Analysis of sequence variations in 59 microRNAs in hepatocellular carcinomas

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Mutation Research 638 (2008) 205–209

Short communication

Analysis of sequence variations in 59 microRNAs in hepatocellular carcinomas Jine Yang a , Fan Zhou a , Teng Xu a , Hua Deng a , Yi-Yuan Ge a , Changqing Zhang b , Jinqing Li b , Shi-Mei Zhuang a,b,∗ a

Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-Sen University, PR China b State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-Sen University, PR China Received 29 March 2007; received in revised form 18 June 2007; accepted 16 August 2007 Available online 21 August 2007

Abstract It is well demonstrated that mutations in protein-coding genes play a key role during carcinogenesis. Whether sequence variations in microRNA genes are also associated with tumorigenesis is still unknown and thus require extensive investigations. In the present study, genomic sequences coding for the precursors of 59 microRNA genes were analyzed in both hepatocellular carcinoma (HCC) tissues and liver cancer derived cell lines. In total, four variations in three microRNAs, including miR-106b, miR-192 and let-7a-2, were found in four out of 96 HCC tissues. Further investigation in the corresponding adjacent non-cancerous tissues identified the same sequence variations, suggesting the possibility of germline mutations or natural polymorphisms. Moreover, no variation was found in eight liver cancer derived cell lines. Among four sequence alterations observed in this study, two were located in miR-106b and identified as known single nucleotide polymorphisms, while the other two found in miR-192 and let-7a-2 had not been reported before. In conclusion, our data suggest that mutation of microRNA is a rare event in HCC and thus may not represent a main mechanism underlying hepatocarcinogenesis. © 2007 Elsevier B.V. All rights reserved. Keywords: MicroRNA; Sequence variation; PCR-SSCA; HCC

1. Introduction MicroRNAs (miRNAs) are small noncoding RNA molecules that may regulate the expression of target Abbreviations: CLL, chronic lymphocytic leukemia; HCC, hepatocellular carcinoma; miRNA(s), MicroRNA(s); PCR-SSCA, PCR-based single-strand conformational analysis; pre-miRNA, microRNA precursor; SNPs, single nucleotide polymorphisms; HBV, hepatitis B virus ∗ Corresponding author at: Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-Sen (Zhongshan) University, Guangzhou 510275, PR China. Tel.: +86 20 84112164; fax: +86 20 84112169. E-mail address: [email protected] (S.-M. Zhuang). 0027-5107/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2007.08.007

genes by either suppressing the mRNA translation or inducing the mRNA cleavage [1]. Growing evidences have indicated a strong association between miRNAs and oncogenesis. A large portion of human miRNA genes have been mapped to the chromosome regions frequently altered in cancers [2,3]. Furthermore, tumor specific expression patterns of miRNAs have been observed in different types of human cancers [4–12]. It is well demonstrated that mutations in proteincoding genes play a key role during carcinogenesis. Whether sequence variations of miRNA genes are also involved in tumorgenesis is still unknown. To date, miRNA sequence aberrations in cancer have only been reported in two studies [13,14]. Calin et al. identified

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mutations in five of 42 miRNAs in 11 out of 75 chronic lymphocytic leukemia (CLL) samples. Furthermore, they showed that a germ-line mutation located 7 bp downstream of the miR-16-1 precursor resulted in dramatically reduced expression of mature miR-16-1 both in vitro and in vivo [13]. Controversially, investigation on 15 miRNA genes in a panel of 91 human cancer cell lines suggest that sequence alterations in miRNA genes are unlikely to affect the miRNA processing although some of them resulted in significant changes in the predicted secondary structure of primary miRNA [14]. Obviously, more extensive investigations on different types of cancers are required to elucidate the functional implication of sequence variations in miRNA. In the present study, we analyzed the sequences of 59 miRNA genes in both hepatocellular carcinoma (HCC) tissues and liver cancer derived cell lines. The miRNAs analyzed are selected by virtue of their abnormal expression or their localization to the sites of frequent chromosomal instability in human cancers, or their high level of expression in the liver. Among 96 patients, a total of four variations in three miRNAs were identified in four patients, both in their tumor tissues and the corresponding adjacent non-cancerous tissues. However, no variation was found in eight liver cancer derived cell lines. These results suggest that sequence variation of miRNA is a rare event in HCC.

2. Materials and methods 2.1. Tissue samples and cell lines Tumors and their corresponding adjacent non-cancerous liver tissues were obtained from 96 patients who were diagnosed with HCC and underwent hepatectomy between March 2000 and September 2003 at Cancer Center, Sun Yat-Sen University (Guangzhou, China). All nonneoplastic and neoplastic samples were histologically confirmed. Tissues were snap frozen in liquid nitrogen after surgical resection and then stored at −80 ◦ C until analysis. All subjects were unrelated ethnic Han Chinese and resided in Guangzhou and the surrounding regions. Informed consent was obtained from all study participants. This study was approved by the Institute Research Ethics Committee at Cancer Center, Sun Yan-Sen University (approval number: YJ2003001). The liver cancer cell lines were obtained from either American Type Culture Collection (Hep3B, HepG2, Huh7 and SK-HEP-1), or cell bank of Chinese Academy of Science, Shanghai (QGY7703, BEL7402 and BEL7404), or the Institute of Liver Cancer, Zhongshan Hospital, Fudan University (MHCC97H). 2.2. Sequence analysis Total genomic DNA was isolated by a standard protocol with proteinase digestion, phenol–chloroform extraction and ethanol precipitation. The miRNAs analyzed in this study are listed in Table 1. The first screen included 55 HCC tissues and

Table 1 List of the mciroRNA genes analyzed in this study and their chromosomal locations miRNAa

Location

miRNAb

Location

miRNAc

Location

let-7a-3 let-7f-2 miR-1-1 miR-1-2 miR-18b miR-24-2 miR-101-1 miR-101-2 miR-127 miR-130a miR-130b miR-137 miR-143 miR-146b miR-193b miR-199a-1 miR-199a-2 miR-224 miR-335 miR-365-1 miR-368

22q13.31 xp11.22 20q13.33 18p11.1 Xq26.2 19p13.12 1p3.3 9p24.1 14q32.31 11q12 22q11.21 1p21.3 5q32 10q24.32 16p13.12 19p13.2 1q24.3 xq28 7q32.2 16p13.12 14q32.31

miR-29b-1 miR-29b-2 miR-30b miR-30c-1 miR-30c-2 miR-34b miR-34c miR-106a miR-106b miR-122a miR-124-1 miR-124-2 miR-125a miR-125b-1 miR-126 miR-144 miR-148a miR-148b miR-192 miR-194-1 miR-194-2

7q32.3 1q32.3 8q24.22 1p34.2 6q13 11q23.1 11q23.1 Xq26.2 7q22.1 18q21.31 8p23.1 8q12.3 19q13.41 11q24.1 9q34.3 17q11.2 7p15.2 12q13.2-13.3 11q13.1 1q41 11cen

let-7a-1 let-7a-2 let-7f-1 miR-17 miR-18a miR-20a miR-21 miR-22 miR-23b miR-24-1 miR-34a miR-92-1 miR-99a miR-125b-2 miR-142 miR-195 miR-212

9,17 11q24.1 9q22.32 13q31 13q31.3 13q31.3 17q23.2 17p13.3 9q22.32 9q22.32 1p36.22 13q31.3 21q21.1 21q21.1 17q23.2 17p13.1 17p13.3

a b c

Represent miRNAs with abnormal expression level in human cancers. Represent miRNAs highly expressed in the liver. Represent miRNAs located in chromosomal regions, frequently altered in tumor cells.

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the identified variations were further investigated in another 41 HCC tissues and eight liver cancer derived cell lines. Sequence variations were screened by PCR-based single-strand conformational analysis (PCR-SSCA). Primers used were designed using online server primer3 (http://frodo.wi.mit.edu/cgibin/primer3/primer3.cgi). The genomic region including miRNA precursor (pre-miRNA) and the flanking sequences was amplified. Details of these primers would be provided upon request. The PCR reaction, in a volume of 20 ␮l containing 20 ng genomic DNA, 1.5–2.5 mM MgCl2 , 0.5 ␮M of each primer, 0.2 mM dNTPs, 0.5 U Taq DNA polymerase (Fermentas, Hanover, MD), was carried out at 94 ◦ C for 4 min, followed by 35 cycles of 94 ◦ C for 40 s, 55–64 ◦ C for 45 s and 72 ◦ C for 30 s and a final extension of 72 ◦ C for 5 min. The PCR product was mixed with four-fold volume of loading buffer (95% formamide, 20 mM EDTA, 0.05% xylene cyanol and bromophenol blue), denatured at 95 ◦ C for 8 min, quenched on ice, and then resolved on a non-denaturing 6–10% acrylamide gel at 4 ◦ C. DNA was visualized using silver staining. Cases showing aberrant band shift were confirmed by another round of PCR and SSCA. The shifted bands were then excised and applied to direct DNA sequencing. 2.3. Prediction of secondary structure of miRNA precursor The RNAfold web server (http://rna.tbi.univie.ac.at/cgibin/RNAfold.cgi) was used to predict the secondary structure of pre-miRNA [15]. The default parameters were employed. The sequence applied for predition analysis included premiRNA and 60 bp flanking sequences at each end of the precursor. In all cases, the folding structures with minimal free energy were depicted.

3. Results and discussion To investigate whether mutations in miRNA genes play a role in the development of HCC, genomic sequences coding for the precursors of 59 miRNAs were analyzed in the present study (Table 1). The miRNAs studied were selected by virtue of their abnormal expression in human cancers [4,7,9,10], or their high levels of expression in the liver [16–18], or their localization to the chromosomal regions frequently altered in tumor cells [3]. The analyses were initially performed in 55 HCCs by PCR-SSCA and direct DNA sequencing. A total of four variations in three miRNAs, including miR-106b, miR-192 and let-7a-2, were observed in four HCCs. Further investigation in the corresponding adjacent non-cancerous tissues identified the same sequence alterations, suggesting the possibility of germline mutations or natural polymorphisms. However, further screening of the miR-106b, miR-192 and let-7a-2

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genes in another 41 HCCs and eight liver cancer derived cell lines did not reveal more variations. Sequence analysis in tumor tissues from case 19 and case 64 identified two known single nucleotide polymorphisms (SNPs), located 40 bp (−40A/G, GeneBank accession no. rs1527423) and 43 bp (−43C/T, GeneBank accession no. rs2307353) upstream of the miR-106b precursor, respectively. Moreover, these two SNPs are closely linked (−43−40CA/TG) in both samples. Hospital record showed that these two patients were diagnosed with HCC at age 24 and 48, respectively, and both of them had infection of hepatitis B virus (HBV). In order to examine whether the variations identified in the present study affect the structure of pre-miRNA, RNAfolder was used to predict the secondary structures for the wildtype and variant sequences of miR-106b. However, no differences were observed. A new variation in let-7a-2, 32bp (+32G/A) downstream of the precursor was found in tumor tissue from case 60. The patient was a 55-year-old woman. Notably, her mother was diagnosed with colorectal cancer, indicating a family history of cancer. More interestingly, this case had no evidence of hepatitis B or C virus infection. In China, 80% or more of the HCC cases are associated with HBV infection [19] and in our study population the association was found to be approximately 90%. Therefore, this sequence variation may represent a germline mutation. However, prediction of secondary structure by RNAfolder revealed that the variation in let7a-2 did not change the base-pairing of the stem-loop structure. Another new variation was identified at 48 bp (−48C/T) upstream of the miR-192 precursor in case 4. The patient was 42 years old and had HBV infection. The RNAfolder predicted that the minor T allele variant disrupted the base-pairing at the stem region and induced conformation changes of miR-192 precursor (Fig. 1). Calin et al. had found that a sequence transition at 7 bp (+7C/T) downstream of the miR-16-1 precursor did result in lower expression level of mature miRNA [13]. The functional implication of miR-192 variation identified here needs further investigation. To date, 36 sequence variations in 29 miRNA coding genes have been identified in epithelial cancer derived cell lines, CLL patients and healthy Japanese [13,14,20]. Among these miRNA genes, eight of them including let7a-1, let-7a-2, let-7a-3, miR-21, miR-29b-2, miR-122a, miR-30c-2 and miR-143 were also analyzed in our study. However, no sequence alterations in these miRNA genes were observed in our set of hepatocellular carcinomas. Actually, sequence change in miR-30c-2 was only found in 0.0006% of healthy Japanese [20] and the variation

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Fig. 1. Computer-predicted secondary structure of the miR-192 precursor. The mature miRNA sequence is shadowed. The arrow indicates the 5 or 3 end of the precursor. The arrowhead identifies the site with sequence variation. The asterisk indicates the major structure change in the precursor of T allele variant.

frequencies in miR-29b-2 and miR-122a in CLL patients were all under 4% [13]. Except miR-143, which showed alternations in 13 out of 96 (13.5%) cancer cell lines, the variations in let-7a-1, let-7a-2, let-7a-3, miR-21 were all less than 3% [14]. Therefore, a possible explanation of why we could not find the same changes would be that the variations occur rarely and thereby they are difficult to be found in a limited number of samples. Alternatively, the frequency of variations may vary in different ethnic populations. Finally, the difference may also result from

the different mechanisms underlying the development of HCC and other types of cancer. In conclusion, our data suggest that mutation of miRNA is a rare event in HCC and thus may not represent a main mechanism underlying hepatocarcinogenesis. Acknowledgments This work was supported by grants from “973” program (2005CB724600), the National Natural Science

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