MicroRNA-373 (miR-373) post-transcriptionally regulates large tumor suppressor, homolog 2 (LATS2) and stimulates proliferation in human esophageal cancer

E XP E RI ME N TAL C ELL R E SE A RC H 315 ( 2 0 0 9 ) 25 2 9– 2 5 38

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

MicroRNA-373 (miR-373) post-transcriptionally regulates large tumor suppressor, homolog 2 (LATS2) and stimulates proliferation in human esophageal cancer Kuen-Haur Lee a , Yih-Gang Goan b,c , Michael Hsiao d , Chien-Hsing Lee e , Shu-Huei Jian e , Jen-Tai Lin f , Yuh-Ling Chen g,⁎, Pei-Jung Lu e,⁎ a

Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan c Department of Surgery, National Yang-Ming University, Taipei, Taiwan d Genomics Research Center, Academia Sinica, Taipei, Taiwan e Institute of Clinical Medicine, National Cheng Kung University, No. 1 University Rd. Tainan, Taiwan f Division of Urology Surgery, Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan g Institute of Oral Medicine, National Cheng Kung University, No. 1 University Rd. Tainan, Taiwan b

A R T I C L E I N F O R M AT I O N

AB ST R AC T

Article Chronology:

LATS2 is a member of the LATS tumor suppressor family. It has been implicated in regulation of the

Received 26 January 2009

cell cycle and apoptosis. Frequent loss of heterozygosity (LOH) of LATS2 has been reported in

Revised version received 19 May 2009

human esophageal cancer. But, the LATS2 gene expression and its regulatory mechanism in

Accepted 2 June 2009

esophageal cancer remain unclear. The present study has shown that LATS2 protein expression was

Available online 6 June 2009

mediated by miR-373 at the post-transcriptional level and inversely correlated with miR-373 amounts in esophageal cancer cell lines. Furthermore, we demonstrated that the direct inhibition

Keywords:

of LATS2 protein was mediated by miR-373 and manipulated the expression of miR-373 to affect

Esophageal cancer

esophageal cancer cells growth. Moreover, this correlation was supported by data collected ex vivo,

LATS2

in which esophageal cancer tissues from esophageal squamous cell carcinoma (ESCC) patients

MicroRNA

were analyzed. Finally, by miRNA microarray analysis, four miRNAs including miR-373 were over-

MiR-373

expressed in ESCC samples. Our findings reveal that miR-373 would be a potential oncogene and it

Proliferation

participates in the carcinogenesis of human esophageal cancer by suppressing LATS2 expression. © 2009 Elsevier Inc. All rights reserved.

Introduction Esophageal cancer (EC) is among the ten most frequent malignancies and the sixth most common causes of cancer related death in the world [1]. The incidence of EC varies worldwide, ranging from over 130 per 100,000 populations in endemic regions such as northern China and Kazakhstan to around 4 per 100,000 popula-

tions in Western countries [2]. Although surgical resection can remarkably improve outcomes in a substantial number of esophageal cancer patients, the prognosis of esophageal cancer patients is poor. The overall 5-year survival rate of EC patients is only 5–10% and about 75% of patients die of disease within the first year after diagnosis [3]. In Asia, the major histologic types of esophageal cancer are squamous cell carcinoma (SCC). Several

⁎ Corresponding authors. Yuh-Ling Chen is to be contacted at Fax: +886 6 2359885. Pei-Jung Lu, fax: +886 6 3028162. E-mail addresses: [email protected] (Y.L. Chen), [email protected] (P.-J. Lu). Abbreviations: miRNA, microRNA; LATS2, large tumor suppressor, homolog 2; ESCC, esophageal squamous cell carcinoma; RT-PCR, reverse transcription-polymerase chain reaction; pre-miR, precursor microRNA; miCHIP, miRNA chip 0014-4827/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.06.001

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previous conventional cytogenetic analyses of ESCC were tried to identify significant genetic factors involving carcinogenesis of ESCC and intend to improve the dismal outcomes of ESCC patients, but all were still inconclusive [4–9]. Therefore, much research has been dedicated to identifying the genetic factors involved in esophageal cancer carcinogenesis. LATS2 gene has been mapped onto chromosome 13q11-12, a hot spot region for loss of heterozygosity in breast, ovary, liver, and lung cancers [10–13]. Over-expression of mouse Lats2 in vras-transformed NIH3T3 cells has been shown to inhibit G1/S transition via down-regulation of cyclin E/CDK2 kinase activity [14], and induction of apoptosis via down-regulation of apoptosis inhibitors such as Bcl-2 and Bcl-xL [15]. These observations suggest that LATS kinases act as tumor suppressors by inhibition of cell cycle progression or induction of apoptosis. Ishizaki et al. [16] have demonstrated LATS2 gene polymorphic variations in only one out of 60 ESCC tumor tissues. However, the expression level and molecular mechanism of LATS2 remain to be clarified in esophageal cancer. MicroRNAs (miRNAs) are naturally occurring small non-coding RNA molecules. These small 18 to 25 nucleotide non-coding transcripts modulate protein expression by completely or partially complementary binding to the 3′-untranslated region of target mRNAs causing degradation of mRNA or inhibition of translation [17]. Previous studies have shown that miRNAs play important roles in essential processes, such as differentiation, cell growth, and cell death [18,19]. Currently emerging results revealed that miRNAs are involved in cancer pathogenesis. For example, frequent alterations of miRNA expression have been found in a variety of human malignancies [20,21], including esophageal cancer [22,23]. One recent study from Dr. Voorhoeve's [24] group highlighted miR373-mediated regulation of LATS2 protein in testicular germ cell tumors. On the base of these observations, the aim of this study was to assess the role of LATS2 in esophageal cancer and whether LATS2 could be regulated by miR-373 in vitro and ex vivo. Furthermore, we would examine whether manipulated the expression of miR-373 affects the esophageal cancer cells growth through inhibition of LATS2 expression.

Materials and methods Cell lines, cultures, and ESCC tumor tissues The esophageal cancer cell lines (CE48T, CE146T, KYSE70, KYSE150, KYSE170 and KYSE510) were kindly provided by Dr Y. C. Wang (Department of Pharmacology, National Cheng Kung University, Tainan, Taiwan). The human esophageal cancer cell lines KYSE50 and CE81T were purchased from the Health Science Research Resources Bank (Osaka, Japan) and the Bioresource Collection and Research Center (Taipei, Taiwan), respectively. The KYSE50 cells were maintained in Dulbecco's modified Eagle medium (DMEM) medium supplemented with 2% fetal calf serum, sodium pyruvate, 100 units/ml penicillin, and 100 g/ml streptomycin, and 1% L-glutamine. The human esophageal squamous cell carcinoma CE81T cell line was maintained in DMEM containing 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 0.1 mM non-essential amino acid. Both cell lines were cultured in a humidified incubator with 5% CO2 at 37 °C. Human esophageal tissue specimens were obtained from Kaoh-

siung Veterans General Hospital (Kaohsiung, Taiwan). The fresh tumor tissue samples were dissected from the main tumor part of each surgically removed specimen in the operating room. All patients gave informed consent before to collect a tissue sample during their planned surgery. These samples were snap frozen in liquid nitrogen and stored at − 80 °C until investigation.

MiRNA array hybridization and analysis The small RNAs were prepared from esophageal cancer cell lines and human esophageal tumor and normal specimens using mirVana™ miRNA Isolation Kit (Ambion, Austin, TX, USA). The N7 position of guanine of isolated small RNA nucleic acids was labeled with Cy3 or Cy5 using the ULS™ Small RNA Labeling Kit (Kreatech Biotechnology, Amsterdam, The Netherlands) according to the manufacturer's instructions. The label samples were incubated at 85 °C for 15 min, and then spun down to collect the contents of the tube before proceeding with purification using the KREApure columns (Kreatech Biotechnology, Amsterdam, The Netherlands). ULS-labeled small RNAs were purified by a KREApure, and stored at −70 °C, or used for miRNA array hybridization. miRNA arrays were generated on glass slides using the mirVana miRNA Array Probe Set (Ambion, Austin, TX, USA). The 210 mature miRNA complementary oligonucleotides were assembled and integrated into our miCHIP. Each probe was printed in duplicate. After hybridization, the miRNA arrays were scanned and analyzed using a GenePix 4000A array scanner (Axon Instruments Union City, CA, USA). Normalization was performed by expressing each miRNA replicate relative to a control miRNA added to each sample, thus allowing comparisons between chips. The quantification data were the ratios of Cy3/Cy5 fluorescence intensity for each specific miRNA.

Construction of the LATS2 3′-UTR-luciferase plasmid and reporter assays The LATS2 3′-UTR target site was amplified by PCR using the primers Fwd-5′-GCAAGCTTATGGGGGCCAGGCAC-3′ and Rev-5′-GGCACTAGTCATTATTGCACAGAGATTT-3′ and cloned into downstream of the luciferase gene in the pMIR-REPORT luciferase vector (Ambion, Austin, TX, USA). This vector was sequenced and named WT-3′UTR. Site-directed mutagenesis of the miR-373 target-site in the LATS2-3′UTR was carried out using Quick change-mutagenesis kit (Stratagene, Heidelberg, Germany) and named Mut.1st-3′UTR and Mut.2nd-3′UTR, respectively, in which used WT-3′UTR as a template. For reporter assays, the cells were transiently transfected with wildtype or mutant reporter plasmid and pre-miR-373 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Reporter assay was performed at 36 h post-transfection using BriteLite™ plus reporter gene assay system (Perkin Elmer Shelton, CT, USA).

Reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNA was extracted from human esophageal cancer cell lines by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using 1 μg of total RNA as a template and random hexamer as a primer. The LATS2 cDNA was amplified by PCR using LATS2 specific paired primers: forward: 5′-AGAAAGGGAGCCATGTCAGA-3′ and Reverse: 5′-TCACCTTCAGCTGGGTTTCT-

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3′, which should give a product of 300 bps; PCR conditions for LATS2 were performed by denaturing the DNA at 94 °C for 5 min, followed by 30 cycles of amplification: 94 °C for 30 s, 60 °C for 30 s, 72 °C for 60 s, and a final extension step at 72 °C for 10 min. The GAPDH was used as an internal control according to the manufacturer's instruction (Ambion, Austin, TX, USA). Amplified fragments were separated on a 1.0% agarose gel and visualized with ethidium bromide staining.

Quantitative RT-PCR assays for mature miRNA The miR-quantitative RT-PCR of esophageal cancer cell lines and human esophageal tissue specimens were obtained by using mirVana™ qRT-PCR miRNA Detection Kit (Ambion, Austin, TX, USA). The reverse transcription reactions were performed in a reaction containing 50 ng small RNA, 1 μl 1× mirVana RT Primer, 2 μl mirVana 5× RT Buffer, 0.4 μl ArrayScript enzyme mix and nuclease-free water added up to 10 μl, with incubation at 37 °C for 30 min, and at 95 °C for 10 min. A total of 25 μl in each PCR reaction contains 10 μl miRNA reverse transcription products with 15 μl PCR master mixture (5 μl mirVana 5× PCR buffer, 2.5 μl SYBR Green I, 0.2 μl SuperTaq polymerase, 0.5 μl mirVana PCR primers and nuclease-free water added up to 15 μl), with incubation at 95 °C for 3 min, 35 cycles of 95 °C, 15 s and 60 °C, 30 s for data collection by LightCycler™ system (Roche Molecular Systems, Indianapolis, IN, USA). miRNA expression was normalized against house-keeping (hk) RNA, 5S rRNA levels. Relative expressions were calculated using the formula 2− 2ΔCt values (ΔCt = CtmiRNA − Cthk).

Western blot analysis Esophageal cancer cell lines and human esophageal tissue specimens were placed in lysis buffer [20 mM Imidazole–HCl (pH 6.8), 100 mM KCl, 2 mM MgCl2, 20 mM EGTA (pH 7.0), 300 mM sucrose, 1 mM NaF, 1 mM Na-vanadate, 1 mM Na molybetadate, and 0.2% Triton X-100] for 1 h at 4 °C. The protein samples were electrophoresed by using SDS-PAGE (7.5%) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was then soaked in the blocking solution (1× TBS, 0.05% Tween-20, 5% non-fat dried milk) for overnight, then incubated with antilats2 (1:500) (CycLex, Nagano, Japan) and anti-actin monoclonal antibody at 4 °C overnight, respectively. Finally, the membrane was then incubated with the secondary antibody (biotinylated antimouse IgG, 1:1,000, PerkinElmer Life Sciences, Boston, USA) for 1 h and washed with TBST three times. The enhanced chemiluminescence (ECL) system Western blotting detection reagents (Perkin-Elmer Life Science, Boston, USA) were used according to the manufacturer's recommendations.

MiRNA and anti-miR transfection and cell proliferation assay The transfection of esophageal cancer cells was performed using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Lipofectamine 2000 transfections were carried out in 60 mm plates containing 1.2 μl of 30 nM or 1.8 μl 60 nM miRNA precursors, 990 μl Opti-MEM (GIBCO™, Invitrogen, Carlsbad, CA, USA) and 10 μl of lipofectamine 2000 reagent. The cells were incubated with the transfection-complexes for 6 h. Then 3 ml fresh cell growth medium was added and incubated in the incubator

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for 16–18 h. For the CE81T cell transfection, we used MicroPorator, a pipette-type electroporation system (NanoEnTek Inc., Seoul, Korea). In case of MicroPoration, cells were dissociated by a brief treatment with trypsin-EDTA, and counted with C-Chip™ microchip type hemocytometer (NanoEnTek Inc.). The anti-miR-373 oligonucleodite (100 nM) was introduced into each 5 × 105 dissociated cells in 10 μl volume according to the manufacturer's instructions (2 pulses with 20 ms duration at 1400 V; Digital Bio Technology). Electroporated cells were then seeded into 6-well culture dishes containing 2 ml of culture media. After 24 h of recovery, the cells were subjected to cell proliferation experiments. Cell proliferation was assessed using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay. After transfection, cells (1× 105/well) were plated in a 96-well plate and incubated at 37 °C, and cell proliferation was assessed at indication time.

Statistical analysis The differences between groups were analyzed using the unpaired one-tail Student's t test. Data are expressed as the mean ± standard error from at least 3 separate experiments performed in triplicate.

Results LATS2 protein expression was mediated by miR-373 at the post-transcriptional level and inversely correlated with miR-373 amounts in esophageal cancer cell lines We first determined expression levels of LATS2 (mRNA and protein) in eight different esophageal cancer cell lines (CE48T, CE81T, CE146T, KYSE50, KYSE70, KYSE150, KYSE170 and KYSE510). We found that each cell line expresses different levels of LATS2 protein (Fig. 1A), but almost the same level of LATS2 mRNA was detected (Fig. 1B). That is, there was no strict correspondence between LATS2 protein and mRNA levels. For example, high levels of LATS2 protein in KYSE70 and KYSE150 cell lines were with almost the same levels of LATS2 mRNA to compare with that in other esophageal cancer cell lines. This phenomenon could be due to a post-transcriptional regulation mediated by miRNAs. A hallmark of miRNAs' action is their ability to bind to the 3′UTR of specific target mRNAs thereby inhibiting its translation, which ultimately leads to reduced protein levels. The LATS2 protein expression was reported to be regulated by miR-373 [24]. Therefore, the expression levels of miR-373 in eight different esophageal cancer cell lines were examined. In these cell lines, low endogenous miR-373 (for example, KYSE70 or KYSE150, Fig. 1C, lanes 5 and 6), with a high amount of LATS2 protein was observed (Fig. 1A, lanes 5 and 6), whereas cell lines with high miR-373 (for example, CE81T or KYSE50, Fig. 1C, lanes 2 and 4) showed low amounts of LATS2 protein (Fig. 1A, lanes 2 and 4). Across all eight cell lines tested, we found the inverse correlation between LATS2 protein levels and miR-373. For miR-373 and LATS2 mRNA; however, there was no correlation. We next investigated whether the 3′-UTR of LATS2 was a functional target of miR-373 in ESCC cell lines. MiR-373 was predicted to bind two sites in the 3′-UTR of LATS2. We first cloned a reporter plasmid driven by the CMV basal promoter that harbored the 432 nt wild-type 3′-UTR of LATS2 at the 3′-position of the luciferase reporter gene (Fig. 1D, marked as WT 3′UTR). To identify

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Fig. 1 – LATS2 and miR-373 correlate inversely in eight esophageal cancer cell lines. (A) The expression of LATS2 protein in eight esophageal cancer cell lines was analyzed by Western blotting. The number below the actin image was the density ratio of LATS2/β-Actin (NIH-Image J). (B) LATS2 mRNA-expression in eight esophageal cancer cell lines was analyzed by RT-PCR. The number below the GAPDH image was the density ratio of LATS2/GAPDH (NIH-Image J). Fold differences in LATS2 protein were higher than in LATS2-mRNA. (C) miR-373-expression was analyzed by miR-quantitative RT-PCR. Across all eight cell lines tested, the inverse correlation between miR-373 and LATS2 protein levels. (D) Diagram of LATS2-3′ UTR containing reporter constructs. WT represents wild-type. Mut represents mutant. (E) The indicated vectors were transfected in KYSE70 cell line. The luciferase activities are shown. SD is from three independent experiments ***P < 0.001.

which miR-373 binding sites of LATS2-3′UTR was critical for miR-373 binding. The two conserved targeting regions for miR-373 binding were specifically mutated (Fig. 1D, marked as Mut.1st-3′UTR and Mut.2nd-3′UTR), respectively. KYSE70 cells transiently transfected with the WT 3′UTR-reporter construct and pre-miR-373 led to a significant decrease of reporter activity when compared to the control (Fig. 1E, 40% decrease compared with lanes 2, P < 0.001). In addition, we also performed Mut.1st-3′UTR, Mut.2nd-3′UTR and cotransfection with Mut.1st-3′UTR and Mut.2nd-3′UTR luciferase reporter assay (Fig. 1E). The luciferase activity of the reporter that carried Mut.1st-3′UTR reporter construct was abolished about 25% of miR-373 mediating LATS2-3′UTR luciferase activity suppression by a

simultaneous transfection with pre-miR-373 (Fig. 1E, compare lanes 3 and 5). However, the luciferase activity of Mut.2nd-3′UTR was abolished about 50% of miR-373 mediating LATS2-3′UTR luciferase activity suppression (Fig. 1E, compare lanes 3 and 7). The activity of the reporter was with synergistic effect on the abolishment of miR373 mediating decrease of reporter activity in co-transfection with Mut.1st-3′UTR and Mut.2nd-3′UTR in the cells (Fig. 1E, compare lanes 3 and 9) which was abolished about 63% of miR-373 mediating LATS2-3′UTR luciferase activity suppression. This result indicated that miR-373 target site 2 of LATS2-3′UTR plays a more important role than miR-373 target site 1 in miR-373 mediating LATS2 protein expression.

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MiR-373 mediated LATS2 protein expression in cultured esophageal cancer cells To determine whether transfection of cell lines with anti-miR-373, or pre-miR-373 oligonucleotides, affects LATS2 protein expression, we performed miR-quantitative RT-PCR to detect miR-373 expression and immunoblotting to detect LATS2 protein expression. In CE81T and KYSE50 esophageal cancer cells characterized by a high miR-373 expression (Fig. 1C), down-regulation of endogenous miR-373 with

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anti-miR-373 oligonucleotides (Figs. 2A, C) led to a significant increase in LATS2 protein (Figs. 2B, D, upper panel) without any change in LATS2-mRNA (Figs. 2B, D, bottom panel). Furthermore, to determine whether over-expression of miR-373 in low-miR-373 expressing cells will have vice versa effects on LATS2 protein expression, KYSE70 and KYSE150 cells were transiently transfected with pre-miR-373, or control-miR oligonucleotides (Figs. 2E, G). The transfection was efficient with an almost 15–25 fold expression of miR-373 as compared to the control in KYSE70 cells (Fig. 2E), and an

Fig. 2 – miR-373 regulates LATS2 expression at the post-transcriptional level. (A–B) KYSE50 cells were transfected with 30 nM and 60 nM control-anti-miR or anti-miR-373 oligonucleotides, and analyzed for miR-373, LATS2-mRNA and protein expression. (C–D) 100 nM of control-anti-miR or anti-miR-373 oligonucleotides was introduced into CE81T cells by electroporation. After 2 days, total RNA was isolated and analyzed for miR-373, LATS2-mRNA and protein expression. (E–H) KYSE70 and KYSE150 cells were transfected with 30 nM and 60 nM control-miR or pre-miR-373 oligonucleotides, respectively. After 2 days, total RNA was isolated and analyzed for miR-373 and LATS2 mRNA as described in Materials and methods. Proteins from the same experiment were used to detect LATS2 protein expression by Western blotting.

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almost 8–14 fold expression of miR-373 was observed in KYSE150 cells (Fig. 2G). There was a significant reduction of LATS2 protein amounts in pre-miR-373-transfected cells (Figs. 2F, H, upper panel), whereas LATS2 mRNA was almost unaltered in pre-miR-373transfected cells (Figs. 2F, H, bottom panel). These data suggest that miR-373 specifically down-regulates LATS2 at the posttranscriptional level.

MiR-373 enhanced the cell proliferation of esophageal cancer cells It has been demonstrated that LATS2 is an essential mitotic regulator required for the coordination of cell division [14]. Our results have shown that the LATS2 protein level was modulated by miR-373 in esophageal cancer. To investigate whether miR-373 modulates cell proliferation in esophageal cancer cells, we assayed its effect on cell proliferation activity. The proliferation activity of CE81T, KYSE50, KYSE70 and KYSE150 cells transfected with different concentrations of anti-miR-373, pre-miR-373, and other control anti-miR or control miR oligonucleotides was determined by MTT assay, respectively. As shown in Figs. 3A and B, both CE81T and KYSE50 treated with anti-miR-373 oligonucleotides had a significant decrease in cell viability compared with the control anti-miR oligonucleotides transfected cells. In contrast, over-

expression of pre-miR-373 in KYSE70 and KYSE150 cells resulted in an increase of cell number (Figs. 3C, D). These results indicated that esophageal cancer cell growth can be modulated through miR373 mediated LATS2 repression.

The level of LATS2 protein expression was inversely correlated with miR-373 expression in ESCC Next, we sought to corroborate a negative regulation of endogenous LATS2 protein by endogenous miR-373 ex vivo. Resected tumor and corresponding normal tissues of 25 patients with esophageal cancer were analyzed for LATS2-protein expression. The LATS2protein quantitative results were shown in Fig. 4A. The decrease of LATS2 expression levels in 17 NT pairs (68% down-regulation) (P < 0.05) where tumor tissues ranged from 0.20 to 0.87-folds was defined as <1, the expression level equal (n = 3) and increase of LATS2-protein expression (n = 5) in NT pairs tumor tissues were defined as =1 and >1, respectively. MiR-quantitative RT-PCR analysis was then performed to quantitatively measure the amount of miR-373 in 23 NT pairs of ESCC specimens. The results in Fig. 4B showed that the mature form of hsa-miR-373 was up-regulated in about 48% (11 of 23 NT pairs) of ESCC tumor tissues examined. The level of LATS2 protein expression inversely correlated with miR-373 expression in ESCC was shown in Fig. 4C. Five representative

Fig. 3 – Effects of miR-373 on cell proliferation of esophageal cancer cells. (A–B) The growth-inhibitory function of anti-miR-373, KYSE50 and CE81T cells were transfected with indicative concentration of control anti-miR or anti-miR-373. Cell viability assay was measured by MTT assay at 0, 24, 48 and 72 h. (C–D) The growth-promoting effect of pre-miR-373 was measured by MTT assay. Cell viability assay was performed at 0, 24, 48 and 72 h after transfection of KYSE70 and KYSE150 cells with 30 or 60 nM of control miR or pre-miR-373. P < 0.001; P < 0.01.

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Fig. 4 – The inverse correlation of miR-373 and LATS2 in ESCC. (A) The expression level of LATS2 protein was quantified in 25 paired esophageal cancer tissues and their adjacent non-tumor tissues. The expression of LATS2 protein was decreased in about 68% (17 of 25 NT pairs) (P < 0.05) of ESCC tumor tissue specimens. (B) The expression of miR-373 was examined in 23 NT pairs ESCC tissues by miR-quantitative RT-PCR, N: normal, T: tumor. The increase of miR-373 levels in 11 NT pairs ranging from 1.02 to 6.64 folds was defined as T > N, the expression level equal (n = 3) and decrease of miR-373 expression (n = 9) were defined as T = N and T < N, respectively. (C) The level of LATS2 protein expression was inversely correlated with miR-373 expression in the representation of five pair ESCC specimens. (D) Pearson's correlation scatter plot of the fold changes of miR-373 and LATS2 protein in total 16 NT pairs of miR-373 expression in proportion to LATS2 expression samples (r = − 0.91).

examples have shown that ESCC tumor tissues with higher miR-373 levels than their adjacent non-tumor tissues (sample Nos. 1–3) have lower LATS2 protein expression in tumor tissues and higher LATS2 protein level in non-tumor tissues. In contrast, ESSCC tumor tissue with the same or lower miR-373 levels than their adjacent non-tumor tissues (sample Nos. 4 and 5) has similar or higher LATS2 protein expression in tumor tissues. The fold changes of miR373 and LATS2 protein in a total 16 NT pairs of miR-373 expression in proportion to LATS2 expression samples were further examined by Pearson's correlation scatter plot. As shown in Fig. 4D, there was a negative inverse correlation between miR-373 and LATS2 protein expression (r = − 0.91). This ex vivo data support the notion that LATS2 is negatively regulated by miR-373 and modulation of LATS2 protein level by miR-373 may explain why the down-regulation of LATS2 can cause tumor formation.

MiRNA let-7d, 330, 340, and 373 were over-expressed in ESCC Finally, to examine the human miRNA expression profiling in ESCC and to identify whether one of these ESCC specific miRNAs was

miR-373, we initially performed microarray analysis (miCHIP contained 210 complementary oligonucleotide spots) for 210 human mature miRNAs in five pairs of ESCC tumors and their adjacent non-tumor esophageal tissues. The esophageal cancer tissue miRNAs were labeled with cys-3 dye and miRNAs obtained from adjacent non-tumor tissues were labeled with cys-5 dye and subjected for the miCHIP analysis, as described in Materials and methods. The cys-5 red fluorescence indicated the miRNA expression level in non-tumor esophageal tissues, whereas the cys-3 green fluorescence indicated the expression level of miRNA in tumor tissues. We first performed miCHIP analysis using a pool of three ESCC samples, and miRNA expression profiling of ESCC tumor was obtained and compared with that obtained from its non-tumor part after normalization by the internal blank shown in Fig. 5A. The potential ESCC specific onco-miRNA was defined as at least a 1.5fold increase in ESCC tumor (tumor ≧ 1.5-fold normal). Accordingly, our results showed that 139 miRNAs were highly expressed in the esophageal tumor parts when compared with non-tumor parts (Fig. 5B). We then performed another two miCHIP analyses using two individual ESCC specimens and showed that 45 and 34

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potential ESCC specific onco-miRNAs were identified in ESCC specimen No. 245 and No. 278, respectively (Fig. 5B). After crossexamination of the 139 potential ESCC specific onco-miRNAs from the pool of three ESCC specimens, 45 miRNAs identified from No. 245 and 34 miRNAs identified from No. 278, we found that only four miRNAs, let-7d, miR-330, miR-340, and miR-373, show over-

Table 1 – The differential expression of miRNAs in ESCC tissues compared with esophageal normal tissues. miRNAs

hsa-let-7d Pool a No. 245 No. 278 hsa-mir-330 Pool No. 245 No. 278 hsa-mir-340 Pool No. 245 No. 278 hsa-mir-373 Pool No. 245 No. 278 a

miRNA relative Location Host gene expression folds (T/N)

Target

9q22.2

mlncRNA

Unknown

19q13.3

EML2

Unknown

5q35.3

RNF130

Unknown

19q13.4

Unknown

LATS2

3.8 9.9 1.6 3.1 1.5 1.8 2.5 1.6 4.0 4.6 1.7 7.6

Pool of three ESCC specimens.

expression (tumor ≧ 1.5-fold normal) in all five ESCC miRNAs examined (Fig. 5B and Table 1). Indeed, these data implied that miR-373 specifically over-expressed in ESCC may play roles in pathogenesis or phenotypic behavior of esophageal cancer.

Discussion

Fig. 5 – Identification of human miRNAs over-expression in esophageal cancers. (A) miRNA extracting and profiling was performed as described in the Materials and methods section by hybridization of miRNA-specific probes. The samples from esophageal cancer tissue miRNAs were labeled with cys-3 and miRNAs obtained from adjacent non-tumor tissues were labeled with cys-5 and subjected for the miCHIP analysis. Representative chip images and spots for selected miRNAs that have increased expression are illustrated. (B) 139 oncogenic miRNA spots over-expressed in ESCC tumor tissues compare with adjacent non-tumor tissues in pool of three samples, 45 and 34 potential ESCC specific oncogenic miRNAs were identified in ESCC specimens No. 245 and No. 278, respectively. After cross-examination of the 139 potential ESCC miRNAs from the pool of three ESCC specimens, 45 miRNAs identified from No. 245 and 34 miRNAs identified from No. 278, we found that only four miRNAs show over-expression.

This study was to evaluate the relationship between LATS2 and miR-373 in esophageal cancer. Our first findings de-monstrated that miR-373 post-transcriptionally downregulates LATS2 expression in vitro in esophageal cancer cells. We also demonstrated that miR-373 promoted cell proliferation through LATS2 mediated pathway. The ex vivo relevance of these findings is supported by the data from resected tissues of ESCC patients which show that high levels of miR-373 significantly correlate with low levels of LATS2 protein. In ESCC, DNA copy number decreases of 13q were reported commonly in Asian studies [9]. Two tumor suppressor genes have been identified on chromosome arm 13q associated with ESCC: RB1 on band 13q14.2 and BRCA2 on band 13q12. However, frequent LOH in the absence of mutations in the RB1 and BRCA2 genes in ESCC patients suggests that other unknown gene(s) on chromosome arm 13q may be involved in the development of ESCC [25]. Our study demonstrated that the expression of LATS2 was downregulated in 68% of ESCC tumor tissues. We also demonstrated that the LATS2-protein expression inversely correlated to miR-373 expression in ESCC. In a recent study, Voorhoeve et al. [24] have demonstrated that miR-372 and miR-373 are novel oncogenes in testicular germ cell tumors by mediating LATS2 expression. Importantly, these observations for different tumors suggest that the functions of miR-373 involved in cancer development through inhibition of LATS2 expression. MiR-373 has also been reported to promote tumor metastasis and induce expression of genes with complementary promoter sequences. Huang et al. [26] found that miR-373 and miR-520c stimulated cancer cell migration and invasion in vitro and significantly up-regulation of miR-373 in

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clinical breast cancer metastasis samples that correlated inversely with CD44 expression. Place et al. [27] found that miR-373 targeted the E-cadherin and CSDC2 gene promoters and induced of Ecadherin and CSDC2 gene expression. These data suggested that altered expression of miR-373 not only promotes tumor cell proliferation by inhibiting the LATS2-protein expression but it is also involved in tumor migration and invasion by mediating gene expression. MiRNAs have recently been suggested to play important roles in cancers as >50% of miRNA genes reside in cancer-associated genomic regions and their expressions have been found to be dysregulated in various cancers. Thus far, only two studies have examined miRNA profiles in esophageal cancer. Guo et al. [23] identified miRNA profiles in esophageal cancer patients and they identified that the high expression of miR-103/107 correlated with poor survival. Feber et al. [22] found that miR-203 and miR-205 were expressed 2- to 10-fold lower in squamous cell carcinoma and adenocarcinomas than in normal epithelium; the miR-21 expression was 3- to 5-fold higher in both tumors than in normal epithelium. In our study, we identified 4 up-regulated miRNAs in 5 different pairs of ESCC tumor tissues to compare with adjacent non-tumor tissues. Interestingly, 2 of the 4 significantly up-regulated miRNAs (miR-330 and miR-373) reside together on chromosome 19q13. The coordinate up-regulation of these miRNAs that resides within the same cluster suggests that this region of chromosome 19 may be amplified. Yen et al. [9] also found that frequent (≧20% of samples) gain abnormalities occurred on chromosome arm 19q (28%) in CGH studies of patients with ESCC in Taiwan. Thus, the up-regulation of miR-373 was associated with gain of chromosome 19q arm. In conclusion, we described here that the miR-373 expression was found to be inversely correlated with LATS2 expression in the esophageal cancer cell lines and ESCC patients. By the luciferase reporter assay, we found that miR-373 target site 2 of LATS2-3′UTR plays a more important role than miR-373 target site 1 in miR-373 mediating LATS2 protein expression. Further characterization the dysregulation of miR-373, in which sensitized cells to proliferation through the inhibition of its target protein-LATS2. By miRNA microarray analysis, we also confirmed the over-expression of miR-373 in ESCC patients. These findings suggested that dysregulation of the expression of miR-373 may dysregulate LATS2 expression resulting in dysregulating cellular processes which may ultimately lead to tumorigenesis of ESCC.

Acknowledgments This work was supported by National Cheng Kung University Hospital grants NCKUH-9701004 and NCKUH-9801004 (P.J. Lu).

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