Decreased Expression of miR216a Contributes to Non–Small-Cell Lung Cancer Progression Free

Lung cancer is the leading cause of cancer-related mortality worldwide (1–3). Among lung cancers, non–small-cell lung cancer (NSCLC) accounts for approximately 80% of lung cancer cases, and more than 70% of patients with NSCLC present with advanced disease (4). Furthermore, the overall 5-year survival rate for NSCLC is only 16% (4). Although the leading causes of death in patients with lung cancer are metastasis and the development of chemoresistance (5–7), the molecular mechanisms underlying the initiation of these events are unclear. Therefore, a better understanding of these mechanisms will facilitate the development of novel therapeutic targets and strategies for the treatment of NSCLC (8).

MicroRNAs (miRNA) comprise a family of small, noncoding RNA molecules that play important roles in many normal biologic processes, including cell proliferation, differentiation, apoptosis, and organ development. Most miRNAs function by negatively regulating gene expression by directly binding to the 3′-untranslated region (3′-UTR) of a target gene mRNA, which induces mRNA cleavage or translational repression (9, 10). Interestingly, recent studies suggest that miRNAs also play important roles in cancer initiation and progression. Indeed, accumulating evidence suggests that certain miRNAs undergo aberrant regulation during carcinogenesis, leading to therapeutic resistance and metastasis in many cancers, including lung cancer (11). For example, miR31, miR135b, and miR494 have been identified as being functionally associated with cancer cell proliferation, invasion, and metastasis (12–14). In addition, these studies have demonstrated that inhibition of these miRNAs can suppress cancer progression, indicating that miRNAs may be an effective therapeutic target for cancer treatment.

In addition, a number of miRNAs that are deregulated in lung cancer have been revealed by microarray profiling in lung cancer tissues compared with normal tissues, including miR216a (15, 16). However, there have been no reports describing the biologic role of miR216a in lung cancer. Here, we demonstrate that decreased levels of miR216a are closely related to poor clinical outcomes in patients with NSCLC. Our data indicate that miR216a is a tumor-suppressor miRNA that inhibits NSCLC growth by suppressing cell proliferation and inducing apoptosis. The overexpression of miR216a inhibits NSCLC metastasis and enhances the sensitivity of NSCLC cells to cisplatin (diamminedichloroplatinum, CDDP) treatment. In addition, we demonstrate that miR216a exerts its tumor-suppressing activity by directly targeting eukaryotic initiation factor 4B (eIF4B) and Zinc finger E-box- binding homeobox 1 (ZEB1).

Fetal bovine serum (FBS), sodium bicarbonate, l-glutamine, nonessential amino acids, insulin, epidermal growth factor, transferrin, hydrocortisone, 3-(4,5-dimethylthiazol-2-yl)-2 and 5-diphenyltetrazolium bromide (MTT), puromycin, CDDP, anti-caspase 3 antibody, anti-actin antibody, anti-flag antibody, and cell culture medium were purchased from Sigma. The QuantiTect SYBR Green PCR Kit was purchased from Qiagen. A miR216a expression vector, miR216a inhibitor (Vector Systems, Inc), eIF4B expression plasmid, shRNA of eIF4B, ZEB1 expression plasmid and shRNA of ZEB1 were obtained from GeneCopoeia. Protein A/G beads and antibodies against to eIF4B, E-cadherin, Vimentin, Ki67, and cleaved PARP were purchased from Santa Cruz Biotechnology. ZEB1 antibody was obtained from Novus Biologicals. Dual-Luciferase Assay Kit and Lipofectamine 2000 were obtained from Promega and Invitrogen, respectively. TRIzol, Opti MEM, High-Capacity cDNA Reverse Transcription Kit, miRNA expression reporter vector, pre-miR216a, miR216a primer set, and antisense nucleotides of miR216a were purchased from Life Technologies. Chamber Slide and Invasion Assay Kits were obtained from BD Biosciences.

NSCLC cell lines HOP-62, H522, H23, and A549 were cultured in RPMI-1640 medium supplemented with 10% FBS. Normal lung bronchial epithelial cell NL20 was cultured in Ham's F12 medium supplemented with 1.5 g/L sodium bicarbonate, 2.7 g/L glucose, 2.0 mmol/L l-glutamine, 0.1 mmol/L nonessential amino acids, 5 μg/mL insulin, 10 ng/mL epidermal growth factor, 1 μg/mL transferrin, 500 ng/mL hydrocortisone, and 4% FBS.

Frozen lung cancer specimens (collected before treatment) from 191 patients with newly diagnosed NSCLC at the General Hospital of the People's Liberation Army were analyzed for the expression of miR216a. Specimens from patients with stage I–IIIA NSCLC were collected during surgery. Specimens from patients with stage IIIB–IV NSCLC were collected via biopsy. Fresh samples were divided into two aliquots. One aliquot was snap frozen in liquid nitrogen immediately following collection and stored at −80 °C for RNA extraction, and the second aliquot was paraffin-embedded for histologic evaluation. Only tissues containing more than 70% tumor cells were used for mRNA isolation. The patients' characteristics are summarized in Table 1. The collection and use of tissue samples were approved by the ethical review committees of the appropriate institutions.

Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol. Mature miR216a and the RNU6 endogenous control were analyzed using the TaqMan microRNA Assay Kit. The relative expression of miR216a was normalized against RNU6 expression using the |$2^{- {\rm \Delta}C_{\rm t}}$| method, and the miR216a expression fold change in lung cancer samples matched to nontumor control samples was evaluated using the |$2^{- {\rm \Delta \Delta}C_{\rm t}}$| method. On the basis of the mean fold change of miR216a expression, patients were divided into high- (fold change > mean) and low- (fold change < mean) miR216a expression groups (17).

For analysis of other genes expression, RT and PCR were performed with a High-Capacity cDNA Reverse Transcription Kit and QuantiTect SYBR Green PCR kit, respectively. The primer sequences for genes were defined as follows: eIF4B forward, 5′-AGCGTCAGCTGGATGAGCCAA-3′; reverse, 5′-TGTCCTCGACCGTTCCCGTT-3′; ZEB1 forward, 5′-AGCAGTGAAAGAGAAGGGAATGC-3′; reverse, 5′-GGTCCTCTTCAGGTGCCTCAG-3′; GAPDH forward, 5′-GCAGGGGGGAGCCAAAAGGGT-3′; and reverse, 5′-TGGGTGGCAGTGATGGCATGG-3′.

Putative target genes of miR216a were predicted using TargetScanHuman 6.2, and several genes that were reported as oncogenes in lung cancer were selected from the predicted target genes. Then, a luciferase reporter assay was used to identify genes that are directly regulated by miR216a. Next, the correlation between miR216a and target gene expression was investigated. Finally, genes were selected whose expression was inversely correlated with miR216a expression in NSCLC.

3′-UTR segments of ZEB1 and eIF4B that were predicted to interact with miR216a were amplified by PCR from human genomic DNA and inserted into the MluI and HindIII sites of the miRNA Expression Reporter Vector. For the luciferase reporter experiments, Hela cells were seeded into 24-well cell culture plates at a concentration of 1 × 10⁴ per well. Next day, the cells were transfected with the indicated reporter plasmids containing firefly luciferase and either the pre-miR-216 or control oligonucleotides (Ctrl. oligo) or antisense nucleotides of miR216a (ASO miR216a). The Renilla luciferase plasmid was cotransfected as a transfection control. Cells were lysed 36 hours after transfection, and luciferase activity was measured by a Dual-Luciferase Assay System according to the manufacturer's protocol. The luciferase activity was normalized by the activity of Renilla luciferase. Each experiment was repeated at least three times in triplicate.

Western blotting and immunohistochemical assays were performed as described previously (18). For immunofluorescence (IF) assays, cells were transfected with the indicated oligonucleotides. The following day, transfected cells were seeded onto chamber slides. Following 24 hours of incubation, the IF assay was performed as described previously (19). For the RIP-Chip assay, Ago2-flag plasmid and pre-miR216a or control oligonucleotides were cotransfected into A549 cells. Following 48 hours of incubation, cells were harvested and subjected to the RIP-Chip assay. The RIP-Chip assay was performed as described by Keene and colleagues (20).

Cells were transfected with the indicated nucleotides using Lipofectamine 2000. After 6 hours of transfection, cells were plated into 96-well (4 × 10³ cells per well) and 6-well (5 × 10⁵ cells per well) plates for MTT and proliferation assay, respectively. For MTT assay, cells were incubated with the indicated drug for 48 hours. The cell viability was measured using MTT according to the manufacturer's protocol. For proliferation assay, cell counts were estimated by trypsinizing the cells and performing analysis in triplicate with a Coulter counter at the indicated time points.

After 24 hours of transfection, cells were seeded in 6-well cell culture plates. Cells were incubated with indicated drugs. Twenty-four hours later, cells were harvested, then, stained with annexin V and 7-aminoactinomycin D (7-AAD) according to the manufacturer's protocol, followed by flow cytometric analysis.

Cells were transfected with the indicated nucleotides or/and plasmid for 24 hours, and then 10,000 cells in growth medium without serum were seeded in the upper wells of BD Chambers. The lower wells contained the same medium with 10% serum. After 24 hours, the cells that had invaded to the lower side of the chamber were fixed with 2.5% glutaraldehyde, stained with 0.1% crystal violet, and counted.

Indicated cells were transfected with miR216a expression vector or antisense-miR216a expression vector. After 48 hours of transfection, cells were incubated with 2 μg/mL of puromycin for 1 week. The expression of miR216a was measured after 7 days of treatment with puromycin and frozen down in aliquots for later use.

Stably expressing miR216a or miR216a-antisense cells were used to generate the lung cancer xenograft model. For the subcutaneous tumor growth assay, 2 × 10⁶ cells in 0.1 mL of phosphate-buffered saline (PBS) were subcutaneously injected into male nude mice (n = 8 per group). After 5 weeks, these mice were sacrificed and the tumor weights were measured.

For the orthotopic tumor implantation assays, 1 × 10⁵ cells in 20 μL of PBS containing 10 ng of Matrigel were injected into the pleural cavity of 5-week-old SCID mice (n = 8 per group). The mice were sacrificed by carbon dioxide anesthesia 28 days after implantation and the lungs were removed and fixed in 10% formalin. The lung surface nodules were counted under microscopy.

For intravenous injections of the tumor cells, 5 × 10⁵ cells were suspended in 0.1 mL of PBS and injected into the lateral tail vein of nude mice (8 mice per group). At 4 weeks after injection, all mice were killed, and lung surface tumor foci were counted.

All data are presented as mean ± standard deviation (SD), and significant differences between treatment groups were analyzed by a Student t test or one-way analysis of variance (ANOVA) and Duncan multiple range test using SAS statistical software version 6.12 (SAS Institute). The survival rate of patients with NSCLC was calculated using Kaplan–Meier survival analysis. The significance of multiple predictors of survival was assessed using Cox regression analysis. Differences were considered statistically significant at a P value of less than 0.05.

To investigate miR216a expression in NSCLC, we performed real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). As shown in Fig. 1A and B, the expression of miR216a was significantly decreased in human NSCLC cell lines and human primary NSCLC tissues compared with normal lung epithelial cell lines (NL20 and AALE) and nontumor human lung tissues, respectively. Our clinical data indicate that the expression level of miR216a is inversely associated with tumor metastasis and clinical stage in patients with NSCLC (Table 1). In addition, Kaplan–Meier survival analysis demonstrates that the expression level of miR216a is correlated with NSCLC patient survival (Fig. 1C). Data show that patients in high-miR216a group have a low number of deaths and longer median overall survival time compared with patients in low-miR216a group. Importantly, multivariable analyses show that miR216a is an independent predictor of NSCLC patient prognosis (Table 2). Taken together, these results suggest that miR216a may play an important role in NSCLC progression.

Next, we investigated the effects of miR216a on NSCLC cell growth by overexpressing or inhibiting miR216a in several NSCLC cell lines (Supplementary Fig. S1A). As shown in Fig. 2A and supplementary Fig. S1B, overexpression of miR216a significantly suppressed NSCLC cell growth. Conversely, depletion of miR216a promoted NSCLC cell growth. We further confirmed these results using a clonogenic assay in A549 and H23 cells (Supplementary Fig. S1C). Results show that the overexpression of miR216a inhibited A549 colony formation, whereas the inhibition of miR216a stimulated H23 colony formation when compared with the control. These results parallel those obtained from MTT assays. Finally, we confirmed these in vitro results using a xenograft model. Consistent with the results obtained from in vitro experiments, the overexpression or inhibition (Supplementary Fig. S2A) of miR216a suppressed (A549 xenograft tumor) or stimulated (H23 xenograft tumor) tumor growth, respectively, when compared with the control group (Fig. 2B).

Tumor growth is regulated by cell proliferation and apoptosis; thus, we examined the effect of miR216a on NSCLC cell proliferation and apoptosis. As expected, in vitro results indicate that miR216a overexpression significantly inhibited cell proliferation (Fig. 2C and supplementary Fig. S1D) and induced apoptosis (Supplementary Fig. S2B) in NSCLC cells. In contrast, the inhibition of miR216a stimulated cell proliferation of NSCLC cells (Fig. 2C and supplementary Fig. S1D) and inhibited apoptosis (Supplementary Fig. S2B). Consistent with these data, the expression of cell proliferation marker protein Ki67 was also significantly decreased in the miR216a-overexpressing A549 xenograft model samples compared with the vector control (Fig. 2D). Conversely, the inhibition of miR216a significantly increased Ki67 expression in the H23 xenograft model compared with the vector control group (Fig. 2D). In addition, the expression of cleaved PARP and caspase-3, which are both proapoptotic proteins, was measured in the tumor samples from the xenograft model. As shown in Supplementary Fig. S2C, the expression of cleaved PARP and caspase-3 was significantly increased following miR216a overexpression in A549 xenograft tumor samples and decreased following miR216a inhibition in H23 xenograft tumor samples. Together, these data suggest that miR216a inhibits NSCLC growth by inhibiting cell proliferation and inducing apoptosis.

The data from human specimens demonstrate that miR216a expression is significantly decreased in primary tumors from patients with NSCLC with metastasis compared with tumor samples from patients with no metastasis, suggesting that miR216a may be involved in NSCLC metastasis. Therefore, we investigated the effects of miR216a on NSCLC metastasis. Western blotting results revealed that the overexpression of miR216a increased E-cadherin expression and decreased vimentin expression in A549 cells. In contrast, the inhibition of miR216a led to a reduction in E-cadherin expression and an increase in vimentin expression in H23 cells (Supplementary Fig. S3A). Furthermore, we confirmed these results using IF analysis, and we observed similar results to those obtained via Western blotting (Supplementary Fig. S3B). Moreover, the overexpression of miR216a suppressed HOP62 cell invasion in vitro, whereas miR216a inhibition stimulated invasion in H522 cells (Fig. 3A). In addition, we investigated whether miR216a could induce similar effects on metastasis in vivo using an orthotopic NSCLC model. A549 cells that stably express miR216a were injected into the pleural space of SCID mice, and lung surface tumor nodules were counted 28 days after implantation. The results show that overexpression of miR216a suppressed lung metastasis formation, whereas the inhibition of miR216a promoted lung metastasis (Fig. 3B). These results were confirmed using another model system. A549 cells stably expressing miR216a or H23 cells stably expressing antisense miR216 were injected into nude mice through the tail vein, and these mice were sacrificed 1 month after injection. Similar to the results observed using the orthotopic model, overexpression of miR216a (A549 cells) significantly reduced the number of lung metastasis, whereas the inhibition of miR216a (H23 cells) increased the number of lung metastasis compared with the control group (Fig. 3C).

Using miRNA target prediction algorithms, we identified eIF4B and ZEB1 as tentative targets of miR216a (Fig. 4A). As shown in Fig. 4B, the expression levels of eIF4B and ZEB1 were significantly decreased following the ectopic expression of miR216a; this decrease in expression was observed at both the mRNA and protein level. In contrast, the inhibition of miR216a led to an increase in the expression of eIF4B and ZEB1 at both the mRNA and protein level (Fig. 4B). To determine whether the regulation of eIF4B- and ZEB1-luciferase expression depends on the binding of their complementary 3′-UTR sequences to the miR216a seed sequence, a three-nucleotide mutation was inserted into the eIF4B and ZEB1 3′-UTRs, as indicated in Fig. 4A. Ectopic expression of miR216a significantly repressed luciferase activity in the wild-type 3′-UTR setting. In contrast, both 3′-UTR mutations completely abrogated the effect of miR216a overexpression on luciferase activity in Hela cells (Fig. 4C). Furthermore, we confirmed these results using an anti-Ago2 RIP-Chip assay. Results indicate that miR216a increased the interaction between the Ago2 complex and eIF4B or ZEB1 mRNA (Supplementary Fig. S4). In addition, the expression of miR216a was inversely correlated with ZEB1 and eIF4B expression in (Fig. 4D and Supplementary Fig. S5A) NSCLC patient specimens. Cumulatively, these data suggest that miR216a negatively regulates the expression of eIF4B and ZEB1 by directly targeting their 3′-UTR sequences.

To determine whether eIF4B and ZEB1 directly contribute to miR216a function, eIF4B and ZEB1 were overexpressed in miR216a-overexpressing A549 cells. Cells were then analyzed for cell growth, proliferation, apoptosis, and metastasis. As expected, exogenously expressed eIF4B and ZEB1 blocked the inhibition of colony formation, cell proliferation, metastasis and induction of apoptosis by miR216a (Fig. 5). Moreover, our clinical data show that NSCLC patient survival was inversely correlated with eIF4B and ZEB1 expression (Supplementary Fig. S5B). Taken together, these data suggest that eIF4B and ZEB1 are important players responsible for the effects of miR216a on NSCLC cell growth and metastasis.

We also evaluated other target genes of miR216a, including PTEN and SMAD7; these proteins have been reported to be regulated by miR216a in liver cancer (21). However, our data indicated that the overexpression of miR216a did not alter the expression of these proteins in NSCLC cells (Supplementary Fig. S6A). Finally, we investigated the effect of miR216a on eIF4B and ZEB1 expression in HepG2 liver cancer cells. Data demonstrate that miR216a decreased the expression of eIF4B but had no effect on ZEB1 expression (Supplementary Fig. S6A). However, clinical data suggest that miR216a and eIF4B expression are not inversely correlated in patients with liver cancer (Supplementary Fig. S6B).

Recent evidence indicates that eIF4B and ZEB1 play important roles in chemoresistance, and that these two proteins are miR216a target genes, suggesting that miR216a may also play a role in lung cancer chemoresistance. Therefore, we investigated the effect of miR216a on NSCLC chemoresistance by overexpressing or inhibiting miR216a in NSCLC cells. As expected, overexpression of miR216a significantly enhanced CDDP-induced inhibition of NSCLC cell growth (Fig. 6A) and apoptosis (Fig. 6B and C). In contrast, inhibition of miR216a blocked CDDP-induced cell growth inhibition (Fig. 6A) and apoptosis (Fig. 6B and C). Furthermore, we determined whether eIF4B and ZEB1 directly contribute to the function of miR216a with respect to chemosensitivity and found that the restoration of eIF4B and/or ZEB1 (Supplementary Fig. S7) can block the overexpressed miR216a–enhanced cell growth inhibition effects of CDDP (Fig. 6D).

Here, we found that miR216a was indeed downregulated in NSCLC. Importantly, we found that levels of miR216a were inversely correlated to NSCLC metastasis, clinical stage, and chemoresistance. In addition, a series of in vitro and in vivo experiments demonstrated that decreased expression of miR216a stimulates NSCLC growth, metastasis, and chemoresistance. Metastasis, TNM stage (22), and chemoresistance (23) are important prognostic factors and are associated with poor survival in patients with lung cancer. In fact, Kaplan–Meier survival analysis revealed that patients with NSCLC whose primary tumors displayed low-miR216a expression had decreased overall survival. These data suggest that decreased expression of miR216a significantly contributes to NSCLC progression via affecting tumor growth, metastasis, and chemoresistance. In addition, multivariate analysis indicates that miR216a is an independent prognostic factor for NSCLC patient survival, suggesting that miR216a may be a useful biomarker for predicting NSCLC disease progression.

We also provide insights about the biologic effects of miR216a in NSCLC. Our data provide the first evidence that the inhibition of miR216a stimulates NSCLC cell growth by increasing cell proliferation and inhibiting apoptosis in vitro and in vivo. Increased cell proliferation and defects in apoptosis are implicated in the progression of virtually all types of human cancers (24, 25). Thus, one strategy for cancer treatment is the inhibition of cell proliferation and induction of apoptosis. Moreover, accumulating evidence shows that inhibition of cell proliferation and induction of apoptosis can suppress tumor growth and metastasis (24, 26). Our data demonstrate that overexpression of miR216a significantly inhibits NSCLC cell proliferation and induces apoptosis in vitro and in vivo. Furthermore, our in vivo data show that miR216a significantly inhibits tumor growth and lung metastasis, suggesting that miR216a functions as a tumor suppressor and that overexpression of miR216a may be a novel strategy for NSCLC treatment. In contrast with our results, Xia and colleagues (21) indicated increased miR216a expression in liver cancer and showed that miR216a functions as a cancer promoter, causing drug resistance and recurrence of liver cancer by targeting PTEN and SMAD7. It is believed that some individual miRNAs may function as a tumor promoter or a tumor suppressor in different tumor types (27–29). miRNAs act by regulating target genes, and a single miRNA can target hundreds of genes while function as both a tumor promoter and suppressor (30); thus, one miRNA can play different roles by targeting different genes. Even the same miRNA in the same tumor can play different roles at different stages of cancer progression. For example, miR200s inhibits local invasion by targeting ZEB1/2 but also promotes lung metastatic colonization by targeting Sec23a in breast cancer (31). In our study, we did not identify any significant miR216a-dependent downmodulation of PTEN or SMAD7 expression in NSCLC, suggesting that these two genes are not major targets of miR216a in NSCLC. In addition, our data demonstrate that eIF4B and ZEB1 are not major targets of miR216a in liver cancer.

As described above, miRNAs can play multiple roles by targeting different genes. Thus, investigating their target genes remains important for understanding the molecular mechanisms by which miRNAs promote or suppress cancer progression. In this study, we identified eIF4B and ZEB1 as miR216a target genes. Our data demonstrate that the restoration of miR216a expression in lung cancer cells leads to the suppression of eIF4B and ZEB1 expression; conversely, the inhibition of miR216a further upregulates eIF4B and ZEB1 expression. Luciferase reporter gene experiments show that miR216a directly targets the 3′-UTR of eIF4B and ZEB1. Furthermore, decreased eIF4B and ZEB1 expression was observed in the miR216a high-expression NSCLC specimens. The transcription factor ZEB1 and translation initiation factor eIF4B are reported to play important roles as oncogenes, and studies have shown that ZEB1 and eIF4B can stimulate cancer progression by increasing cell proliferation and metastasis and inhibiting apoptosis (32–36). Our data indicate that the restoration of ZEB1 and eIF4B blocked the miR216a overexpression-induced inhibition of cancer growth, cell proliferation, metastasis, and the induction of apoptosis. In addition, our clinical data show that expression of eIF4B and ZEB1 is inversely correlated with miR216a expression, and that patients with high expression of ZEB1 or eIF4B have a decreased survival rate. Taken together, these data suggest that eIF4B and ZEB1 are major players responsible for the effects of miR216a on NSCLC cell growth, proliferation, apoptosis, and metastasis.

CDDP is a platinum-coordinated complex that is widely used as a first-line chemotherapeutic agent for the treatment of human NSCLC (37). However, studies have shown that continuous infusion or multiple administrations of CDDP often results in the development of drug resistance, which often leads to treatment failure as demonstrated by tumor growth or tumor relapse (38, 39). Recently, studies have established that miRNAs are also involved in the development of chemoresistance (40–42). Our data indicate that decreased miR216a expression contributes to CDDP resistance in NSCLC cells. In contrast, overexpression of miR216a dramatically enhances the sensitivity of NSCLC cells to CDDP treatment. In addition, we demonstrate that miR216a-regulated CDDP resistance in NSCLC cells is due to the negative regulation of its downstream target genes, eIF4B and ZEB1. As described above, eIF4B and ZEB1 function as oncogenes in tumors, and studies have shown that eIF4B (36) and ZEB1 (34) contribute to the development of chemoresistance. However, the expression levels of eIF4B (15.3% of patients with NSCLC) and ZEB1 (8.6% of patients with NSCLC) are increased in a small percentage of patients with NSCLC. Therefore, miR216a might not be a predictor of patient outcome following CDDP therapy in patients with NSCLC.

In summary, we combined clinical and experimental studies to determine the role of miR216a in the progression of NSCLC. Our work provides new insights about the mechanisms of miR216a activity in tumor growth, metastasis, and chemoresistance. Our findings may also aid in the development of potential therapeutics for the treatment of NSCLC.

No potential conflicts of interest were disclosed.

Conception and design: C.-X. Xu, H. Jin

Development of methodology: R.-T. Wang, M. Xu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.-T. Wang, M. Xu, Z.-G. Song

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.-T. Wang, M. Xu, Z.-G. Song, H. Jin

Writing, review, and/or revision of the manuscript: M. Xu, C.-X. Xu, H. Jin

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.-T. Wang

Study supervision: C.-X. Xu, H. Jin

This study was supported by the China International Medical Foundation (CIMF-F-H001-240).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.