H1650, PC-9 and HCC827 lung cancer cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These cell lines were cultured in RPMI containing 10% fetal bovine serum. All cells were cultured at 37°C in an incubator with 5% CO₂.

Paraffin-embedded tissue specimens were collected at the General Hospital, Jinan Command of the People's Liberation Army (Jinan, China) between October 2008 and May 2016 (Table I). All the patients involved were diagnosed with adenocarcinoma via biopsy or surgery. Tumor histology and subtypes were classified according to the World Health Organization criteria (26). Eastern Cooperative Oncology Group performance status (ECOG-PS) was used to assess the functional status of patients (27). In addition, the majority of the selected patients were treated with first-line EGFR-TKI treatment. Primary drug resistance was defined as progression within 3 months after the use of EGFR-TKIs, and the sensitive group had a PFS duration of >6 months. Informed consent was obtained from all patients, and the study was approved by the Ethics Committee of the General Hospital (Jinan Command of the People's Liberation Army, Jinan, China).

Gefitinib was purchased from LC Laboratories (cat. no. G-4408; New Boston, MA, USA), dissolved in dimethyl sulfoxide to a final concentration of 10 mM and stored at −20°C. Anti-Bim antibodies used for western blotting were purchased from Cell Signaling Technology, Inc. (cat. no. 2819; Danvers, MA, USA). Anti-HuR and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All other chemicals were acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Small interfering RNA (siRNA) against HuR was used for RNA interference. The siRNA target sequence for HuR was: 5′-UUUGUCAUGGUCACAAAGCTT-3′. Briefly, cells were plated at a density of 5×10⁵ cells/well in 12-well plates. Using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), cells were transfected with 80 nmol/l siRNA duplex mixture (GeneChem Co., Ltd., Shanghai, China) for 48 h.

The HuR gene sequence was amplified by polymerase chain reaction (PCR) using a PrimeScript™ One Step RT-PCR Kit (Takara Bio, Inc., Otsu, Japan) and then subcloned into a lentiviral expression vector (GV365; GeneChem Co., Ltd.). The PCR primers used for HuR amplification were as follows: HuR forward, 5′-GAGGATCCCCGGGTACCGGTCGCCACCATGTCTAATGGTTATGAAGAC-3′ and reverse, 5′-TCCTTGTAGTCCATACCTTTGTGGGACTTGTTGGTTTTG-3′. The thermocycling conditions used for PCR were as follows: Initial denaturation for 5 min at 98°C; followed by 30 cycles of denaturation for 10 sec at 98°C, annealing for 10 sec at 55°C and extension for 90 sec at 72°C; followed by a final extension for 8 min at 72°C. Lentiviral packaging plasmids (pHelper 1.0; GeneChem Co., Ltd.) were used for the packaging and production of lentiviral particles. GV365 has a green fluorescent protein (GFP) marker for positive selection. Briefly, H1650 cells were transduced with lentiviral particles as previously described (28), and H1650 cells that stably expressed high levels of HuR were obtained and subsequently named GV365-H1650 cells. Subsequently, the expression of GFP was observed using fluorescence microscopy, while the expression of HuR was detected using reverse transcription-quantitative PCR (RT-qPCR) and western blotting.

As aforementioned, HCC827 cells were transfected with siRNA targeting HuR for 48 h, and the resultant cells were subsequently names named siHCC827 cells. H1650, GV365-H1650, siHCC827 and HCC827 cells were cultured in 96-well flat-bottomed microliter plates and were allowed to adhere at 37°C overnight in 5% CO₂ (~1×10⁴ cells/well). After cellular adhesion, gefitinib was added at a final concentration of 0-40 µM. After 48 h of incubation, the cytotoxic effects were assessed using the Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer's protocol. Each sample was plated in triplicate.

Apoptosis was examined by performing Annexin V and dye exclusion assays. Briefly, cells were simultaneously stained with Annexin V-allophycocyanin (APC; blue staining) and propidium iodide (PI; red staining). This assay was conducted to differentiate between intact (APC⁻/PI⁻), early apoptotic (APC⁺/PI⁻) and late apoptotic (APC⁺/PI⁺) cells. Comparative experiments were performed by bivariate flow cytometry using a FACScan (BD Biosciences, San Jose, CA, USA). Cell Quest software (version 3.3; BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the obtained data of the cell populations, for which debris was gated out.

Total RNA was isolated from H1650, GV365-H1650, siHCC827 and HCC827 cells using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in accordance with the manufacturer's instructions. Isolated RNA was reverse-transcribed into cDNA using PrimeScript™ RT reagent kit with gDNA Erase (Takara Bio, Inc.). To investigate the expression of target genes, qPCR was performed in triplicate using an ABI Step One Plus Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR^® Premix Ex Taq™ (Takara Bio, Inc.) in a 25 µl reaction with 900 nM primers. qPCR thermocycling conditions were as follows: Initial denaturation for 30 sec at 95°C; followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec. The PCR primers for HuR, Bim or β-actin were as follows: HuR forward, 5′-ACATGACCCAGGATGAGTTACGAAG-3′, and reverse, 5′-TCGCGGTCACGTAGTTCACAA-3′; Bim forward, 5′-TGATTCTTGCAGCCACCCTG-3′, and reverse, 5′-GGGGAACAAGGGCCAAGAAA-3′; β-actin forward, 5′-TGGCACCCAGCACAATGAA-3′, and reverse, 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. The relative quantification of HuR and Bim expression was calculated using the 2^−ΔΔCq method (29), and normalized to β-actin expression.

Cells were lysed with lysis buffer (Beyotime Institute of Biotechnology), and the precipitated cell debris was discarded by centrifugation at 4°C for 15 min (1,200 x g). The protein concentration was then determined using a Bradford protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein samples (30-50 µg/lane) were separated on a 2.5% SDS-PAGE gel and then transferred to polyvinylidene difluoride membranes. Following this, membranes were blocked with 5% (w/v) non-fat dry milk in 1X Tris buffered saline with 0.1% Tween 20 for 3 h at 37°C and then incubated overnight at 4°C with primary antibodies against the following proteins: HuR (cat. no. sc-5261; 1:1,000) and GAPDH (cat. no. sc-20358; 1:1,000) were purchased from Santa Cruz Biotechnology, Inc.; and antibodies against Bim (cat. no. 2819; 1:2,000) were procured from Cell Signaling Technology, Inc. Following this, the membranes were washed and incubated with the following secondary antibodies for 1 h at 25°C: HRP-tagged rabbit anti-mouse IgG (cat. no. sc-358920; 1:2,000; Santa Cruz Biotechnology, Inc.), HRP-conjugated donkey anti-goat IgG-HRP (cat. no. sc-2020; 1:5,000; Santa Cruz Biotechnology, Inc.) and HRP-conjugated mouse anti-rabbit IgG (cat. no. A01827-200; 1:5,000; Genscript, Piscataway, NJ, USA). The blots were visualized using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology, Shanghai, China). Images were captured using Scion image software (version 4.0.3.2; Scion Corporation, Frederick, MD, USA).

Formalin-fixed, paraffin-embedded tissues collected from patients with NSCLC (n=81) were sectioned at 5 mm. Briefly, tissue sections were incubated at 60°C for 2 h, deparaffinized, rehydrated in a descending alcohol series and subsequently blocked with 3% hydrogen peroxide for 30 min at room temperature. Following incubation with sodium citrate buffer (0.01 M) at room temperature for 30 min, the sections were then preincubated in 10% normal goat serum (Beyotime Institute of Biotechnology) at room temperature for 30 min to prevent non-specific staining. Subsequently, the sections were incubated overnight at 4°C with antibodies against HuR (cat. no. sc-5261; 1:1,000; Santa Cruz Biotechnology, Inc.) and Bim (cat. no. 2819; 1:1,000; Cell Signaling Technology, Inc.). Following this, sections were then incubated with corresponding horseradish peroxidase (HRP)-tagged secondary antibodies (cat. no. sc-358920, 1:1,000; and cat. no. sc-2020, 1:2,000; Santa Cruz Biotechnology, Inc.) for 30 min at room temperature. Two pathologists individually examined the levels of IHC signaling under a light microscope (magnification, x200). The algorithm output returns a number of quantitative measurements, namely the intensity and percentage of positive staining present (30). Subsequently, the staining intensity and percentage of positive staining were categorized into 4 and 5 classes, respectively, following the determination of cut-off values, according to a previously published protocol (30). The intensity of staining was categorized as 0 (no staining), 1+ (weak staining), 2+ (moderate staining) and 3+ (strong staining). In each sample, five high-power fields were selected, and the specimens were categorized into five semi-quantitative classes based on percentage of positive staining as follows: 0 (<5% stained cells), 1 (6-25% stained cells), 2 (26-50% stained cells), 3 (51-75% stained cells) and 4 (>76% stained cells). The products of percent positive and intensity scores yielded final IHC scores of <2 (negative) and >2 (positive).

A total of 14 healthy female BALB/c nude mice (aged 4 weeks old, weighing 14.6-21.8 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and subsequently housed at 21°C and 50-55% humidity with a 14/10 h light/dark cycle under germ-free (GF) conditions at the Laboratory Animal Research Center in General Hospital, Jinan Command of the People's Liberation Army. Mice were permitted to acclimatize for 1 week post-arrival. Mice received a standard laboratory chow and water ad libitum. For experiments assessing the effect of drug treatment on tumor growth, 10 female BALB/c nude mice were selected. The mice were randomized into two groups, including the transfection and control groups. In the transfection and control groups, GV365-H1650 cells or non-transfected H1650 cells were respectively injected into the back of BALB/c nude mice. The xenograft size was measured every 3 days, and the tumor volume was determined as follows: (length x width²)/2. A total of 2 weeks post-tumor implantation (all tumors were ≥500 mm³ in volume), gefitinib (5 mg/kg) was administrated once a day via intragastric administration for a total of 15 days. Animal welfare and relevant experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals (Animal Scientific Procedures, SAC/TC281), and were approved by the Ethics Committee of the General Hospital, Jinan Command of the People's Liberation Army.

The correlation of HuR and Bim staining with the different clinicopathological features of patients was evaluated using the χ² test. Univariate survival analysis was calculated using the Kaplan-Meier Method. Significance of the data was analyzed by the log-rank test. A P-value of <0.05 was considered to indicate a difference that was statistically significant, and all P-values were two-sided. All statistical calculations were performed using the SPSS statistical software (version 18.0; SPSS, Inc., Chicago, IL, USA).

The associations between HuR and Bim expression levels and clinicopathological characteristics of patients with NSCLC were determined using χ² tests. There was an evident difference in terms of the sex between the two groups of patients. Female patients accounted for 18.5% of cases in the resistant group, whereas they comprised 57.4% of the sensitive group cases, and this different was statistically significant (P=0.01; Table I). However, there were no significant differences between the EGFR-TKI-sensitive group and EGFR-TKI-resistant group with respect to the age, ECOG-PS (27), mutation type, tumor stage, surgery or chemoradiotherapy history, and brain metastases (P>0.05; Table I) (31).

For HuR staining, 81.5% (22 out of 27) cytoplasm-negative and 18.5% (5 out of 27) cytoplasm-positive specimens were identified in the EGFR-TKI-resistant group, whereas 20.4% (11 out of 54) specimens were cytoplasm-negative and 79.6% (43 out of 54) were cytoplasm-positive in the EGFR-TKI-sensitive group. These results indicated that IHC staining for HuR was higher in the EGFR-TKI-sensitive group as compared with that in the EGFR-TKI-resistant group (P<0.001; Table II). For Bim staining, 70.4% (19 out of 27) cytoplasm-negative and 29.6% (8 out of 27) cytoplasm-positive specimens were detected in the EGFR-TKI-resistant group, whereas 7.4% (4 out of 54) cases were cytoplasm-negative and 92.6% (50 out of 54) were cytoplasm-positive in the EGFR-TKI-sensitive group. These findings indicated that IHC staining for Bim was also higher in the EGFR-TKI-sensitive group as compared with that in the EGFR-TKI-resistant group (P<0.001; Table II). Therefore, the altered expression of HuR and Bim may modulate the therapeutic effect of EGFR-TKIs in NSCLC and serve as an important index for the treatment of this disease.

The correlation of Bim expression with HuR expression in NSCLC patients was analyzed by performing χ² tests. In patients with a positive expression of HuR, the positive rate of Bim was significantly higher (95.8 vs. 4.2%, P<0.01). In addition, the Bim negative expression rate was significantly higher in patients with negative HuR expression (63.6 vs. 36.4%, P<0.05), proving the existence of correlation between HuR and Bim expression (Table III). Notably, among the 27 cases with interpretable HuR and Bim staining, negative Bim expression was associated with negative cytoplasmic HuR expression (P=0.01; Table IV). However, Bim status was not correlated with the sex, age or mutation type (P>0.05; Table IV).

In the EGFR-TKI-sensitive group, the mPFS was 12.8 months for patients with positive Bim expression and 9.7 months for patients with negative Bim expression. The mPFS of patients with positive Bim expression was significantly higher in comparison with that of patients with negative Bim expression, based on Kaplan-Meier analysis (P=0.0036; Fig. 1). By contrast, the mPFS was 16.5 months for patients with positive HuR expression and 9.7 months for patients with negative HuR expression in the EGFR-TKI-sensitive group; thus, patients with positive HuR expression exhibited a significantly longer mPFS based on Kaplan-Meier analysis (P=0.0008; Fig. 1).

To determine whether HuR and Bim expression were implicated in gefitinib resistance, three EGFR-mutant lung cancer cell lines, namely PC9, HCC827 and H1650, were used in the present study. CCK-8 assays were performed to determine the half maximal inhibitor concentration (IC₅₀) for gefitinib. The IC₅₀ value for gefitinib in the H1650 cells was 21.28 µM. By contrast, HCC827 and PC-9 cells exhibited significantly lower IC₅₀ values for gefitinib (0.05 and 0.004 µM, respectively; Fig. 2A and B). Western blot analysis revealed an evident downregulation of HuR and Bim levels in gefitinib-resistant H1650 cells (Fig. 2C). The determination of mRNA levels for these genes led to similar conclusions. Indeed, H1650 cells exhibited reduced HuR levels by 10-fold and reduced Bim levels by 5-fold, as compared with those in parental HCC827 cells (Fig. 2D). According to the differential expression levels of HuR, in subsequent experiments, expression levels of HuR were downregulated in HCC827 cells via siRNA interference. Meanwhile, expression levels of HuR were upregulated in H1650 cells transfected with HuR lentiviral vectors.

The results of the current study revealed that HuR and Bim were upregulated in HCC827 cells compared with their expression levels in H1650 cells. Since HCC827 cells had the lowest IC₅₀ value for gefitinib, it is hypothesized that altered expression of HuR/Bim serves an important role in modulating gefitinib sensitivity in EGFR-mutant lung cancer cells. The study next examined HCC827 cells to determine whether lower expression of HuR promotes gefitinib resistance. In HCC827 cells, HuR expression was down-regulated via siRNA interference. As expected, RT-qPCR and western blotting data indicated that HuR expression decreased significantly following HuR knockdown as compared with the levels in control cells (Fig. 3A-C). Furthermore, a CCK-8 assay kit was used to detect gefitinib sensitivity at 48 h after transfection, revealing that the cell viability increased in HuR-knockdown HCC827 cells compared with that in control HCC827 cells. The results revealed that the cell viability of siHuR-HCC827 cells was markedly increased compared with control HCC827 cells (Fig. 3D). Annexin V/PI staining also demonstrated a marked decrease in gefitinib-induced apoptosis in the HuR-knockdown HCC827 cells compared with that in control cells (Fig. 4).

The effect of HuR knockdown in HCC827 cells on Bim expression was also examined. Western blotting and RT-qPCR were used to detect the expression of Bim protein and mRNA in HCC827 cells, respectively. The expression levels of Bim protein and mRNA were significantly decreased in the HuR-knockdown group in comparison with that in the control group (P<0.01; Fig. 3).

As mentioned earlier, the expression of HuR was lower in H1650 cells. Next, these cells were used to determine whether a higher expression of HuR was able to restore gefitinib sensitivity by manipulating Bim expression using a HuR lentiviral expression vector. HuR and Bim expression levels in infected and control cells were then examined using western blotting and RT-qPCR. Western blotting indicated that the protein expression of HuR and Bim was increased with lentiviral infection (Fig. 5A). HuR and Bim mRNA levels were also significantly higher in the H1650 infected group as compared with those in the control group (P<0.01; Fig. 5B and C). The results revealed that the cell viability of GV365-H1650 cells was markedly decreased compared with the control H1650 cells (Fig. 5D). Furthermore, Annexin V/PI staining indicated that HuR overexpression increased the apoptosis rates when compared with those in control cells (Fig. 6).

Prior to the administration of gefitinib treatment, xenograft tumors originating from the HuR-overexpressing H1650 stable clone and control clone exhibited continued and rapid growth. As shown in Fig. 7A, the size of tumors in the two groups 15 days post-treatment grew rapidly, with no significant difference between the two groups without gefitinib. By contrast, when gefitinib treatment was administered for 15 days in mice with xenograft tumors, the tumor burden induced by HuR-overexpressing H1650 cells was evidently suppressed by gefitinib in comparison with that in the control group (Fig. 7B). In addition, suppression of the tumor burden in the transfection + gefitinib group continued for 15 days. However, the tumor burden induced by the control + gefitinib clone was not evidently changed following gefitinib treatment. Tumor volumes in the transfection + gefitinib group were significantly smaller in comparison with those in the control + gefitinib group (Fig. 7C and D). The growth trend of xenograft tumors in each group exhibited an upward trend; however, the growth increase in the transfection + gefitinib group was significantly slower compared with that of the control + gefitinib group (Fig. 7E; Table V). IHC analysis further revealed that HuR expression was significantly increased and that Bim expression was simultaneously elevated in transfection + gefitinib group following gefitinib treatment (Fig. 7F and G).

HuR and Bim expression levels were next measured by western blotting and RT-qPCR in the transplanted tumors. The levels of HuR and Bim were significantly higher in the transduction + gefitinib group as compared with the control + gefitinib group following gefitinib treatment (Fig. 8). These results strongly support the mechanism implied by the cell model, and suggest that HuR-mediated Bim overexpression confers EGFR-TKI resistance.