MicroRNA-21 is overexpressed in human cholangiocarcinoma and regulates programmed cell death 4 and tissue inhibitor of metalloproteinase 3

Abstract

Cholangiocarcinomas (CCAs) are aggressive cancers, with high mortality and poor survival rates. Only radical surgery offers patients some hope of cure; however, most patients are not surgical candidates because of late diagnosis secondary to relatively poor accuracy of diagnostic means. MicroRNAs (miRs) are involved in every cancer examined, but they have not been evaluated in primary CCA. In this study, miR arrays were performed on five primary CCAs and five normal bile duct specimens (NBDs). Several miRs were dysregulated and miR-21 was overexpressed in CCAs. miR-21 differential expression in these 10 specimens was verified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). To validate these findings, qRT-PCR for miR-21 was then performed on 18 additional primary CCAs and 12 normal liver specimens. MiR-21 was 95% sensitive and 100% specific in distinguishing between CCA and normal tissues, with an area under the receiver operating characteristic curve of 0.995. Inhibitors of miR-21 increased protein levels of programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinases 3 (TIMP3). Notably, messenger RNA levels of TIMP3 were significantly lower in CCAs than in normals.

Conclusions: MiR-21 is overexpressed in human CCAs. Furthermore, miR-21 may be oncogenic, at least in part, by inhibiting PDCD4 and TIMP3. Finally, these data suggest that TIMP3 is a candidate tumor suppressor gene in the biliary tree.

Figure 1. Array data and qRT-PCR data for miR-21 in 10 primary tissue specimens

X-axis, specimens: N1-5, normal bile duct specimens; T1-5, primary CCAs. Y-axis, miR-21 values from array and qRT-PCR data. Array and qRT-PCR data are ratios to specimen N1. Solid bars, array data; open bars, qRT-PCR values.

Figure 2

Figure 2a. qRT-PCR data for miR-21 in 20 primary CCAs and 14 normal tissues. Blue triangles, normal tissues; red diamonds, cancers. Y-axis, miR-21 qRT-PCR values as ratios to the N1 normal specimen; p-value obtained by Student’s unpaired t-test. Figure 2b. ROC curve built Using miR-21 qRT-PCR data from all 34 specimens. X-axis, 1–specificity; Y–axis, sensitivity. The area under the curve equalled 0.995 (Asymptotic 95% confidence interval: 0.981–1.008).

Figure 2

Figure 2a. qRT-PCR data for miR-21 in 20 primary CCAs and 14 normal tissues. Blue triangles, normal tissues; red diamonds, cancers. Y-axis, miR-21 qRT-PCR values as ratios to the N1 normal specimen; p-value obtained by Student’s unpaired t-test. Figure 2b. ROC curve built Using miR-21 qRT-PCR data from all 34 specimens. X-axis, 1–specificity; Y–axis, sensitivity. The area under the curve equalled 0.995 (Asymptotic 95% confidence interval: 0.981–1.008).

Figure 3. Western blot using: a. anti-PDCD4 antibody and b. anti-TIMP3 antibody

NT, untreated cells; NSI, cells treated with control nonspecific miR inhibitor; 21i, cells treated with specific miR-21 inhibitor. Lower panels, beta-actin.

Figure 3. Western blot using: a. anti-PDCD4 antibody and b. anti-TIMP3 antibody

NT, untreated cells; NSI, cells treated with control nonspecific miR inhibitor; 21i, cells treated with specific miR-21 inhibitor. Lower panels, beta-actin.

Figure 4

Figure 4a. mRNA qRT-PCR for PDCD4. X-axis, normal (blue triangles) and cancerous (red diamonds) primary specimens. Y-axis, PDCD4 mRNA qRT-PCR value relative to specimen N1. p-value, Student’s unpaired t-test. Figure 4b. PDCD4 mRNA qRT-PCR data after treatment with specific vs. nonspecific miR inhibitors. Solid bars, PDCD4 mRNA levels in cells treated with control nonspecific miR inhibitor. Open bars, PDCD4 mRNA levels in cells treated with specific miR-21 inhibitor. Experiments were performed in triplicate in HuCCT1 as well as in TFK1 CCA cell lines.

Figure 5. TIMP3 mRNA qRT-PCR data

X-axis, normal (blue triangles) and cancerous (red diamonds) primary specimens. Y-axis, TIMP3 mRNA qRT-PCR levels. P-value, Student’s unpaired T-test.