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• W2010111184 abstract "The key of cell migration process on solid substrates is phosphorylation of myosin light chain-2 (MLC2), which is implicated in a variety of intracellular functions. The previous data show that MLC2 interacts with a novel human gene, myofibrillogenesis regulator 1 (MR-1). Here, we reported that MR-1 was specially overexpressed in human hepatoma HepG2 cells. Transient treatment of cells with small interfering RNA (siRNA) against MR-1 or stable transfection of cells with plasmid expressing MR-1-siRNA led to inhibitions of cell proliferation, migration, and adhesion. Following down-regulation of MR-1, the phosphorylations of MLC2, focal adhesion kinase (FAK), and Akt were dramatically decreased, and the formation of stress fiber was destroyed by MR-1-siRNAs in hepatoma HepG2 cells. In addition, exogenous MR-1-induced as well as inherent phosphorylations of FAK and Akt were decreased by MLC kinase (MLCK) inhibitor, and F-actin polymerization inhibitor also decreased phosphorylations of FAK and Akt. Correspondingly, MR-1-enhanced migration of cells was also inhibited by these two inhibitors. These indicated that MLC2 activation and intact actin cytoskeleton were pivotal for MR-1 function. In vivo data showed that MR-1-siRNA markedly inhibited growth of human HepG2. This study suggested that overexpression of MR-1 was associated with cancer cell proliferation and migration through MLC2 and that MR-1 might be a potential cancer therapeutic target. The key of cell migration process on solid substrates is phosphorylation of myosin light chain-2 (MLC2), which is implicated in a variety of intracellular functions. The previous data show that MLC2 interacts with a novel human gene, myofibrillogenesis regulator 1 (MR-1). Here, we reported that MR-1 was specially overexpressed in human hepatoma HepG2 cells. Transient treatment of cells with small interfering RNA (siRNA) against MR-1 or stable transfection of cells with plasmid expressing MR-1-siRNA led to inhibitions of cell proliferation, migration, and adhesion. Following down-regulation of MR-1, the phosphorylations of MLC2, focal adhesion kinase (FAK), and Akt were dramatically decreased, and the formation of stress fiber was destroyed by MR-1-siRNAs in hepatoma HepG2 cells. In addition, exogenous MR-1-induced as well as inherent phosphorylations of FAK and Akt were decreased by MLC kinase (MLCK) inhibitor, and F-actin polymerization inhibitor also decreased phosphorylations of FAK and Akt. Correspondingly, MR-1-enhanced migration of cells was also inhibited by these two inhibitors. These indicated that MLC2 activation and intact actin cytoskeleton were pivotal for MR-1 function. In vivo data showed that MR-1-siRNA markedly inhibited growth of human HepG2. This study suggested that overexpression of MR-1 was associated with cancer cell proliferation and migration through MLC2 and that MR-1 might be a potential cancer therapeutic target. Cancer progression from a primary tumor to secondary metastasis is a highly complex process involving alteration of gene expression, acquisition of cell motility, interaction with extracellular matrix (ECM), 3The abbreviations used are:ECMextracellular matrixFAKfocal adhesion kinaseMLC2myosin light chain-2MLCKmyosin light chain kinaseMR-1myofibrillogenesis regulator-1siRNAsmall interfering RNAshRNAshort hairpin RNART-PCRreverse transcription-PCRGAPDHglyceraldehyde-3-phosphate dehydrogenasedTdeoxythymineFNfibronectin change in cell adhesion, and expression of ECM-degrading protease (1Kassis J. Lauffenburger D.A. Turner T. Wells A. Semin. Cancer Biol. 2001; 11: 105-117Crossref PubMed Scopus (149) Google Scholar). Cell migration is a critical step in tumor metastasis. Cancer cells move within tissues during invasion and metastasis by their own motility, and cell migration involves multiple processes that are regulated by various signaling molecules (2Yamazaki D. Kurisu S. Takenawa T. Cancer Sci. 2005; 96: 379-386Crossref PubMed Scopus (513) Google Scholar). It results from a dynamic interplay between the substrate and cytoskeleton protein located at the focal adhesion complex (3Acconcia F. Manavathi B. Mascarenhas J. Talukder A.H. Mills G. Kumar R. Cancer Res. 2006; 66: 11030-11038Crossref PubMed Scopus (36) Google Scholar). extracellular matrix focal adhesion kinase myosin light chain-2 myosin light chain kinase myofibrillogenesis regulator-1 small interfering RNA short hairpin RNA reverse transcription-PCR glyceraldehyde-3-phosphate dehydrogenase deoxythymine fibronectin It is known that cells exert force propelling the cell forward by contraction of the actin cytoskeleton through activation of myosin II (4Wakatsuki T. Wysolmerski R.B. Elson E.L. J. Cell Sci. 2003; 116: 1617-1625Crossref PubMed Scopus (129) Google Scholar). The actin-myosin II interaction in non-muscle cells is regulated by the phosphorylation of MLC2 at serine-19 (5Wilson A.K. Pollenz R.S. Chisholm R.L. de Lanerolle P. Cancer Metastasis Rev. 1992; 11: 79-91Crossref PubMed Scopus (19) Google Scholar). MLC2 dephosphorylation can induce apoptosis (6Fazal F. Gu L. Ihnatovych I. Han Y. Hu W. Antic N. Carreira F. Blomquist J.F. Hope T.J. Ucker D.S. de Lanerolle P. Mol. Cell. Biol. 2005; 25: 6259-6266Crossref PubMed Scopus (67) Google Scholar), and inhibitor of MLCK can abrogate MLC2 phosphorylation, cell polarization, and migration (7Gutjahr M.C. Rossy J. Niggli V. Exp. Cell Res. 2005; 308: 422-438Crossref PubMed Scopus (52) Google Scholar). MLC2 is also involved in the activation of mid-G1 phase cyclin D1 expression (8Roovers K. Assoian R.K. Mol. Cell. Biol. 2003; 23: 4283-4294Crossref PubMed Scopus (90) Google Scholar, 9Roovers K. Klein E.A. Castagnino P. Assoian R.K. Dev. Cell 2003. 2003; 5: 273-284Scopus (46) Google Scholar). It has been reported that hyperphosphorylated MLC2 induces stress fiber formation and integrin clustering that link cell surface cytoskeletal proteins such as FAK to actin (10Swant J.D. Rendon B.E. Symons M. Mitchell R.A. J. Biol. Chem. 2005; 280: 23066-23072Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 11Sawhney R.S. Cookson M.M. Omar Y. Hauser J. Brattain M.G. J. Biol. Chem. 2006; 281: 8497-8510Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). FAK is a member of the focal adhesions that mediates integrin-mediated signal transduction associated with a variety of cellular functions including cellular proliferation, migration, and adhesion (12Gilmore A.P. Romer L.H. Mol. Biol. Cell. 1996; 7: 1209-1224Crossref PubMed Scopus (462) Google Scholar). Downstream signaling pathways implicated in FAK signaling include the Jun N-terminal kinases (JNK) survival pathway that inactivates the tumor suppressor p53 regulating cell death pathway (13Ilić D. Almeida E.A. Schlaepfer D.D. Dazin P. Aizawa S. Damsky C.H. J. Cell Biol. 1998; 143: 547-560Crossref PubMed Scopus (437) Google Scholar), the death-associated protein kinase (14Wang W.J. Kuo J.C. Yao C.C. Chen R.H. J. Cell Biol. 2001; 159: 169-179Crossref Scopus (141) Google Scholar), and the phosphoinositide 3-kinase/Akt pathway regulating cell viability (15Chen H.C. Guan J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152Crossref PubMed Scopus (478) Google Scholar, 16Khwaja A. Rodriguez-Viciana P. Wennström S. Warne P.H. Downward J. EMBO J. 1997; 16: 2783-2793Crossref PubMed Scopus (940) Google Scholar). Previously, we have identified a novel human gene, MR-1, from a human skeletal muscle cDNA library (GenBank™ accession number AF417001) (17Li T.B. Liu X.H. Feng S. Hu Y. Yang W.X. Han Y. Wang Y.G. Gong L.M. Acta Biochim. Biophys. Sin. 2004; 36: 412-418Crossref Google Scholar). MR-1 is located on human chromosome 2q35 (GenBank accession number AC021016), and there are three alternatively spliced forms of MR-1 encoding three isoforms (18Liu X.H. Li T. Sun S. Xu F.F. Wang Y.G. Am. J. Physiol. 2006; 290: H279-H285Google Scholar). MR-1 is composed of three distinct exons, in which exon 3 is unique when compared with other two genes, and encodes a 142-amino acid protein with a hydrophobic transmembrane structure between amino acids 75 and 92. Yeast two-hybrid screening and in vitro GST pulldown revealed that MR-1 protein interacts with three proteins involved in muscle contraction such as MLC2 (17Li T.B. Liu X.H. Feng S. Hu Y. Yang W.X. Han Y. Wang Y.G. Gong L.M. Acta Biochim. Biophys. Sin. 2004; 36: 412-418Crossref Google Scholar), indicating that MR-1 might be associated with cell migration and growth process through MLC2. In this study, we investigated whether MR-1 was related to the proliferation and migration of human cancer cells. We observed that MR-1 was overexpressed in human cancer cells. Our data demonstrated for the first time that the inhibitions of proliferation, migration, and adhesion of hepatoma HepG2 cells by siRNA against MR-1 were associated with the interdiction of MLC2/FAK/Akt signaling pathway. SiRNA Preparation and Treatment—21-Nucleotide siRNAs were synthesized by Ribo Technology Company (Beijing, China) using 2′-ACE protection chemistry. Two siRNA sequences targeting MR-1 were 5′-ACC GUG UGA AGC AGA UGA AdTdT-3′ and 5′-CCU AGG CUA UUG ACU GUU AdTdT-3′, and the mock siRNA sequence was 5′-UUC UCC GAA CGU GUC ACG UdTdT-3′. Human hepatoma HepG2 cells were cultured in the MEM-EBSS medium (Invitrogen) supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin and 10! heat-inactivated fetal bovine serum at 37 °C in a humidified atmosphere containing 5! CO2. At 24 h before transfection at 50–80! confluence, HepG2 cells were trypsinized and diluted to appropriated concentration with fresh medium without antibiotics. Transfection of siRNA was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For drug treatment, HepG2 cells were treated with MLCK inhibitor ML-7 (Cell Signaling Technology, Beverly, MA) or F-actin polymerization inhibitor cytochalasin D (Sigma) for 24 h. Construction of siRNA-expressing and MR-1-expressing Plasmid—The DNA sequence to knock down expression of MR-1 was 5′-CCT AGG CTA TTG ACT GTT A TTCAAGAGA TAA CAG TCA ATA GCC TAG G-3′, and the mock sequence was 5′-TTC TCC GAA CGT GTC ACG T TTCAAGAGA ACG TGA CAC GTT CGG AGA A-3′. pCD-shRNA was reconstructed from pcDNA3.0 (Invitrogen) in our laboratory, in which cytomegalovirus promoter was cut off and replaced with H1 promoter producing the transcription of siRNA. The above mentioned DNA sequences were ligated into pCD-shRNA to form plasmid pCD-MR-1 and plasmid pCD-mock. The MR-1 cDNA encoding MR-1 was amplified by PCR from mRNA and subcloned into pcDNA3.1 expression vector (Invitrogen) to form plasmid pcDNA3.1-MR-1. Transfection and Selection of Stably Transfected HepG2/MR-1- Cells—HepG2 cells at 70–80! confluence were transfected with 2 μg of pCD-MR-1, pCD-mock, pcDNA3.1-MR-1, and pcDNA3.1 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For stable transfection, the cells were exposed to 400 μg/ml G418 (Invitrogen) after 1 day of transfection. After growth for 3 weeks, cells were plated at a lower density in MEM-EBSS with 250 μg/ml G418 and 10! fetal bovine serum in 96-well plates until a single colony was formed. Single cloned cells named HepG2/MR-1--sc and HepG2/mock-sc cells were isolated and grown up. In addition, mix cloned populations named HepG2/MR-1--mc were also cultured for experiments. RT-PCR and Western blot were used to test for MR-1 level of HepG2/MR-1-. Western Blot—Whole cell lysates were used for immunoblotting as described previously (19Liu T.G. Yin J.Q. Shang B.Y. Min Z. He H.W. Jiang J.M. Chen F. Zhen Y.S. Shao R.G. Cancer Gene Ther. 2004; 11: 748-756Crossref PubMed Scopus (54) Google Scholar). β-Actin was used as loading control. Anti-MR-1 polyclonal antibody was a generous gift from Dr. Tianbo Li (Institute of Medicinal Biotechnology, Beijing, China). Other antibodies were anti-FAK, anti-phospho-FAK (Tyr-925), anti-Akt, anti-phospho-Akt (Tyr-473), anti-MLC2 and anti-phospho-MLC2 (Ser-19) (Cell Signaling Technology), anti-β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA), and horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Electrochemiluminescence was performed according to the manufacturer's instructions with ChemiImager 5500 imaging system (Alpha Innotech Co.). RT-PCR Analysis—Total mRNA was extracted from the cells by TRIzol reagent (Invitrogen) with an extra step of acid phenol extraction. RT-PCR was carried out using a SuperScript™ one-step RT-PCR kit (Invitrogen) as described previously (19Liu T.G. Yin J.Q. Shang B.Y. Min Z. He H.W. Jiang J.M. Chen F. Zhen Y.S. Shao R.G. Cancer Gene Ther. 2004; 11: 748-756Crossref PubMed Scopus (54) Google Scholar). Oligonucleotide primers used were as follows: MR-1 P1, 5′-TAT CCT CCT CTT CAT CCT CAC C-3′; MR-1 P2, 5′-AGG CAC GAA CTG GAA TCT GG-3′; GAPDH P1, 5′-CGG AGT CAA CGG ATT TGG TCG TAT-3′; GAPDH P2, 5′-GTC TTC ACC ACC ATG GAG AAG GCT-3′; β-actin P1, 5′-CCC AGG CAC CAG GGC GTG ATG GT-3′; β-actin P2, 5′-GGA CTC CAT GCC CAG GAA GGA A-3′. GAPDH and β-actin mRNA was analyzed as internal control. A measure of 1 μg of total RNA was reverse-transcribed to synthesize cDNA at 50 °C for 30 min, and then the cDNA was subjected to PCR amplification with specific primers in 25-μl mixtures. PCR comprised 30 cycles with denaturing at 94 °C for 15 s, annealing at 57 °C for 30 s, and extension at 72 °C for 40 s in each cycle using an MJ PCR system (Bio-Rad). The PCR products were then subjected to 2! agarose gel electrophoresis. Quantitative Real-time RT-PCR Analysis—Quantitative real-time RT-PCR was performed using specific sense and antisense primers in a 25-μl reaction volume containing 12.5 μl of Absolute™ QPCR SYBR Green mix (Invitrogen), 0.25 pmol of each primer, and 0.5 μg of mRNA. Oligonucleotide primers used were as follows: for MR-1 P1, 5′-caa cac ggg cga gta tga ga-3′; MR-1 P2, 5′-ctg ggc caa acc tga gga c-3′; CLCA1 P1, 5′-TCA TCA GGA AAT GGA GCT GTC-3′; CLCA1 P2, 5′-TCA TCA GGA AAT GGA GCT GTC-3′; KIAA1486 P1, 5′-GCT GAG CAC CTC ATC AGA GAC-3′; KIAA1486 P2, 5′-GGG TCT TGG TTT AGC TGA CG-3′; IMMT P1, 5′-GGA TAT AAA TAC TGC CTA TGC CAG A-3′; IMMT P2 5′-CTT CCT CTT CAG CAA CTG CAT-3′. The amplification number of cycles was 40, and the reaction took place for 3 min at 50 °C, 15 s at 95 °C, and 30 s at 63 °C, with an initial step of 95 °C for 3 min. Cell Migration Assays—Cell migration was measured as the ability of cells to migrate through a Transwell filter (8-μm pores, Costar, Cambridge, MA). Cells suspended in serum-free MEM-EBSS containing 0.1! bovine serum albumin were applied to the upper chamber. MEM-EBSS containing 20! fetal bovine serum and 10 μg/ml fibronectin (FN) was added to the lower chamber. After the cells were incubated at 37 °C for 3 h, cells that migrated to the lower side of the upper chamber were stained with hemotoxylin, and the number of cells per microscopic field (×300) was counted under microscope. Cell Adhesion Assay—Cells were washed in serum-free MEM-EBSS containing 0.2! trypsin inhibitor and resuspended in culture medium. 100 μl of suspended cells was added to each well of 96-well plates coated with 10 μg/ml FN and blocked with 1 μg/ml bovine serum albumin. The plates were incubated for the appropriate periods of time at 37 °C in CO2 incubator. Non-adherent cells were removed by washing with phosphate-buffered saline, and attached cells were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide assay was processed as described previously (20Masuda T. Wada K. Nakajima A. Okura M. Kudo C. Kadowaki T. Kogo M. Kamisaki Y. Clin. Cancer Res. 2005; 11: 4012-4021Crossref PubMed Scopus (53) Google Scholar). For Western blot assay, cells were allowed to adhere to FN-coated plates for 2 h, and then proteins were collected. Analysis of Cell Spreading Morphology—Cells were suspended for 1 h in serum-free MEM-EBSS containing 1! bovine serum albumin and then replanted on 10 μg/ml FN (Merck)-coated 96-well plates. Cells were allowed to spread for the indicated times at 37 °C and then photographed using a Nikon upright scope equipped with a camera. F-actin Cytoskeleton Organization Detection—Cells were planted in six-well plates on glass coverslips. After the indicated treatment, cells were fixed in 3.7! paraformaldehyde for 20 min. Fixed cells were washed twice with phosphate-buffered saline, permeabilized by treatment with 0.5! Triton X-100 in phosphate-buffered saline for 5 min, and then stained with 5 μg/ml rhodamine-conjugated phalloidin (Merck) in phosphate-buffered saline for 10 min in the dark. Pictures were taken with Nikon fluorescence microscope. Cell Proliferation and Tumorigenicity Assay—For cell proliferation assay, 1 ml of cells was seeded at a density of 1000/ml in 24-well plates with 10! fetal bovine serum medium. The medium was changed regularly. The cell number was counted every 24 h for 6 days. For tumorigenicity assay, various cells (5 × 106) were injected subcutaneously into the right flank of nude mice. Bidimensional tumor measurements were made every 4 days, and the average of these measurements was used to calculate tumor volume. Statistical Analysis—Data are expressed as the arithmetic mean ± S.D. Statistical analysis was performed using the t test. p < 0.05 was considered statistically significant. Overexpression of MR-1 in Human Cancer Cells—To examine whether MR-1 was particularly expressed in human cancer cells, we first measured mRNA levels of MR-1 by RT-PCR assay in various human cancer cell lines, including liver cancer (HepG2, SMMC-7721, and BEL-7402), breast cancer (MCF-7), colon cancer (HT-29 and HCT116), fibrosarcoma (HT1080), and lung cancer (A549, PG, and PAa) cells, and in normal human cell lines, including liver L02 cells, lung fibroblast 2BS cells, and human umbilical vein endothelial cells. As shown in Fig. 1A, mRNA levels of MR-1 in human cancer cells were higher than in human normal cells, indicating that MR-1 might be an oncogene. Furthermore, a Western blot assay showed that protein levels of MR-1 were higher in hepatoma cells when compared with normal cells (Fig. 1B). Both mRNA and protein levels of MR-1 were superlatively high in hepatoma HepG2 cells. In the rest of this study, we chose hepatoma HepG2 cells to validate MR-1 as a therapeutic oncogene. Inhibition of Cell Proliferation by Down-regulation of MR-1—RNA interference technology has been successfully used to identify gene functions. To avoid the off-target effects, we designed and synthesized two siRNAs against MR-1, MR-1-siRNA-1 and MR-1-siRNA-2, to investigate the function of MR-1. RT-PCR and Western blot analysis showed that transcription and expression of MR-1 in HepG2 cells were dramatically down-regulated by both MR-1-siRNAs after 36 h transfection. (Fig. 2A). In the next experiment, we found that following down-regulation of MR-1, the ability of cell growth was obviously decreased in MR-1 siRNA-treated cells when compared with control and mock siRNA-treated cells (Fig. 2B). Furthermore, MR-1-siRNA-2 silence sequence were ligated into an expression plasmid to create an expressing MR-1-siRNA plasmid pCD-MR-1, and at the same time, a negative control plasmid pCD-mock was also created. Here, we investigated whether MR-1-siRNA-2 sequence expressed by pCD-MR-1 has off-target effects on the other genes. A BLAST search of MR-1-siRNA-2 sense chain and antisense chain in the National Center for Biotechnology Information (NCBI) was performed. The results showed that there were two transcripts, chloride channel regulator (CLCA1) and KIAA1486, complementary to the MR-1-siRNA-2 sense chain with query coverage of 73!, and three transcripts of inner membrane protein, mitochondrial (IMMT) complementary to the MR-1-siRNA-2 antisense chain with query coverage of 63!. The real-time RT-PCR assay showed that mRNA level of MR-1 was significantly decreased after a 36-h treatment of MR-1-siRNA-2, whereas CLCA1, KIAA1486, and IMMT were no changed (Fig. 2C), indicating that MR-1-siRNA-2 was specific for MR-1 knockdown without off-target effects and was suitable for the following experiments. To generate HepG2/MR-1- cells with stable knockdown of MR-1, pCD-MR-1 and pCD-mock were stably transfected into HepG2 cells, respectively. RT-PCR and Western blot assays showed that transcription and expression of MR-1 were knocked down in both HepG2/MR-1--sc and HepG2/MR-1--mc cells (Fig. 2D). It was observed that the ability of cell proliferation was markedly reduced in HepG2/MR-1--sc. In addition, HepG2/MR-1--mc cells also showed decreased proliferation ability when compared with parental cells (Fig. 2E). These data suggested that MR-1 might play an essential role in cancer cell growth. Inhibition of Cell Migration, Adhesion, and Spreading by Down-regulation of MR-1—It is well known that contraction of the actin cytoskeleton through activation of myosin II exerts force to propel cells to move. The actin-myosin II interaction is regulated by the phosphorylation of MLC2 at Ser-19 (4Wakatsuki T. Wysolmerski R.B. Elson E.L. J. Cell Sci. 2003; 116: 1617-1625Crossref PubMed Scopus (129) Google Scholar). Based on the interaction of MR-1 and MLC2, we suppose that MR-1 probably affect cell migration. The results showed that MR-1-siRNA-1 and MR-1-siRNA-2 both significantly reduced the migration of HepG2 cells with inhibitory rate of 52.9 and 62.7! (Fig. 3A). Similarly, the ability of the migration of MR-1 knockdown HepG2/MR-1--sc and HepG2/MR-1--mc cells was retarded when compared with that of parental and mock cells with inhibitory rates of 56.6 and 39.5! (Fig. 3B). Cell adhesion and spreading on ECM is a key step in cell migration process. As the cells adhere and spread, they generate tractions and thereby migration on the substrate. As shown in Fig. 3C, the cell adhesion ability on FN was decreased in MR-1-siRNA-treated HepG2 cells. Similarly, HepG2/MR-1--sc and HepG2/MR-1--mc cells also partially lost adhesion ability (Fig. 3D). To test cell spreading efficiency, cells were plated on FN-coated cell culture dishes. A striking difference in the appearance of cell spreading on FN was observed between HepG2/MR-1--sc and parental cells. HepG2 and HepG2/mock-sc cells began to spread at 60 min and were normally flattened at 120 min, whereas HepG2/MR-1--sc cells exhibited a delayed spreading and a limited extension on FN (Fig. 3E). These results confirmed that MR-1 was important for cell migration, adhesion, and spreading. Phosphorylation of MLC2, FAK, Akt, and Stress Fiber Formation by MR-1—To clarify the mechanism of action of MR-1, we examined whether the phosphorylation of MLC2 at Ser-19 was affected by MR-1. As shown in Fig. 4A, after treatment of MR-1-siRNAs, MLC2 phosphorylation was reduced. It is known that phosphorylation of MLC2 controls myosin II activity (21Niggli V. Schmid M. Nievergelt A. Biochem. Biophys. Res. Commun. 2006; 343: 602-608Crossref PubMed Scopus (35) Google Scholar) and subsequent organization of the actin stress fibers (22Hakuma N. Kinoshita I. Shimizu Y. Yamazaki K. Yoshida K. Nishimura M. Dosaka-Akita H. Cancer Res. 2005; 65: 10776-10782Crossref PubMed Scopus (36) Google Scholar). Therefore, rhodamine-conjugated phalloidin was used to stain the cells for measurement of F-actin (23Yu D.H. Qu C.K. Henegariu O. Lu X. Feng G.S. J. Biol. Chem. 1998; 273: 21125-21131Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). The results of the fluorescent image in Fig. 4B showed abundances of organized stress fibers in control and mock siRNA-treated cells. By contrast, MR-1-siRNA-treated HepG2 cells displayed mass spots of F-actin staining and no organized stress fiber formation, and there were numerous microspikes at the cell periphery (Fig. 4B). The finding indicated that MR-1 could strengthen the formation of actin stress fibers. Based on the above results, we further detected phosphorylation of FAK, a key kinase in the formation of focal adhesions, which is dependent upon activation of myosin II (24Chrzanowska-Wodnicka M. Burridge K. J. Cell Biol. 1996; 133: 1403-1415Crossref PubMed Scopus (1417) Google Scholar). The data showed that the phosphorylation of FAK at Tyr-925, representation of FAK activity, were both dramatically decreased in MR-1-siRNA-1-treated and MR-1-siRNA-2-treated cells (Fig. 4A), indicating the stimulating function of MR-1 on FAK activity. Our results also showed that Tyr-473 phosphorylation of Akt, a survival signaling factor activated by FAK (25Sonoda Y. Watanabe S. Matsumoto Y. Aizu-Yokota E. Kasahara T. J. Biol. Chem. 1999; 274: 10566-10570Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), was decreased in MR-1-siRNA-treated cells (Fig. 4A). At the same time, the levels of total MLC2, FAK, and Akt were kept invariable. As shown in Fig. 4C, the phosphorylations of FAK and Akt were dramatically down-regulated in the suspension cells. Cell adhesion on FN stimulated the phosphorylations of FAK and Akt, which were also significantly inhibited by MR-1-siRNA-1 and MR-1-siRNA-2. The levels of total FAK and Akt were kept invariable in various cells (Fig. 4C). These results showed that MLC2, FAK, and Akt might be involved in MR-1-regulated adhesion and migration. Initiation of MLC2-FAK-Akt Pathway by MR-1—To investigate the MR-1-mediated signaling pathway, we further tested the key molecules to be involved. The results showed that exogenous expression of MR-1 by transfection of pcDNA3.1-MR-1 enhanced phosphorylations of MLC2, FAK, and Akt, which were blocked by MLCK inhibitor ML-7. Here, MR-1 levels were not affected by ML-7 treatment, indicating that MLC2 was downstream of MR-1 and upstream of FAK and Akt (Fig. 5A). Furthermore, to test whether the formation of stress fibers stimulated the activation of FAK, we examined the activations of FAK and Akt using specifically the actin polymerization inhibitor cytochalasin D. Fig. 5B showed that the phosphorylations of FAK and Akt were inhibited by cytochalasin D. However, MLC2 phosphorylation was increased. These data indicated that the integrity of the actin cytoskeleton was an essential upstream factor for FAK activation and subsequent Akt activation in the MR-1-mediated signaling pathway. In addition, exogenous MR-1 enhanced the migration ability of cells by 28.77!, which was blocked by ML-7 and cytochalasin D (Fig. 5C). This result also supported the fact that MLC2 and stress fibers functioned as key downstream factors of MR-1. In Vivo Reduction of HepG2/MR-1- Tumorigenicity—The above data raised a possibility that hepatoma HepG2/MR-1--sc was not easily formed and developed in the animal model. To validate this possibility, human hepatoma HepG2, HepG2/mock-sc and HepG2/MR-1--sc cells were injected subcutaneously into the flanks of athymic nude mice, and tumor volumes were measured. Tumor masses resected from mice on day 20 after injection are shown in Fig. 6A. After 4 days of tumor inoculation, hepatoma HepG2 and HepG2/mock-sc were formed and grew quickly (Fig. 6B). However, the tumorigenicity of HepG2/MR-1--sc was dramatically limited (Fig. 6B). On day 20, the average size of HepG2, HepG2/mock and HepG2/MR-1--sc was 1156, 885, and 103 mm3 respectively, and the percentage of growth inhibition was 91.1! when compared with HepG2. After the completion of human genome sequencing and significant advances in genomics and proteomics, discovering the oncogene for therapeutic intervention of cancer remains as a future challenge. The present study demonstrates that MR-1 may be a novel human oncogene overexpressed in human cancer cell lines (Fig. 1). In vitro study showed that the proliferation, migration, and adhesion of hepatoma HepG2 cells are strongly inhibited by down-regulation of MR-1 either stably or transiently, using RNA interference technology. Furthermore, in vivo study further proves that MR-1 is essential for tumorigenicity. These results suggest that MR-1 is a potential tumor biomarker and therapeutic target. Understanding the signaling molecules involved in MR-1 bioactivity is important for targeting therapeutic purposes. Previous data have shown that MR-1 protein interacts with MLC2 (17Li T.B. Liu X.H. Feng S. Hu Y. Yang W.X. Han Y. Wang Y.G. Gong L.M. Acta Biochim. Biophys. Sin. 2004; 36: 412-418Crossref Google Scholar). Our data show that phosphorylation of MLC2 is significantly decreased by the inhibition of MR-1 expression and stimulated by exogenous MR-1 (Figs. 4A and 5A). Moreover, the effect of MR-1 on migration appears to be mediated by MLC2, based on the ability of the MLCK inhibitor ML-7 to block the stimulatory effect of exogenous MR-1 on HepG2 migration (Fig. 5A). Therefore, we identify MLC2 as a key mediator of MR-1 bioactivity. It has been known that phosphorylation of MLC2 controls the activity of myosin II, which has multiple functions in cells, including stimulation of cell motility as a key component of focal adhesion formation and stress fiber formation (24Chrzanowska-Wodnicka M. Burridge K. J. Cell Biol. 1996; 133: 1403-1415Crossref PubMed Scopus (1417) Google Scholar, 26Wilkinson S. Paterson H.F. Marshall C.J. Nat. Cell Biol. 2005; 7: 255-261Crossref PubMed Scopus (315) Google Scholar, 27Giannone G. Dubin-Thaler B.J. Rossier O. Cai Y. Chaga O. Jiang G. Beaver W. Döbereiner H.G. Freund Y. Borisy G. Sheetz M.P. Cell. 2007; 128: 561-575Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 28Romer L.H. Birukov K.G. Garcia J.G. Circ. Res. 2006; 98: 606-616Crossref PubMed Scopus (213) Google Scholar) and prevention of apoptosis as a cell survival regulator (29Connell L.E. Helfman D.M. J. Cell Sci. 2006; 119: 2269-2281Crossref PubMed Scopus (48) Google Scholar). Moreover, our data suggest that the inhibition of MR-1 expression suppresses H" @default.
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