A total of 80 LAD tissues and matched adjacent normal tissues were collected from patients treated at the First Affiliated Hospital of Chinese PLA General Hospital (Beijing, China) between August 2010 and August 2013. The age of these patients range from 45 to 75 years (mean age: 55 years). The sex ratio of these patients is 38 males and 42 females). Inclusion criteria: Patients with lung adenocarcinoma who have never received radiotherapy or chemotherapy. Exclusion criteria: Patients who were diagnosed as pneumonia in addition to LAD or those whose course of disease was not visited and recorded. The present study was a retrospective study. The LAD diagnosis of all tissues had been histopathologically confirmed, and frozen in liquid nitrogen. All patients provided written informed consent for use of tissue in the present research. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Chinese PLA General Hospital (Beijing, China).

The human LAD cell lines, SPC-A1, NCI-H441 and NCI-H1975, and the normal lung epithelial cell, BEAS-2B, were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). BEAS-2B cells were incubated in complete medium [RPMI-1640+10% fetal bovine serum (FBS; Lonza Group, Ltd., Basel, Switzerland)]. LAD cells were cultured and incubated in medium (DMEM+10% FBS). All mediums were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). All cell lines were incubated in a humidified atmosphere at 37°C with 5% CO₂. The complete medium was replaced every 2–3 days.

The LAD cell lines (500 cells/well) were transfected with 50 nM small interfering RNA (si)-CHRF or si-negative control (NC) using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The si-CHRF expression vector and matched scrambled control vectors were synthesized and purchased from GeneCopoeia, Inc. (Rockville, MD, USA). To obtain the optimal transfection efficiency, we used two siRNAs to knock down CHRF. They are si-CHRF#1 and si-CHRF#2. The siRNA sequences used are as follows: si-CHRF#1: 5′-TGCCTCTCTAGAGAGCAGC-3′; si-CHRF#2: (5′-CCGATCTGACATGACTGCG-3′. A total of 48 h after transfection, the cells were harvested for RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. All experiments were performed in triplicate.

Total RNA was extracted from LAD tissues or cells using TRIzol (Thermo Fisher Scientific, Inc.), according to manufacturer's instructions. RNA samples were stored at −80°C. A One-Step SYBR RT-PCR kit (Takara Bio, Inc., Otsu, Japan) was used to determine the expression of CHRF using a 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Each reaction contained 2 µl total RNA template, 1 µl forward primer, 1 µl reverse primer and 16 µl reaction mixture. The thermocycling conditions were as follows: Stage 1 (reverse transcription reaction), 1 cycle of 42°C for 5 min, 95°C for 10 sec; Stage 2 (PCR reaction), 40 cycles of 95°C for 5 sec, 60°C for 34 sec; Stage 3 (dissociation curve analysis), 95°C for 15 sec, 40 cycles of 65°C for 1 min and 95°C for 14 sec. The primers used for CHRF and GAPDH were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The primers are as follows: Human CHRF forward primer: 5′-AGATTCACATGGTATCCTGAAC′; reverse: 5′-TAGTCTGGCCACATTTTGTCTC-3′. GAPDH forward primer: 5′-TGTGTCCGTCGTGGATCTGA-3′; reverse: 5′-CCTGCTTCACCACCTTCTTGA-3′. All experiments were performed in triplicate. The qPCR quantification was conducted according to the 2^−∆∆Cq method (18).

A total of either SPCA-1 or NCI-H441 2×10⁴ cells/well were seeded in 96-well plates. A total of 20 µl MTT solution (0.5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to each well, followed by incubation at 37°C for 4 h. The cell culture medium was then carefully aspirated, and the formazan crystals were dissolved in 0.2 ml dimethyl sulfoxide. Absorbance was measured at 490 nm on a SpectraMax M5 microplate reader. All experiments were performed in triplicate.

Quantification of apoptosis was performed using an AnnexinV-FITC Apoptosis Detection kit (Beijing Biosea Biotechnology Co., Ltd., Beijing, China) 48 h after transfection, according to the manufacturers' protocol. The results were analyzed using CellQuest software v.0.9.13 (BD, Franklin Lakes, NJ, USA). Cells in the right lower quadrant were considered to be apoptotic. For cell cycle analysis, 1×10⁶ cells were fixed. To fix cell, 0.5 ml of cold PBS was used to resuspend cells. Next, the resuspended cells were added into 1.2 ml of 99.7% absolute ethyl alcohol (The final concentration is 70%). Finally, the resuspended cells were fixed at 4°C overnight. The cells were then stained with propidium iodide (50 µg/ml) at 4°C for 30 min in the dark. The cell cycle distribution was analyzed by using FlowJo 7.6.1 (FlowJo, LLC, Ashland, Oregon, USA). All experiments were performed in triplicate.

Cell invasion ability of SPCA-1 and NCI-H441 cells was assessed using Costar transwell chambers (Corning Incorporated, Corning, NY, USA) containing polycarbonate membranes (6.5 mm in diameter with a pore size of 8 µm), according to the manufacturer's protocol. The transwell membranes were each coated with 80 µl Matrigel (500 ng/µl; BD Biosciences, Franklin Lakes, NJ, USA), and incubated at 37°C for 4 h. A total of 2×10⁵ cells were added to the upper compartment of each well in 200 µl of PFHM-II Protein-Free Hybridoma Medium (Gibco: Thermo Fisher Scientific, Inc.); supernatant complete medium of cells (0.5 ml) was added to the bottom chamber. Following incubation for 24 h at 37°C, the cells which had invaded to the lower chamber were stained with hematoxylin and eosin (Thermo Fisher Scientific, Inc.). Cells were fixed with 95% ethanol for 10 min at 37°C and then stained with 19% hematoxylin for 20 min and 0.5% eosin for 3 min at 37°C., Then it was counted under a light microscope at a magnification of ×1,000 and the number was counted within randomly nine field for each experiment. All experiments were performed in triplicate.

Cell proteins were isolated using 400 µl of RIPA buffer (Thermo Fisher Scientific, Inc.). Gels were scanned and quantified by densitometry using the Quantity-One 4.4 software (Bio-Rad, Laboratories, Inc., CA, USA). The protein was quantified by using the Bradford method (Bio-Rad, Laboratories, Inc.). The sample solution containing 50 µg proteins were separated by 10% SDS-PAGE, then transferred into nitrocellulose membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 1% Tween (TBS) for 1 h, then incubated with the following primary antibodies overnight at 4°C: Phosphorylated (p-)PI3K (cat no. ab125633, 1:2,000), total PI3K (cat no. ab127617, 1:2,000), p-AKT (cat no. ab38449, 1:2,000), total Akt (cat no. ab126580; 1:2,000) and GAPDH (cat no. ab8245; 1:2,000) (all from Abcam, Cambridge, UK). The secondary antibody (anti-mouse IgG) conjugated to horseradish peroxidase (cat no. 7076, 1:300; Cell Signaling Technology, Inc., Danvers, MA, USA) was incubated with the membranes for 1 h at 37°C. Protein binds were visualized using an enhanced chemiluminescence reagent chromogenic substrate (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. All experiments were performed in triplicate.

All statistical analysis was performed using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA). The data were analyzed and presented as the mean ± standard deviation. Data between two groups were analyzed by using the paired-student t-test. Multiple comparisons were made by one-way ANOVA with the Least Significant Difference post hoc test. Correlations between CHRF expression and clinicopathological features of LAD patients were analyzed by Pearson χ² test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test was used to compare differences between patient groups. P<0.05 was considered to indicate a statistically significant difference.

To determine the role of the lncRNA, CHRF, in the development and progression of lung adenocarcinoma (LAD), RT-qPCR was performed to measure the expression of CHRF in 80 pairs of LAD tissues and adjacent normal tissues. The results revealed that CHRF was significantly increased in LAD tissues compared with adjacent normal tissues (Fig. 1A, P<0.01). The expression level of CHRF was also measured in LAD cell lines and the normal lung epithelial BEAS-2B cells. As demonstrated in Fig. 1B, the level of CHRF was increased in LAD cells compared with normal epithelial cells (P<0.01). The level of CHRF was highest in SPC-A1 and NCI-H441 cells, which were selected for use in subsequent experiments. These results suggest that CHRF may serve a crucial role in the development of LAD.

In order to explore the association between CHRF and various clinicopathological characteristics of LAD patients, the mean value of CHRF expression in LAD tissue was used as a cutoff value (patients exhibiting expression higher than the cutoff value were classified into high expression and patients exhibiting expression lower than or equal to the cutoff value were classified into low expression) and all patients were divided into a high expression group (n=41) and a low expression group (n=39). As presented in Table I, high expression of CHRF was significantly associated with advanced TNM stage, lymph node metastasis and large tumor size (P<0.05). However, there was no evident association between the expression of CHRF and other characteristics, including age, gender, smoking and differentiation (P>0.05).

The Kaplan-Meier and the log-rank test were used to investigate the clinical relevance of CHRF in LAD. Patients exhibiting high expression of CHRF had a markedly lower overall survival probability than those with low expression of CHRF (Fig. 2). Univariate and multivariate Cox regression analyses indicated that the expression of CHRF, tumor size and lymph metastasis were associated with the overall survival time of LAD patients. Therefore, lncRNA CHRF may act as an independent prognostic marker for the overall survival time of LAD patients (Table II).

Loss-of-function experiments were performed to investigate the biological function of CHRF in LAD. The results of RT-qPCR analysis revealed that the expression of CHRF was downregulated in si-SPCA-1 and si-NCI-H441 cells compared with the si-NC group (P<0.01; Fig. 3A). MTT assays demonstrated downregulation of CHRF expression significantly reduced cell proliferation both in si-SPCA-1 and si-NCI-H441 cells (P<0.01; Fig. 3B). Flow cytometry revealed that knockdown of CHRF caused cell cycle arrest most often in the G₀/G₁ phase, and induced cell apoptosis (P<0.01; Fig. 3C and D). These results indicate that knockdown of CHRF reduced cell proliferation by affecting cell cycle and apoptosis.

Transwell assays were performed to further explore the effect of CHRF on the progression of LAD. This demonstrated that cells transfected with si-CHRF were less migratory compared with si-NC cells (P<0.01; Fig. 4A). Furthermore, the transwell invasion assay demonstrated that knockdown of CHRF significantly reduced the invasive ability of LAD cells (P<0.01; Fig. 4B). These data suggest that CHRF functioned as an oncogene in the development and progression of LAD.

To explore the potential mechanism through which CHRF affects the progression of LAD, western blotting was performed to determine the effect of downregulated CHRF on the PI3K/Akt signaling, which has been demonstrated to be ectopically activated in human cancers, increasing cell proliferation and metastasis (19). Western blotting revealed that knockdown of CHRF dramatically reduced the protein level of p-PI3K and p-Akt in LAD (P<0.01; Fig. 5). These data indicate that the PI3K/Akt signaling pathway may function in the proliferation and metastasis of LAD cells induced by CHRF.