MicroRNA-21 Regulates the Invasion and Metastasis in Cholangiocarcinoma and May Be a Potential Biomarker for Cancer Prognosis

Cholangiocarcinoma (CCA) is a highly malignant tumor derived from bile duct epithelial cells (Sawanyawisuth, 2009). Its represents 10% to 15% of the total hepatobiliary tumors and its incidence andmortality are increasing worldwide. Most patients with bile duct cancer are diagnosed when the disease is advanced, the overall prognosis is poor, usually only survive for several years (Blechacz et al., 2008; Blechacz et al., 2011). However, in recent years, multiple studies have been conducted to investigate the genes and their products that are involved in cholangiocarcinoma metastasis (Andersen et al., 2 012; Shen et al., 2010). MicroRNAs are a class of naturally occurring small non-coding RNAs that control gene expression by targeting mRNAs for translational repression or cleavage (Pillai et al., 2005; Iorio et al., 2012). Recently, the role for MicroRNAs in the establishment and progression of cholangiocarcinoma has become evident,and some


Introduction
Cholangiocarcinoma (CCA) is a highly malignant tumor derived from bile duct epithelial cells (Sawanyawisuth, 2009).Its represents 10% to 15% of the total hepatobiliary tumors and its incidence andmortality are increasing worldwide.Most patients with bile duct cancer are diagnosed when the disease is advanced, the overall prognosis is poor, usually only survive for several years (Blechacz et al., 2008;Blechacz et al., 2011).However, in recent years, multiple studies have been conducted to investigate the genes and their products that are involved in cholangiocarcinoma metastasis (Andersen et al., 2 012;Shen et al., 2010).
MicroRNAs are a class of naturally occurring small non-coding RNAs that control gene expression by targeting mRNAs for translational repression or cleavage (Pillai et al., 2005;Iorio et al., 2012).Recently, the role for MicroRNAs in the establishment and progression of cholangiocarcinoma has become evident,and some

MicroRNA-21 Regulates the Invasion and Metastasis in Cholangiocarcinoma and May Be a Potential Biomarker for Cancer Prognosis
Qiang Huang 1& , Lei Liu 1& , Chen-Hai Liu 1 , Hao You 1 , Feng Shao 1 , Fang Xie 1 , Xian-Sheng Lin 1 , San-Yuan Hu 2 *, Chuan-Hai Zhang 1   miRNAs have been identified as oncogenes or tumor suppressor genes in cholangiocarcinoma (Li et al., 2012;Zhang et al., 2012).Specifically, microRNA-21 stands out as the microRNA most often overexpressed in very diverse types of malignancy.Moreover, microRNA-21 may play an important role in many cancer-related processes such as apoptosis, invasion, and metastasis (Meng et al., 2007;Markou et al., 2008;Slaby et al., 2007).Florin et al also found microRNA-21 was always overexpressed in cholangiocarcinoma cell (Florin et al., 2009).However, the precise role played by microRNA-21 in cholangiocarcinoma invasion and metastasis is unknown.In a bioinformatic analysis, RECK has been known to contain the microRNA-21 binding site, which acts as a tumor suppressor gene regulating various aspects associated with cancer cell invasion, metastasis (Liu et al., 2010;Han et al., 2011).In this study, based on these various aspects of the microRNA-21 function, we hypothesized that microRNA-21 played an important role in the progression of cholangiocarcinoma and potential regulated RECK expression.Furthermore, we evaluated whether or not microRNA-21 as a biomarker for earlier diagnosis and predicting the survival of cholangiocarcinoma patients.

Human tissues
Surgical specimens from patients who had been surgically treated for cholangiocarcinoma at Anhui Provincial Hospital, Hefei,form 1 January 2005 to 1 January 2010 were employed.All without radiotherapy or chemotherapy before operation.All tumors were clinically and histologically diagnosed as cholangiocarcinoma.Inclusion criteria for all cases included: (i) unambiguous histology and absence of mixed tumour types; (ii) sufficient viable tissue available for RNA extraction; (iii) absence of any treatment prior to surgery;and (iv) age of tissue block less than 7 years (Jaime et al., 2011).Eventually leaving 41 cases specimens meet the requirements,there were 25 cases of men,16 women, from 32 to 80 years old(The median age 60±2.6 years).The clinicopathological characteristics of the patients are given in Table 1.From each block, 15 slices of 5 µm each were collected in one 1.5 mL tube for RNA extraction and microRNA analysis.

Cell lines and cultures
The cholangiocarcinoma RBE cell were obtained from the Cell Bank of the Chinese Academy of Sciences.All cells were grown in RPMI 1640 medium and with 10% fetal bovine serum at 37°C in a humidified atmosphere of 95% air and 5% CO 2 .

In situ hybridization of microRNA
In situ hybridization was done according to the manufacturer's protocol for formalin-fixed, paraffin embedded tissue.The probes were 3', 5'-labeled with digoxigenin with the DIG tailing kit (Exiqon).The staining was carried out as previously described.After deparaffinization, the specimens were subjected to proteinase K (40 μg/ml) digestion for 20 mins.The postfixed tissues were subsequently incubated overnight with the locked nucleic acid-modified probes.For the immunodetection, tissues were incubated overnight at 4℃ in anti-DIG-AP FAB fragment (1:1000).The final visualization was carried out with BCIP/NBTP.The stained sections were reviewed and scored under a light microscope (Olympus America) independently by two investigators.The staining intensity and percentage of tissue staining were recorded (staining intensity: 1= no staining or weak staining, 2 = intermediate staining, 3 = strong staining; percentage:1 = <10%, 2 = 10-30%, 3 = >30%).Both scores multiplied,If more than 3 points that case was considered to be high expressed,otherwise it is low expressed (Lorenzo et al.,2007).

RNA Extraction and Real-time RT-PCR
Total RNA was extracted as described previously (Rosenfeld et al., 2007).Briefly, the samples was incubated several times in xylene at 60℃ to remove excess paraffin and then washed several times with ethanol.
Protein degradation was performed by incubation of the sample in a proteinase K solution at 55℃ for a few hours.The RNA was extracted using acid phenol/chloroform and then precipitated with ethanol, DNAse was introduced to digest DNA.The expression of mature microRNAs was determined by real-time PCR using the TaqMan microRNA assay.U6 small nuclear RNA was used as an internal control.The following primers were used: microRNA-21, 5'-ACACTCCAGCTGGGTAGCTTATCAGACTGATG-3 ' ( f o r w a r d ) ; 5 ' -C T C A A C T G G T G T C G T G G A -3'(reverse); andU6,5'-CTCGCTTCGGCAGCACA-3'(forward); 5'-AACGCTTCACGAATTTGCGT-3' (reverse).To quantify expression of RECK Mrna was quantified by real-time PCR.Glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA was used as an internal control.PCR primers used were as follows, RECK,5'-TTCAGAACCCCAACTACTG-3' (forward) and 5'-ATGCAAGATGCTTTGATGC-3' (reverse); and for GAPDH, 5'-TGCACCACCAACTGCTTAGC-3' (forward) and 5'-GGCATGGACTGTGGTCATGAG-3' (reverse).All PCRs were done in triplicates.

Transfections and In vitro Invasion and metastasis Assays
Transfection with microRNA-21, lipo (Liposome control group), microRNA-21-NC (Negative control group) and microRNA-21-IN (microRNA-21 inhibition group) was performed with Lipofectamine 2000 (Invitrogen) acording to the manufacturers' instructions.Cells were collected for experiments after were transfected in 48 h.Cell We used a Transwell insert to determine the effect of microRNA-21 on cholangiocarcinoma cell invasion and metastasis in vitro.Cells transfected with either microRNA-21 or microRNA-21-IN plated on transwell chambers precoated with Matrigel.Medium containing 10% fetal bovine serum in the lower chamber served as the chemoattractant.After the cells were incubated for 24 h at 37°C in a humidified incubator, the noninvading cells were removed with cotton swabs.The invasive cells attached to the lower surface of the membrane insert were fixed in 100% methanol at room temperature for 2 mins and stained with toluidine blue.The number of invasive cells on the lower surface of the membrane was then counted under a microscope.

Western blotting
For isolating the proteins, cells harvested in 6-well plates were washed once in PBS and lysed in the lysis buffer.SDS-PAGE and Western blotting were done according to standard procedures.Western blotting of GAPDH on the same membrane was used as a loading control.The signals were detected by secondary antibodies labeled with ECL Detection System, and signal intensity was determined by Labworks 4.6.

Statistical analysis
These analyses were performed with SPSS Version 17.0 statistical software package.Differences between two groups were compared using Pearson's chi-square test for qualitative variables and the Mann-Whitney U test.The overall Survival and relapse-free Survival curves were calculated using the Kaplan-Meier method and analyzed

F i g u re 1 . D e t e c t i o n o f m i c ro R N A -2 1 i n Cholangiocarcinoma Tissues by in Situ Hybridization.
The potisive rate for microRNA-21 was significantly higher in lymph node metastasis group than in that without (P<0.05;Figure 1A,B).The potisive rate for microRNA-21 was higher in the Perineural invasion group than that without (P<0.05;Figure 1C,D) (Magnification: ×400)

In situ hybridization showed microRNA-21 expression in cholangiocarcinoma
To visualize microRNA-21 expression in different tumor, we performed in situ hybridization in formalinfixed, paraffin-embedded tissue (Figure 1).The potisive rate for microRNA-21 expression was significantly higher in the cholangiocarcinoma with lymph node group (Figure 1B) than that without (Figure 1A).The potisive rate for microRNA-21 was higher in the Perineural invasion group (Figure 1D) than that without (Figure 1C); microRNA-21 expressions was significantly higher in tumors showing in tumor differentiation degree,lymph node metastasis, perineural invasion (P<0.05).It has nothing to do with others factor,such as gender, age, tumor location, tumor type (P>0.05)(Table 1).

qRT-PCR data for microRNA-21 validate In situ hybridization findings
Total RNA was isolated from cholangiocarcinoma tissues, and the microRNA-21 levels were determined by real-time PCR (Figure 2).The correlation of T/N ratios for microRNA-21 expression with the clinicopathologic factors listed in Table 1 was examined.The patients with Perineural invasion or lymph node metastasis were significantly higher than those without them (P=0.038,0.010; Figure 2A,B).

microRNA-21 can effect cell invasion and metastasis in cholangiocarcinoma
The four cell groups microRNA-21 levels were determined by real-time PCR (Figure 3A).We next assayed whether microRNA-21 can change the capacity of cholangiocarcinoma cells for invasion and metastasis.RBE cells were suitable for the invasion and metastasis assay, because they showed good invasion to the Matrigel membranes.The cell invasion and metastasis assay showed that knockdown of microRNA-21 resulted in decreased RBE cell invasion and metastasis rate compared with the control cells (P<0.05, Figure 3 B, C).In contrast, ectopic expression of microRNA-21 in RBE cells resulted in significant increased cell invasion and metastasis in transwell assays (P< 0.05, Figure 3 D, E).In addition,we found overexpression or knockdown of miRNA-21 have on obviously effect on cell proliferation or cell death in the firest 48h.

RECK, a putative target of microRNA-21
Bioinformatic analyses found that the RECK gene,among a number of others, may be a potential microRNA-21 target.We chose RECK for further analysis because it was previously implicated in promoting cholangiocarcinoma progression, specifically, its can effect cell invasion and metastasis ability (Ziyan et al.,  2011).Indeed, we found that RBE cells transfected with microRNA-21-IN had dramatically increased RECK levels by 10 fold, compared with the control cells (P<0.001, Figure 4).

Association of microRNA-21 expression with overall survival and relapse-free survival
We then performed Kaplan-Meier analyses to determine whether microRNA-21 expression was associated with overall survival and relapse-free survival in the cholangiocarcinoma patients.These Patients who had high microRNA-21 expression presented mean 3-year overall survival of 15%, whereas patients who had low microRNA-21 express presented mean 3-year overall survival of 33%.The difference was statistically significant (P = 0.046, Figure 5A).Similarly,a statistically significant association of microRNA-21 with relapse-free survival was also found (P = 0.036, Figure 5B).

Discussion
The current studies showed that a number of miRNAs were differentially expressed in cancer and normal tissues.In this regard, microRNA-21 has been suggested to function as an oncogene because it is overexpressed in many types of malignancy such as lung cancer (Liu et al., 2012), ovarian cancer (Iorio et al., 2007), colon cancer (Slaby et al., 2007), gastric cancer (Chan et al., 2008) and esophageal squamous cell carcinoma (Yukiharu Hiyoshi et al., 2009).Particularly, it had a high expression in the more aggressive cholangiocarcinoma cell and tissue samples (Florin et al., 2009).In vitro, to visualize microRNA-21 expression in different tumors, we performed in situ hybridization and real-time quantitative PCR in formalin-fixed, paraffin-embedded tissue (Figure 1, 2).The results showed the microRNA-21 expression in patients with lymph node metastasis or Perineural invasion was significantly higher than that in those without them,as suggested that abnormal expression of DOI:http://dx.doi.org/10.7314/APJCP.2013.14.2.829 Role of MicroRNA-21 in Cholangiocarcinoma Invasion and Metastasis 0

F
Mature microRNA-21expression was analyzed by TaqMan-quantitative real-time PCR and normalized to U6 expression.The ratios of tumor to normal tissue for microRNA-21expression were presented as relative T/N ratio of microRNA-21 expression.**P < 0.05.(P=0.038,0.010; Figure 2A,B) A B

Table 1 . Correlation of Clinicopathologic Parameters and MicroRNA-21 Expression
using the Log-rank test.The level of statistical significance was set at P≤0.05.