuclear receptor subfamily 0, group B member 2, oestrogen-related receptor c, yin-yang 1, and activator protein 1, were reported involved in miR-206 regulation. 34,35 In this study, we showed that HGF induced a decrease in miR-1-3p/miR-206 expression in both PC-9 and HCC-827 cells. In previous study, it has been reported that HGF stimulation enhanced Nrf2 activity in myotubes, 36  Epithelial-mesenchymal transition is another mechanism of acquired EGFR-TKI resistance in lung cancers. In vitro studies showed that the mesenchymal phenotype is more resistant to EGF-TKI than the epithelial phenotype. 45 Activated HGF/c-Met pathway drives a mesenchymal phenotype in liver cancer has been reported. 46 In our study, both morphologic observation and molecular marker detection by Western blot and immunofluorescence stain showed that HGF stimulation induced EMT in PC-9 and HCC-827 cells. We observed an elongated cell morphology, loss of E-cadherin and increase in vimentin and snail expression. Whereas transfection of miR-1-3p and miR-206 caused HGF-expressed PC-9 and HCC-827 cells to undergo mesenchymal-epithelial transition, the reverse of EMT. Together these findings indicate that suppressing EMT is another critical factor that miR-1-3p and miR-206 overcoming HGF-induced gefitinib resistance. Previous study reported that miR-1 regulated EMT by directly target Slug gene in prostate cancer. 47 However, whether EMT-related genes are target directly by miR-1-3p and miR-206 need further experimental verification.
In summary, we demonstrated in vitro and in vivo that miR-1-3p and miR-206 can restore HGF-induced gefitinib resistance in EGFR activating lung cancer cells. The effects are mediated by inhibition of Akt/Erk pathways and EMT.

ACKNOWLEDG EMENTS
This work has been supported by Natural Science Foundation of

CONFLI CTS OF INTEREST
The authors declare no conflict of interest. F I G U R E 8 Proposed models on the inhibitory role of miR-1-3p/ miR-206 in HGF-induced gefitinib resistance. As depicted in the model, miR-1-3p/miR-206 overcome HGF-induced gefitinib resistance by targeting c-Met-Akt/Erk pathway and epithelialmesenchymal transition (EMT) in lung cancer with EGFR activating mutation