MiR-181a Targets PHLPP2 to Augment AKT Signaling and Regulate Proliferation and Apoptosis in Human Keloid Fibroblasts.

BACKGROUND/AIMS: Keloids are fibrous overgrowths induced by cutaneous injury. MicroRNAs (miRNAs) have recently emerged as post-transcriptional gene repressors and participants in a diverse array of pathophysiological processes leading to skin disease. The purpose of the current study was to explore the precise functions of miR-181a in human keloid development and the underlying mechanisms. METHODS: A miRNA microarray analysis was performed to compare expression profiles between keloid and normal skin tissues. Quantitative real-time PCR was conducted to estimate miR-181a expression. Cell proliferation was determined using the cell counting kit-8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU) assays, and cell cycle and apoptosis were detected with flow cytometry. Direct targets of miR-181a were identified using the luciferase reporter assay. RESULTS: miR-181a was significantly upregulated in human keloid tissues and fibroblasts, compared with their control counterparts. Overexpression of miR-181a enhanced keloid fibroblast DNA synthesis and proliferation and inhibited apoptosis, whereas miR-181a suppression triggered the opposite effects. Moreover, miR-181a suppressed the expression of PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) through direct interactions with its 3'UTR region and subsequently enhanced AKT activation. Overexpression of PHLPP2 without its 3'UTR attenuated the effects of miR-181a on cell proliferation and apoptosis in keloid fibroblast cells. Furthermore, miR-181a mimics increased normal skin fibroblast proliferation. CONCLUSIONS: Our results highlight a novel pathway mediated by miR-181a, which may be effectively used as a therapeutic target for treatment of keloids.

Establishing a gene trap system mediated by T-DNA(GUS) in rice.

Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promoterless beta-glucuronidase (GUS) reporter gene in the T-DNA region. Transformation of these two plasmids into the rice variety Zhonghua 11 (Oryza sativa ssp. japonica cv.), mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 T(0) plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistant (hyg(r)) homozygotes were screened and the copy number of the T-DNA inserts was determined in the GUS-positive transgenic plants. The flanking sequences of the T-DNA were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of T-DNA were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calli induced from the seeds of the T(1) generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. The results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.

[Preliminary screening of target genes of rice transcription factor OsBP-73].

Previous data showed that a 31-bp (from -840 bp to -810 bp) DNA fragment located at the 5' upstream region of rice waxy gene could interact with nuclear protein extracted from developing endosperm of rice. When this 31 bp DNA sequence was used as a bait to screen a rice cDNA library with a yeast one-hybrid system, three groups of cDNA clones were isolated. One of them is pC73, the correspondent rice gene of pC73 was named as OsBP-73 (Oryza sativa binding protein). A pull-down assay was made to identify the target genes of transcription factor by using genomic DNA and recombinant p73 protein. The cDNA fragment containing DNA-binding domain of OsBP-73 was cloned into expression vector pET28-c(+) (Fig.1) to produce protein p73, fused with a his(6)-tag, from E. coli BL21 (DE3) (Fig.2). The p73 was purified with Ni-NTA under native condition (Fig.3). The target genes of p73 were identified in rice genome-wide by using a pull-down assay, and 22 candidate genes were obtained (Figs.4 and 5, and Table 1). The obtained results show that putative light-repressible receptor protein kinase and GAMYB-binding protein could serve as targets of the OsBP-73, suggesting that OsBP-73 might be involved in light signal transduction.

OsRRM, a Spen-like rice gene expressed specifically in the endosperm.

We used the promoter trap technique to identify a rice plant, named 107#, in which the beta-glucuronidase (GUS) reporter gene was expressed specifically in the endosperm. A single copy of the T-DNA was inserted into the plant genome, and a candidate gene OsRRM was identified by the insertion. The OsRRM promoter directed GUS expression specifically in rice endosperm, analogous to the GUS expression pattern observed in 107#. OsRRM is a single-copy gene in rice and encodes a nuclear protein containing 1005 amino-acid residues with two RNA recognition motifs and one Spen paralog and ortholog C-terminal domain. Western blot analysis confirmed that the OsRRM protein was specifically expressed in rice endosperm. Ectopic expression of OsRRM in transgenic plants led to abnormalities, such as short stature, retarded growth and low fructification rates. Our data, in conjunction with the reported function of Spen genes, implicated OsRRM in the regulation of cell development in rice endosperm.

[Establishment of promoter trapping system mediated by activator/dissociation (beta-glucuronidase) construction in rice].

The coding region of Bar gene, the left border of Ds element, the coding region of GUS gene, the transposase of Ac element, the right border of Ds element and the promoter of Ubi gene were inserted into the T-DNA region of vector pCAMBIA1300 in turn to construct plasmid p13B. The orientations of the ubiquitons' promoter, Ac transposase and Bar are identical but opposite to that of the GUS gene (Fig.1). The plasmid p13B was then introduced into the calli of Oryza sativa subsp. japonica cv. Zhonghua 11 by Agrobacterium tumefaciens-mediated transforming to trap genes in rice. Eighteen independent transgenic lines were obtained and propagated. T(2) generations of 18 independent transgenic lines were screening by herbicide (Basta) (Fig.2) and the herbicide-resistant plants obtained were analyzed by PCR (Fig.3). Ds element transposed in an inheritable manner was found in 37 plants, in which 5 plants showed GUS activity (Fig.4).

A rice quantitative trait locus for salt tolerance encodes a sodium transporter.

Many important agronomic traits in crop plants, including stress tolerance, are complex traits controlled by quantitative trait loci (QTLs). Isolation of these QTLs holds great promise to improve world agriculture but is a challenging task. We previously mapped a rice QTL, SKC1, that maintained K(+) homeostasis in the salt-tolerant variety under salt stress, consistent with the earlier finding that K(+) homeostasis is important in salt tolerance. To understand the molecular basis of this QTL, we isolated the SKC1 gene by map-based cloning and found that it encoded a member of HKT-type transporters. SKC1 is preferentially expressed in the parenchyma cells surrounding the xylem vessels. Voltage-clamp analysis showed that SKC1 protein functions as a Na(+)-selective transporter. Physiological analysis suggested that SKC1 is involved in regulating K(+)/Na(+) homeostasis under salt stress, providing a potential tool for improving salt tolerance in crops.

[Expression of OsBP-73 gene requires involvement of its intron in rice].

It has been demonstrated in our previous work that rice OsBP-73 gene contains two exons interrupted by a 2471 bp intron. Here it was reported that the 5' flanking region of the ATG translation start codon in the first exon of OsBP-73 gene (from -1818 to +215) can not direct GUS gene expression in resistant rice calli or transgenic rice. When the complete OsBP-73 intron and its flanking region (from -1818 to +2844) are constructed in frame with GUS coding region, GUS activity can be detected in the resistant rice calli and transgenic rice. Experimental data also show that the complete OsBP-73 intron itself has no promoter activity. These results suggest that the OsBP-73 intron sequence is involved in directing the GUS gene expression. Another experiment demonstrates that the complete intron sequence can enhance the activity of OsEBP-89 gene promoter.

[Inducible expression of OsEBP-89 gene in rice].

OsEBP-89 gene was an EREBP (ethylene responsive elements binding protein) transcription factor from rice (Oryza sativa). Northern blot analysis revealed that the expression of OsEBP-89 gene can be induced by ACC, 2,4-D, ABA, BR, JA, GA, 6-BA and salt. We found an ethylene responsive element-like ERE (ethylene responsive element), named IVC box, in the promoter region (-552 to -510) of OsEBP-89 gene. EMSA results showed the IVC box could specifically bind with nuclear proteins extracted from immature rice seeds. When the rice suspension cell harboring IVC/35S mini-promoter (-46)/GUS chimeric construct (pIVC/GUS) was treated by ACC, GUS activity could be induced. However, two plasmids with OsEBP-89 promoter/GUS (pPSG) and IVC box-deleted OsEBP-89 promoter/GUS (pSphSG) were transformed to rice, when both transgenic suspension cell treated with ACC the GUS expression was also inducible. This result indicated that there are more than one ethylene-responsive elements located in the promoter region of the OsEBP-89 gene.

Expressional analysis of an EREBP transcription factor gene OsEBP-89 in rice.

OsEBP-89 gene encodes an ethylene responsive element binding protein (EREBP) transcription factor from rice (Oryza sativa). Northern blot analysis revealed that OsEBP-89 was expressed in root, stem, seeds, flowers and leaves of rice. Histochemical assay showed that GUS expressed mainly in phloem of vascular tissues of the root and stem transition region (RST), basal part of sheath roots, stem node and basal part of adventitious roots, also in endosperm of seeds in transgenic rice harboring OsEBP-89/GUS construct (pNSG). A sequence of region from C279 to C97 was found to play an important role for OsEBP-89 genes expression though promoter deletion assay. The possible function of OsEBP-89 gene was discussed.

[Rapidly obtaining the markerless transgenic rice with reduced amylose content by co-transformation and anther culture].

The plasmid p13W8 carrying antisense fragment of waxy gene and plasmid pCAMBIA1300 containing hpt gene were introduced into rice by Agrobacterium tumefaciens-mediated co-transformation, and 86 transgenic plants were obtained, 32 of them showed positive bands for antisense waxy gene by PCR analysis, the waxy-positive plant frequency is 37.2%. The segregation of antisense fragment of waxy gene and hpt gene was observed by PCR using hpt gene primers and waxy gene primers respectively in 29 T(1) population. One hundred and eighty-three plants containing only the antisense fragment of waxy gene were identified in 1 264 T(1) plants, the waxy-positive plant frequency is 14.4% (Table 1). The amylose content of seeds derived from transgenic plants with only the antisense fragment of waxy gene were determined, varying degrees of reduction in amylose content were found in some plants (Table 2). Four T(1) plants with reduced amylose content were selected through anther culture. Thirty-four anther culture plants seed normally, 23 of them were shown to contain only the antisense fragment of waxy gene (Table 3) by PCR analysis, and the amylose content was reduced to 5%-12% (Table 4). It took only one and half years to obtain the stably inherited markerless transgenic rice with reduced amylose content by co-transformation and anther culture technique.

OsBP-73, a rice gene, encodes a novel DNA-binding protein with a SAP-like domain and its genetic interference by double-stranded RNA inhibits rice growth.

The SAP domain is a recently defined DNA binding domain that forms a helix-extended-helix structure. SAP proteins have been implicated in nuclear architecture and/or RNA metabolism. In this paper, we describe the cloning and characterization of a rice gene, OsBP-73, encoding a 375 amino acid protein with a SAP-like domain. We identified the binding sequence of OsBP-73 by gel retardation assays and southwestern blotting. Northern blot analysis demonstrated that OsBP-73 is weakly expressed in root, leaf and immature seed. OsBP-73 gene expression was also examined by histochemical studies of transgenic rice plants carrying an OsBP-73 5'/GUS reporter gene. The reporter gene is mainly expressed in the tissues with high cell division activities, such as root tip, stem node, panicle and immature seed. Genetic interference of OsBP-73 gene expression by double-stranded RNA strikingly inhibits the whole plant growth but does not affect the passage from the juvenile to adult phase. These results suggest that OsBP-73 may play an important role in the regulation of cell proliferation.

An interaction between a MYC protein and an EREBP protein is involved in transcriptional regulation of the rice Wx gene.

We previously demonstrated that a 31-bp nucleotide sequence located upstream of the rice Wx gene played an important role in its expression. We further showed that this cis-acting regulator interacts with nuclear proteins extracted from developing rice endosperm. We used the 31-bp sequence as bait in a yeast one-hybrid system to isolate several cDNA clones from a rice cDNA expression library. One of these cDNAs encodes a MYC protein, designated OsBP-5, which is 335 amino acids long and contains a putative basic helix-loop-helix-ZIP DNA-binding domain. This domain exhibits 50% amino acid sequence identity with the R/B proteins that regulate the expression of genes involved in anthocyanin biosynthesis in plants. The results of electrophoretic mobility shift assays (EMSAs) and Southwestern gel blots indicate that this protein binds specifically to the CAACGTG motif within the 31-bp sequence. However, by itself, the OsBP-5 protein is unable to trans-activate a lacZ reporter gene controlled by the 31-bp sequence when tested in a yeast expression system. Interestingly, OsBP-5 can trans-activate this reporter gene when another protein, OsEBP-89, a member of the EREBP family of transcription factors, is present. Furthermore, in vitro pull-down experiments show that a protein isolated from developing rice endosperm interacts with the OsBP-5 protein, and Western blots confirm that the interacting protein is OsEBP-89. The formation of a supershift band in EMSAs also indicates that two proteins interact with each other. Interference of OsBP-5 gene expression by double-stranded RNA reduces the amylose content in mature seed of transgenic rice plants but has no visible effect on their phenotype. These results suggest that the OsBP-5 and OsEBP-89 proteins act synergistically, perhaps as a heterodimer, to regulate the transcription of the rice Wx gene.

The OsEBP-89 gene of rice encodes a putative EREBP transcription factor and is temporally expressed in developing endosperm and intercalary meristem.

The AP2/EREBP transcription factors play important roles in plant development and in the responses of plants to biotic and abiotic stresses. All members of the EREBP subfamily described to date are from dicotyledonous plants. In this paper, we describe the cloning and characterization of a rice gene, OsEBP-89, encoding a protein 326 amino acids long with a typical EREBP domain; this is the first report of an EREBP transcription factor in a monocotyledonous plant. Except for the EREBP domain, the OsEBP-89 protein does not have substantial sequence similarities to other members of the subfamily. The DNA-binding activity of the EREBP domain was confirmed by electrophoretic mobility-shift assays. An activation domain rich in acidic amino acids was identified by using a yeast one-hybrid system. Two putative nuclear-localization signals were also identified. The results of northern blot hybridization experiments showed that the transcript of the OsEBP-89 gene accumulates primarily in immature seeds, roots, and leaves (low levels). More detailed information about the pattern of OsEBP-89 gene expression was obtained by histochemical studies of transgenic rice plants carrying an OsEBP-89 5'/GUS reporter gene. The reporter gene was expressed in the endosperm starting at 7 days after pollination and in the intercalary meristem of plants. Expression of OsEBP-89 was induced in roots of rice seedlings by treatment with ACC, NaCl, or 2,4-D. Two cis-acting elements, an endosperm motif and a primary PERE, are present upstream of the OsEBP-89 coding region and may be involved in regulating its expression. Collectively, these results suggest that the OsEBP-89 gene is a new member of the EREBP subfamily and may be involved in ethylene-dependent seed maturation and shoot development of rice.