The 57 LAD samples and corresponding NT samples were prospectively collected from patients of the First Affiliated Hospital of Wenzhou Medical University, China, from August 2013 to August 2014. The clinical data of these cases are shown in Table I. The diagnosis of adenocarcinoma was confirmed by histopathology. TNM clinical stage based on the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC) in 2002. The LAD and matched NT samples were snap-frozen in liquid nitrogen immediately after resection. We have followed prognosis of 35 LAD patients by telephone or other means, the longest follow-up time was 28 months. According to the expression level of RPLP0P2, the survival data are divided into the high and low expression group. This study was approved by the Institutional Ethics Review Board of the First Affiliated Hospital of Wenzhou Medical University, and all patients provided written informed consent for this study.

Total RNA was extracted from frozen LAD tissues by using TRIzol reagent (Invitrogen). According to the manufacturer's instructions, total RNA was reverse-transcribed into cDNA using an RT Reagent kit (Takara, Shanghai, China). RPLP0P2 and GAPDH mRNA expression in LAD tissues were measured by quantitative PCR by using SYBR Premix Ex Taq in ABI 7000 instrument. RPLP0P2 sense, 5′-AAAAACGATCAACGAACCTT-3′ and antisense, 5′-AATCGTCTCTGCTTTTCTTG-3′; GAPDH sense, 5′-TGACTTCAACAGCGACACCCA-3′ and antisense, 5′-CACCCTGTTGCTGTAGCCAAA-3′; LRRC10B sense, 5′-AAGCCACCGTGCCTCCA-3′ and antisense, 5′-TCCCTCGTCCCGTTATTGC-3′. Total RNA (2 mg) was transcribed to cDNA. PCR was performed in a total reaction volume of 20 µl, including 10 µl of SYBR Premix Ex Taq (2X), 2 µl of cDNA template, 1 µl of PCR forward primer (10 mM), 1 µl of PCR reverse primer (10 mM), and 6 µl of double-distilled water. The quantitative real-time PCR reaction included an initial denaturation step of 10 min at 95°C; 40 cycles of 5 sec at 95°C, 30 sec at 60°C; and a final extension step of 5 min at 72°C. All experiments were performed in triplicate, and all samples were normalized to GAPDH. The median in each triplicate was used to calculate relative lncRNA concentrations (ΔCt = Ct median lncRNA - Ct median GAPDH), and 2^−ΔΔCt in expression was calculated (17).

Five human LAD cell lines (SPCA-1, NCI-H1299, A549, NCI-H441, LTEP-a2) were all purchased from the Cell Bank of the Chinese Academy of Sciences and were cultured with complete medium (containing 10% fetal serum and 90% RPMI-1640) set at 37°C, 5% CO₂ and complete medium was changed at least once every two days.

A549 cells were transfected overexpression vector targeting RPLP0P2 as well as a negative control (GeneChem, Shanghai, China). Transfection was accomplished by seeding 2x10⁵ cells into a 6-well plate, and after 24 h, the medium was aspirated and incubated with transfection complex according to the manufacturer's instructions and MOI values (MOI=20). The A549 cells were infected by lentivirus for 72 h and the overexpression efficiency was detected by qPCR.

Migration and invasion assay was performed with 8.0-µm pore inserts (Millipore, USA) in a 24-well plate. For migration assay, 2x10⁴ cells were seeded into the upper compartment of the Transwell inserts. The invasion assay was performed with Matrigel-coated filters (Sigma Corp., USA). Cells were allowed to incubate for 24 and 48 h, respectively. Migrated and invaded were fixed by methanol and stained by 0.1 % (w/v) crystal violet, then bleached with 33% acetic acid and absorbance value measured at 570 nm on a microplate reader. Each experiment was performed in triplicate.

Cell viability was evaluated by Cell Counting kit-8 (CCK-8; Corning, Inc., USA) abiding by the manufacturer's instructions. Briefly, 3,000 cells were resuspended and seeded into a 96-well plate supplemented in the presence of 10% FBS and cultured for a week. The next day, the RPLP0P2 overexpression cells were incubated with CCK-8 for 1 h and the absorbance was measured at 450 nm using a multifunctional microplate reader (Tecan) at day 1, 3, 5 and 7. This experiment was done in quadruplicate cells.

The cells were harvested by centrifugation and fixed by 70% ethanol at 4°C overnight. The cells were resuspended with 400 µl PBS (containing 2 mg/ml RNA enzymes) and incubated at 37°C for 30 min, then added 400 µl propidium iodide (0.1 mg/ml) for 10 min and detected DNA content by a flow cytometry analyzer (Cytomics FC 500; Beckman Coulter). The results were analyzed using MultiCycle software.

The 96-well plates were processed with 50 µl FN (50 µg/ml), and no processed wells were the CON group. These wells were added into 2x10⁴ cells/well and stained by 0.1% (w/v) crystal violet, then dissolved with 2% SDS and detected at OD550 nm. This experiment was done in quadruplicate cells.

Differences in variables among groups were tested using the one-way ANOVA for the normal distribution or Kruskal-Wallis test for the non-normal distribution. A comparison between the two groups was performed by least significant difference (LSD) test or Student's t-test or Mann-Whitney U test. Survival analysis was performed using Chi-square test. P<0.05 was considered to be statistically significant.

According to Table I, RPLP0P2 expression level of LAD is 0.287 (0.131–2.96) and significantly lower than its adjacent cancer tissues (Mann-Whitney U =2.120, P=0.0029). We showed that the RPLP0P2 level of LAD with lymph node metastasis was significantly lower than that of LAD without lymph node metastasis group (Mann-Whitney U=9.102, P=0.011). RPLP0P2 expression levels among different clinical stages were not different (Kruskal-Wallis test =7.124, P=0.154). The expression of RPLP0P2 was not relative to the histology differentiation (Kruskal-Wallis test=3.235, P=0.676), smoking (Mann-Whitney U=321.00, P=0.165), or gender (Mann-Whitney U=287.00, P=0.423). LRRC10B mRNA expression level of LAD was significantly higher than its adjacent cancer tissues (Mann-Whitney U=1.530, P=0.000). Pearson correlation analysis showed that RPLP0P2 expression levels were negatively correlated to LRRC10B mRNA levels (Pearson correlation =−0.754, P=0.0021) (Fig. 1).

The overall survival time of LAD low expression RPLP0P2 group (median 10 months) was significantly lower than that of the high expression (median 26 months) (χ²=18.81, P<0.0001) (Fig. 2).

Compared to normal human bronchial epithelial BEAS-2B cell line, we detected the expression levels of RPLP0P2 from five LAD cell lines (including A549, NCI-H441, NCI-H1299, SPCA-1, LETP-a2) by qPCR. It was shown that the expression levels of RPLP0P2 from LETP-a2, SPCA-1, NCI-H441 cells were highly expressed wherein LETP-a2 was the highest and that of SPCA-1, NCI-H441 were moderately expressed, while that of A549 and NCI-H1299 cells the lowest (Fig. 3). Therefore, A549 cells were lentivirus-mediated transfection RPLP0P2 overexpression cells.

The expression levels of RPLP0P2 in three A549 cell groups were different (F=117.00, P<0.0001) and that of overexpression A549 cells was higher than that of the NC group (t=10.34, P=0.0005), control group (t=10.81, P=0.0004) (Fig. 4). The LRRC10B mRNA expression levels of three A549 cell groups were different (F=29.11, P=0.0008) and that of RPLP0P2 overexpression A549 cells was lower than that of NC group (t=5.909, P=0.0042), or control group (t=6.056, P=0.0037). While RPLP0P2 expression levels were not changed significantly after LRRC10B siRNA (F=0.4489, P=0.6582). These experiments hinted that LRRC10B may be a downstream gene regulated by RPLP0P2.

The OD570 value of three A549 cell groups was not different (F=1.262, P=0.3488), the OD570 value of RPLP0P2 overexpression A549 cells was similar to that of NC group (t=1.715, P=0.153), and control group (t=0.7292, P=0.506) (Fig. 5). Thus, the cell migration ability of A549 cells did not change after RPLP0P2 was overexpressed. According to Fig. 6, the OD570 value of the three A549 cell groups was not different (F=0.9129, P=0.4507) the OD570 value of RPLP0P2 overexpression A549 cells was similar to that of NC group (t=0.9879, P=0.3791), and control group (t=1.196, P=0.2979).

Fig. 7 shows the OD450 nm of different A549 groups gradually increased with the change of time. Compared with day 1, the OD450 nm of day 3 (P<0.05, P<0.05, P<0.05), day 5 (P<0.001, P<0.001, P<0.01), day 7 (P<0.001, P<0.001, P<0.001) were significantly increased. Compared with appropriate days of control and NC group, the OD450 nm of 1 and 3 days in RPLP0P2 overexpression group had no statistically significant difference (P>0.05), while that of the 5 days (P<0.05) and the 7 days (P<0.01) significantly reduced, it indicates that cell proliferation ability of A549 was significantly reduced after RPLP0P2 overexpression. Fig. 8 shows that after RPLP0P2 was overexpressed, S phase (P=0.0002 and P=0.0001) and G2/M phase cells (P=0.0004 and P=0.0006) of A549 cells significantly reduced, while apoptosis and G0/G1 phase cells (P=0.0003 and P=0.0007) obviously increased compared to control and NC groups. The cell cycle results further confirmed that the RPLP0P2 expression level was associated with the ability of cell proliferation. The OD550 of control group (P<0.05), NC group (P<0.05) were higher than RPLP0P2 overexpression group among CON group in Fig. 9. Compared to CON group, the OD550 of control group, NC group from FN processing significantly increased while that of RPLP0P2 overexpression group was not different. Therefore, it was shown that RPLP0P2 lowered the adhesion capacity of A549 cells after overexpression.