Loss of connective tissue growth factor as an unfavorable prognosis factor activates miR-18b by PI3K/AKT/C-Jun and C-Myc and promotes cell growth in nasopharyngeal carcinoma

Abstract

Connective tissue growth factor (CTGF) has different roles in different types of cancer. However, the involvement and molecular basis of CTGF in tumor progression and prognosis of human nasopharyngeal carcinoma (NPC) have almost never been reported. In this study, we observed that downregulated CTGF expression was significantly associated with NPC progression and poor prognosis. Knockdown of CTGF markedly elevated the ability of cell proliferation in vivo and in vitro. Subsequently, we discovered that the reduction of CTGF increased the expression of miR-18b, an oncomir-promoting cell proliferation. Further, we discovered that attenuated CTGF-mediated upregulation of miR-18b was dependent on the increased binding of transcription factors Jun proto-oncogene (C-Jun) and v-Myc myelocytomatosis viral oncogene homolog (C-Myc) to miR-18b promoter region via phosphoinositide 3-kinase (PI3K)/AKT pathway. Finally, we further found that miR-18b directly suppressed the expression of CTGF in NPC. In clinical fresh specimens, miR-18b was widely overexpressed and inversely correlated with CTGF expression in NPC. Our studies are the first to demonstrate that reduced CTGF as an unfavorable prognosis factor mediates the activation of miR-18b, an oncomir directly suppresses CTGF expression, by PI3K/AKT/C-Jun and C-Myc and promotes cell growth of NPC.

Figure 1

Reduced expression of CTGF was correlated with poor prognosis in NPC. (a) Using qPCR examination, CTGF mRNA expression was markedly decreased in 62 NPC tissues compared with 20 nasopharynx tissues (P=0.0036). (b) CTGF protein expression was progressively decreased in 37 atypical hyperplasia and 213 NPC samples compared with 49 normal nasopharynx tissues and 21 squamous epithelium by immunohistochemistry assay. (1) CTGF expression in normal epithelium, 1–2: strong expression; (2) CTGF expression in squamous epithelium, 3: strong expression; (3) CTGF expression in atypical hyperplasia tissue, 4: weak expression; (4) CTGF expression in NPC, 5: strong expression; 6: weak expression; 7: negative expression. (c) Lower expression levels of CTGF reduced the overall survival time of 173 NPC patients with clinical prognosis information.

Figure 2

Stable suppression of CTGF expression stimulated the proliferation of NPC cells and speeded up the transition of cell cycle from G1 to S. (a) (A1) Polyclonal cells of lentivirus-mediated shRNA-CTGF-A and B, and PLV-Ctr were screened by GFP using FACS cytometry assay. (A2) Expression of CTGF was suppressed in shRNA-CTGF-A and B compared with PLV-Ctr cells by western blot. (b) In vitro proliferative ability of NPC cells was significantly restored in CTGF-suppressed cells compared with PLV-Ctr cells by MTT assay. (c) When compared with PLV-Ctr cells, tumor weight of shRNA-CTGF-A and B cells was markedly increased in vivo. (d) When compared with PLV-Ctr cells, tumor volume of shRNA-CTGF-A and B cells was markedly increased in vivo. (e) Downregulated CTGF expression stimulated cell cycle progression from G1 to S in shRNA-CTGF-A and B cells (P<0.001). (f) Specific siRNA was used to suppress the expression of CTGF in NPC 6-10B and HONE1 cells by western blot examination. (g) Cell growth ability was enhanced in si-CTGF-treated NPC cells (*P<0.05)

Figure 3

CTGF inhibited the expression of miR-18b, and miR-18b directly targets CTGF in NPC. (a) MiR-18b was notably upregulated in SiRNA-CTGF-treated 6-10B NPC cells by miRNA arrays analysis. (b) Real-time PCR confirmed the reliability of miRNA arrays in SiRNA-CTGF-treated HONE1, 6-10B and shRNA-6-10B-treated cells. (c) CTGF expression was repressed or elevated with the transfection of miR-18b mimics and inhibitor, respectively, at 100 nm in NPC 6-10B cells. (d) Luciferase reporter assay was used to determine whether miR-18b could directly target the 3′UTR of CTGF in NPC cells. Lanes 1 and 2: a significant decrease of luciferase activity was indicated in miR group. Lanes 5 and 6: the activity of mt 3′UTR vector was unaffected by a simultaneous transfection with miR-18b. Lanes 3 and 4: co-transfection with miR-18b inhibitor and wt 3′UTR vector led to a marked increase of luciferase activity. Data are presented as mean±S.D. for three independent experiments (*P<0.05)

Figure 4

C-Jun and C-Myc directly positively modulated miR-18b expression in NPC. (a) siRNA (fragment 1) was used to suppress the expression of C-Jun in NPC 6-10B cells. (b) The expression of C-Myc was suppressed by its specific siRNA (fragment 2) in NPC 6-10B cells. (c) Knocking down C-Jun downregulated the expression of miR-18b in NPC 6-10B cells. (d) Reduced C-Myc expression downregulated the expression of miR-18b in NPC 6-10B cells. (e and f) Crosslinked chromatin preparation from mock and pcDNA3.1-C-Myc or pcDNA3.1-C-Jun-transfected 6-10B cells was immunoprecipitated with anti-C-Myc, anti-C-Jun, or a normal rabbit IgG. The AP1 binding sites on the immunoprecipitated DNA was determined by quantitative RT-PCR. Amplification of input chromatin (input) before immunoprecipitation was served as positive controls for chromatin extraction and PCR amplification. Chromatin immunoprecipitation using a nonspecific antibody (normal human IgG) served as negative controls. QPCR analysis indicated that C-Jun and C-Myc could bind more miR-18b promoter region than that in control group in NPC 6-10B cells (*P<0.05)

Figure 5

Reduced CTGF activated PI3K/AKT-mediated expression of C-Jun and C-Myc in NPC. (a) Suppressing the expression of PI3K by its specific inhibitor Ly294002 reduced C-Jun and C-Myc expression by inactivating pPI3K/AKT in NPC 6-10B cells. (b) Reduced CTGF stimulated the expression of C-Jun and C-Myc by activating phosphorylated PI3K/AKT, but not their total proteins in NPC 6-10B cells

Figure 6

High expression of miR-18b promotes cell proliferation and is negatively correlated with the expression of CTGF in NPC. The ability of cell proliferation was markedly elevated after increasing miR-18b mimics expression in NPC. (b) Transduction of miR-18b inhibitor suppressed cell growth in NPC cells. (c) miR-18b expression was dramatically elevated in NPC tissues and NPC cells compared with nasopharynx tissues. (d) miR-18b was negatively correlated with the expression of CTGF in NPC samples. (e) CTGF protein expression was suppressed in xenograft tumor specimens after stably knocking down CTGF expression. (f) Reduced CTGF expression induced the miR-18b expression in xenograft tumor specimens. Data are presented as mean±S.D. for three independent experiments (*P<0.05)