Lung cancer is the leading cause of cancer-related fatalities worldwide, and non-small cell lung cancer (NSCLC) is the main pathological type. MicroRNAs (miRNAs or miRs) are a class of small non-coding RNAs, which are involved in tumor initiation and progression. miR-223 is a tumor suppressor miRNA that has been reported in various types of cancer, including lung cancer. In the present study, to characterize the biological behavior of miR-223 in NSCLC, we established an miR-223 overexpression model in erlotinib-resistant PC-9 (PC-9/ER) cells by infection with lentivirus to induce the overexpression of miR-223. As a result, miR-223 enhanced the sensitivity of the PC-9/ER cells to erlotinib by inducing apoptosis
Lung cancer remains a leading cause of cancer-related mortality worldwide. Non-small cell lung cancer (NSCLC) accounts for 80–85% of all cancer cases, with an overall 5-year survival rate of <20% (
miRNAs are single-stranded RNAs, 20–22 nucleotides in length (
IGF-1R is a member of the insulin receptor (IR) family, which is well known for its role in the resistance of NSCLC cells to EGFR-TKIs (
The lung adenocarcinoma PC-9 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in RPMI-1640 medium, with 10% fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 U/ml penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA). All cells were cultured at 37°C in a humidified atmosphere of 95% air/5% CO2. Erlotinib (Sigma-Aldrich) was dissolved by dimethyl sulfoxide (DMSO) and stored at −20°C. The concentration used to maintain the resistance of PC-9/ER cells to erlotinib was 0.1
The PC-9 cell line harboring exon 19 del mutation acquired erlotinib resistance following 6 months of continuous drug exposure (1
The lentiviral vector GV259, used to induce miR-223 οverexpression, was packaged and purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China). The PC-9/ER cells (5×104) were infected with 2×106 lentivirus-transducing units in the presence of 10
Cell proliferation assay was carried out according to the instructions of the manufacturer of CCK-8 (Dojindo). Briefly, the PC-9 and PC-9/ER cells were seeded in 96-well plates at a density of 3.5×103 cells/well with 100
Total RNA was extracted from the cells using RNAiso Plus reagent (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's instructions. The expression of miR-223 was detected by one-step RT-qPCR with SYBR Premix Ex Taq (Takara Bio., Inc.). The specific primers of miR-223 and IGF-1R were designed by Shanghai GeneChem Co., Ltd. The relative expression levels were calculated by means of the 2−ΔΔCt method, relative to the internal controls, U6 RNA for miRNA and β-actin for genes, respectively. The following primers were used: IGF-1R forward, 5′-GGCATACCTCAACGCCAATA-3′ and reverse, 5′-CAGCCCTTTCCCTCCTTT-3′; β-actin forward, 5′-GTGAAGGTGACAGCAGTCGGTT-3′ and reverse, 5′-GAAGTGGGGTGGCTTTTAGGA-3′. All the RT-qPCR reactions were run in triplicate and repeated in 3 independent experiments.
Total protein was extracted from the cells using radio immunoprecipitation assay (RIPA) buffer and was quantified by a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Haimen, China). The proteins were separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then blocked for 1 h at room temperature with 5% BSA and were subsequently incubated overnight at 4°C with the following antibodies: Polyclonal rabbit anti-IGF-1R (cat. no. 3027), monoclonal rabbit anti-phosphorylated (p-)IGF-1R (cat. no. 3918), polyclonal rabbit anti-Akt (cat. no. 4691), polyclonal rabbit anti-p-Akt (cat. no. 4060), monoclonal rabbit anti-S6 (cat. no. 2217), monoclonal rabbit anti-p-S6 (cat. no. 4858) and polyclonal mouse anti-β-actin (cat. no. 3700; 1:1,000) (Cell Signaling Technology, Inc., Beverly, MA, USA). Following washing 3 times with Tris-buffered saline containing Tween-20 (1:500), the membranes were incubated with secondary antibodies-anti-rabbit IgG (cat. no. SA00001-2; 1:5,000) and anti-actin antibody (cat. no. 20536-1-AP; 1:5,000) (both from Proteintech Group, Chicago, IL, USA) for 1 h at room temperature as previously described (
A total of 5×105 cells was resuspended in phosphate-buffered saline (PBS) following exposure to 1
To induce exogenous IGF-1 expression, the PC-9/ER-miR-223 and PC-9/ER-EV cells were seeded in 6-well plates with RPMI-1640 medium containing 10% FBS. Once the cells reached 70% confluence, they were washed twice with serum-free medium to remove the FBS. Following overnight serum starvation, the cells were stimulated for 30 min at 37°C, 5% CO2 in serum-free medium containing 100 ng/ml of IGF-1 (cat. no. CYT-216; ProSpec, Rehovot, Israel), as previously described (
Nude mice (male, 4–6 weeks old) were purchased from the Chinese Academy of Medical Sciences (Beijing, China). They were housed in a laminar flow room under specific pathogen-free conditions at room temperature (22±2°C) and humidity (<40%) with free access to food and water. In tumor growth assay, a total of 18 mice was randomly divided into 6 groups. A total of 2×106 PC-9, PC-9/ER, PC-9/ER-miR-223 and PC-9/ER-EV cells was subcutaneously injected into the lower quadrant of the rats. When the tumor volumes reached approximately 100–150 mm3, erlotinib (60 mg/kg) was intragastrically administered from the 14th day to each mouse daily for 14 days. Moreover, the mice in the PC-9/ER-miR-223 and PC-9/ER-EV groups received a peritoneal perfusion of IGF-1 (50
Quantitative data are presented as the means ± standard deviation (SD). The Student's t-test was used to analyze data following testing for normal distribution and homoscedasticity. Statistical analysis was performed and the half inhibitory concentration (IC50) of erlotinib was calculated using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). A value of P<0.05 was considered to indicate a statistically significant difference. All the experiments were independently repeated at least 3 times.
Lentiviral gene transfer is capable of inducing a stable gain- and loss-of-function phenotype of cells. It is a critical tool to explore miRNA function in cell culture and animal models (
The CCK-8 assay revealed the sensitivity of the PC-9 and PC-9/ER cells (or miR-223 and EV) groups to erlotinib. The PC-9/ER cells were clearly more resistant to erlotinib than the PC-9 cells. The over-expression of miR-223 in the PC-9/ER cells markedly enhanced the sensitivity to erlotinib. The IC50 values of the PC-9, PC-9/ER, PC-9/ER-miR-223 and PC-9/ER-EV cells were: 0.25, 5.16, 1.19 and 5.29, respectively (
IGF-1R can be activated by the ligand IGF-1. The concentration and exposure time to IGF-1 were determined as follows: 60 min was the most appropriate duration to induce the re-expression of p-IGF-1R in the PC-9 cell line. The most appropriate concentration was 100 ng/ml (data not shown). We thus performed the following experiments under this condition. The levels of p-IGF-1R were significantly increased following stimulation with IGF-1 (
To further confirm the above findings, an
miRNAs have been reported to play important roles in tumor initiation and progression (
In our previous study, we found that IGF-1R is a target of miR-223 (
Despite the promising effects of TKIs, the acquired resistance must be overcome. The persistent activation of downstream signaling pathways, particularly PI3K/Akt, is sufficient to confer resistance to cells against EGFR-TKIs by bypassing EGFR blocking (
Techniques to inhibit IGF-1R have been investigated as promising therapeutic strategies against resistance to TKIs in NSCLC. To date, IGF-1R antibodies, including dalotuzumab (MK-0646), have been reported to be safe and able to reduce IGF-1R signaling in phase I and II clinical trials. The clinical activity of IGF-1R inhibitors has been demonstrated with sustained responses in a number of patients. However, in adult patients with tumors, including NSCLC, breast cancer and pancreatic cancer, have failed to reveal the clinical benefit of IGF-1R inhibitors in the overall patient population (
In conclusion, the present study hypothesized that combined EGFR/IGF-1R inhibition may improve the efficacy of targeted molecular therapy in erlotinib-resistant NSCLC. IGF-1R is a valid target for selected tumor types, including erlotinib-resistant lung cancer with a low expression of miR-223. By contrast, miR-223, an evolutionarily conserved miRNA, represents a potential biomarker for erlotinib-resistant NSCLC. Thus, the overexpression of miR-223 in TKI-resistant NSCLC may prove to be beneficial. The present study described the role of the IGF/IGF-1R system, and proposed additional novel strategies for targeting this system. Strategies to target this system have also been proposed previously (
The authors would like to express their gratitude to Mr. Dian-Gang Chen (Cancer Institute of PLA, Xinqiao Hospital, Third Military Medical University, China) for kindly providing the H1975 cell line and instructing the experimental skills. We should express our gratitude to the premium language editing service of Spandidos Publications. The present study was supported by the National Natural Science Foundation of China (grant no. 81172070), the Chongqing Science and Technology Key Project Fund (grant no. 2011AB5032), the Military Clinical Key Projects of New and High Technology (grant no. 2010gxjs070) and the Natural Science Foundation of Chongqing (grant no. cstc2012jjA10096).
Lentivirus-mediated miR-223 overexpression and insulin-like growth factor-1 receptor (IGF-1R) expression in the PC-9 cell line. (A) The cells were sorted by fluorescence-activated cell sorting. (B) The infection efficiencies of PC-9/ER-miR-223 and PC-9/ER-EV cells were observed by fluorescence microscopy, based on the green fluorescent protein fluorescent signal after 72 h. (C) miR-223 detection in the PC-9/ER cells by RT-qPCR. (D) The mRNA expression of IGF-1R was analyzed by RT-qPCR. (E) Western blot analysis of IGF-1R. (F) Histogram of panel (E) (n=3 experiments; *P<0.05).
Overexpression of miR-223 enhances cell sensitivity to erlotinib by inducing apoptosis. (A) The sensitivity of the PC-9, PC-9/ER, miR-223 and EV-infected PC-9/ER cells to erlotinib was determined by cell counting kit-8 (CCK-8) assay. IC50 of groups PC-9, PC-9/ER, PC-9/ER-miR-223 and PC-9/ER-EV: 0.25, 5.16,1.19 and 5.29. (B) The sensitivity to erlotinib of the PC-9/ER-miR-223 cells was assessed by CCK-8 assay in the presence or absence of insulin-like growth factor-1 (IGF-1) (100 ng/ml). (C and D) miR-223 enhanced cell apoptosis compared with the EV group following erlotinib treatment for 48 h as determined by flow cytometry. The apoptotic ratios were 18.92±2.123 vs. 11.13±1.127%. The data are expressed as the means ± SD (n=3 experiments; *P<0.05).
miR-223 inhibits insulin-like growth factor-1 receptor (IGF-1R)/Akt/S6 signaling by targeting IGF-1R and these inhibitory effects are reversed by exogenous IGF-1. (A and B) The protein levels of IGF-1R and p-IGF-1R in the miR-223- or EV-infected cells were detected at 0, 15, 30 and 60 min following stimulation with IGF-1. Statistical analysis of p-IGF-1R protein bands was performed following quantification using imaging analysis software. (C) Western blotting was performed to detect the expression levels of IGF-1R, p-IGF-1R, AKT, p-AKT, S6 and p-S6 in the PC-9 cell line following stimulation with IGF-1 for the indicated groups. Statistical analysis of the (D) p-IGF-1R, (E) p-AKT and (F) p-S6 protein bands was performed following quantification using imaging analysis software. The data are expressed as the means ± SD (n=3 experiments; *P<0.05).
miR-223 improves the tumor response to erlotinib in tumor xenografts. (A) Representative nude mice bearing xenografts in 6 groups. All the mice received erlotinib via intragastric administration for 14 days. (B) Growth curves of tumors in 6 groups (n=3 mice per group; *P<0.05). (C) Survival curves of nude mice in 6 groups (n=30). Continuous intragastric administration until morbidity or death (n=5 mice per group; *P<0.05).