miR-141 Inhibits Proliferation and Migration of Colorectal Cancer SW480 Cells.

BACKGROUND: This study was designed to determine the molecular function of miR-141 and the underlying mechanisms in colorectal cancer (CRC). MATERIALS AND METHODS: SW480 cells in which miR-141 was up- or down-regulated were established. Reverse transcription, quantitative polymerase chain reaction and Western blotting were used to examine the microRNA and protein expression. Cell-cycle progression was analyzed by flow cytometry. Proliferation marker Ki-67 was evaluated by immunofluorescence. Transwell assay was conducted to determine the migration rates of cells. Subcutaneous xenograft models were used to examine the effect of miR-141 on tumorigenicity. Human mitogen-activated protein kinase (MAPK) and receptor tyrosine kinase (RTK) pathway phosphorylation array assays were used to interrogate MAPK and RTK pathway activation. RESULTS: miR-141 directly targeted zinc finger E-box-binding homeobox 1/2 (ZEB1/2). We first determined the expression levels of ZEB1 and ZEB2 in miR-141-expressing cells and miR-141-knockdown cells and found that inhibition of miR-141 significantly increased the expression of ZEB2. In vitro study revealed that miR-141 overexpression inhibited the expression of Ki-67. Furthermore, overexpression of miR-141 led to a significant reduction in the proliferation of SW480 cells via induction of cell-cycle arrest at the G1 stage. In contrast, inhibition of miR-141 markedly promoted the proliferation of SW480 cells by promoting cell-cycle progression. Moreover, overexpression of miR-141 significantly inhibited SW480 cell migration in vitro. In addition, overexpression of miR-141 significantly reduced tumor size and weight, and inhibited the growth of SW480 cell-derived tumor in nude mice. Notably, overexpression of miR-141 also suppressed the liver metastasis of SW480 cells in nude mice. Using RTK and MAPK arrays, we found increased phosphorylation of hepatocyte growth factor receptor (HGFR/c-MET) following inhibition of miR-141, but phosphorylation of P53, AKT, ERK1/2, P38 and mTOR, etc., in SW480 cells was not affected by miR-141. CONCLUSION: Our results suggest that miR-141 functions as a tumor suppressor through ZEB2 and HGFR in CRC cells.

Laminin-ß1 impairs spatial learning through inhibition of ERK/MAPK and SGK1 signaling.

Laminin is a major structural element of the basal lamina consisting of an α-chain, a ß-chain, and a γ-chain arranged in a cross-like structure, with their C-terminal inter-coiled. Laminin is abundantly expressed in the hippocampus of mature brain and is implicated in several psychiatric disorders, but its possible role involved in learning and memory function is not known. This issue was examined here. Our results revealed that water maze training significantly decreased laminin-ß1 (LB1) expression in the rat hippocampal CA1 area. Transfection of LB1 WT plasmid to hippocampal CA1 neurons impaired water maze performance in rats. Meanwhile, it decreased the phosphorylation level of ERK/MAPK and protein kinase serum- and glucocorticoid-inducible kinase-1 (SGK1). By contrast, knockdown of endogenous LB1 expression using RNA interference (LB1 siRNA) enhanced water maze performance. Meanwhile, it increased the phosphorylation level of ERK/MAPK and SGK1. The enhancing effect of LB1 siRNA on spatial learning and on the phosphorylation of ERK/MAPK and SGK1 was blocked by co-treatment with the MEK inhibitor U0126 at a concentration that did not apparently affect spatial learning and ERK/MAPK phosphorylation alone. Further, the enhancing effect of LB1 siRNA on spatial learning and SGK1 phosphorylation was similarly blocked by co-transfection with SGK1 siRNA at a concentration that did not markedly affect spatial learning and SGK1 expression alone. These results together indicate that LB1 negatively regulates spatial learning in rats. In addition, LB1 impairs spatial learning through decreased activation of the ERK/MAPK-SGK1 signaling pathway in the rat hippocampus.

Serum- and glucocorticoid-inducible kinase 1 (SGK1) increases neurite formation through microtubule depolymerization by SGK1 and by SGK1 phosphorylation of tau.

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a member of the Ser/Thr protein kinase family that regulates a variety of cell functions. Recently, SGK1 was shown to increase dendritic growth but the mechanism underlying the increase is unknown. Here we demonstrated that SGK1 increased the neurite formation of cultured hippocampal neurons through microtubule (MT) depolymerization via two distinct mechanisms. First, SGK1 directly depolymerized MTs. In vitro MT depolymerization experiments revealed that SGK1, especially N-truncated SGK1, directly disassembled self-polymerized MTs and taxol-stabilized MTs in a dose-dependent and ATP-independent manner. The transfection of sgk1 to HeLa cells also inhibited MT assembly in vivo. Second, SGK1 indirectly depolymerized MTs through the phosphorylation of tau at Ser214. An in vitro kinase assay revealed that active SGK1 phosphorylated tau Ser214 specifically. In vivo transfection of sgk1 also phosphorylated tau Ser214 in HEK293T cells and hippocampal neurons. Further, sgk1 transfection significantly increased the number of primary neurites and shortened the length of the total process in cultured hippocampal neurons. These effects were antagonized by the cotransfection of the tauS214A mutant plasmid. Dexamethasone, a synthetic glucocorticoid, mimics the effect of sgk1 overexpression. Together, these results suggest that SGK1 enhances neurite formation through MT depolymerization by a direct action of SGK1 and by the SGK1 phosphorylation of tau.

Serum- and glucocorticoid-inducible kinase (SGK) is a target of the MAPK/ERK signaling pathway that mediates memory formation in rats.

We have previously demonstrated that serum- and glucocorticoid-inducible kinase (SGK) plays a causal role in facilitating memory formation of spatial learning in rats, but the SGK signaling pathway involved in spatial memory formation is not known. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) also plays an important role in memory formation. We therefore examined whether SGK is a downstream target of the MAPK/ERK signaling cascade and whether ERK signaling to SGK mediates spatial memory formation in rats. Results from an in vitro kinase assay revealed that ERK directly phosphorylates SGK at Ser78, but not at Thr256 and Ser422, whereas inhibition of ERK by PD98059 significantly decreased SGK phosphorylation at Ser78, Thr256 and Ser422 following spatial training. Prior administration of PD98059 also antagonized the enhancing effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator that also causes ERK activation, on SGK phosphorylation and cAMP response element binding protein (CREB) phosphorylation. Moreover, TPA-induced SGK phosphorylation and CREB phosphorylation was abolished by prior SGKS78A mutant DNA transfection. By contrast, SGKS78A mutant DNA transfection to hippocampal area CA1 did not affect spatial memory formation, whereas SGKT256A mutant DNA transfection to area CA1 significantly impaired spatial memory formation. ERK was known to regulate sgk mRNA expression, but in the present study we have demonstrated that SGK is also a downstream target of the ERK signaling cascade; ERK directly phosphorylates SGK at Ser78 and indirectly activates SGK at Thr256 and Ser422 through unknown intermediate molecules. Furthermore, ERK activation of SGK is involved in spatial memory formation in rats.

Focal adhesion kinase is required, but not sufficient, for the induction of long-term potentiation in dentate gyrus neurons in vivo.

Tyrosine kinase phosphorylation plays an important role in the induction of long-term potentiation (LTP). Focal adhesion kinase (FAK) is a 125 kDa nonreceptor tyrosine kinase that shows decreased phosphorylation in fyn mutant mice, and Fyn plays a critical role in LTP induction. By examining the role of FAK involved in LTP induction in dentate gyrus in vivo with medial perforant path stimulation, we found that both FAK and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) phosphorylation were increased significantly 5 and 10 min after LTP induction, whereas cAMP-responsive element binding protein (CREB) phosphorylation was increased 40 min later. Transfection of the dominant-negative FAK mutant construct HA-FAK(Y397F) impaired LTP, whereas transfection of the constitutively activated form HA-FAK(Delta1-100) reduced the threshold for LTP induction. Transfection of HA-FAK(Delta1-100) by itself did not induce long-lasting potentiation. Further, transfection of the HA-FAK(Y397F) construct decreased FAK, MAPK/ERK, and CREB phosphorylation, and the inhibition of MAPK/ERK decreased CREB phosphorylation. Moreover, blockade of NMDA receptor (NMDAR) did not decrease FAK, MAPK/ERK, and CREB phosphorylation although LTP induction was blunted by NMDAR antagonist. These biochemical changes were not associated with low-frequency stimulation either. Immunoprecipitation results revealed that tyrosine phosphorylation of NR2A and NR2B as well as the association of phosphorylated FAK with NR2A and NR2B was increased with LTP induction. These results together suggest that FAK is required, but not sufficient, for the induction of LTP in a NMDAR-independent manner and that MAPK/ERK and CREB are the downstream events of FAK activation. Further, FAK may interact with NR2A and NR2B to modulate LTP induction.