The Function Of Novel Gene MR-1 In Human Hepatoma Cells

Myofibrillogenesis regulator-1 (MR-1) is a novel human gene identified from a human skeletal muscle cDNA library, which interacts with contractile proteins in cells. Previous study has shown that MR-1 is involved in myocardial hypertrophy and muscle cell contraction. Moreover, MR-1 is overexpressed in myocardial mast cells and leukemia cells. This study suggests that MR-1 is overexpressed in cancer cells and is associated with cell proliferation, adhesion and migration. MR-1 may be a novel cancer therapeutic target.1. MR-1 was over expressed in cancer cells and was located at the cytoplasmTo examine whether MR-1 was particularly expressed in human cancer cells, we examined the levels of MR-1 mRNA in several human cancer cell lines and normal human cell lines by RT-PCR assay. We found that MR-1 mRNA levels of human cancer cell lines were higher than those of human normal cell lines. Furthermore, Western blot assay showed that the levels of MR-1 protein were higher in hepatoma cells compared with normal cells. These data indicated that MR-1 might be a cancer-related protein.To detect the localization of MR-1 in cancer cells, pEGFPN1-MR-1 vector, expressing the fusion protein of MR-1-EGFP, was constructed. The vector was transfected into HepG2 cells to determine the Subcellular localization of MR-1. The results showed that the fusion protein of MR-1-EGFP was located at the cytoplasm.2. Inhibition of cell proliferation, adhesion and migration by MR-1-siRNATo detect the function of MR-1, we used RNA interference to inhibit the expression of MR-1 in HepG2 cells. We investigated the rates of proliferation and colony formation of MR-1-siRNA-treated HepG2 cells. It was found that MR-1-siRNA could markedly inhibit the cell growth rate. Colony formation was also inhibited by MR-1-siRNA with inhibitory rate of 66.81%. In transwell migration assay the number of cells that transferred membrane was only 37.27% in MR-1-siRNA treated cells as compared to control cells. In addition, it was shown that adhesion ability on FN was decreased after treatment of MR-1-siRNA. 3. Abilities of cell spreading, proliferation, adhesion and migration were inhibited in HepG2/MR-1(-) cellsTo investigate a role of MR-1 in HepG2 cells, we used RNAi technology to establish MR-1 knockdown HepG2/MR-1(-) cells through stable transfecting pCD-sh-MR-1 that could transcript MR-1 -shRNA.We detected the proliferation ability of HepG2/MR-1(-) cell line compared with HepG2 cells. The data showed that the proliferation rate of HepG2/MR-1(-) cells were decreased compared with HepG2 cells and the inhibitory rate of colony formation was 31.77%. In transwell migration assay, it was found that the number of HepG2/MR-1(-) cells migrating through the membrane was 43.45% as compared to HepG2 cells. In adhesion assay, it was shown that adhesion ability of HepG2/MR-1(-) on FN was decreased compared with HepG2 cells. In addition, the spreading rate of HepG2/MR-1(-) cells on FN was reduced and the spreading morphology was changed, indicating that MR-1 could stimulate cell spreading.We further tested the tumorigenicity of HepG2/MR-1(-) cells. In vivo experiments showed that HepG2/MR-1(-) cells were transplanted subcutaneously in nude mice and the growth rate was significantly decreased within 20 days compared with HepG2 cells. The inhibitory rate of HepG2/MR-1(-) tumor growth at day 20 was 91.01%.4. Cell migration, adhesion and spreading excluding proliferation ability were enhanced in BEL-7402/MR-1(+) cellsBEL-7402/MR-1(+) cell line, over expressing extrinsic MR-1, was established through stable transfecting pcDNA3.1-MR-1 vector.We first investigated the proliferation and colony formation rate of BEL-7402/MR-1(+) cells growth. It was shown that the proliferation ability of BEL-7402/MR-1(+) cells was not altered obviously. However, the migration ability of BEL-7402/MR-1(+) cells was enhanced. Wound-healing assays showed that the ability of BEL-7402/MR-1(+) cells across the scratches was enhanced after 24 h and 48 h culture compared with parent cells. Tanswell migration assay showed that migration ability of BEL-7402/MR-1(+) cells was strengthened by 29.7% compared with BEL-7402 cells. In addition, BEL-7402/MR-1(+) cells displayed increased adhesion rate and enhanced spreading ability on FN. 5. Mechanism of action of MR-1 in cell proliferation, adhesion and migrationPrevious data have shown that MR-1 interacts with MLC2. It has been reported that phosphorylation of MLC2 controls the activity of myosin II, which has multiple functions within the cells, including stimulation of cell motility as a key component of focal adhesion formation and stress fiber formation. FAK, a key kinase in formation of focal adhesions, which is dependent upon activation of myosin II regulates cell viability through phosphoinositide 3-kinase (PI3K)/Akt pathway. Based on these, we explored the mechanism of the action of MR-1.(1) Inhibition of MLC2/FAK/Akt phosphorylation and stress fiber formation by MR-1-siRNATo clarify the mechanism of action of MR-1, we examined whether the phosphorylation of MLC2 at Ser-19 was affected by MR-1. It was shown that after treatment of MR-1-siRNA, MLC2 phosphorylation was reduced in HepG2 cells, while total MLC2 was no changed. Rhodamin-conjugated phalloidin was used to stain the cells for measurement of F-actin. The results showed that MR-1-siRNA-treated HepG2 cells displayed mass spots of F-actin staining and no organized stress fiber formation, meaning impair of cell contraction force. We next detected the effect of MR-1-siRNA on the activity of FAK. In addition, phosphorylation of Akt, a FAK substrate, was detected. It was shown that phosphorylations of FAK at Tyr-925 and Akt at Tyr-473 were significantly decreased by MR-1-siRNA. Moreover, we further demonstrated that FN-stimulated phosphorylations of FAK and Akt were obviously increased in the attached cells compared with those in the suspended cells, which was also inhibited by MR-1-siRNA.(2) Initiation of MLC2-FAK-Akt pathway by MR-1We further clarified MR-1-mediated signaling transduction pathway. Firstly, MR-1-siRNA-induced inhibition of FAK/Akt activation could be mimicked by MLCK inhibitor ML-7 that caused a significant decrease of FAK/Akt phosphorylation, indicating that MLC2 was upstream of FAK/Akt in this signaling pathway. Secondly, actin polymerization inhibitor cytochalasin D significantly reduced FAK/Akt phosphorylation, and whereas increased MLC2 phosphorylation, suggesting that the formation of stress fiber was downstream of MLC2 and upstream of FAK/Akt. Both inhibitors reduced ability of cell proliferation and migration. In addition, PI3K inhibitor LY294002 decreased the phosphorylations of Akt, MLC2 and FAK, indicating that Akt could inhibit activation of MLC2 through certain mechanism, which formed a reciprocal communication between FAK and cytoskeleton signaling protein. MR-1 level was no change after treatment of these inhibitors. Moreover, exogenous MR-1 could simulate phosphorylations of MLC2, FAK and Akt, which was blocked by ML-7, meaning that MR-1 was an upstream regulator of this pathway.(3) Effect of MR-1 on human proliferation and migration related gene expressionTo further understand the function of MR-1, gene chip technology was carried out to identify MR-1-regulated genes. In MR-1 knockdown HepG2 cells, the expressions of 326 genes were altered, of which 192 down-regulated genes and 134 up-regulated genes. In BEL-7402/MR-1(+) cells, 1318 genes from full human whole genome were altered, of which 980 down-regulated genes and 338 up-regulated genes. It was found that alteration of MR-1 expression induced diverse expression of many genes related to proliferation, mobility and metastasis of cancer cells. Microarray analysis showed that a series of signal transduction-related genes were altered by MR-1, including of cytoskeleton regulation, MAPK signaling, cell cycle regulation, Wnt signal and so on.In conclusion, our data demonstrate that MR-1 is overexpressed in several human cancer cells and plays an important role in cancer cell growth, adhesion and migration through the mechanism of activation of MLC2-FAK-Akt signaling pathway. MR-1 might be a novel cancer therapeutic target.