Cell cycle arrest and reversion of Ras-induced transformation by a conditionally activated form of mitogen-activated protein kinase kinase kinase 3

Signal-induced proliferation, differentiation, or stress responses of cells depend on mitogen-activated protein kinase (MAPK) cascades, the core modules of which consist of members of three successively acting kinase families (MAPK kinase kinase [MAP3K], MAPK kinase, and MAPK). It is demonstrated here that the MEKK3 kinase inhibits cell proliferation, a biologic response not commonly associated with members of the MAP3K family of kinases. A conditionally activated form of MEKK3 stably expressed in fibroblasts arrests these cells in early G1. MEKK3 critically blocks mitogen-driven expression of cyclin D1, a cyclin which is essential for progression of fibroblasts through G1. The MEKK3-induced block of cyclin D1 expression and of cell cycle progression may be mediated via p38 MAPK, a downstream effector of MEKK3. The MEKK3-mediated block of proliferation also reverses Ras-induced cellular transformation, suggesting possible tumor-suppressing functions for this kinase. Together, these results suggest an involvement of the MEKK3 kinase in negative regulation of cell cycle progression, and they provide the first insights into biologic activities of this kinase.     

miR-124和miR-520b下调Eps8表达并抑制HeLa细胞增殖

Eps8是一个多功能的信号分子,参与肌动蛋白重排、受体内吞和肿瘤的发生发展. 为了寻找靶向Eps8的microRNAs (miRNAs)并研究其在宫颈癌中的调控作用,本文采用软件预测获得4个可能调控Eps8表达的miRNAs. 利用双荧光素酶报告系统和Western印迹研究发现,miR-124 和miR-520b结合到人EPS8 mRNA的3′非翻译区(untranslated region, UTR)并有效抑制Eps8蛋白的表达. 进一步细胞存活检测、MTT法和克隆形成实验分析显示,miR-124和miR-520b过表达显著抑制HeLa细胞的生长和增殖.而且,miRNA调节的Eps8下调能提高HeLa细胞对化疗药物顺铂的敏感性. 同时证明,miR-124和miR-520b激活肿瘤抑制基因p53与下游基因p21报告基因转录活性,也相应地上调了p53与p21的蛋白表达. 这些结果提示,miR-124和miR-520b下调癌基因EPS8表达,从而抑制HeLa细胞增殖,负调控宫颈癌细胞生长.     

Upregulated microRNA-29a by hepatitis B virus X protein enhances hepatoma cell migration by targeting PTEN in cell culture model

Hepatitis B virus X protein (HBx) plays important roles in the development of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) contribute to cancer development by acting as oncogenes or tumor suppressors. Previously, we reported that HBx was able to promote the migration of hepatoma HepG2 cells. However, the regulation of miRNAs in the development of HBV-related HCC is poorly understood. In the present study, we reported that miR-29a was a novel regulator of migration of hepatoma cells mediated by HBx. Our data showed that the expression of miR-29a was dramatically increased in p21-HBx transgenic mice, HBx-transfected hepatoma HepG2-X (or H7402-X) cells and HepG2.2.15 cells that constitutively replicate HBV. However, our data showed that miR-29a was upregulated in 4 of the 11 clinical HCC samples. We found that the overexpression of miR-29a promoted the migration of HepG2 cells, while a specific miR-29a inhibitor could partially abolish the enhanced migration of HepG2-X cells. Moreover, we identified PTEN was one of the target genes of miR-29a in HepG2 cells. The deletion of the miR-29a-binding site was able to abolish the role of miR-29a in suppression of luciferase activity of the PTEN 3'UTR reporter. Meanwhile, the overexpression of PTEN was able to reverse the promoted migration of HepG2 cells mediated by miR-29a. Moreover, our data showed that the modulation of Akt phosphorylation, a downstream factor of PTEN, was involved in the cell migration enhanced by miR-29a, suggesting that miR-29a is responsible for the cell migration through its target gene PTEN. Thus, we conclude that miR-29a is involved in the regulation of migration of hepatoma cells mediated by HBx through PTEN in cell culture model.     

MEKK2 associates with the adapter protein Lad/RIBP and regulates the MEK5-BMK1/ERK5 pathway

MEKK2 and MEKK3 are two closely related mitogen-activated protein kinase (MAPK) kinase kinases. The kinase domains of MEKK2 and MEKK3 are nearly identical, although their N-terminal regulatory domains are significantly divergent. By yeast two-hybrid library screening, we have identified MEK5, the MAPK kinase in the big mitogen-activated protein kinase 1 (BMK1)/ERK5 pathway, as a binding partner for MEKK2. MEKK2 expression stimulates BMK1/ERK5 activity, the downstream substrate for MEK5. Compared with MEKK3, MEKK2 activated BMK1/ERK5 to a greater extent, which might correlate with a higher affinity MEKK2-MEK5 interaction. A dominant negative form of MEK5 blocked the activation of BMK1/ERK5 by MEKK2, whereas activation of c-Jun N-terminal kinase (JNK) was unaffected, showing that MEK5 is a specific downstream effector of MEKK2 in the BMK1/ERK5 pathway. Activation of BMK1/ERK5 by epidermal growth factor and H2O2 in Cos7 and HEK293 cells was completely blocked by a kinase-inactive MEKK3 (MEKK3kin(-)), whereas MEKK2kin(-) had no effect. However, in D10 T cells, expression of MEKK2kin(-) but not MEKK3kin(-) inhibited BMK1/ERK5 activity. Two-hybrid screening also identified Lck-associated adapter/Rlk- and Itk-binding protein (Lad/RIBP), a T cell adapter protein, as a binding partner for MEKK2. MEKK2 and Lad/RIBP colocalize at the T cell contact site with antigen-loaded presenting cells, demonstrating cotranslocation of MEKK2 and Lad/RIBP during T cell activation. MEKK3 neither binds Lad/RIBP nor is recruited to the T cell contact with antigen presenting cell. MEKK2 and MEKK3 are differentially associated with signaling from specific upstream receptor systems, whereas both activate the MEK5-BMK1/ERK5 pathway.     

Involvement of cholesterol in hepatitis B virus X protein-induced abnormal lipid metabolism of hepatoma cells via up-regulating miR-205-targeted ACSL4

Hepatitis B virus X protein (HBx) plays crucial roles in the development of hepatocellular carcinoma (HCC). The abnormal lipid metabolism is involved in the hepatocarcinogenesis. We previously reported that HBx suppressed miR-205 in hepatoma cells. In this study, we supposed that HBx-decreased miR-205 might contribute to the abnormal lipid metabolism according to the bioinformatics analysis. Interestingly, we showed that the expression levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) were negatively associated with those of miR-205 in clinical HCC tissues. Then, we validated that miR-205 was able to inhibit the expression of ACSL4 at the levels of mRNA and protein through targeting its 3′UTR. Strikingly, we found that HBx was able to increase the levels of cellular cholesterol, a metabolite of ACSL4, in hepatoma cells, which could be blocked by miR-205 (or Triacsin C, an inhibitor of ACSL4). However, anti-miR-205 could increase the levels of cholesterol in the cells. Moreover, we demonstrated that the levels of cholesterol were increased in the liver of HBx transgenic mice in a time course manner. Functionally, oil red O staining revealed that HBx promoted lipogenesis in HepG2 cells, which could be abolished by miR-205 (or Triacsin C). However, anti-miR-205 was able to accelerate lipogenesis in the cells. Interestingly, the treatment with Triacsin C could remarkably block the role of anti-miR-205 in the event. Thus, we conclude that miR-205 is able to target ACSL4 mRNA. The HBx-depressed miR-205 is responsible for the abnormal lipid metabolism through accumulating cholesterol in hepatoma cells.     

MicroRNA-221 regulates osteosarcoma cell proliferation,apoptosis, migration,and invasion by targeting CDKN1B/p27

MicroRNAs (miRNAs, miR) are of critical importance in growth and metastasis of cancer cells; however, the underlying functions of miRNAs in osteosarcoma (OS) remain largely unknown. This study was aimed to elucidate the role of miR-221 in regulating the biological behavior of OS cells. The proliferation ability was examined by cell counting kit-8 (CCK-8) and cell cycle assay. The abilities of cell migration, invasion, and apoptosis were monitored by transwell assay and flow cytometry, respectively. The effect of miR-221 on cyclin-dependent kinase inhibitor 1B (CDKN1B) expression was evaluated by luciferase assays, real-time polymerase chain reaction, and Western blot analysis. We found that miR-221 was elevated in OS cell lines compared with the normal osteoblastic cell line. Transfection of the miR-221 inhibitor into MG63 and U-2OS cell lines obviously suppressed cell proliferation, migration, and invasion, which is accompanied with cell cycle arrest in G0/G1 phase. Furthermore, luciferase reporter assays indicated that CDKN1B is directly targeted by miR-221 in OS cells. Knockdown of CDKN1B inhibited the effects of miR-221 inhibitor, along with decreased Bax and caspase-3 and increased cyclin E, cyclin D1, Bcl-2, Snail, and Twist1 expression. The results suggested that miR-221 might act as a potentially useful target for treatment of OS.     

MiR-23a-3p promoted G1/S cell cycle transition by targeting protocadherin17 in hepatocellular carcinoma

MiR-23a-3p has been shown to promote liver cancer cell growth and metastasis and regulate that of chemosensitivity. Protocadherin17 (PCDH17) is a tumor suppressor gene and plays an essential part in cell cycle of hepatocellular carcinoma (HCC). This study aimed at evaluating the effects of miR-23a-3p and PCDH17 on HCC cell cycle and underlining the mechanism. The level of miR-23a-3p was up-regulated, while PCDH17 level was down-regulated in HCC tissues compared to adjacent tissues. For the in vitro studies, high expression of miR-23a-3p down-regulated PCDH17 level; increased cell viability; promoted G1/S cell cycle transition; up-regulated cyclin D1, cyclin E, CDK2, CDK4, p-p27, and p-RB levels; and down-regulated the expression of p27. Dual luciferase reporter assay suggested PCDH17 was a target gene of miR-23a-3p. MiR-23a-3p inhibitor and PCDH17 siRNA led to an increase in cell viability and the number of cells in the S phase and up-regulated cyclin D1 and cyclin E levels, compared with miR-23a-3p inhibitor and NC siRNA group. For the in vivo experiments, high expression of miR-23a-3p promoted tumor growth and reduced PCDH17 level in the cytoplasm. These results indicated that high expression of miR-23a-3p might promote G1/S cell cycle transition by targeting PCDH17 in HCC cells. The miR-23a-3p could be considered as a molecular target for HCC detection.

     

Role of MicroRNA-182 in Posterior Uveal Melanoma: Regulation of Tumor Development through MITF, BCL2 and Cyclin D2

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play central roles in diverse pathological processes. In this study, we investigated the effect of microRNA-182 (miR-182) on the development of posterior uveal melanomas. Initially, we demonstrated that miR-182 expression was dependent on p53 induction in uveal melanoma cells. Interestingly, transient transfection of miR-182 into cultured uveal melanoma cells led to a significant decrease in cell growth, migration, and invasiveness. Cells transfected with miR-182 demonstrated cell cycle G1 arrest and increased apoptotic activity. Using bioinformatics, we identified three potential targets of miR-182, namely MITF, BCL2 and cyclin D2. miR-182 was shown to have activity on mRNA expression by targeting the 3' untranslated region of MITF, BCL2 and cyclin D2. Subsequent Western blot analysis confirmed the downregulation of MITF, BCL2 and cyclin D2 protein expression. The expression of oncogene c-Met and its downstream Akt and ERK1/2 pathways was also downregulated by miR-182. Concordant with the findings that miR-182 was decreased in uveal melanoma tissue samples, overexpression of miR-182 also suppressed the in vivo growth of uveal melanoma cells. Our results demonstrated that miR-182, a p53 dependent miRNA, suppressed the expression of MITF, BCL2, cyclin D2 and functioned as a potent tumor suppressor in uveal melanoma cells.     

Altered p27(Kip1) phosphorylation, localization, and function in human epithelial cells resistant to transforming growth factor beta-mediated G(1) arrest

p27(Kip1) is an important effector of G(1) arrest by transforming growth factor beta (TGF-beta). Investigations in a human mammary epithelial cell (HMEC) model, including cells that are sensitive (184(S)) and resistant (184A1L5(R)) to G(1) arrest by TGF-beta, revealed aberrant p27 regulation in the resistant cells. Cyclin E1-cyclin-dependent kinase 2 (cdk2) and cyclin A-cdk2 activities were increased, and p27-associated kinase activity was detected in 184A1L5(R) cells. p27 from 184A1L5(R) cells was localized to both nucleus and cytoplasm, showed an altered profile of phosphoisoforms, and had a reduced ability to bind and inhibit cyclin E1-cdk2 in vitro when compared to p27 from the sensitive 184(S) cells. In proliferating 184A1L5(R) cells, more p27 was associated with cyclin D1-cdk4 complexes than in 184(S). While TGF-beta inhibited the formation of cyclin D1-cdk4-p27 complexes in 184(S) cells, it did not inhibit the assembly of cyclin D1-cdk4-p27 complexes in the resistant 184A1L5(R) cells. p27 phosphorylation changed during cell cycle progression, with cyclin E1-bound p27 in G(0) showing a different phosphorylation pattern from that of cyclin D1-bound p27 in mid-G(1). These data suggest a model in which TGF-beta modulates p27 phosphorylation from its cyclin D1-bound assembly phosphoform to an alternate form that binds tightly to inhibit cyclin E1-cdk2. Altered phosphorylation of p27 in the resistant 184A1L5(R) cells may favor the binding of p27 to cyclin D1-cdk4 and prevent its accumulation in cyclin E1-cdk2 in response to TGF-beta.     

The Effect of GADD45a on Furazolidone‐Induced S‐Phase Cell‐Cycle Arrest in Human Hepatoma G2 Cells

In this study, overexpression of GADD45a induced by furazolidone in HepG2 cells could arouse S‐phase cell cycle arrest, suppress cell proliferation, and increase the activities of cyclin D1, cyclin D3, and cyclin‐dependent kinase 6 (CDK6). To the opposite, GADD45a knockdown cells by RNAi could reduce furazolidone‐induced S‐phase cell cycle arrest, increase the cell viability, decrease the activities of cyclin D1, cyclin D3, and CDK6; however, cyclin‐dependent kinase 4 (CDK4) showed no change. Moreover, data from our current studies show that cyclin D1, cyclin D3, and CDK6 are target genes functioning at the downstream of the GADD45a pathway induced by furazolidone. These results demonstrate that the GADD45a pathway is partially responsible for the furazolidone‐induced S‐phase cell cycle arrest. GADD45a influences furazolidone‐induced S‐phase cell cycle arrest in human hepatoma G2 cells via cyclin D1, cyclin D3, and CDK6, but not CDK4.     

Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT

The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.     

Phosphorylation of Thr-516 and Ser-520 in the Kinase Activation Loop of MEKK3 Is Required for Lysophosphatidic Acid-mediated Optimal I��B Kinase �� (IKK��)/Nuclear Factor-��B (NF-��B) Activation

MEKK3 serves as a critical intermediate signaling molecule in lysophosphatidic acid-mediated nuclear factor-κB (NF-κB) activation. However, the precise regulation for MEKK3 activation at the molecular level is still not fully understood. Here we report the identification of two regulatory phosphorylation sites at Thr-516 and Ser-520 within the kinase activation loop that is essential for MEKK3-mediated IκB kinase β (IKKβ)/NF-κB activation. Substitution of these two residues with alanine abolished the ability of MEKK3 to activate IKKβ/NF-κB, whereas replacement with acidic residues rendered MEKK3 constitutively active. Furthermore, substitution of these two residues with alanine abolished the ability of MEKK3 to mediate lysophosphatidic acid-induced optimal IKKβ/NF-κB activation.