Livin, a member of the inhibitor of apoptosis protein (IAP) family, is expressed at a high level in lung adenocarcinoma and influences the progression of cancer, and its response to chemotherapy and radiotherapy. Aberrant microRNA (miRNA) expression has also been associated with cancer initiation and development. However, the clinical significance of Livin and its relationship with miRNAs in lung adenocarcinoma are still unclear. In the present study, the expression level of Livin in 90 pairs of lung adenocarcinoma and their adjacent tissues were detected by immunohistochemistry staining. Spearman correlation and Kaplan-Meier, univariate and multivariate analyses were applied to evaluate the correlation between the expression of Livin and clinical characteristics. With the integration of bioinformatics analysis and dual-luciferase reporter gene assays, we identified the miRNA that can target Livin mRNA. The functional effects of miRNA-mediated Livin knockdown were assessed by Cell Counting Kit-8 (CCK-8) and apoptosis assays, and cell cycle analysis. The present study revealed that Livin was upregulated in lung adenocarcinoma tissues and may be associated with the poor prognosis in lung adenocarcinoma patients. The overexpression of Livin is partly caused by the downregulation of miR-198. Further exploration revealed that miRNA-198-mediated silencing of Livin significantly inhibited cell growth and enhanced apoptosis of A549 cells, accompanied by marked upregulation of caspase-3. Finally, we observed that the miR-198 overexpression and Livin neutralization had similar effects on improving cisplatin chemosensitivity in A549 cells. Overall, these findings suggest that Livin has the potential to become a biomarker for predicting the prognosis of lung adenocarcinoma and may provide a promising strategy for assisting chemotherapy of lung adenocarcinoma through the miR-198/Livin/caspase-3 regulatory network.
Lung cancer is the most common cancer worldwide, as well as the major cause of cancer-related deaths (
Livin, an inhibitor of apoptosis protein (IAP), is selectively overexpressed in certain tumors including lung adenocarcinoma (
MicroRNAs (miRNAs) are small, non-coding RNA molecules that post-transcriptionally regulate protein expression by complementary base pairing (
In the present study, the expression level of Livin in lung adenocarcinoma tissues vs. adjacent tissues was detected and the correlation between the expression of Livin and the clinical outcome was investigated. Bioinformatics analysis suggested that 2 out of 61 DEMs, namely miR-198 and miR-515-5p, were more likely to interact with Livin. Then, we identified the interaction between miRNA-198 and Livin using human lung adenocarcinoma cell line A549. Furthermore, we found that forced expression of miR-198 suppressed tumor cell proliferation and induced apoptosis of lung adenocarcinoma via the downregulation of Livin and the activation of caspase-3. Finally, the effects of the downregulation of Livin and the overexpression of miR-198 on A549 cell sensitivity to the cisplatin was detected using Cell Counting Kit-8 (CCK-8) assays.
The paraffin section of lung adenocarcinoma and their pericarcinous tissues (2 cm away from the tumor edge) were obtained from patients who had undergone surgery at the Department of Thoracic Surgery of The First Hospital of China Medical University from July 2004 to June 2009. The follow-up data of 90 cases were cut-off in August 2014, and the overall survival for the corresponding patients was calculated from the day of surgery to the day of death or to the last follow-up. The experiments were approved by the Ethics Committee of the First Hospital of China Medical University.
To assess the relationship between the expression of Livin and prognosis, formalin-fixed paraffin-embedded sections from specimens of lung adenocarcinoma and the adjacent tissues were evaluated by immunohistochemistry staining. The 4–5 µm paraffin-embedded slides were deparaffinized with xylene and rehydrated in graded alcohols. After high-pressure antigen retrieval, the activity of endogenous peroxidase was blocked with 3% hydrogen peroxide in ethanol for 10 min. Subsequently, the slides were washed with phosphate-buffered saline (PBS) and reacted with a rabbit primary antibody against Livin (dilution 1:500; Abcam, San Francisco, CA, USA) for 1 h. Then, incubation followed with an HRP-labeled anti-rabbit IgG (Nichirei, Tokyo, Japan) for 30 min at room temperature. The sections were stained with 3,3-diaminobenzidine (DAB) used as the chromogen. Finally the sections were counterstained with hematoxylin.
The degree of immunostaining was evaluated and scored by two independent pathologists blinded to the clinical information. Both cytoplasmic and nuclear immunostaining was considered to be positive staining. The proportion of positive cells was categorized as following: 5%, score 0; >6–25%, score 2; >26–50%, score 3; and >51%, score 4. The other score was set on the basis of the extent of staining: no staining as 1; light yellow staining as 2; medium brown staining as 3; brown staining as 4. The average of the two scores was interpreted as either low expression (average <4) or high expression (average ≥4).
The information of miRNA microarray was downloaded from the NCBI Gene Expression Omnibus (GEO;
The human lung adenocarcinoma cell line A549 was obtained from the Cell Bank of the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China) and was propagated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (both from Invitrogen, Carlsbad, CA, USA), 100 IU/ml penicillin and 100 µg/ml streptomycin, at 37°C in an atmosphere of 5% CO2.
All the miRNA inhibitors, miRNA mimics, nonsense sequence as miRNA negative control (NC), short hairpin RNA of Livin (sh-Livin) and short hairpin RNA negative control (NC-shRNA) were chemically synthesized by GenePharma (Shanghai, China). All of the transfections in the present study were transient, using JetPRIME reagent (PolyPlus Transfections SA, Illkirch, France) according to the manufacturers protocol. The cells were not harvested for the subsequent assays until 48 h after RNA oligonucleotide transfection.
Total RNA was extracted from A549 using TRIzol reagent (Takara, Otsu, Japan). Complementary DNA (cDNA) of miR-198 and miR-515-5p were obtained with TransScript® miRNA First-Strand cDNA Synthesis (TransGen Biotech, Beijing, China). The transfection efficiency of miRNAs was assessed by quantitative real-time PCR (qPCR) with SYBR-Green qPCR Master Mix (Takara) on an ABI 7500 Fast System thermocycler (Applied Biosystems, Foster City, CA, USA). All the experiments were conducted in accordance with the manufacturer's instructions. Triplicate reactions were performed and the data were normalized to U6 and calculated with the 2−ΔΔCt method. The involved primers are described as follows: miR-198 forward, 5′-GCCAACTGGTCCAGAGGG-3′; miR-515-5p forward, 5′-TTCTCCAAAAGAAAGCACTTTCTG-3′; U6 forward, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; the universal reverse primers of miRNAs from the kit.
Forty-eight hours after transfection, the cells were lysed using cell lysis buffer supplemented with protease inhibitors and phenylmethylsulfonyl fluoride (PMSF) (Beyotime, Zhejiang, China). Total proteins were extracted by centrifugation at 12,000 × g and 4°C for 20 min. The protein concentration was assessed using the BCA protein assay kit (Beyotime) and equal amounts of total proteins were separated in 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h with 5% non-fat milk powder in Tris-Buffered saline containing 0.5% Tween-20, and then incubated with a primary antibody overnight at 4°C, followed by washing and incubation with a secondary antibody for 2 h at room temperature. Finally, the membranes were detected by enhanced chemiluminescence (ECL) plus western blot detection reagents (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The antibodies (Thermo Fisher Scientific, Inc.) were used according to the manufacturers instructions, and were as follows: primary antibodies against GAPDH (ab9485), Livin (ab97350), caspase-3 (ab32351) and the HRP-conjugated secondary antibody (ab6721).
To confirm whether miR-198 or miR-515-5p can interact with Livin mRNA, pGLO Dual-Luciferase miRNA Target Expression Vector pmirGLO-wt-Livin (wild-type) and pGLO-mut-Livin (mutant type) of miR-198, pGLO-wt-Livin (wild-type) and pGLO-mut-Livin (mutant type) of miR-515-5p were constructed by GeneChem (Shanghai, China). The pGLO Dual-Luciferase miRNA Target Expression Vector plasmids (mutant or wild-type) and the internal control
Cell proliferation was detected using CCK-8 (KeyGen Biotech, Jiangsu, China). The A549 cells in the logarithmic phase with or without transfection were seeded in 96-well plates at a density of 5×103 cells/well. CCK-8 (10 µl/well) was added at various time-points (0, 24, 48 and 72 h) and incubated at 37°C for 2 h. The absorbance of each well at 450 nm was measured using a microplate reader (Bio-Rad, Hercules CA, USA) to assess the number of viable cells. To decrease random errors, all of the procedures were replicated at least three times.
Flow cytometric analyses were utilized to assess cell apoptosis and cell cycle distribution using a flow cytometer (Bio-Rad). The A549 cells were synchronized before the cell cycle assays. After 48 h of transfection, the cells, which were cultured in 6-well plates and prepared for apoptosis examination and cell cycle distribution, were harvested by trypsin and detected using an Annexin V-FITC/PI apoptosis detection kit and a Cell Cycle Detection kit (both from KeyGen Biotech) respectively, according to the manufacturers recommendations. Each experiment was performed at least in triplicate.
Before each experiment, the chemotherapeutic drug cisplatin (F.H. Faulding & Co, Mulgrave Victoria, Australia) was dissolved in 0.9% sodium chloride, and then diluted with fresh medium. The concentration of cisplatin was set as 0.01, 0.1 and 1 µmol/l. The cell proliferation rate 48 h after transfection and drug treatment was detected by CCK-8 assay kits. Furthermore, the cell proliferation rate treated with 1 µmol/l of cisplatin was assessed every 24 h for a period of 72 h.
The results of immunohistochemical staining were analyzed using Chi-square test, Kaplan-Meier analysis, Spearman rank correlation and Cox regression analysis. The result of the Kaplan-Meier survival analysis was compared with the log-rank test. Other experiments were analyzed using Student's t-test and expressed as the mean ± standard deviation (SD). Statistical analyses were processed using SPSS 23.0 software (SPSS, Inc., Chicago, IL, USA) and p<0.05 was considered as statistically significant.
The expression of Livin in cancerous and pericancerous tissues is shown in
Forty-five putative miRNAs generated by two databases, were identified as the possible miRNAs targeting Livin (
The aberrant expression of miR-198 and miR-515-5p in lung adenocarcinoma was reported as previously described (
The effects of overexpressing or silencing Livin in A549 cells have been previously demonstrated in detail (
Livin has been demonstrated to be capable of inhibiting apoptosis by binding to caspase family members directly with its BIR domain, and then suppressing caspase activities (
In addition, we determined the correlation between Livin and lymph node status, which had been reported by Lin
Livin RNA interference has been identified to enhance the chemosensitivity to chemotherapeutic drugs in NSCLC cells (
To the best of our knowledge, this is the first study elucidating the relationship between Livin and the clinicopathological features in lung adenocarcinoma patients. Livin is a member of the IAPs family, and is also known as baculoviral IAP repeat-containing protein 7 (BIRC7). A high level of Livin has been found in various tumors, including NSCLC (
Nonetheless, to date, only Yuan
In general, we demonstrated that Livin, an apoptotic pathway suppressor, can enhance the growth of lung adenocarcinoma, which may be partly due to the downregulation of miR-198. Livin has the potential to be a biomarker for predicting the prognosis of lung adenocarcinoma and focusing on the miR-198/Livin/caspase-3 regulatory network may become a promising strategy for drug resistant lung adenocarcinoma therapy.
The present study was supported by the National Natural Science Foundation of China (grant no. 81372876), and the Liaoning Province Natural Science Foundation (grant no. 2013021041).
Immunohistochemistry staining of Livin protein. High expression of Livin in lung adenocarcinoma tissues: (A) magnification, ×100 and (B) magnification, ×200; low expression of Livin in adjacent tissues: (C) magnification, ×100 and (D) magnification, ×200.
Kaplan-Meier analysis of the overall survival (months) in 90 lung adenocarcinoma patients with different Livin expression (log-rank test; p<0.05).
Bioinformatics analysis. Cluster analysis for the differentially expressed miRNAs between lung adenocarcinoma tissues (cancer group) and adjacent tissues (control group). Sixty-one significant differentially expressed miRNAs (|logFC| >1, adjusted p<0.05) are highlighted in green (downregulation) and red (upregulation).
Direct regulation of Livin by miR-198 in A549 cells. (A) The expression of miR-198 and miR-515-5p was evaluated by qRT-PCR in A549 cells 48 h after transfection to confirm the transfection efficiency. U6 was used as an internal control; *p<0.05. (B) The effects of miRNA mimics and inhibitors on Livin expression in A549 cells was detected using western blotting 48 h after transfection. GAPDH was used as a loading control. (C) The putative miR-198 and miR-515-5p binding sites in the 3′-UTR of Livin mRNA. (D and E) Dual-luciferase reporter assays using vectors encoding the putative miR-198 and miR-515-5p target sites in the Livin 3′-UTR for both the wild-type and mutant type. Normalized data were calculated as
Effect of miR-198 on the A549 cell line. (A) Cell proliferation was determined using CCK-8 assays after transfection with miR-198. The results revealed that miR-198 inhibited cell growth compared with the control groups; *p<0.05. (B) Caspase activation was revealed after miR-198 transfection for 48 h. (C and D) Double staining with Annexin V-FITC and PI was used to assess apoptosis after miR-198 transfection for 48 h. The results revealed that the apoptotic rate in the miR-198 group was higher than that of the control groups; *p<0.05. (E and F) Flow cytometric analysis of the cell cycle revealed that the miR-198 group had a lower proportion of S phase cells and a higher proportion of G2/M phase cells than in the control groups, which means that upregulation of miR-198 may cause cell cycle arrest, *p<0.05.
Influence of miR-198 overexpression and silencing of Livin on A549 cell sensitivity to cisplatin. (A) Evidently the survival rate was dose-dependent, and both miRNA-198 and Livin-shRNA enhanced the sensitivity to cisplatin. (B) The proliferation curve of NC-shRNA + miRNA-198 and Livin shRNA + NC groups with the treatment of 1 µmol/l of cisplatin. The proliferation rate of the blank group was significantly higher than that of the NC-shRNA + miRNA-198 and Livin-shRNA + NC groups. However, A549 cells cotransfected with Livin-shRNA and NC had a similar effect on the sensitivity to cisplatin as miR-198 with NC-shRNA (*p<0.05).
Differential expression of Livin in cancerous and pericancerous tissues.
Livin expression | |||||
---|---|---|---|---|---|
Tissues | n | High(%) | Low(%) | Chi-square value | P-value |
Cancerous | 90 | 52 (57.8) | 38 (42.2) | 34.545 | <0.0001 |
Pericarcinous | 90 | 14 (15.6) | 76 (84.4) |
Statistically significant (p<0.05).
Correlation between Livin expression and clinicopathological characteristics.
Livin expression | |||||
---|---|---|---|---|---|
Variables | High | Low | Total | rs | P-value |
Sex | −0.059 | 0.579 | |||
Female | 25 | 16 | 41 | ||
Male | 27 | 22 | 49 | ||
Age (years) | −0.104 | 0.327 | |||
≤60 | 26 | 15 | 41 | ||
>60 | 26 | 23 | 49 | ||
Grade | 0.161 | 0.129 | |||
1 | 4 | 8 | 12 | ||
2 | 43 | 27 | 70 | ||
3 | 5 | 3 | 8 | ||
T stage | 0.087 | 0.413 | |||
T1 | 9 | 8 | 17 | ||
T2 | 28 | 22 | 50 | ||
T3 | 11 | 6 | 17 | ||
T4 | 4 | 2 | 6 | ||
N stage | 0.239 | 0.023 |
|||
N0 | 19 | 23 | 42 | ||
N1 | 16 | 8 | 24 | ||
N2 | 12 | 6 | 18 | ||
N3 | 5 | 1 | 6 | ||
M stage | 0.033 | 0.755 | |||
M0 | 50 | 37 | 87 | ||
M1 | 2 | 1 | 3 | ||
TNM stage | 0.175 | 0.099 | |||
I | 16 | 15 | 31 | ||
II | 11 | 13 | 24 | ||
III | 23 | 9 | 32 | ||
IV | 2 | 1 | 3 |
Statistically significant (p<0.05). TNM, tumor-node-metastasis.
Univariate and multivariate analyses of the factors correlated with overall survival of lung adenocarcinoma patients.
Univariate analysis | Multivariate analysis | |||||
---|---|---|---|---|---|---|
Variables | HR | 95% CI | P-value | HR | 95% CI | P-value |
Livin expression | 0.466 | 0.281–0.773 | 0.003 |
0.568 | 0.339–0.950 | 0.031 |
Sex | 1.393 | 0.866–2.243 | 0.172 | |||
Age | 1.107 | 0.690–1.776 | 0.674 | |||
T stage | 1.541 | 1.135–2.092 | 0.006 |
1.170 | 0.799–1.715 | 0.419 |
N stage | 1.788 | 1.404–2.276 | <0.001 |
1.565 | 1.107–2.211 | 0.011 |
M stage | 0.700 | 0.171–2.863 | 0.620 | |||
TNM stage | 1.562 | 1.223–1.995 | <0.001 |
1.031 | 0.679–1.564 | 0.886 |
Grade | 1.969 | 1.218–3.182 | 0.006 |
1.697 | 0.953–3.022 | 0.072 |
Statistically significant (p<0.05). HR, hazard ratio; CI, confidence interval; TNM, tumor-node-metastasis.
The possible miRNAs targeting Livin.
hsa-miR-940 | hsa-miR-323-5p |
hsa-miR-548b-3p | hsa-miR-24 |
hsa-miR-198 | hsa-miR-324-5p |
hsa-miR-25* | hsa-miR-148b* |
hsa-miR-214 | hsa-miR-652 |
hsa-miR-455-3p | hsa-miR-525-5p |
hsa-miR-516a-5p | hsa-miR-582-5p |
hsa-miR-296-3p | hsa-miR-183 |
hsa-miR-141 | hsa-miR-139-5p |
hsa-miR-188-5p | hsa-miR-486-3p |
hsa-miR-515-3p | hsa-miR-551a |
hsa-miR-552 | hsa-miR-598 |
hsa-miR-572 | hsa-miR-601 |
hsa-miR-593* | hsa-miR-541 |
hsa-miR-596 | hsa-miR-296-5p |
hsa-miR-149* | hsa-miR-432* |
hsa-miR-548d-3p | hsa-miR-615-3p |
hsa-miR-942 | hsa-miR-654-5p |
hsa-miR-874 | hsa-miR-660 |
hsa-miR-222* | hsa-miR-7 |
hsa-miR-519e | hsa-miR-512-3p |
hsa-miR-423-5p | hsa-miR-589* |
hsa-miR-662 |