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

Abstract

Cholangiocarcinomas (CCA) are aggressive cancers, with a high mortality and poor survival rate. Only radical surgery offers patients some hope of cure; however, most patients are not surgical candidates because of the late diagnosis secondary to relatively poor accuracy 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 5 primary CCAs and 5 normal bile duct specimens (NBD). Several miRs were dysregulated, and miR-21 was overexpressed, in CCAs. miR-21 differential expression in these 10 specimens was verified with 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 (ROC) 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 (mRNA) 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.

Background

Cholangiocarcinoma, an epithelial cancer of the biliary tree¹, is the second most common primary hepatic malignancy², with a United States incidence of 0.82–0.95/100,000³. While the rate of extrahepatic CCA has remained stable, intrahepatic CCA has increased by more than 100% in 40 years⁴. Survival in CCA is dismal, usually measured in months⁵. The only potentially curative treatment for CCA is surgical resection⁶. Unfortunately, the vast majority of patients are diagnosed at late stages, when surgery is not a viable option^7,8. This late diagnosis stems from a paucity of disease-specific symptoms in early stages². Moreover, in earlier stages of CCA, diagnostic techniques are plagued by low specificity². These considerations demand a better understanding of cholangiocarcinogenesis, with an emphasis on biomarkers for earlier diagnosis.

MicroRNAs (miRs) are noncoding RNAs 18–25 nucleotides in length⁹. The effects of miRs are mediated by binding to target mRNAs and: 1) suppressing translation or 2) degrading miR-bound mRNA¹⁰. Groundbreaking research in CCA cell lines revealed important miR differences among CCA and normal cholangiocytic cell lines¹¹. MiR-21 was overexpressed in CCA cell lines¹¹, and this same pioneering study identified the gene PTEN as a potential miR-21 target. Further cutting-edge research found miR-29 downregulated in the CCA cell line, KMCH, and identified Mcl-1 as a target. Finally, in additional studies, downregulation of miR-370 was discovered in the CCA cell lines, MzChA-1 and KMCH-1¹². These seminal in vitro studies laid a paramount foundation for understanding the impact of miRs on cholangiocarcinogenesis. In the current study, we sought to gain insight into miR dysregulation in primary human CCAs. We hypothesized that molecular findings from direct in vivo analyses would complement insights gained from previous fruitful in vitro studies in helping us to understand this deadly disease.

Materials and Methods

Human tissues

The first 5 CCA specimens were obtained at surgery performed at Johns Hopkins University. We also collected 5 primary normal bile duct (NBD) specimens from surgical resections performed for pancreatic cancer. These patients underwent a Whipple’s procedure. Bile ducts contained in the excision specimens were confirmed histologically to be free of tumor. After the initial stage of the study, an additional number of 18 patients were included. These patients underwent resections for cholangiocarcinoma. From 12 patients, paired normal and cancer tissues were obtained. From 6 patients, only cancer tissues were obtained. Therefore, a total number of 30 specimens were added to the study: 18 cancer and 12 normal tissues. The normal tissue was confirmed histologically to not contain any cancer. Informed consent for the surgical procedure and for using the specimens for research was obtained from all patients under approved Johns Hopkins University IRB protocols.

Cell lines

CAK-1 and HuCCT1 were generated at Johns Hopkins University from extrahepatic CCAs ¹³. TFK1 was established from a common bile duct CCA resected surgically ¹⁴.

DNA and RNA extraction

Total RNA was isolated using TRIzol reagent (Invitrogen).

MicroRNA arrays

100 ng of total RNA for each specimen was used for miR arrays. We employed an Agilent Human miR array chip (Agilent, Santa Clara, CA) containing 15,000 probes corresponding to 470 unique human miRs. Data was extracted using Feature Extraction Software 9.3 and GeneSpring software (Agilent). A raw array value below 5 was considered background-level. Array data was normalized to the control small RNA species printed on the slide. Data from miRs with at least 5 of 10 values above background were used for further analyses. Data was analyzed using Significance Analysis of Microarrays (SAM, Stanford University). A false discovery rate (FDR) less than 2% was considered acceptable.

Quantitative qRT-PCR (qRT-PCR) for miR expression

We performed miR qqRT-PCR to confirm the expression of candidate miRs. TaqMan MiR Assays, Human (Applied Biosystems, Foster City, CA) were used. Cycle passing threshold (Ct) was recorded and normalized to RNU6B expression. Relative expression was calculated as 2^{Ct_miR-21-Ct_RNU6B}. PCR reactions were carried out in duplicate. All qRT-PCR values were calculated as ratios to the U6-normalized qRT-PCR miR-21 value in the first normal specimen. For calculating the sensitivity and specificity of miR-21 to diagnose CCAs vs. non-cancerous tissues, a cut-off of 4.5 was used.

Quantitative qRT-PCR for mRNA expression

iQ SYBR Green Supermix (BIO-RAD) was used. Primer sequences are available in Supplementary Table 1. PCR products were confirmed by melting curve analysis. Beta-actin was used to normalize mRNA expression levels. Relative expression was calculated as 2^{Ct_target gene-Ct_beta-actin}. PCR reactions were carried out in duplicate.

Transfection of miR-21 inhibitor

Synthesized RNA duplexes of miR-21 inhibitor were purchased from Dharmacon (Lafayette, CO). 30~50% confluent cells were transfected with 60nM of miR-21 inhibitor, or inhibitor-negative control using Lipofectamine RNAi MAX (Invitrogen). RNA and proteins were harvested 72 hours after transfection. To document that miR-21 inhibition does not affect the level of unrelated microRNA species, we measured the level of an unrelated microRNA, miR-590-5p. While the HuCCT1 cells treated with miR-21 inhibitor showed a significant decrease in the level of miR-21 compared to the untreated cells, the level of miR-590-5p was the same in the cells treated with miR-21 inhibitor and in the mock condition (Supplementary Figure 1). To document that miR-21 transfection does not affect the level of unrelated proteins, we verified that the level of p21 were not affected. Supplemental Figure 2 shows that transfection with miR-21 inhibitor does not affect the levels of p21 in the TFK1 cholangiocarcinoma cells.

Western blotting

Cells were lysed in Laemmli sample buffer (Bio-Rad, Hercules, CA) supplemented with a protease inhibitor (Complete, EDTA-free (Roche)). Protein concentration was measured using a BCA Protein Assay kit (Pierce, Rockford, MA). Cell lysates (50μg) were electrophoresed on 10–20% polyacrylamide gels (Bio-Rad) and transferred to Immobilon-PSQ membranes (Millipore, Bedford, MA). The membranes were blocked with TBS containing 5% skim milk and 0.1% Tween-20, then incubated with the primary antibody. Anti-PDCD4, anti-TIMP3 and anti-p21 antibodies produced in rabbit (Sigma-Aldrich, St. Louis, MO 63178 for PDCD4 and TIMP3 and Zymed, San Francisco, CA) were used according to the manufacturer’s instructions. The membranes were incubated after washing with the secondary antibody, HRP-conjugated goat anti-rabbit IgG (Calbiochem, Gibbstown, NJ) and analyzed using enhanced chemiluminescence-plus reagent (GE Healthcare, Buckinghamshire, UK).

Results

MicroRNA arrays identify differentially expressed miRs in primary CCA specimens

Microarrays were performed on 5 NBD and 5 CCAs. 221 miRs exhibited at least 50% of values above background. With the FDR set at 1.44, SAM identified 20 over- and 112 underexpressed miRs in CCAs vs. NBDs. This dysregulation imbalance agreed with previous elegant in vitro CCA studies¹¹. The top 10 miRs in each category are listed in Table 2. MiR-200b, overexpressed in CCA cell lines Mz-ChA-1 and TFK¹¹, was not overexpressed in our primary CCAs. Similarly, although overexpressed in cell lines¹¹, miR-141 was not overexpressed in our primary CCAs. Conversely, miRs -93, -25, -21, and -27a, were found overexpressed both in CCA cell lines¹¹ and in our primary CCAs. Of note, the miR 25-93-106b cluster was recently found overexpressed and involved in gastric cancer¹⁵. Interestingly, miR-106b, of the same family with miRs -93 and -25, was also overexpressed in our primary CCAs. Among miRs underexpressed in primary CCAs, miR-560 has not been previously described in human cancers. In contrast, miR-370, was found underexpressed in both our primary CCAs and in cell lines¹². Analogously, miR-198, underexpressed in our primary CCAs, is underexpressed in hepatocellular carcinomas (HCCs)¹⁶.

Table 2.

Table 2a. MiRs overexpressed in primary CCAs vs. normal tissues and b. MiRs underexpressed in primary CCA vs. normal tissues. The ten most overexpressed miRs and the ten most underexpressed miRs in CCA vs. normal specimens are listed. Score(d), SAM score.

Gene ID	Score(d)	Fold Change
hsa-miR-106b	6.567	3.494
hsa-miR-21	5.596	7.765
hsa-miR-107	4.601	2.089
hsa-miR-106a	4.512	2.416
hsa-miR-93	4.235	2.484
hsa-miR-27a	3.760	2.323
hsa-miR-19a	3.539	2.366
hsa-miR-103	3.431	2.005
hsa-miR-17-5p	3.387	1.943
hsa-miR-25	3.200	2.102

Gene ID	Score(d)	Fold Change
hsa-miR-560	−6.551	0.053
hsa-miR-370	−5.495	0.045
hsa-miR-198	−5.252	0.077
hsa-miR-188	−4.941	0.030
hsa-miR-662	−4.847	0.061
hsa-miR-191*	−4.634	0.063
hsa-miR-512-3p	−4.493	0.050
hsa-miR-520e	−4.431	0.017
hsa-miR-513	−4.286	0.035
hsa-miR-494	−4.271	0.048

MiR-21 is overexpressed in human CCA specimens

In our array data, miR-21 was the most fold overexpressed microRNA and had the second-highest SAM score (Table 2 and Figure 1). Its average expression was 7.7-fold greater in CCAs than in NBDs. This ratio resembles the in vitro difference reported for miR-21 in Mz-ChA-1 (2.74 fold) and in TFK (4.35 fold) ¹¹ and agrees with similar findings in HCC tissues¹⁷. Moreover, a similar fold difference (4.7 to 10-fold) was reported in HCC cell lines as well as in HCC vs. normal liver tissues (2 to 65-fold)¹⁸.

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.

qRT-PCR data for miR-21 validate array findings

Our qRT-PCR data closely matched our array data (Figure 1). The Pearson correlation coefficient between array and qRT-PCR was 0.91. The average expression of miR-21 measured by qRT-PCR was 5.9 fold higher in CCAs vs. NBDs, confirming our array data.

Differential qRT-PCR expression for miR-21 is validated prospectively (Figure 2a)

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).

To prospectively validate miR-21 overexpression in human CCA, we verified its expression in the remaining 30 specimens included in the study: 18 CCAs and 12 normal tissues. Normal tissues displayed uniformly low expression of miR-21, with a standard deviation (SD) of 1.34. In contrast, CCAs displayed more variable expression (SD 11.19). No correlation was found between the level of miR-21 expression and cancer location (extrahepatic vs. intrahepatic), TNM stage or histologic grade. All but one cancer displayed miR-21 levels greater than the highest value in normal specimens. The fold difference between cancer and normal groups was 5.4, consistent with our comparison of 5 cancers vs. 5 normal tissues.

MiR-21 accurately discriminates between CCA and normal bile duct

miR-21 correctly diagnosed 22 of 23 cancers and 17 of 17 normal specimens, yielding a sensitivity of 95% and specificity of 100%. The area under the ROC curve (AUROC) was 0.995 (Figure 2b).

MiR-21 targets Programmed Cell Death 4 (PDCD4) in CCA

By in-silico searches, we identified PDCD4 as a potential miR-21 target. PDCD4 is downregulated in HCC tissues¹⁹ and is involved in TGF-beta1-induced-apoptosis in an HCC cell line¹⁹, however, it has not been previously implicated in cholangiocarcinogenesis. By extension, we hypothesized that PDCD4 may be a tumor-suppressor-gene in CCA, and that its expression could be regulated by miR-21. Transfecting the CCA cell lines CAK1, TFK1 and HuCCT1 with a miR-21 inhibitor (miR-21i) resulted in a dramatic increase in PDCD4 protein levels (Figure 3a). This finding strongly suggests that PDCD4 is regulated by miR-21 in CCA.

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.

MiR-21 regulates PDCD4 at the level of protein translation in CCA specimens

To elucidate the mechanism by which miR-21 regulates PDCD4, we measured mRNA levels of PDCD4 in normal liver and CCA specimens. Figure 4a shows that PDCD4 mRNA was approximately equal in CCA and normal tissues (mean level 4.32 in normal vs. 4.4 in CCAs). These findings argue that miR-21 inhibits PDCD4 protein production, rather than degrading its mRNA. Figure 4b shows that there was no effect on PDCD4 mRNA levels following miR-21i transfection of HuCCT1 and TFK1 CCA cells. This finding further suggests that, in CCA, miR-21 regulates PDCD4 at the level of protein translation. After we performed our own experiments, a similar miR-21–PDCD4 interaction was just recently observed in the human embryonic kidney cell line HEK-293T²⁰.

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.

Tissue Inhibitor of Metalloproteinases 3 (TIMP3) mRNA is statistically significantly underexpressed in CCAs

By employing in-silico searches, TIMP3 was also identified as a miR-21 target in CCA. The sole study suggesting TIMP3’s involvement in cholangiocarcinogenesis reported that 8.9% of 79 intrahepatic CCA tissues displayed TIMP3 promoter hypermethylation²¹. In contrast, 42% of HCCs analyzed showed TIMP3 hypermethylation²², suggesting that this mechanism may be more important in HCCs. Moreover, upregulation of TIMP3 expression in the HCC cell line HCC-7721, inhibits invasion in-vitro and metastasis in nude mice²³. To assess the expression of TIMP3 in our CCA specimens, we performed qRT-PCR of TIMP3 mRNA. Figure 5 shows that TIMP3 mRNA was significantly higher in normal than in CCA tissues. This finding strongly suggests, for the first time, that TIMP3 is an important tumor-suppressor-gene in cholangiocarcinogenesis.

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.

MiR-21 targets TIMP3 in CCA

Based on the qRT-PCR assays for TIMP3 mRNA and miR-21, we hypothesized that miR-21 is a major regulator of TIMP3. To test this hypothesis, we transfected TFK1 and HuCCT1 with NSI or miR-21i and performed qRT-PCR for TIMP3 mRNA. For both TFK1 and HuCCT1, TIMP3 did not amplify to 50 cycles in both untreated and NSI-treated cells. Upon treating with miR-21i, the mRNA for TIMP3 was amplified at cycle 42 in TFK1 cells and 35 in HuCCT1 cells. We then transfected CAK1, TFK1 and HuCCT1 with miR21i or NSI. Figure 3b shows that miR-21 inhibition resulted in a remarkable increase in TIMP3 protein levels. These results show, for the first time, that miR-21 is an important regulator of TIMP3 protein level in CCA. A recent study performed in glioma cell lines demonstrated that miR-21 does not directly bind to the TIMP3 3′UTR. This finding suggests that the interaction between miR-21 and TIMP3 in glioma cell lines is likely indirect²⁴.

Discussion

Previous seminal microRNA research in CCA has been performed in cell lines^{11, 12, 25}. Interestingly, work from Dr. Tushar Patel’s laboratory revealed that microRNA profiles are not identical in CCA cell lines¹¹. This finding suggests that different miRs exert unique carcinogenetic contributions, depending on the particular environment in which they occur. Therefore, in the first stage of our study, we used human primary CCA specimens rather than cell lines, in an attempt to appreciate the impact that miRs may exert on cholangiocarcinogenesis in vivo. Our results confirmed many, although not all, of the profiling results found in cell lines. Among miRs overexpressed in primary human CCA tissues, miR-21 had also been reported in previous cell line research¹¹, perhaps because of its paramount involvement in the cholangiocarcinogenesis. Although miR-21 has been reported as overexpressed in a variety of tumors^{18, 24, 26–29}, it has not been reported in primary human CCA tissues. Moreover, our study suggests that miR-21 is uniformly overexpressed in human CCA, in view of our finding that it was 95 % sensitive and 100% specific in diagnosing human CCA. This finding may address the existing difficulty of diagnosing CCA by current means, such as imaging.

PDCD4 was initially identified as an upregulated apoptosis-related protein³⁰, suggesting its role as a tumor-suppressor-gene³¹. While PDCD4 was recently reported as a potential tumor-suppressor in hepatocarcinogenesis¹⁹, our work is the first reporting its involvement in cholangiocarcinogenesis. Moreover, this is the first report of PDCD4 being inhibited by miR-21 in CCA. The interaction between miR-21 and PDCD4 was previously reported in breast cancer^{20, 32, 33} and colon cancer³⁴ suggesting that this interplay may be a general carcinogenetic pathway, rather than a tissue-specific mechanism. Furthermore, since the mRNA levels of PDCD4 were similar in human CCA and normal specimens, we conclude that miR-21-induced PDCD4 inhibition is posttranscriptional (Figure 4a). Confirming our findings, a similar level of mRNA for PDCD4 was found in colon cancer and normal specimens in a previous study³⁴. As shown before in Colo206f, HeLa and HEK-293T cells, miR-21 directly binds to its binding site in the 3′UTR of PDCD4^{20, 32–34}.

Matrix metalloproteinases play a crucial role in cancer invasion and metastasis³⁵. Among the 4 members of the family, TIMP3 is uniquely pro-apoptotic³⁵. For example, the adenoviral transfer of TIMP3 into HeLa, HT1080 fibrosarcoma cells, and melanoma cells reduces their invasiveness and stimulates apoptosis^{36, 37}. More recently, herpes simplex-mediated transfer of TIMP3 into neuroblastoma and malignant peripheral nerve tumor xenografts reduced tumor growth and reduced density of the tumor vascular network³⁸. Nonetheless, there are no previous reports regarding the roles of TIMP3 in CCA. A singular manuscript reported low-level promoter methylation of TIMP3 in intrahepatic CCA²¹. Our manuscript is the first to report 1) lower levels of mRNA for TIMP3 in human CCA specimens compared to normal human specimens; 2) an inhibitory effect of miR-21 on TIMP3 in CCA cell lines. Taken together, these findings provide support for our hypothesis that TIMP3 is a tumor-suppressor-gene in CCA and that its activity is closely regulated by miR-21.

Supplementary Material

Supp Fig 1. Supplementary Figure1.

HuCCT1 cells treated with miR-21 inhibitor showed a significant decrease in the level of miR-21 compared to the untreated cells. There were similar levels of miR-590-5p in the cells treated with miR-21 inhibitor and in the mock condition. Y-axis displays the fold difference relative to the mock condition.

Supp Fig 2. Supplementary Figure 2.

miR-21 inhibitor does not affect the levels of p21 in the TFK1 cholangiocarcinoma cells. NT – non-treated cells, NSI – cells treated with micro-RNA inhibitor negative control (nonspecific inhibitor) and 21i – cells treated with miR-21 inhibitor.

Supp Tab. Supplementary Table 1.

The forward and reverse primer sequences for mRNA qRT-PCR for PDCD4 and TIMP3, respectively, are listed.

Table 1. Clinicopathologic data for CCAs.

Location – intrahepatic (I) or extrahepatic (E). Two cancers originated in the perihilar region and were labeled extrahepatic-Klatskin tumor (E-K) to differentiate them from the distal extrahepatic cancers (E).

	Location	Age	Gender	Size	Stage	Differentiation
T1	I	58	F	5.5 cm	T1NxMx	NA
T2	I	73	M	4.5cm	T3N1Mx	M
T3	I	77	F	7cm	T3N0Mx	NA
T4	E	84	F	2.2cm	T3N1MX	P
T5	I	66	F	3.2cm	T1N0M0	M
T6	E	64	M	1.5 cm	T3N1M0	M
T7	E-K	46	F	3.5 cm	T3NxMx	M
T8	E	84	F	3.5 cm	T3N1Mx	P
T9	I	57	M	8 cm	T1N0M0	M
T10	E	66	M	NA	T3N1Mx	M
T11	E	66	M	2.5cm	T3N1Mx	P
T12	E	85	F	4cm	T3N1Mx	M
T13	E	70	M	2cm	T3N1Mx	P
T14	I	61	M	6cm	T3N1Mx	M-P
T15	E	72	M	NA	T3N1M0	M
T16	I	63	F	NA	NA	M
T17	I	71	F	19cm	T3N0Mx	P
T18	I	54	F	5.4cm	T4N0M1	M-P
T19	I	36	F	0.3cm	T1N0MX	M-P
T20	I	49	F	3cm	T2NXMX	M
T21	I	50	F	7cm	T1N0Mo	M
T22	E	47	M	3cm	T3N1Mx	M-P
T23	E-K	54	M	4cm	T2N0Mx	W

Age – age at surgery. Gender – female (F) and male (M). Size – measured in centimeters (cm) at the time of resection. Staging – TNM staging. Differentiation – well (W), moderately (M), moderately-poorly (MP) and poorly (P) differentiated. NA – not available

Abbreviations

CCA

Cholangiocarcinoma

miR-21

MicroRNA-21

NBD

normal bile duct specimens

PDCD4

programmed cell death 4

TIMP3

tissue inhibitor of metalloproteinases 3

PTEN

phosphatase and tensin homolog deleted on chromosome 10

SAM

Significance Analysis of Microarrays

FDR

false discovery rate

qRT-PCR

quantitative reverse transcriptase polymerase chain reaction

ROC

Receiver Operating Characteristic

Ct value

Cycle passing threshold

SD

Standard deviation

NSI

Non-specific miR inhibitor

miR21i

miR-21 inhibitor

HCC

Hepatocellular Cancer