MiR-21 promotes intrahepatic cholangiocarcinoma proliferation and growth in vitro and in vivo by targeting PTPN14 and PTEN - PubMed (original) (raw)

MiR-21 promotes intrahepatic cholangiocarcinoma proliferation and growth in vitro and in vivo by targeting PTPN14 and PTEN

Li-Juan Wang et al. Oncotarget. 2015.

Abstract

Intrahepatic cholangiocarcinoma (ICC) constitutes the second-most common primary hepatic malignancy. MicroRNAs (miRNAs) play important roles in the pathogenesis of ICC. However, the clinical significance of miR-21 levels in ICC remains unclear. Here, we investigated the role of miR-21 in ICC and found that its expression was significantly upregulated in serum of ICC patients. Serum miR-21 levels robustly distinguished ICC patients from control subjects. Further experiments showed that inhibition of miR-21 suppressed ICC cell proliferation in vitro and tumor growth in vivo. Specifically, inhibition of miR-21 induced cell cycle arrest and apoptosis. Moreover, PTPN14 and PTEN were identified as direct and functional targets of miR-21. Finally, we showed high expression levels of miR-21 were closely related to adverse clinical features, diminished survival, and poor prognosis in ICC patients. This study revealed functional and mechanistic links between miR-21 and tumor suppressor genes, PTPN14 and PTEN, in the pathogenesis of ICC. MiR-21 not only plays important roles in the regulation of cell proliferation and tumor growth in ICC, but is also a diagnostic and prognostic marker, and a potential therapeutic target for ICC.

Keywords: PTEN; PTPN14; intrahepatic cholangiocarcinoma; miR-21; tumorigenesis.

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Figures

Figure 1

Figure 1. Expression of miR-21 in culture media of ICC cell lines and serum samples

(A) and (B) MiR-21 levels in the media of both HUCCT1 and RBE increased with increased cell counts and longer incubation intervals. (C) Serum levels of miR-21 in normal controls and ICC patients. Boxes represent interquartile range, and the horizontal line across each box indicates median value. (D) Serum miR-21 yielded an area under the curve (AUC) value of 0.9081, with 87.8% sensitivity and 90.5% specificity in distinguishing ICC patients from normal control subjects. (E) Comparison of serum miR-21 levels from all ICC patients (n=74). (F) Comparison of serum miR-21 in 17 ICC patients who underwent palliative resection. (G) Comparison of serum miR-21 levels in 57 ICC patients who underwent potentially curative surgeries.

Figure 2

Figure 2. Inhibition of miR-21 inhibits malignant phenotypes of ICC cells in vivo and in vitro

(A) Expression of miR-21 in HUCCT1 and RBE with increasing amounts of miR-21 inhibitor (20 and 50 nM) was validated by RT-qPCR. (B) and (C) Cell growth analyzed by MTT assays. (D) and (E) Representative results of colony formation. (F) and (G) Representative results of soft agar colony formation. The number of colonies counted was that of an entire well and the error bars represent mean ± SD from three independent experiments. (H) and (I) Effects of miR-21 on cell apoptosis of HUCCT1 and RBE cells. (J), (K), and (L) Effects of miR-21 on cell cycle of HUCCT1 and RBE cells. (M) and (N) RBE-Vector and RBE-miR-21 inhibited cells were injected into the hind limbs of nude mice (n = 5). Tumor volumes were measured on the indicated days. Data points are presented as the mean tumor volume ± SD. (O) RT-qPCR analysis of miR-21 expression in tissues of resected tumor formed from RBE-Vector and RBE-miR-21 inhibitor.

Figure 3

Figure 3. PTPN14 and PTEN are direct targets of miR-21

(A) and (B) MiR-21 and its putative binding sequence in the 3′-UTR of PTPN14 and PTEN; diagrammatic representation of the luciferase reporter plasmids with WT and MT PTPN14 and PTEN 3′-UTR. (C) Relative luciferase activity in 293T cells after transfection with WT or MT PTPN14 or PTEN 3′-UTR plasmids co-transfected with miR-21 inhibitors. (D), (E), and (F) MiR-21 inhibitors promoted the expression level of PTPN14 and PTEN at the mRNA level and protein level in HUCCT1 and RBE cells. Three independent experiments were performed in duplicate. Data are presented as mean ± SD. Two-tailed Student's t test was used. * P< 0.05.

Figure 4

Figure 4. Both gain- and loss-of-function studies showed that both PTPN14 and PTEN abrogate the suppressive roles of miR-21 in ICC cell proliferation and cell growth in vivo and in vitro

(A), (B) Cell proliferation; (C) Colony formation assay; (D) Soft agar colony formation assay; (E) Cell apoptosis assay; (F), (G) Cell cycle assay; and (H) Tumorigenesis assay indicate silencing PTPN14 and/or PTEN could rescue the inhibitory effects of miR-21-suppressors on HUCCT1 and RBE cells.

Figure 5

Figure 5. Clinical significance of miR-21, PTPN14 and PTEN in ICC

(A) In situ hybridization for miR-21 and IHC for PTPN14 and PTEN in the tissues of ICC patients. Positive representation of miR-21, PTPN14, and PTEN are on the left half of Figure 5A, negative presentation of miR-21, PTPN14, and PTEN are on the right half of Figure 5A. Magnification: x 200; Scale bar: 50 μm. (B) and (C) Kaplan-Meier plots representing probabilities of progression-free and overall survival in ICC patients according to expression level of miR-21. (D) and (E) Kaplan-Meier plots representing probabilities of progression-free and overall survival in ICC patients according to expression level of PTPN14. (F) and (G) Kaplan-Meier plots representing probabilities of progression-free and overall survival in ICC patients according to expression level of PTEN. (H) and (I) Kaplan-Meier plots representing the probabilities of progression-free and overall survival in ICC patients according to expression level of PTPN14 and PTEN.

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