miR-544a Stimulates endometrial carcinoma growth via targeted inhibition of reversion-inducing cysteine-rich protein with Kazal motifs.

Endometrial carcinoma (EC) is a female-specific malignant tumor. Although current treatments can achieve good outcomes and improve patient survival, there remains a high incidence of treatment-induced infertility, a serious side effect that is unacceptable to those of childbearing age. Studies have demonstrated that micro ribonucleic acids (microRNAs or miRNAs) such as miR-544a regulate tumor-related gene expression. However, whether miR-544a is involved in the progression of EC is unknown. This study aimed to investigate the biological functions and underlying mechanisms of miR-544a in EC in vivo and in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed miR-544a overexpression in EC tissue and cell lines, which was associated with a decreased in overall survival as revealed by Kaplan-Meier analysis. Functionally, the miR-544a inhibitor restricted the proliferation [detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay], invasion, and migration (detected by transwell assay) of human endometrial adenocarcinoma cells (HEC-1B and Ishikawa) and facilitated cell apoptosis (detected by flow cytometry assay). Western blotting analysis revealed that the miR-544a inhibitor decreased the expressions of matrix metalloproteinase (MMP)-2 and MMP-9 and elevated the levels of cleaved caspase3 and cleaved poly (ADP-ribose) polymerase. Furthermore, animal experiments indicated that the miR-544a antagonist (antagomir-544a) suppressed tumor growth significantly in a mouse xenograft model. The mechanistic, qRT-PCR, and immunohistochemical indications were that a reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and miR-544a had inverse expression changes in EC. Bioinformatics analysis revealed RECK as a potential target for miR-544a, and this was verified by the dual-luciferase reporter assay. Subsequently, in vitro experiments, including transwell assay, MTT assay, flow cytometry assay, and Western blotting analysis, demonstrated that RECK exerted antitumor effects on EC, which were negatively regulated by miR-544a. Taken together, our study findings suggested miR-544a as a valuable target in EC therapy.

L-Tetrahydropalmatine enhances the sensitivity of human ovarian cancer cells to cisplatin via microRNA-93/PTEN/Akt cascade.

PURPOSE: Το evaluate the effect of L-Tetrahydropalmatine (L-THP) on the sensitivity of a cisplatin resistant ovarian cancer (OC) cell line. As miR-93 is reported to be overexpressed in OC and cisplatin resistance, we also evaluated its pathway in OC. METHODS: The levels of miR-93 were evaluated using RT-PCR and Luciferase assay was performed to confirm the target of miR-93. The extent of apoptosis was evaluated by Annexin V and propidium iodide (PI) staining, whereas Hoechst 33258 staining was done for identifying the number of apoptotic cells. RESULTS: The cisplatin-resistant A2780/DDP cell line showed lower survival rate compared to control when incubated with L-THP along with cisplatin. L-THP caused G0/G1 cell cycle arrest and increased the sensitivity to cisplatin. Furthermore, we found that the levels of miR-93 in cisplatin-resistant cells were highly expressed compared to parental cells. L-THP suppressed the expression of miR-93 and increased the levels of PTEN, a crucial tumor suppressor in OC. It was further observed that the cells transfected with PTEN siRNA showed increased survival compared with the control group and this phenomenon could be reversed by the AKT inhibitor Triciribine. The A2780 cells treated with PTEN siRNA showed similar survival rate to the cells with miR-93 overexpression. CONCLUSION: The findings of this study suggested L-THP increased the sensitivity of ovarian cancer cells to cisplatin via modulating miR-93/PTEN/AKT pathway in A2780/DDP ovarian cancer cell line.

The role of Dby mRNA in early development of male mouse zygotes.

Ejaculated mammalian spermatozoa contain a complex yet specific population of mRNA. However, the possible roles that mRNA has in early zygotic and embryonic development remain unclear. We found that Dby mRNA is selectively retained in capacitated mouse spermatozoa, and is transferred into the oocyte during fertilization by reverse transcription-polymerase chain reaction even though no DBY protein expression is detected. The cellular location of Dby mRNA is seen in the post-acrosome region, and it comprises nearly half of the mouse spermatozoa in in situ hybridization. In contrast, transcripts of the control gene, Smcy, are not detected in capacitated mouse spermatozoa, although the H-Y antigen encoded by Smcy is expressed on the surface of the spermatozoa. In our microinjection experiment, the zygotic development rate of the as-Dby male pronucleus injection group was significantly lower than that of the as-Smcy male pronucleus injection group (35.9% vs. 95%, P = 0.001) and the as-Dby female pronucleus injection group (35.9% vs. 93.8%, P = 0.001). The rate of male-developed zygotes was also lower than that of the as-Smcy male pronucleus injection group (17.4% vs. 57.9%, P = 0.002) and the as-Dby female pronucleus injection group (17.4% vs. 54.1%, P = 0.002). Thus, we conclude that Dby mRNA is selectively retained in capacitated mouse spermatozoa, and it has an important role in the early zygotic development of male mouse zygotes. This might imply that spermatozoa mRNA is involved in early zygotic and embryonic stages of reproduction.

Nicotine induces cyclooxygenase-2 and prostaglandin E(2) expression in human umbilical vein endothelial cells.

Nicotine is a major component of cigarette smoking which may be involved in the progress of atherogenesis. In order to explain the mechanism of nicotine-induced endothelium dysfunction, we investigated the effects of nicotine on cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expression in human umbilical vein endothelial cells (HUVECs). Nicotine treatment increased the expressions of COX-2 at mRNA and protein level in a dose-dependent manner, following prostaglandin E(2) (PGE(2)) release enhancement. Pyrrolidine dithiocarbamate (PDTC, NF-kappaB inhibitor) and alpha-Bungarotoxin (alpha-Btx, nicotinic acetylcholine receptor antagonist) attenuated the nicotine-induced COX-2 expression and PGE(2) production. Furthermore, nicotine-induced ICAM-1 expression was reduced by NS-398 (selective COX-2 inhibitor). Taken together, the present study demonstrated that nicotine-induced COX-2 expression through NF-kappaB activation which mediated by nicotinic acetylcholine receptor and the induction of COX-2 was related to ICAM-1 expression.

[Characterization of the mRNA profile in ejaculated spermatozoa from healthy fertile men].

OBJECTIVE: To explore the complexity of mRNA in the ejaculated sperm from healthy fertile men. METHODS: Semen samples were collected from 10 healthy fathers. The swim-up method was adopted to purify the sperm from possible contamination of somatic cells and the spermatozoal total RNA extracted by Trizol was used for SAGE library analysis. RESULTS: A totle of 21 052 SAGE raw tags were sequenced from 877 clones and 2 712 unique tags that occurred at least twice in the library were given further analysis. 19.7% of the unique tags had no match in the existing SAGE map, representing novel genes. Molecular function analysis revealed 67% of unique tags related to protein binding or nucleic acid binding categories, 41% to catalytic activity, 13% to message transducer activity, and 10% to transporter, structural and transcription regulator activity, respectively. CONCLUSION: There exists a complex repertoire of mRNAs in the ejaculated spermatozoa from fertile men.

Effects of testosterone and 17-beta-estradiol on TNF-alpha-induced E-selectin and VCAM-1 expression in endothelial cells. Analysis of the underlying receptor pathways.

This study investigated the effects of testosterone and 17-beta-estradiol on tumor necrosis factor-alpha (TNF-alpha)-induced endothelial expression of E-selectin and vascular cell adhesion molecule-1 (VCAM-1) and the potential roles of hormone receptors involved in these actions. Human umbilical vein endothelial cells (HUVEC) were stimulated with TNF-alpha in the presence or absence of testosterone or 17-beta-estradiol, and the expression of E-selectin and VCAM-1 was investigated. As shown by Western blot analysis, co-administration with testosterone or 17-beta-estradiol increased the expression of E-selectin and VCAM-1 induced by TNF-alpha at 6 h and 3 h, respectively. Similarly, RT-PCR analysis revealed a significant increase in the amount of mRNA for E-selectin and VCAM-1 after co-administration with testosterone or 17-beta-estradiol in TNF-alpha-stimulated HUVEC. The presence of mRNA and proteins for androgen receptor and estrogen receptor alpha in HUVEC was verified by RT-PCR and Western blot. Flow cytometric analysis showed that preincubation with androgen receptor antagonist cyproterone and estrogen receptor antagonist tamoxifen completely abrogated the upregulating effects of testosterone and 17-beta-estradiol on TNF-alpha-induced E-selectin and VCAM-1 expression, respectively. Expression of TNF receptors in TNF-alpha-stimulated HUVEC was not influenced by testosterone and 17-beta-estradiol. The data indicate that both testosterone and 17-beta-estradiol increase TNF-alpha-induced E-selectin and VCAM-1 expression in endothelial cells via a receptor-mediated system, and expression of TNF receptors are not changed in these actions. The implications of these results for the facilitory effects of both sex hormones on immune reactions are discussed.