MicroRNA profiling during germline differentiation of mouse embryonic stem cells.

MicroRNAs are new classes of small non—coding regulatory RNAs which control degradation or suppress translation of its target mRNAs by sequence complementarity. Mature microRNAs are enriched in embryonic stem cells and play important roles in controlling stem cell self—renewal as well as control of differentiation. There is significant evidence that microRNAs are involved in the regulation of stem cell differentiation. The male mouse Embryonic Stem Cell line C57BL6/J with normal karyotype 46, XY was used for profiling microRNA expression in undifferentiated mouse embryonic stem cells (mESCs) and mESCs which were differentiated to germ line cells to determine and compare differences in microRNA expression before and after differentiation. Also, testis tissue samples of a 5—day—old mouse and a mature mouse was used as in vivo control. Profiling was performed by quantitative real—time PCR using locked nucleic acid microRNA—specific LNATM—enhanced primers. After data analysis and comparison of results profiled microRNAs expression, three microRNAs, mmu—miR—21, mmu—miR—21* and mmu—miR—16 showed 50.31, 43.76 and 46.77—fold change increase of expression, respectively, in differentiated mESCs in comparison with undifferentiated state with significant p—value (Average p—value p<0.001 for each members of microRNAs). Expression of Let—7 microRNA family increased in differentiated state when compared with undifferentiated mESCs (Average p—value<0.0001 for each members of family). The levels of expression all other profiled microRNAs were significantly higher in undifferentiated in comparison with differentiated mESCs and their expression was down regulated after differentiation. (Average p—value <0.003 for each members of microRNAs).

Tumour regression induced by co-administration of MIP-3α and CpG in an experimental model of colon carcinoma.

CCL20/macrophage inflammatory protein-3α (MIP-3α) represents one of the potent chemoattractive proteins for dendritic cells (DCs). Herein, we investigated whether in vivo genetic modification of tumour cells aimed at intratumoural production of MIP-3α might lead to accumulation of DCs in tumour tissue. Mice injected with CT26, received recombinant adenovirus (Ad) vectors (AdMIP-3α) expressing MIP-3α protein. This was complemented by injections of CpG. Interestingly, MIP-3α gene therapy combined with CpG injections resulted in specific cytotoxicity. This was associated with significant suppression of tumour growth rate. These findings demonstrate the potential of strategies that utilize in vivo overexpression of chemokines.

Expression of splice variants of cancer-testis genes ODF3 and ODF4 in the testis of a prostate cancer patient.

The outer dense fiber (ODF) genes encode proteins that co-assemble along the axoneme of the sperm tail. Recently, it was demonstrated that some ODF genes are aberrantly expressed in tumors, including prostate adenocarcinoma, basal cell carcinoma, and chronic myeloid lymphoma. We cloned ODF3 and ODF4 cDNA from the testis of a patient suffering from prostate adenocarcinoma and found two alternative splice variants of these genes.

Elevated expression levels of testis-specific genes TEX101 and SPATA19 in basal cell carcinoma and their correlation with clinical and pathological features.

BACKGROUND: Basal cell carcinoma (BCC) is characterized by a low rate of metastasis, slow growth and strong stroma dependency, with significant morbidity and public health burden. Cancer-testis (CT) genes are specifically expressed in normal testis, fetal ovary and different types of cancers. Testis immune privileged status makes CT genes promising candidates as cancer markers, vaccines and immunotherapy. OBJECTIVES: To find new CT genes as cancer markers and candidate genes for immunotherapy and to correlate pathological and clinical features with their expression in patients with BCC. METHODS: By means of digital differential display, seven testis-specific genes were selected. Their expression patterns were analysed in 78 BCC and 15 normal skin samples using semiquantitative reverse transcription-polymerase chain reaction. Pathological and clinical characteristics were determined using appropriate methods. RESULTS: SPATA19, TEX101, ODF1, ODF2 and ODF3 were expressed in 56.6%, 38.2%, 2.6%, 17.4% and 2.6% of BCCs but not in normal skin samples. ODF4 and PASD1 were not expressed in any BCC samples. TEX101 and SPATA19 expression in high-risk BCCs was higher than in low-risk tumours (P < 0.001). SPATA19 expression was correlated with a history of cancer radiotherapy (P < 0.001). Significant associations were found between expression of TEX101 with nodular subtype, ODF2 with infiltrating subtype, and ODF1 with tumours located on the neck. Among gene expressors, 42.1% co-expressed two genes and 5.3% co-expressed three genes. CONCLUSIONS: We report five new CT antigens, of which SPATA19 and TEX101 may be possible targets for cancer immunotherapy and novel markers for early detection of BCC.

Association of TGIFLX/Y mRNA expression with azoospermia in infertile men.

HOX genes are well-known to encode transcriptional regulatory proteins that play essential roles in directing embryonic development. TGIFLX/Y contains two genes, TGIFLX (X-linked) and TGIFLY (Y-linked), which are specifically expressed in human adult testes. The function(s) of these genes in normal and abnormal development are unknown. To investigate the potential role(s) of the TGIFLX/Y gene in infertile males, a nested reverse transcriptase polymerase chain reaction (RT-PCR) was performed on testicular samples from 110 patients with nonobstructive azoospermia. Although the only 51 (46.4%) of the 110 patients had detectable levels of TGIFLY expression, none of the patients with various spermatogenesis defects showed any of the TGIFLX gene expression found in normal testes. These results suggest that the function of TGIFLX may be required for the regulation of spermatogonial stem cell specification and proliferation. While functional similarity has been demonstrated among some homeobox genes, these results may refute the suggestion of redundancy between TGIFLX and TGIFLY. Furthermore, TGIFLX might be a potential biomarker candidate for male infertility assessment.

Statistical analysis of six STR loci located in MHC region in Iranian population for preimplantation genetic diagnosis.

We studied allele frequency and other statistical parameters of six short-tandem repeat (STR) loci: D6S265, D6S439, D6S105, D6S82-1, MICA, and MOGd, which are located in major histocompatibility complex (MHC) region (6p21.3) in 101 Iranian individuals. STR polymorphisms were analysed by PCR and polyacrylamide gel electrophoresis and subsequent comparison with standard (allelic) ladders. Exact tests of Hardy-Weinberg equilibrium were performed for the six loci. All loci, except the MOGd (P = 0.4291), were in Hardy-Weinberg equilibrium. These data may be useful in PGD-HLA matching and forensic science.

Identification and characterisation of a novel gene, TSGA10, expressed in testis.

We describe the isolation of a novel gene, TSGA10, by differential mRNA display which is expressed solely in adult human testis. It seems likely that the gene is expressed during spermatogenesis possibly in spermatocytes. The gene is composed of 19 exons extending over more than 80 kb. The complete cDNA contains an open reading frame of 2094 nucleotides, which appears to encode a novel protein. It has been mapped by polymerase chain reaction on a panel of somatic cell hybrids and by fluorescence in situ hybridization to chromosome 2q11.2.

Cloning, characterization, and mapping of the gene encoding the human G protein gamma 2 subunit.

G proteins play vital roles in cellular responses to external signals. The specificity of G protein-receptor interaction is mediated mostly by the gamma-subunit and the individual members of the gamma-subunit multigene family would hence be expected to each have a particular expression profile. In an experiment designed to isolate genes expressed predominantly in human testis we identified a cDNA fragment corresponding to the gamma2 gene. Although the protein sequence of the gamma2 subunit has previously been published, the cDNA sequence, expression pattern, genomic structure, and localisation of the human GNG2 gene have not been described. We report the complete sequence of the GNG2 cDNA which is 1066 bp long and contains an open reading frame encoding a protein of 71 amino acids. This protein is 100% homologous to the bovine, mouse, and rat G protein gamma2 subunit. The gene structure is very similar to that of other Ggamma-subunit genes in that there are two introns, one located in the 5' UTR and the other within the ORF. We show that this gene is expressed in a range of foetal tissues as well as adult testis, adrenal gland, brain, white blood cells and lung but not in adult liver, muscle, sperm, prostate gland nor in the testes of two different infertile patients. There is evidence that GNG2 is expressed in malignant tissues. Using two independent methods, we have mapped the human GNG2 gene to chromosome 14q21.