Chinese-German Journal of Clinical Oncology

February 2009, Vol. 8, No. 2, P77–P80

DOI  10.1007/s10330-008-0160-6

Expression profiles of miRNAs in human pancreatic cancer cell lines* Shineng Zhang, Haijun Zuo, Zhong Yu, Fengting Huang, Wa Zhong Department of Gastroenterology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China Received: 19 December 2008 / Revised: 29 December 2008 / Accepted: 5 January 2009 Abstract  Objective: To analyze initially the differences of miRNAs expression profiles in human pancreatic cancer cell lines by microarray technique. Methods: A total of 743 probes were designed according to the known miRNAs sequences of human, mice, and rats. miRNAs microarray was manufactured and its credibility was verified. Total RNAs were extracted and miRNAs were separated from human pancreatic cancer cell lines (SW1990, Capan-2, BxPC-3, Aspc-1, and Panc1) and immortal human pancreatic duct epithelial cell line H6C7. They were labeled with T4 RNA ligase, then were hybridized with microarray. Through array scan and analysis, miRNAs expression profiles in pancreatic cancer were obtained. The results were verified by Northern blotting and RT-PCR. Results: A total of 63 miRNAs related to pancreatic cancer were found to be differentially expressed in 5 pancreatic cancer cell lines, including 25 down-regulated and 38 up-regulated miRNAs. Expressions of mir-21 and let-7 were also confirmed. Conclusion: The results suggested that miRNAs expression profiles could be found in pancreatic cancer cells. Key words  pancreatic cancer; miRNAs; expression profiling

Pancreatic cancer is one of the most malignant tumors with a low survival rate. The majority of patients with diagnosed pancreatic cancer present with an already unresectable tumor stage and frequently need chemotherapy and radiotherapy. The median survival time following diagnosis is only 3–5 months [1]. However, little improvement in therapeutic options has been made in the last decade, requiring a urgent need for the knowledge of tumorigenesis and development of new therapies. The discovery of microRNAs (miRNAs) has broadened our understanding of the mechanisms that regulate gene expression with the addition of an entirely novel level of regulatory control. These small noncoding transcripts of 18–25 nucleotides modulate protein expression by binding to complementary or partially complementary target mRNAs and thereby targeting the mRNA for degradation or translational inhibition. miRNAs control various biological processes, including cell differentiation, cell proliferation, apoptosis, stress resistance, and fat metabolism [2–4] . Mounting evidence in recent years has shown that miRNAs play key roles in tumorigenesis due to abnorCorrespondence to: Shineng Zhang. Email: [email protected] * Supported by a grant from the Natural Science Foundation of Guangdong Province, China (No. 8151008901000139).

mal expression and mutations in miRNAs. Some miRNAs possess oncogenic or tumor suppressor activity. The first study documenting abnormalities in miRNA expression in tumors identified miR-15a and miR-16-1, which are located in a frequently deleted region in B-cell chronic lymphocytic leukemia [5]. The let-7 family, which is down-regulated in lung cancer when RAS is frequently mutated [6], negatively regulates RAS [7]. miR-155 is overexpressed in Burkitt’s lymphoma [8], breast cancer [9], and lung cancer [10]. Its overexpression in transgenic mice leads to preleukemic pre-B-cell proliferation [11]. All these findings suggest that altered miRNA expression contributes to tumorigenesis. In this study, to determine if there are changes in miRNA expression in a well characterized set of human pancreatic cancer cells we analyzed RNAs from 5 pancreatic cancer cell lines and 1 immortal human pancreatic duct epithelial cell line using miRNA microarrays containing probes targeting 576 human mature miRNAs.

Materials and methods Cell lines Human pancreatic cancer cell lines including SW1990, Capan-2, BxPC-3, Aspc-1 and Panc1 were preserved in our lab and cultured in RPMI1640 media (Gibco, USA)

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with 10% fetal calf serum. The immortalized pancreatic ductal cell H6C7 was provided by Prof. Ming-sound Tsao (Ontario Cancer Institute, Toronto University, Canada), and cultured in DMEM/F-12 media (Gibco, USA). These cells were all cultured at 37 ℃ in a 5% CO2 atmosphere. Preparation of miRNAs microarray Mammalian miRNAs microarray was produced by Boao Biology Co., Ltd, Beijing, China. Mammalian miRNAs microarray V2.0 aimed directly at 576 human mature miRNAs, 238 rat mature miRNAs and 358 mice mature miRNAs, and the intersection was selected [12, 13]. A total of 743 probes were designed (the data was from Sanger miRNA database of 2006: miRBase8.2). These probes were spotted by SmartArrayTM Biochip Microarrayer in a 75 mm × 25 mm glass slide after chemical modification. The samples spotted in microarray also included human U6, and tRNA was used as the internal standard and 8 artificial probes with 30 bases were used as the external standard, including Zip5, Zip13, Zip15, Zip21, Zip23, Zip25, Y2, and Y3. To realize the quality control of microarray, Hex was used as the positive control of spotting, and 50% DMSO was used as the negative control of hybridization. Separation and fluorescent labelling of micromolecular RNAs The total cellular RNA was extracted by one-step Trizol, and then condensed through isopropyl alcohol deposition. It was measured by spectrophotometer and then the quality of total RNA was checked by formaldehyde denaturing gel electrophoresis. MiRNAs were separated from 50–100 μg total RNA by PEG method, and fluorescently labelled with T4RNA ligase. Then it was precipitated with dehydrated alcohol, and dried finally. Microarray hybridization and data analysis The fluorescently-labeled micromolecular RNA was dissolved in 16 μL hybridization solution (15% formamide, 0.2% SDS, 3 × SSC, 50 × Denhardt’s), at 42 ℃ and stayed overnight. It was washed in solution with 0.2% SDS and 3 × SSC at 42 ℃ for 4 min, then washed in solution with 0.2% SSC at room temperature for 4 min. After drying the glass slide, it was scanned immediately by LuxScan 10K/A dual pathways laser scanner (Boao Biological Company, Beijing, China), then the array images were analyzed by LuxScan 3.0 image analysis software (Boao Biological Company, Beijing, China). The differential expression sites were screened by significance analysis of microarrays (SAM, version 2.1), and the screening conditions included: FDR was limited in 5%, and Fold change was not less than 1.5 times. Northern blotting 100 µg from each of six total RNAs was extracted re-

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spectively, and separated with 8 mol/L urea denatured polyacrylamide gel (15%). Then they were transferred to nylon membrane by semi-dry transition system, and fixed by UV cross linking method. After prehybridization, probes labelled by [γ-32P]ATP were added, and then hybridized for 24 h at 42 ℃. Then washing membrane, solarization and radioautography were carried out. The oligonucleotide probes used in our study included: mir-21: 5’-TCAACATCAGTCTGATAAGCTA-3’; let-7: 5’-TACTATACAACCTACTACCTCAATTTGGC-3’; U6: 5’-GCAGGGGCCATGCTAATCTTCTCTGTATCG-3’. RT-PCR 5 µg from each of six total RNAs was extracted respectively. Specific primers which were complementary with miRNA precursors and retroviridase Superscript III were used for reverse transcription. Then, 1 μL reverse transcript was used for PCR amplification of miRNA precursors. The primers were as follows: mir-21, sense: 5’-GCTTATCAGACTGATGTTGACTG-3’, antisense: 5’-CAGCCCATCGACTGGTG-3’; let-7, sense: 5’-CCTGGATGTTCTCTTCACTG-3’, antisense: 5’-GCCTGGATGCAGACTTTTCT-3’; U6, sense: 5’-CTCGCTTCGGCAGCACA-3’, antisense: 5’-AACGCTTCACGAATTTGCGT-3’. The size of PCR product was checked by 15% non-denaturing polyacrylamide gel.

Results Separation and purification of cell miRNAs The D260/D280 values of miRNAs from six kinds of cells were between 1.8 and 2.1, which were consistent with the request of miRNA array. Result of microarray hybridization The miRNAs microarrays of human, mice, and rats were successfully manufactured. The whole point lattice was divided into 8 deuto-lattice, which had 20 rows and 18 columns. The distant of points was 245 μm, and the diameter of point was about 150 μm. Each probe repeated for three times (Fig. 1). The array images after scanning were analyzed and the data was normalized as a whole. Through comparison, we found that there were 63 miRNAs differentially expressed between human pancreatic cancer cells and immortal human pancreatic duct epithelial cells, 25 of which were lowly expressed and 38 were highly expressed. It was showed by preliminary bioinformatics analysis that the expressions were related to cell proliferation, apoptosis and cell regulation of signal transduction gene. Among them, mir-21 and let-7 expressed in 5 pancreatic cancer cells similarly, and there were different levels of up-regulation and down-regulation.

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Fig.  1  Scanning images after miRNAs hybridization. a: H6C7 cell; b: Capan-2 cell

Cluster analysis of miRNAs expressions The images after hybridization obtained by scanning were managed by data normalization and logarithmic transformation, then cluster analysis was carried out by Cluster 3.0 (Stanford University, USA) (Fig. 2). Verification on the expression differences of mir-21 and let-7 by Northern blotting and RT-PCR Pre-miRNA and mature miRNA (70–80 nt) from total RNA were detected by Northern blotting. There were different levels of high expression of mir-21 in 5 pancreatic cancer cell lines, however, let-7 was lowly expressed (Fig. 3). mir-21 and let-7 precursors were amplified by specific primers, using U6 as the internal control. The results were similar between experiments of three times, and the detection results of array were coincident essentially (Fig. 4).

Discussion Since miRNAs were found, their roles in gene regulatory networks had received considerable attention. To explore the relationship between miRNAs and diseases has become a research hot spot in biomedical field currently. Now, it is clear that there may be abnormal expression of miRNAs in a variety of tumors, and there may be different miRNAs expression profiles in different tumor types. MiRNAs may be a new oncogene or tumor suppressor gene. There are lots of research methods for miRNAs. Microarray technique is a new method developed from

Fig.  3  Expressions of mir-21 and let-7 in cells detected by Northern blotting

Fig.  4  Expressions of mir-21 and let-7 in cells detected by RT-PCR

90’s of the last century, and a large number of biological molecules can be detected and analyzed one time, with characteristics of high automation and throughput. The miRNAs expression profiles of pancreatic cancer, chronic pancreatitis and normal pancreatic tissues were compared by Bloomston et al [14–16] with array technique, and the results showed that there were specific miRNAs expressions in pancreatic cancer tissues, in which the expressions of mir-196a-2, mir-221, and mir-21 and so on were up-regulated, while let-7 was down-regulated. In our study, the miRNAs expression differences of 5 human pancreatic cancer cells and immortal human pancreatic duct epithelial cells were compared by the newest miRNA array, and the results showed that a total of 63 miRNAs were found

Fig.  2  Cluster analysis of miRNAs expressions in cells. Yellow: positive; blue: negative; black: zero; grey: missing

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to be differentially expressed. The preliminary analysis of bioinformatics showed that the expressions were mainly related to cell proliferation, apoptosis, and gene regulation of cell signal transduction. It was detected that in our study the expressions of mir-21 were up-regulated with different degrees in pancreatic cancer cells and the results were further confirmed by Northern blotting and RT-PCR, which were consistent with the up-regulation in pancreatic cancer tissues reported in literatures. Mir-21 was a mRNA which was found earlier, and it was up-regulated and might play an anti-apoptosis factor role in tumors such as glioma, breast cancer, lung cancer and colon cancer and so on. Apoptosis could be increased when mir-21 expression was down-regulated by antisense oligonucleotides which aimed directly at mir-21, indicating mir-21 might play oncogene-like role in the onset and development of tumors [17]. Meanwhile, we also found that let-7, which was one of the earliest miRNAs and complementary with multiple tumor-related genes, was significantly down-regulated in pancreatic cancer cells. Recently, the relationships between miRNAs expression profiles of different cell subgroups and their differentiation potentials in vitro were compared by Yu et al [18] with enriching and screening model of breast cancer stem cells, and they found that the decreased or absent expression of let-7 was the most significant in breast cancer stem cells. The target Ras and HMGA2 gene could be inhibited by let-7, which indicated that let-7 might play a very important role in maintaining the “stem cell characteristic” and tumor formation characteristic of breast cancer stem cells. Target miRNA regulation was studied by some scholars with tumor-associated miRNA antisense technology and RNAi technology, and the potential treatment prospects was shown by the preliminary results [19, 20]. Compared with tumor tissues, interference of tumor interstitial cells might be eliminated using tumor cell lines as the research object. In our study, the miRNAs expression differences of 5 human pancreatic cancer cell lines and 1 immortal human pancreatic duct epithelial cell line were analyzed, and specified miRNAs expression profile was obtained initially. We are studying and analyzing the associated biological function and its clinical significance.

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