Lentivirus-mediated knockdown of NLK inhibits small-cell lung cancer growth and metastasis

Abstract

Nemo-like kinase (NLK), an evolutionarily conserved serine/threonine kinase, has been recognized as a critical regulator of various cancers. In this study, we investigated the role of NLK in human small-cell lung cancer (SCLC), which is the most aggressive form of lung cancer. NLK expression was evaluated by quantitative real-time polymerase chain reaction in 20 paired fresh SCLC tissue samples and found to be noticeably elevated in tumor tissues. Lentivirus-mediated RNAi efficiently suppressed NLK expression in NCI-H446 cells, resulting in a significant reduction in cell viability and proliferation in vitro. Moreover, knockdown of NLK led to cell cycle arrest at the S-phase via suppression of Cyclin A, CDK2, and CDC25A, which could contribute to cell growth inhibition. Furthermore, knockdown of NLK decreased the migration of NCI-H446 cells and downregulated matrix metalloproteinase 9. Treatment with NLK short hairpin RNA significantly reduced SCLC tumor growth in vivo. In conclusion, this study suggests that NLK plays an important role in the growth and metastasis of SCLC and may serve as a potential therapeutic target for the treatment of SCLC.

Keywords: NLK; RNAi; SCLC; migration; proliferation.

Figure 1

Expression patterns of NLK in SCLC and adjacent lung tissues. Note: qRT-PCR analysis of NLK expression in 20 paired SCLC and adjacent lung tissue samples. Abbreviations: NLK, Nemo-like kinase; qRT-PCR, quantitative real-time polymerase chain reaction; SCLC, small-cell lung cancer.

Figure 2

Lentivirus-delivered shRNA targeting NLK downregulated its endogenous expression. Notes: (A) GFP expression in the uninfected and lentivirus-infected NCI-H446 cell lines recorded by fluorescence microscopy. (B) qRT-PCR analysis of NLK knockdown efficiency in the uninfected and lentivirus-infected NCI-H446 cells. (C) Representative immunoblot showing NLK knockdown efficiency determined in the uninfected and lentivirus-infected NCI-H446 cells. The β-actin gene and protein were used as internal controls. Data are mean ± SD (n=3; t-test). *P<0.05. Abbreviations: Con, control; GFP, green fluorescence protein; Lv-shCon, lentivirus-containing non-silencing shRNA; Lv-shNLK, lentivirus-containing NLK shRNA; NLK, Nemo-like kinase; qRT-PCR, quantitative real-time polymerase chain reaction; shRNA, short hairpin RNA.

Figure 3

Depletion of NLK decreased cell proliferation and colony formation. Notes: (A) Growth curves of the uninfected and lentivirus-infected NCI-H446 cell lines determined by CCK-8 assay. (B) Representative colony formation showing clonogenic survival determined in the uninfected and lentivirus-infected NCI-H446 cells. (C) Statistical analysis of colony numbers in the uninfected and lentivirus-infected NCI-H446 cells. Data are mean ± SD (n=3; t-test). *P<0.05. Abbreviations: CCK-8, Cell Counting Kit-8; Con, control; Lv-shCon, lentivirus-containing non-silencing shRNA; Lv-shNLK, lentivirus-containing NLK shRNA; NLK, Nemo-like kinase; shRNA, short hairpin RNA.

Figure 4

Downregulation of NLK induced S-phase cell cycle arrest. Notes: (A) Flow cytometric analysis of cell cycle distributions in the uninfected and lentivirus-infected NCI-H446 cells. (B) Statistical analysis of G1-, S-, G2/M-phase populations in the uninfected and lentivirus-infected NCI-H446 cells. Data are mean ± SD (n=3; t-test). *P<0.05; **P<0.01. Abbreviations: Con, control; Lv-shCon, lentivirus-containing non-silencing shRNA; Lv-shNLK, lentivirus-containing NLK shRNA; NLK, Nemo-like kinase; shRNA, short hairpin RNA.

Figure 5

Downregulation of NLK impeded cell migration. Notes: (A) Representative crystal violet staining showing cell migration determined in the uninfected and lentivirus-infected NCI-H446 cells. (B) Statistical analysis of migrated cells in the uninfected and lentivirus-infected NCI-H446 cells. Data are mean ± SD (n=3; t-test). *P<0.05. Abbreviations: Con, control; Lv-shCon, lentivirus-containing non-silencing shRNA; Lv-shNLK, lentivirus-containing NLK shRNA; NLK, Nemo-like kinase; shRNA, short hairpin RNA.

Figure 6

Downregulation of NLK affected cell cycle and migration regulatory proteins. Notes: Representative immunoblot showing expression alterations of Cyclin A, CDK2, CDC25A, and MMP-9 determined in the uninfected and lentivirus-infected NCI-H446 cells. β-actin protein was used as an internal control. Abbreviations: Con, control; Lv-shCon, lentivirus-containing non-silencing shRNA; Lv-shNLK, lentivirus-containing NLK shRNA; MMP-9, matrix metalloproteinase 9; NLK, Nemo-like kinase; shRNA, short hairpin RNA.

Figure 7

Downregulation of NLK inhibited tumor growth and lung metastasis in vivo. Notes: (A) Growth curves of xenograft tumors in nude mice treated with lentivirus-infected NCI-H446 cells. (B) Statistical analysis of weights of xenograft tumors in nude mice treated with lentivirus-infected NCI-H446 cells. (C) Representative images recorded under light microscope, representing the size of tumors in nude mice. (D) The tumor was confirmed by H&E staining. (E) Incidence of tumor lung metastasis in nude mice induced by injection of NCI-H446 cells, which were stably infected with Lv-shNLK or Lv-shCon. (F) Lung metastases in the mice were confirmed by H&E staining; magnification ×100. Data are mean ± SD (n=3; t-test). *P<0.05; **P<0.01. Abbreviations: Con, control; Lv-shCon, lentivirus-containing non-silencing shRNA; H&E, hematoxylin and eosin; Lv-shNLK, lentivirus-containing NLK shRNA; NLK, Nemo-like kinase; shRNA, short hairpin RNA.