Drought stress affects the growth and development of rice, resulting in severe loss in yield and quality. Ectopic expression of the bacterial RNA chaperone, cold shock protein (Csp), can improve rice drought tolerance. Archaeal TRAM (TRM2 and MiaB) proteins have similar structure and biochemical functions as bacterial Csp. Moreover, DNA replication, transcription and translation of archaea are more similar to those in eukaryotes. To test if archaeal RNA chaperones could confer plant drought tolerance, we selected two TRAM proteins, Mpsy_3066 and Mpsy_0643, from a cold-adaptive methanogenic archaea Methanolobus psychrophilus R15 to study. We overexpressed the TRAM proteins in rice and performed drought treatment at seedling and adult stage. The results showed that overexpression both TRAM proteins could significantly improve the tolerance of rice to drought stress. We further demonstrated in rice protoplasts that the TRAMs could abolish misfolded RNA secondary structure and improve translation efficiency, which might explain how TRAMs improve drought tolerance transgenic rice. Our work supports that ectopic expression of archaeal TRAMs effectively improve drought tolerance in rice.
Droughts ; Ectopic Gene Expression ; Gene Expression Regulation, Plant ; Oryza ; Plant Proteins ; Plants, Genetically Modified ; Stress, Physiological

Previously, we have reported that CKAP2 is involved in the maintenance of centrosome integrity, thus allowing for proper mitosis in primary hepatocytes. To understand this biological process, we identified the mitosis-specific phosphorylation sites in mouse CKAP2 and investigated CKAP’s possible role in cell cycle progression. Because we observed mouse CKAP2 depletion in amplified centrosomes and aberrant chromosomal segregation, which was rescued by ectopic expression of wild-type CKAP2, we focused on the centrosome duplication process among the various aspects of the cell cycle. Among the identified phosphorylation sites, T603 and possibly S608 were phosphorylated by CDK1–cyclin B1 during mitosis, and the ectopic expression of both T603A and S608A mutants was unable to restore the centrosomal abnormalities in CKAP2-depleted cells. These results indicated that the phosphorylation status of CKAP2 during mitosis is critical for controlling both centrosome biogenesis and bipolar spindle formation.
Animals ; Biological Processes ; Cell Cycle ; Centrosome* ; Ectopic Gene Expression ; Hepatocytes ; Mice ; Mitosis ; Phosphorylation*

Olfactory receptors (ORs) in mammals are generally considered to function as chemosensors in the olfactory organs of animals. They are membrane proteins that traverse the cytoplasmic membrane seven times and work generally by coupling to heterotrimeric G protein. The OR is a G protein–coupled receptor that binds the guanine nucleotide-binding G(αolf) subunit and the Gβγ dimer to recognize a wide spectrum of organic compounds in accordance with its cognate ligand. Mammalian ORs were originally identified from the olfactory epithelium of rat. However, it has been recently reported that the expression of ORs is not limited to the olfactory organ. In recent decades, they have been found to be expressed in diverse organs or tissues and even tumors in mammals. In this review, the expression and expected function of olfactory receptors that exist throughout an organism's system are discussed.
Animals ; Cell Membrane ; Ectopic Gene Expression ; GTP-Binding Proteins ; Guanine ; Mammals* ; Membrane Proteins ; Olfactory Mucosa ; Rats

MicroRNAs (miRNAs) are negative regulators of gene expression, and miRNA deregulation is found in various tumors. We previously reported that suppression of adenine nucleotide translocase 2 (ANT2) by short hairpin RNA (shRNA) inhibits hepatocellular carcinoma (HCC) development by rescuing miR-636 expression. However, the tumor-suppressive mechanisms of ANT2 shRNA are still poorly understood in HCC. Here, we hypothesized that miRNAs that are specifically downregulated by ANT2 shRNA might function as oncomiRs, and we investigated the roles of ANT2 shRNA-regulated miRNAs in the pathogenesis of HCC. Our data show that miR-19a and miR-96, whose expression is regulated by ANT2 suppression, were markedly upregulated in HCC cell lines and clinical samples. Ectopic expression of miR-19a and miR-96 dramatically induced the proliferation and colony formation of hepatoma cells in vitro, whereas inhibition of miR-19a and miR-96 reduced these effects. To investigate the in vivo function, we implanted miR-96-overexpressing HepG2 cells in a xenograft model and demonstrated that the increase in miR-96 promoted tumor growth. We also found that miR-19a and miR-96 inhibited expression of tissue inhibitor of metalloproteinase-2. Taken together, our results suggest that ANT2-regulated miR-19a and miR-96 play an important role in promoting the proliferation of human HCC cells, and the knockdown of ANT2 directly downregulates miR-19a and miR-96, ultimately resulting in the suppression of tumor growth.
Carcinoma, Hepatocellular* ; Cell Line ; Ectopic Gene Expression ; Gene Expression ; Hep G2 Cells ; Heterografts ; Humans ; In Vitro Techniques ; MicroRNAs ; Mitochondrial ADP, ATP Translocases ; RNA, Small Interfering* ; Tissue Inhibitor of Metalloproteinase-2

ectopic expression and this is associated with an increased activity of tumor stem cells in these patients. This leads to a high aggressiveness of the tumor and the development of metastatic disease. The aim was to evaluate the prognostic significance of the presence of amplifications of stem genes and their expression in patients with early breast cancer (BC).METHODS: The study included 28 patients with T₁N(x)M₀ BC. We used surgical specimens, including formalin-fixed paraffin-embedded archive materials, for 8 patients. A microarray analysis was performed on high-density DNA chips from CytoScanHDArray to assess the status of copy number aberration (CNA) of stem genes locus. Gene expression was assessed using RT-qPCR.RESULTS: CNA analysis of the studied tumors of patients without chemotherapy showed that 17/18 patients without metastases did not have two or more amplifications of chromosomal regions. Ten patients had visceral metastases. In 9/10 of these patients in the primary tumor there were two or more amplifications of the stem genes locus. Two or more amplifications of stem genes locus were found in 12 patients with stage I. Hematogenous metastases did not develop in all patients. Comparison of metastasis-free survival rates in groups of patients with 1 or without amplifications and with two or more amplifications showed statistically significant differences (P = 0.01).CONCLUSION: Our studies have shown that the presence of clones with two or more amplifications of stem gene in patients with BC T₁N(x)M₀ has a significant prognostic value and determines an unfavorable prognosis for distant metastasis.]]>
Archives ; Breast Neoplasms ; Breast ; Clone Cells ; Drug Therapy ; Ectopic Gene Expression ; Gene Expression ; Humans ; Microarray Analysis ; Neoplasm Metastasis ; Neoplastic Stem Cells ; Oligonucleotide Array Sequence Analysis ; Prognosis ; Survival Rate

Dehydration-responsive element binding proteins (DREBs) are an important class of transcription factors related to plant stress tolerance. Ammopiptanthus mongolicus is an evergreen broadleaf shrub endemic to desert areas of northwest China, and it has a very high tolerance to harsh environments. In order to reveal the functions and mechanisms of the AmDREB1F gene from this species in enduring abiotic stresses, we performed subcellular localization test, expression pattern analysis, and stress tolerance evaluation of transgenic Arabidopsis harboring this gene. The protein encoded by AmDREB1F was localized in the nucleus. In laboratory-cultured A. mongolicus seedlings, the expression of AmDREB1F was induced significantly by cold and drought but very slightly by salt and heat stresses, and undetectable upon ABA treatment. In leaves of naturally growing shrubs in the wild, the expression levels of the AmDREB1F gene were much higher during the late autumn, winter and early spring than in other seasons. Moreover, the expression was abundant in roots and immature pods rather than other organs of the shrubs. Constitutive expression of AmDREB1F in Arabidopsis induced the expression of several DREB-regulated stress-responsive genes and improved the tolerance of transgenic lines to drought, high salinity and low temperature as well as oxidative stress. The constitutive expression also caused growth retardation of the transgenics, which could be eliminated by the application of gibberellin 3. Stress-inducible expression of AmDREB1F also enhanced the tolerance of transgenic Arabidopsis to all of the four stresses mentioned above, without affecting its growth and development. These results suggest that AmDREB1F gene may play positive regulatory roles in response to abiotic stresses through the ABA-independent signaling pathways.
Arabidopsis/metabolism* ; Droughts ; Ectopic Gene Expression ; Fabaceae/genetics* ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism* ; Plants, Genetically Modified/genetics* ; Stress, Physiological/genetics*

Matrix metalloproteinase 2 (MMP2) is a potent protumorigenic, proangiogenic, and prometastatic enzyme that is overexpressed in metastatic cancer. Although there have been various studies on the MMP2 gene, further studies of regulatory factors are required to achieve inhibition of MMP2 enzyme activities. MicroRNAs (miRNAs) play key roles in tumor metastasis. However, the specific functions of miRNAs in metastasis are unclear. In this study, we assessed the function of the microRNA-29 family (miR-29s) in HT1080 human fibrosarcoma cells and examined the regulatory mechanisms of these miRNAs on MMP2 activation. Using miRanda, TargetScan, and PicTar databases, miR-29s were identified as candidate miRNAs targeting MMP2. Gain-of-function studies showed that overexpression of miR-29s could inhibit the invasion of HT1080 cells, suggesting their tumor-suppressive roles in HT1080 cells. In addition, dual luciferase reporter assays indicated that miR-29s could inhibit the expression of the luciferase gene containing the 3'-untranslated region of MMP2 mRNA. Ectopic expression of miR-29s down-regulated the expression of MMP2. Moreover, ectopic expression of miR-29s reduced MMP2 enzyme activity. These results suggested that miR-29s could decrease the invasiveness of HT1080 cells by modulating MMP2 signaling. Taken together, our results demonstrated that miR-29s may serve as therapeutic targets to control tumor metastasis.
Ectopic Gene Expression ; Fibrosarcoma ; Humans ; Humans ; Luciferases ; Matrix Metalloproteinase 2 ; MicroRNAs ; Neoplasm Invasiveness ; Neoplasm Metastasis ; RNA, Messenger

Wnt10b, an endogenous inhibitor of adipogenesis, maintains preadipocytes in an undifferentiated state by suppressing adipogenic transcription factors. We have previously demonstrated that Wnt10b transcription during adipogenesis is negatively regulated by X-box-binding protein 1 (XBP1), an important transcription factor of the unfolded protein response. In this report, we demonstrate that XBP1s can directly induce the transcription of microRNA-148a, which in turn mediates the silencing of Wnt10b mRNA during adipogenic differentiation of 3T3-L1 cells. Stability of Wnt10b mRNA was found to be significantly increased by knockdown of XBP1s. Using computational algorithms, a set of microRNAs was predicted to bind Wnt10b mRNA, of which microRNA-148a was selected as a potential target for XBP1s. Our results revealed that microRNA-148a could bind to the 3′UTR of Wnt10b mRNA. Its ectopic expression significantly suppressed both Wnt10b expression and β-catenin activity. When we altered the expression of XBP1 in 3T3-L1 cells, microRNA-148a levels changed accordingly. A potential XBP1 response element was found in the promoter region of microRNA-148a, and XBP1s directly bound to this response element as shown by point mutation analysis and chromatin immunoprecipitation assay. In addition, a microRNA-148a mimic significantly restored adipogenic potential in XBP1-deficient 3T3-L1 cells. These findings provide the first evidence that XBP1s can regulate Wnt10b by a post-transcriptional mechanism through directly inducing microRNA-148a.
3T3-L1 Cells* ; Adipogenesis* ; Chromatin Immunoprecipitation ; Ectopic Gene Expression ; MicroRNAs ; Point Mutation ; Promoter Regions, Genetic ; Response Elements ; RNA, Messenger ; Transcription Factors ; Unfolded Protein Response

Wnt10b, an endogenous inhibitor of adipogenesis, maintains preadipocytes in an undifferentiated state by suppressing adipogenic transcription factors. We have previously demonstrated that Wnt10b transcription during adipogenesis is negatively regulated by X-box-binding protein 1 (XBP1), an important transcription factor of the unfolded protein response. In this report, we demonstrate that XBP1s can directly induce the transcription of microRNA-148a, which in turn mediates the silencing of Wnt10b mRNA during adipogenic differentiation of 3T3-L1 cells. Stability of Wnt10b mRNA was found to be significantly increased by knockdown of XBP1s. Using computational algorithms, a set of microRNAs was predicted to bind Wnt10b mRNA, of which microRNA-148a was selected as a potential target for XBP1s. Our results revealed that microRNA-148a could bind to the 3′UTR of Wnt10b mRNA. Its ectopic expression significantly suppressed both Wnt10b expression and β-catenin activity. When we altered the expression of XBP1 in 3T3-L1 cells, microRNA-148a levels changed accordingly. A potential XBP1 response element was found in the promoter region of microRNA-148a, and XBP1s directly bound to this response element as shown by point mutation analysis and chromatin immunoprecipitation assay. In addition, a microRNA-148a mimic significantly restored adipogenic potential in XBP1-deficient 3T3-L1 cells. These findings provide the first evidence that XBP1s can regulate Wnt10b by a post-transcriptional mechanism through directly inducing microRNA-148a.
3T3-L1 Cells* ; Adipogenesis* ; Chromatin Immunoprecipitation ; Ectopic Gene Expression ; MicroRNAs ; Point Mutation ; Promoter Regions, Genetic ; Response Elements ; RNA, Messenger ; Transcription Factors ; Unfolded Protein Response

Naive CD4 T cells activated by antigen-presenting cells (APCs) undergo terminal differentiation in the periphery. Multiple mechanisms determine their fates, that is, whether they differentiate into conventional T (Tconv) cells or regulatory T (Treg) cells. The key event during Treg generation is expression of the transcription factor Foxp3, which is the lineage-determining regulator for Treg differentiation and function. Here we show that the transcription factor Batf3 acts as a fate-decision factor with respect to Tconv versus Tregs by restraining Treg differentiation. Batf3 was preferentially expressed in effector CD4 T cells but not in Treg cells, and ectopic expression of Batf3 inhibited Foxp3 induction. Batf3-deficient CD4 T cells favorably differentiated into Treg cells in vitro and in colonic lamina propria. Batf3 KO mice also showed enhanced Treg function in gut-associated immune disease models (for example, ovalbumin tolerance and inflammatory bowel disease models). Batf3 bound to the CNS1 region of the Foxp3 locus and reduced expression of the gene. Thus, Batf3 is a transcriptional suppressor of Treg differentiation.
Animals ; Antigen-Presenting Cells ; Colon ; Ectopic Gene Expression ; Immune System Diseases ; In Vitro Techniques ; Inflammatory Bowel Diseases ; Mice ; Mucous Membrane ; Ovalbumin ; T-Lymphocytes ; T-Lymphocytes, Regulatory* ; Transcription Factors*