Knockdown of SMYD3 by RNA interference down-regulates c-Met expression and inhibits cells migration and invasion induced by HGF

Cancer Letters 280 (2009) 78–85

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Knockdown of SMYD3 by RNA interference down-regulates c-Met expression and inhibits cells migration and invasion induced by HGF Jia-Ning Zou, Shu-Zhen Wang, Jia-Sen Yang, Xue-Gang Luo, Jing-Hang Xie, Tao Xi * School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing 210009, PR China

a r t i c l e

i n f o

Article history: Received 13 November 2008 Received in revised form 21 January 2009 Accepted 9 February 2009

Keywords: SMYD3 RNA interference HGF-c-Met Migration and invasion

a b s t r a c t We previously reported that over-expression of SMYD3, a histone H3-K4 specific di- and tri-methyltransferase, plays a key role in cell viability, adhesion, migration and invasion. In this study, we investigated the mechanisms underlying these phenomena and found that knocking down SMYD3 expression in tumor cells significantly reduced the biological function of HGF and inhibited carcinoma cells migration and invasion. Due to the fact that the proto-oncogene c-Met encodes the high-affinity receptor for HGF, and the HGF-c-Met signaling plays a critical role in the tumor genesis, we further identified the partial correlation between SMYD3 and c-Met. The results showed that high expression of c-Met accompanied with over-expression of SMYD3. Silencing SMYD3 expression in tumor cells by specific shRNAs down-regulated c-Met gene transcription, while over-expressing SMYD3 induced c-Met transcription. Moreover, we demonstrated here that two SMYD3 binding sites within the c-Met core promoter region were significant in the transactivation of c-Met. The present findings provide significant insights into the epigenetic regulatory mechanisms of oncogene c-Met expression, and develop the strategies that may inhibit the progression of cancer migration and invasion. Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.

1. Introduction Recent evidence has accumulated that modifications of histone amino-terminal play key roles in the regulation of chromatin structure, transcription and DNA replication [1– 3]. The covalent modifications regulate not only the chromatin structure but also the interaction with chromatinassociated proteins, which in turn dictate dynamic transitions between transcriptionally active or transcriptionally silent chromatin states [1,4,5]. Histone modifications include acetylation [6], phosphorylation [7], methylation [8] and/or ubiquitination [9]. Research in the past several years has revealed a growing number of histone methyltransferase to promote or inhibit tumor genesis through

* Corresponding author. School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China. Tel.: +86 25 83271389; fax: +86 25 83271249. E-mail address: [email protected] (T. Xi).

their histone methyltransferase activity [10–12]. SET and MYND domain-containing protein 3 (SMYD3) was significantly up-regulated in human colorectal, liver and breast cancers whereas undetectable or very weak in many types of normal human tissues [12,13]. Evidence has indicated that SMYD3 promoted dimethyltransferase and trimethyltransferase in histone H3-K4, which elicits its oncogenic effect via activating transcription of its downstream target genes [12,14]. Enhanced expression of SMYD3 was essential for the growth of many cancer cells [12–15]. We have clarified in our previous work that over-expression of SMYD3 gene affected cell viability, adhesion, migration and invasion [16], knocking down SMYD3 expression in cervical carcinoma cells inhibited cells proliferation and migration/invasion [17,18]. To further explain the mechanism underlying those phenomena and find SMYD3 target genes essential for transformation, one mechanism that has been proposed for SMYD3 transactivating function called attention to us: SMYD3 specifi-

0304-3835/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.canlet.2009.02.015

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cally binds to a DNA elements (50 -CCCTCC-30 or 50 GGAGGG-30 ) in the promoter region of its target genes and di- or tri-methylates nearby H3-K4. The correlation between SMYD3 H3-K4 methylation pattern in special promoter regions and gene transcriptional activity has been confirmed: SMYD3 is associated with high transcriptional activity of Nks2.8 [12], WHT10B [13] and hTERT [19]. The processes of cellular migration and invasion are critical for the ability of tumor cells to metastasize locally and to distant sites. It is the most common cause of death in cancer patients [20]. Today, the study of cellular migration and invasion benefits from the enormous advances in genomics and proteomics, providing thousands of genes and proteins, all well characterized structurally and functionally [21–23]. The proto-oncogene c-Met, a receptor tyrosine kinase, encodes the high-affinity receptor for hepatocyte growth factor (HGF) also known as scatter factor (SF) [24–26]. c-Met was shown to be over-expressed and mutated in a variety of malignancies [25–27]. It has now been established that aberrant HGF-c-Met signaling plays a critical role in epithelial–mesenchymal interaction and regulation of cell migration, invasion, cell proliferation and survival, angiogenesis, morphogenic differentiation, and organization of three-dimensional tubular structures [20,28–31]. Experimental evidence suggests that c-Met activation correlates with poor clinical outcome and the likelihood of metastasis [27,29,32]. Therefore, inhibitors of c-Met tyrosine kinase may be useful for the treatment of a wide variety of cancers that have spread from the primary site [26,28,31]. In our early study, we established the abundance of SMYD3 mRNA in many human cell lines, we noted that the cells over-expressed or repressed SMYD3 were much the same as the cells expressed c-Met or not [12,13,15, 25–27]. Furthermore, the bioinformatics assay showed that four SMYD3 binding sites (50 -GGAGGG-30 ) were present in the promoter region of c-Met, among which there are two SMYD3 binding sites in the core region of c-Met promoter (382 to +89) [33]. We here report that the potential correlation between SMYD3 and HGF-c-Met signal pathway. These findings will help to better understand the regulatory mechanisms of SMYD3 on cell migration and invasion and contribute to the development of treatment of tumors.

2. Materials and methods 2.1. Cell lines and culture conditions Human breast carcinoma cell line MCF-7, human ovarian carcinoma cell line HO-8910, human lung carcinoma cell line A549, human colonic carcinoma cell line HT-29, Human hepatocellular carcinoma cell line HepG2, human cervical carcinoma cell line HeLa, human gastric carcinoma cell line MGC-803, human leukemia cell lines K562 and HL60, human fetal lung fibroblast HFL-1 were purchased from Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human umbilical vein endothelial cell line ECV304 was purchased from China Center for Type Culture Collection,

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Wuhan University (Hubei, China). Human embryonic kidney cell line HEK293 was purchased from Sanger Biology Technology Company (Shanghai, China). Cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Paisley, UK), containing 10% fetal bovine serum (FBS, Gibco), 100 units/mL penicillin, and 100 mg/L streptomycin. 2.2. Gene silencing effect of SMYD3 shRNAs Plasmids expressing shRNAs were prepared as described previously [17,18]. The targeted sequences used for SMYD3 shRNAs were 50 -AACATCTACCAGCTGAAGGTG-30 . The negative control shRNAs with targeted sequences 50 -GTAGATGG TCGACCTTCAC-30 had no significant homology with any known gene. shRNAs plasmids transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and the cells were then harvested for analysis 48 h after transfection. 2.3. Plasmids, transfection and re-transfection The SMYD3 expression plasmid was constructed as described [16]. Cells were transfected with either the SMYD3 expression plasmid (pcSMYD3) or empty control vector (pcDNA3.1) using Lipofectamine 2000 according to manufacturer’s protocol, and the cells were then harvested for analysis 48 h. Forty eight hours after SMYD3 gene silenced by RNAi, pcSMYD3 was re-transfection into the SMYD3 knocked down cells, and the cells were then harvested for analysis another 48 h after transfection. 2.4. Scattering assay Cells were seeded at density of 1  104 cells/well in 24well plates, and the effects of 20 ng/ml HGF were tested for 24 h. All experiments were independently performed three times, and each sample was repeated with three wells. 2.5. Invasiveness assay The invasiveness assay was performed using ThinCertTM cell culture inserts for 24-well plates composed of a polycarbonate membrane containing 8 lm pores (Greiner, Frickenhausen, German). In briefly, cells were seeded on the upper chamber of the ThinCertTM at 2.0  104 cells in 200 ll serum-free DMEM. The lower chamber was filled with 600 ll DMEM containing 10% FBS with or without 20 ng/ml HGF. After being incubated at 37 °C for 20 h, the cells in upper chamber were removed with a cotton swab, and the cells that had migrated to the lower side of the membrane were fixed with methanol: acetic acid (3:1) for 10 min and stained with 10% Giemsa. The number of migrated cells was counted in five randomly chosen fields of three independent experiments at 100 magnification. 2.6. RNA extraction and reverse transcription PCR Total cellular RNA was extracted, and then potential residual genomic DNA was eliminated with RNase-free

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Dnase I (Bio Basic, Ontario, Canada). For each sample, 2 lg of total RNA was reverse transcribed using oligo dT 18 primer and MMLV (Promega, Madison, WI, USA) to synthesize complementary DNA (cDNA) following standard protocols. 1 ll newly synthesized cDNA was used as a template for PCR, which was performed with 1.5 mM MgCl2, 2.5U Taq polymerase, and 0.5 lM primers. The primer sequences were as follows: for SMYD3, 50 -CCCAGTATCTCTTT GCTCAATCAC-30 (forward) and 50 -ACTTCCAGTGTGCCTTC AGTTC-30 (reverse); for internal control glyceraldehyde-3phosphate dehydrogenase (GAPDH), 50 -ATTCAACGGCACAG TCAAGG-30 (forward) and 50 -GCAGAAGGGGCGGAGATGA-30 (reverse); for c-Met, 50 -ACAGTGCATGTCAAC ATCGCT-30 (forward) and 50 - GCTCGGTAGTCTACAGATT-30 (reverse). PCR was performed at 94 °C for 5 min, then 26 cycles at 94 °C for 45 s, at 54 °C for 45 s, and at 72 °C for 1 min; extension was carried out at 72 °C for 10 min. PCR products were electrophoretically on 1.5% agarose gels and fragments were visualized by ethidium bromide staining. 2.7. Western blot analysis Total cellular proteins were extracted with lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholic phenylmethyl-sulfonyl fluoride, 1 lg/mL of aprotinin, and 1 mmol/L of DTT). Protein (40 lg) were separated on a 12% SDS-polyacrylamide gel and transferred electrophoretically onto a nitrocellulose membrane. Blots were blocked for 1 h in blocking buffer (10% non-fat dried milk, 0.5% Tween in TBS) and incubated with anti-human SMYD3 rabbit polyclonal antibody (prepared in our laboratory) overnight at 4 °C, with anti-human c-Met rabbit polyclonal antibody (1:200, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), with anti-human b-actin rabbit polyclonal antibody (1:200, Santa Cruz Biotechnologies) as control. Blots were washed, incubated according to standard procedures and developed with ECL system (Amersham Pharmacia). 2.8. Construction of the c-Met luciferase reporter and luciferase activity assay The c-Met luciferase reporter (pGL3-0.4met-WT) was generated by inserting a deletion DNA fragment which contained the core promoter sequence of the c-Met 50 flanking region from 382 to +89 into pGL3-basic vector (Promega) [34]. Mutant reporter plasmids (pGL3-0.4metMUT) were made by replacing the two SMYD3 binding sequences (50 -GGAGGG-30 to 50 -GGTCGG-30 ) in pGL3-0.4MetWT using the OuickChange Site-Directed Mutagenesis Kit according to the manufacturer’s instructions (Stratagene, La Jolla, CA). Cells cultured in six well plates were transfected with pGL3-0.4Met-WT or mutant variants pGL30.4met-MUT and SMYD3 shRNAs or negative control shRNAs using Lipofectamine 2000. For each set of transfections, pGL3-control containing SV40-derived promoter sequences was used as a positive control. A Renilla luciferase containing plasmid, which is driven by thymidine kinase promoter, was always included in transfection to control transfection efficiency. Luciferase activity was determined by using a dual luciferase reporter assay system (Promega) 48 h after transfection. The promoter-dri-

ven firefly luciferase activity was normalized to the thymidine kinase Renilla activity. 2.9. Statistical analysis The data from the above mentioned experiments were expressed as mean ± SD. The statistical significance of differences was determined using Student’s t test. The minimal level of significance was P < 0.05. 3. Results 3.1. Knockdown of SMYD3 inhibits HGF-stimulated colony scattering The cellular responses to c-Met stimulation by HGF/SF are important in mediating a wide range of biological activities especially in cellular invasion and colony scattering. To investigate the effect of SMYD3 on the motility of tumor cells stimulated by HGF, we down-regulated SMYD3 expression by shRNAs in HepG2, MGC-803 and MCF-7 cells, which are reported to over-express SMYD3 and c-Met gene [12,13,15,25–27]. As shown in Fig. 1, HGF caused a remarkable scattering of HepG2, MGC803 and MCF-7 cells, and knockdown SMYD3 gene expression was effective in blocking HGF-stimulated scattering of these cells. After re-transfection of SMYD3 plasmid vector to the SMYD3 knocked down cells, cells changed their figure from a compact shape to a loose appearance and recovered the ability to move out from their colonies. 3.2. Inhibition of cells invasion induced by HGF in vitro by down-regulation of SMYD3 expression To determine the possible effect of SMYD3 knockdown on the invasive activity of these three cancer cell lines induced by HGF, an in vitro invasion assay was performed. We assayed cells in a ThinCertTM chamber with Matrigel-coated filter. Cells that passed through the filter pores and migrated to the lower surface of the filters were counted. As shown in Fig. 2, the invasive activity of HepG2, MGC-803 and MCF-7 cells was increased 3.2- to 3.5-fold, in the presence of exogenous HGF/SF in the lower chamber. However, SMYD3 knockdown cells significantly inhibited the invasiveness activity of HGF-stimulated cells (P < 0.01). SMYD3 re-transfected cells obviously recovered the invasive activity of SMYD3 knockdown cells (P < 0.01). These results indicated that suppression of SMYD3 inhibits the HGF-stimulated invasiveness activity of our examined tumor cells. 3.3. Partially correlation between the expression of SMYD3 and c-Met SMYD3 is over-expressed in the majority of colorectal carcinoma, hepatocellular carcinoma and breast carcinoma, whereas over-expression of c-Met is also observed in most of these tumors [12,13,15,25–27]. Moreover, the bioinformatics assay showed that two SMYD3 binding sites were present in the promoter region of c-Met gene [33,34]. Thus, we further inquired whether SMYD3 was partially required for c-Met expression. To achieve the goal, we used RT-PCR and Western blot to assay the abundance of SMYD3 and c-Met gene expression in many human cell lines, including major types of human carcinoma cell lines, normal endothelial cells, embryonic cells and fibroblasts. As expected, both the results from RT-PCR and Western blot demonstrated that most human epithelial cancer cell lines and human umbilical vein endothelial cells (ECV304) that were detected over-expressed c-Met accompanying with high level of SMYD3. Some human leukemia cells, fibroblasts and embryonic cells that do not express endogenous c-Met gene were also failed to detect the prominent expression of SMYD3 (Fig. 3). 3.4. SMYD3 expression is important for expression of c-Met and overexpression of SMYD3 up-regulates c-Met expression To further assess the partially correlation between SMYD3 and c-Met, we knocked down SMYD3 expression with specific SMYD3 shRNAs in human hepatocellular carcinoma HepG2, breast carcinoma MCF-7 and gastric carcinoma MGC-803 cell lines. The efficient silencing of SMYD3 expression was verified by both RT-PCR and Western blot analyses. A

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Fig. 1. Inhibition of cell motility by knockdown of SMYD3 gene expression in HepG2, MGC-803 and MCF-7 cells. The cells transfected SMYD3 shRNAs plasmids or negative control shRNAs plasmids, the cells without transfection and the cells re-transfected SMYD3 plasmid vector after RNAi were plated in 24-well plates at a density of 1  104 cells/well and cultured for 24 h in the medium containing HGF or not. significant reduction in both mRNA and protein levels of c-Met was seen following the inhibition of SMYD3 expression in these cells (Fig. 4A). We next ectopically expressed SMYD3 in HFL-1 fibroblasts, HEK293 embryonic cells and K562 leukemia cells, as shown in Fig. 4B, mock control cells and the cells transfection of the mock control vector (pcDNA3.1) expressed no c-Met mRNA, whereas transfection of the SMYD3 expression vector (pcSMYD3) induced c-Met mRNA expression in these three cells lines. And analogical to the results of RT-PCR, the followed Western bolt assay also showed that c-Met protein was found in SMYD3 over-expression cells. These data show that SMYD3 positively regulates c-Met mRNA expression.

3.5. SMYD3 activates the c-Met promoter To determine the mechanisms by which SMYD3 induces c-Met expression, we examined the effect of SMYD3 on the c-Met promoter activity. SMYD3 is known to bind to a putative motif 50 -CCCTCC-30 or 50 -GGAGGG-30 in its target promoters. There are two potential SMYD3 binding sites 50 -GGAGGG-30 within the c-Met core promoter region (Fig. 5A). To examine the significance of SMYD3 binding sites in the transactivation of c-Met, a reporter plasmid containing the wild-type of c-Met core promoter (pGL3-0.4met-WT) and a plasmid with mutant SMYD3 binding sites (pGL3-0.4met-MUT) were transfected into HepG2, MGC803 and MCF-7 cells, respectively. We found that the mutant reporters exhibited significantly lower luciferase activity compared with wild-type reporters, indicating that SMYD3 binding sites is responsible for the transactivation of c-Met in these cells (Fig. 5B). Cotransfection with plasmids expressing SMYD3 shRNAs reduced the luciferase activity of wildtype reporter plasmids compared with a mock plasmid expressing negative control shRNAs, but did not affect the activity of mutant plasmids. These results were highly consistent with inhibitory effects of SMYD3 knocking down on constitutive c-Met gene expression in HepG2, MCF-7 and MGC-803 cells.

4. Discussion Investigation of the molecular basis of migration and invasion is an uncovering strategy for delaying progression of preinvasive carcinoma and treatment of primary tumors and established metastasis. Multiple alterations in the genome of cancer cells underlie tumor development. These

genetic alterations occur in various orders; many of them concomitantly influence invasion, while the other cancer related cellular activities [20,35]. With the progress of the study on tumor epigenetics, a series of histone methyltransferase have been shown to regulate tumorigenesis through their histone methyltransferase activity [10–12]. We previously found out that the SMYD3-transfected NIH3T3 cells enhanced abilities of cell migration and invasion [16]. Then we demonstrated the inducible shRNAs expression system that specifically reduces the SMYD3 expression level and inhibits the migration and invasion of cervical carcinoma cell lines [17,18]. In the study presented here, we firstly observed that SMYD3 gene knockdown by shRNAs markedly inhibited the migration and invasion activity induced by HGF in the examined cells through the scatting assays and ThinCertTM cell culture inserts assays. These finding thus reveal that down-regulation of SMYD3 gene expression in tumor cells restrains the biological activity of HGF. Given the fact that hepatocyte growth factor is also known as scatter factor (HGF/ SF), it is so named because it was identified independently as both a growth factor for hepatocytes and as a fibroblastderived cell motility factor, or scatter factor [24–26]. Signaling via the HGF-c-Met pathway has been shown to lead to an array of cellular responses including proliferation (mitosis), scattering (motility), and branching morphogenesis [26–31]. The identification of HGF-c-Met signal pathway as an influence mechanism of SMYD3 might gain new insights into SMYD3-mediated oncogenic activity in cancer migration and invasion. Much evidence has indicated that several regulated downstream oncogenes and signal transduction factors of SMYD3 might be involved in its modulation of cells migration and invasion [12,14]. To further investigate the potential relationship between SMYD3 and oncogene c-Met, RT-PCR and Western blot were performed in the study to

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Fig. 2. Down-regulation of SMYD3 expression inhibited cells invasive activity induced by HGF. The cells that invaded through the ThincertTM inserts were stained and counted under a light microscope at 100 magnification. Five fields per well were selected for cell counts. Each sample was repeated with three wells and all experiments were independently performed in triplicate. Asterisk denotes a significant difference (**P < 0.01) determined by a Fisher’s protected least-significant test.

Fig. 3. RT-PCR and Western blot analysis of SMYD3 and c-Met in major types of human cell lines.

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Fig. 4. The relativity between SMYD3 and c-Met gene expression. (A) HepG2, MGC-803 and MCF-7 cells were treated with or without shRNAs targeting SMYD3 or control shRNA for 48 h. Cells were harvested for RT-PCR and Western blot analysis. The efficient knocking down of SMYD3 expression was verified by both RT-PCR and Western blot assessments. (B) RT-PCR and Western blot analysis of c-Met gene expression in HEK293, HFL-1, and K562 cells transfected with pcSMYD3 or pcDNA3.1 vector for 48 h. The over-expression of SMYD3 was also verified by both RT-PCR and Western blot assessments.

assay the abundance of SMYD3 and c-Met gene in many human cell lines. Interestingly, it showed that the high level of the expression of oncogene c-Met corresponded to SMYD3 over-expression in our examined cells. This result indicated that SMYD3 should be crucial for the constitutive transcription of c-Met gene. The followed experiments revealed that silencing SMYD3 expression in tumor cells by specific shRNAs down-regulated the mRNA and protein level of c-Met. These findings, together with induced c-Met mRNA expression induced by SMYD3 over-expression in fibroblasts, embryonic kidney cell and leukemia cells strongly suggest that the c-Met expression depends heavily on the presence of SMYD3 in the tumor cells. SMYD3 is identified as a subfamily of SET domain containing proteins with unique domain architecture. This family of proteins is defined by a SET domain that is split into two segments by an MYND domain, followed by a cysteine-rich post SET domain [12,36–38]. This unique domain architecture suggests that there are two

mechanisms to explain the role of SMYD3 in trans-activating function. Firstly, the MYND-type zinc-finger domain in SMYD3 recognizes its binding motif 50 -CCCTCC-30 or 50 -G GAGGG-30 in the promoter region of its target genes. Secondly, SMYD3 contains a SET domain that di- or tri-methylates nearby H3-K4 with assistance from its associated protein HSP90A. Following binding to DNA, SMYD3 recruits RNA polymerase II through an interaction with the RNA helicase HELZ, forming a transcriptional complex and thus results in a wider distribution of H3-K4 methylation across a target gene and activates transcription. The specific SMYD3 binding elements in target DNA (50 CCCTCC-30 or 50 -GGAGGG-30 ) are present in the promoter regions of SMYD3 downstream genes, such as Nks2.8 [12], WHT10B [13], hTERT [19]. It is interesting to note in our study that four 50 -GGAGGG-30 are found to present in the promoter region of c-Met, among which there are two in the core region of c-Met promoter (382 to +89). The results imply that the disruption of the functional

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Fig. 5. SMYD3 activates the c-Met promoter. (A) Sequence of the c-Met core promoter region. Two putative SMYD3 binding sites are underlined. Sequences that were mutated are indicated by dots, above which substituted nucleotides are indicated. Arrow indicates the first nucleotide of the first exon. (B) Transcriptional assay of c-Met containing wild-type or mutant SMYD3 binding element in the presence or absence of SMYD3 shRNA in tumor cells. The promoter activity of c-Met constructs is shown as percentage of the luciferase activity obtained by transfecting the same cell types with pGL3-control, an expression vector containing SV40-derived promoter sequences. Variation in transfection efficiency was normalized by the thymidine kinase-driven Renilla luciferase activity. Bars, ±SD. Asterisk denotes a significant difference (P < 0.05) determined by a Fisher’s protected least-significant test.

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