OBJECTIVE: Oocyte-specific homeobox 4 (Obox4) is preferentially expressed in oocytes and plays an important role in the completion of meiosis of oocytes. However, the Obox4 expression pattern has not been reported yet. In this study, we investigated the subcellular localization of Obox4 using a green fluorescent protein (GFP) fusion expression system. METHODS: Three regions of Obox4 were divided and fused to the GFP expression vector. The partly deleted homeodomain (HD) regions of Obox4 were also fused to the GFP expression vector. The recombinant vectors were transfected into HEK-293T cells plated onto coated glass coverslips. The transfected cells were stained with 4',6-diamidino-2-phenylindol and photographed using a fluorescence microscope. RESULTS: Mutants containing the HD region as well as full-length Obox4 were clearly localized to the nucleus. In contrast, the other mutants of either the N-terminal or C-terminal region without HD had impaired nuclear localization. We also found that the N-terminal and C-terminal of the Obox HD contributed to nuclear localization and the entire HD was necessary for nuclear localization of Obox4. CONCLUSION: Based on the results of the present study, we demonstrated that the intact HD region of Obox4 is responsible for the nuclear localization of Obox4 protein in cells.
Fluorescence ; Genes, Homeobox ; Glass ; Meiosis ; Oocytes

OBJECTIVETo investigate the differently expressed Homeobox genes between adenoid cystic carcinoma of salivary gland and normal gland tissue, and find out the effect of homeobox genes on oncogenesis and differentiation of adenoid cystic carcinoma of salivary gland.

METHODSSix strictly paired specimens including adenoid cystic carcinoma and its surrounding normal gland tissue and two pairs of specimens including cell strain of adenoid cystic carcinoma and its surrounding normal gland tissue were established. Customized Oligo microarray which contains probes of 232 human homeobox genes was used to analyze and conclude two groups of different genes data. RT-PCR technique was used to examine the mRNA expressing level of highly suspected relevant genes of adenoid cystic carcinoma in different specimens. Obvious differently expressed Homeobox genes were found through statistical analyses.

RESULTSIn tissue specimens homeobox genes were found 67 up-regulated and 54 down-regulated, and in cell specimens homeobox genes were found 12 up-regulated and 15 down-regulated. One up-regulated gene and 7 down-regulated genes were found both in tissue and cell specimens, among which EVX1 and PITX1 were the most frequent. RT-PCI showed that there was statistical expressing difference between TGIF, EVX1 and normal gland tissue in ACC-M.

CONCLUSIONAs the key gene to cellular proliferation and differentiation, homeobox genes are closely relevant to the oncogenesis of adenoid cystic carcinoma of salivary gland.

Assessing the cell-type expression pattern of a certain gene can be achieved by using cell-type-specific gene manipulation. Recently, cre-recombinase-dependent gene-silencing tool, pSico has become popular in neuroscientific research. However, pSico has a critical limitation that gene-silenced cell cannot be identified by fluorescence, due to an excision of the reporter gene for green fluorescence protein (GFP). To overcome this limitation, we newly developed pSico-Red, with mCherry gene as a reporter outside two loxP sites, so that red mCherry signal is detected in all transfected cells. When a cell expresses cre, GFP is excised and shRNA is enabled, resulting in disappearance of GFP. This feature of pSico-Red provides not only cell-type-specific gene-silencing but also identification of cre expressing cells. Using this system, we demonstrated for the first time the neuronal expression of the Bestrophin-1 (Best1) in thalamic reticular nucleus (TRN) and TRN-neuron-specific gene-silencing of Best1. We combined adeno-associated virus (AAV) carrying Best1-shRNA in pSico-Red vector and transgenic mouse expressing cre under the promoter of distal-less homeobox 5/6 (DLX5/6), a marker for inhibitory neurons. Firstly, we found that almost all of inhibitory neurons in TRN express Best1 by immunohistochemistry. Using pSico-Red virus, we found that 80% of infected TRN neurons were DLX5/6-cre positive but parvalbumin negative. Finally, we found that Best1 in DLX5/6-cre positive neurons were significantly reduced by Best1-shRNA. Our study demonstrates that TRN neurons strongly express Best1 and that pSico-Red is a valuable tool for cell-type-specific gene manipulation and identification of specific cell population.
Animals ; Dependovirus ; Fluorescence ; Genes, Homeobox ; Genes, Reporter ; Immunohistochemistry ; Mice ; Mice, Transgenic ; Neurons* ; RNA, Small Interfering

OBJECTIVE: To compare the clinical effect of the combined treatment of acupuncture, moxibustion, Chinese herbal medicine and western medication and simple western medication on polycystic ovary syndrome (PCOS) of kidney deficiency and blood stagnation pattern and explore the effect on endometrial receptivity and the expression of serum homeobox gene A10 (HOXA10). METHODS: A total of 60 patients with PCOS of kidney deficiency and blood stagnation pattern were randomized into a combined treatment group and a western medication group, 30 cases in each one. In the western medication group, on the fifth day of menstruation, clomiphene citrate tablets were taken orally, 50 mg each time, once daily, consecutively for 5 days. On the day when the follicle diameter was ≥ 18 mm, chorionic gonadotrophin for muscular injection, a dose of 10 000 U was given. Before sleep, the aspirin enteric-coated tablets were taken orally, 50 mg (except during menstruation). In the combined treatment group, on the base of the treatment as the western medication group, acupuncture and moxibustion were adopted and the Chinese herbal for tonifying the kidney and activating blood circulation was taken orally. The acupoints were Guanyuan (CV 4), Qihai (CV 6), Zusanli (ST 36), Sanyinjiao (SP 6), Zigong (EX-CA 1), etc. Acupuncture was remained for 30 min each time, once every two days and discontinued during menstruation. Chinese herbal was given from the 3rd day of menstruation till the onset of the next menstruation, one dose each day. After consecutive treatment for 3 menstrual cycles in the two groups, the real-time polymerase chain reaction (RT-PCR) method was adopted to determine the expression of serum HOXA10 before and after treatment in the patients of the two groups. The endometrial thickness at ovulatory phase, uterine arterial flow 7 days after ovulation [including uterine arterial pulsatility index (PI), resistance index (RI), peak systolic velocity (PSV)/end diastolic velocity (EDV), meaning S/D], pregnancy rate and the score of Chinese medicine symptoms before and after treatment were compared in the patients between the two groups. RESULTS: ① After treatment, the expression of serum HOXA10 was higher than that before treatment in the patients of the two groups ( CONCLUSION The combined treatment with acupuncture, moxibustion and medication effectively improves endometrial receptivity and uterine arterial flow in the patients with PCOS of kidney deficiency and blood stagnation pattern and increases pregnancy rate. The therapeutic effect is better than the simple western medication and its mechanism is probably related to the regulation of serum HOXA10 expression.
Acupuncture Points ; Acupuncture Therapy ; Female ; Genes, Homeobox ; Homeobox A10 Proteins ; Humans ; Kidney ; Moxibustion ; Polycystic Ovary Syndrome/genetics* ; Pregnancy

PURPOSE: Homeobox (HOX) genes are essential developmental regulators that should normally be in the silenced state in an adult brain. The aberrant expression of HOX genes has been associated with the prognosis of many cancer types, including glioblastoma (GBM). This study examined the identity and role of HOX genes affecting GBM prognosis and treatment resistance. MATERIALS AND METHODS: The full series of HOX genes of five pairs of initial and recurrent human GBM samples were screened by microarray analysis to determine the most plausible candidate responsible for GBM prognosis. Another 20 newly diagnosed GBM samples were used for prognostic validation. In vitro experiments were performed to confirm the role of HOX in treatment resistance. Mediators involved in HOX gene regulation were searched using differentially expressed gene analysis, gene set enrichment tests, and network analysis. RESULTS: The underexpression of HOXA11 was identified as a consistent signature for a poor prognosis among the HOX genes. The overall survival of the GBM patients indicated a significantly favorable prognosis in patients with high HOXA11 expression (31±15.3 months) compared to the prognoses in thosewith low HOXA11 expression (18±7.3 months, p=0.03). When HOXA11 was suppressed in the GBM cell lines, the anticancer effect of radiotherapy and/or temozolomide declined. In addition, five candidate mediators (TGFBR2, CRIM1, TXNIP, DPYSL2, and CRMP1) that may confer an oncologic effect after HOXA11 suppression were identified. CONCLUSION: The treatment resistance induced by the underexpression of HOXA11 can contribute to a poor prognosis in GBM. Further investigation will be needed to confirm the value of HOXA11 as a potential target for overcoming the treatment resistance by developing chemo- or radiosensitizers.
Adult ; Brain ; Cell Line ; Genes, Homeobox ; Glioblastoma* ; Humans ; In Vitro Techniques ; Microarray Analysis ; Prognosis* ; Radiotherapy

Spatially and temporally programmed expression of the Hox genes along the antero-posterior (A-P) axis is essential for correct pattern formation during embryonic development. An accumulating body of evidence indicates the pivotal role of spatial chromatin organization for the coordination of gene regulation. Recently, chromosome conformation capture (3C) technique has been developed and opened a new way to study chromosomal interactions in the nucleus. In this study, we describe 3C method we applied in F9 embryonic teratocarcinoma cells and demonstrate that the chromosomal interactions at Hox loci are successfully detected. Interestingly, at Hoxc loci, the abundance of intrachromosomal interactions with neighboring fragments was drastically decreased when the genes are expressed. These results indicate the possibility of the dynamic pattern of chromosomal interaction in association with the transcriptional regulation of Hox genes.
Axis, Cervical Vertebra ; Chromatin ; Embryonic Development ; Female ; Gene Expression ; Genes, Homeobox ; Pregnancy ; Teratocarcinoma ; Transcriptional Activation

Congenital central hypoventilation syndrome (CCHS) is a disorder of the autonomic nervous system characterized by a decreased response to hypercarbia. CCHS is frequently associated with congenital megacolon; the combination is called Haddad syndrome. CCHS is associated with dysfunction in respiratory features of the autonomic nervous system and with other disorders, including facial deformities, cardiovascular symptoms, and tumors. Patients with CCHS frequently have a mutation in the homeobox protein 2b (PHOX2B) gene. Most mutations involve heterozygous expansion of alanine repeats (GCN). Interestingly, a higher polyalanine repeat number is associated with a more severe clinical phenotype. To clarify the role of PHOX2B in disease pathogenesis, we introduce and review the clinical and molecular features of CCHS and Haddad syndrome.
Alanine ; Autonomic Nervous System ; Congenital Abnormalities ; Genes, Homeobox ; Hirschsprung Disease ; Humans ; Hypoventilation* ; Molecular Biology* ; Phenotype

OBJECTIVE: Although some sporadic reports reveal the link between the homeobox (HOX) genes and ovarian carcinoma, there is no comprehensive analysis of the expression pattern of the class I homeobox genes in ovarian carcinoma that determines the candidate genes involved in ovarian carcinogenesis. METHODS: The different patterns of expression of 36 HOX genes were analyzed, including 4 ovarian cancer cell lines and 4 normal ovarian tissues. Using a reverse transcription-polymerase chain reaction (RT-PCR) and quantification analysis, the specific gene that showed a significantly higher expression in ovarian cancer cell lines than in normal ovaries was selected, and western blot analysis was performed adding 7 ovarian cancer tissue specimens. Finally, immunohistochemical and immunocytochemical analyses were performed to compare the pattern of expression of the specific HOX gene between ovarian cancer tissue and normal ovaries. RESULTS: Among 36 genes, 11 genes had a different level of mRNA expression between the cancer cell lines and the normal ovarian tissues. Of the 11 genes, only HOXB4 had a significantly higher level of expression in ovarian cancer cell lines than in normal ovaries (p=0.029). Based on western blot, immunohistochemical, and immunocytochemical analyses, HOXB4 was expressed exclusively in the ovarian cancer cell lines or cancer tissue specimens, but not in the normal ovaries. CONCLUSION: We suggest HOXB4 may be a novel candidate gene involved in ovarian carcinogenesis.
Aluminum Hydroxide ; Blotting, Western ; Carbonates ; Cell Line ; Female ; Genes, Homeobox ; Ovarian Neoplasms ; Ovary ; RNA, Messenger

This study was purposed to construct the recombinant retroviral vector containing human homeobox gene hoxA10 and to establish the packaging cell lines which stably produce viruses. The whole coding region of hoxA10 gene was amplified by PCR and inserted into the retroviral vector MSCVneo. The recombinant vector was identified by DNA sequencing. The recombinant and control retroviral vectors were transfected into the packaging cell line PT67 by liposome Lipofectamine(TM) 2000. These cell lines stably producing retrovirus were isolated following G418 selection. The viral suspension was harvested and the viral titer was determined by NIH3T3 cells. The results showed that the recombinant retroviral vector was proved to encode hoxA10 genes by sequencing. The cell lines efficiently producing virus were screened by G418 and designated as PT67/MSCVneo and PT67/MSCVneo-hoxA10. The titers of them were 5 x 10(5) CFU/ml and 4 x 10(4) CFU/ml respectively. It is concluded that the recombinant retroviral vector containing homeobox gene hoxA10 and the stably packaging cell lines which efficiently and correctly produce viruses are successfully constructed, which provides a basis for further exploration of the hoxA10 gene function in the regulation of hematopoiesis.
Cell Line ; Genes, Homeobox ; Genetic Vectors ; Homeodomain Proteins ; genetics ; Humans ; Retroviridae ; genetics ; Transfection

In medicolegal investigations, correct identification of the necrophagous fly species collected around and on the corpse is an essential step for estimating the postmortem interval (PMI). Therefore, forensic pathologists and entomologists investigating deaths due to violent crimes need a rapid, easy-to-use protocol to identify fly species found on corpses. A rapid and robust DNA-based tool that can distinguish between various immature and mature species from the Calliphoridae, Muscidae, and Sarcophagidae families would be ideal for such investigations. To date, the DNA barcode initiative is the best approach for identifying species-specific nucleotide sequences. We have developed 3 sequence-characterized amplified region (SCAR)-based identification systems derived from the Abdominal-B homeobox sequences of 17 fly species belonging to the Muscidae and Sarcophagidae. The flies used in this study were collected in Korea. These assay systems can classify 17 forensically important fly species into the dipteran family group and reliably distinguish them from inter- and intraspecific fly species through a 2-step multiplex PCR. This novel approach may also be used as an alternative to conventional DNA-based identification methods.
Base Sequence ; Cadaver ; Crime ; Diptera ; DNA ; Genes, Homeobox ; Humans ; Korea ; Multiplex Polymerase Chain Reaction ; Muscidae ; Sarcophagidae