HPT axis­independent TSHß splice variant regulates the synthesis of thyroid hormone in mice.

Thyroid stimulating hormone (TSH) consists of an α­subunit and a unique ß­subunit. The first in­frame TSHß splice variant produced by the cells of immune system was identified in 2009. The TSHß splice variant and native TSHß exhibit different expression profiles, and research has been conducted to elucidate the role of the TSHß splice variant in different diseases. However, understanding of the fundamental physiological characteristics of the TSHß splice variant is currently limited. To verify whether the TSHß splice variant has the potential to induce thyroid follicular cells to synthesize thyroid hormone, in vivo and in vitro stimulation experiments were conducted in the present study. A total of 60 C57BL/6 mice were divided into control­, 5 and 10 µg TSHß splice variant­treated groups at random. Mice were sacrificed at 0.5, 1 and 4 h after intraperitoneal injection, and serum levels of tri­iodothyronine (T3) and thyroxine (T4) were determined using a radioimmunoassay. Thyroid follicular cells were isolated from the thyroids of mice, and stimulated with 2 µg/ml TSHß splice variant. Supernatants were collected, and the levels of T3 and T4 were detected. The protein expression levels of the sodium­iodide symporter, thyroperoxidase and thyroglobulin in thyroid follicular cells were quantified using western blot analysis. To verify whether the TSHß splice variant expression was regulated by the hypothalamus­pituitary­thyroid (HPT) axis, similar to native TSHß, a total of 60 C57BL/6 mice were equally divided into control, 2 mg/kg T3 intraperitoneal injection and 0.05 mg/kg thyroid­releasing hormone intraperitoneal injection groups at random. Mice were sacrificed at 1 and 4 h after injection. Alterations in the expression of the TSHß splice variant in the pituitary, thyroid, peripheral blood leukocytes and spleen tissues were detected using western blot analysis. The present study demonstrated that the TSHß splice variant is not regulated by the HPT axis and may affect thyroid hormone synthesis. Modifications in the expression of the TSHß splice variant may occur in a uniquely regulated manner to provide peripheral immunological compartments with a source of activated cells, particularly under immune stress.

miR-22-Notch Signaling Pathway Is Involved in the Regulation of the Apoptosis and Autophagy in Human Ovarian Cancer Cells.

microRNA-22 (miR-22) is a brain-enriched regulatory gene which has been reported to be involved in the development of cancers. The Notch signaling pathway exerts important functions in cell growth. This study is designed to investigate the mechanisms of miR-22-Notch signaling pathway in apoptosis and autophagy of human ovarian cancer cells. After over-expressing miR-22 in human ovarian cancer cell lines OVCAR-3 and SKOV3, cell viability is determined by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) method, cell apoptosis is observed by Flow cytometry (FCM), mRNA expression of miR-22 is measured by RNA preparation and RT-PCR, protein expression of Notch1, Hes1, Beclin1 and LC3B-II is analyzed by Western blot. It is suggested that miR-22 expression is heavily decreased in human ovarian cancer cell lines OVCAR-3 and SKOV3. Over-expression of miR-22 potently suppresses cell viability and authophagy while promotes the percentage of apoptotic cancer cells. In addition, the decreased expression level of Notch1 and its targeted gene is detected in miR-22-over-expressed cells. Moreover, followed by the block of the Notch signaling pathway using Notch1 small interference RNA (siRNA), the effects of miR-22 on the apoptosis and autophagy of human ovarian cancer cell lines OVCAR-3 and SKOV3 are obviously blocked. Together, miR-22 inhibits apoptosis and promotes autophagy of human ovarian cancer cells through the suppression of the Notch signaling pathway, indicating a potential use of miR-22 in the ovarian cancer treatment.

Functional human TSHß splice variant produced by plasma cell may be involved in the immunologic injury of thyroid in the patient with Hashimoto's thyroiditis.

Hashimoto's Thyroiditis (HT) is the most common cause of hypothyroidism in areas of the world where iodine levels are sufficient. However, the pathogenesis of HT has not been completely elucidated. The first functional human TSHß splice variant was supposed to be involved in the pathology of Hashimoto's thyroiditis. The question remains as to which kind of intrathyroid cells expresses functional TSHß splice variant and whether there are expression variations of functional TSHß splice variant in the injured thyroid of patient with HT. For the answer to this question, immune-injured thyroids were obtained from 30 patients with HT. Localization study of functional TSHß splice variant in injured thyroid was done by immunofluorescence double staining. Transcription and translation level of functional TSHß splice variant were detected by using qRT-PCR and semi-quantitative immunohistochemistry method, respectively. The correlation between expression level of functional TSHß splice variant and degree of thyroid follicles damage was assessed. It was firstly identified that functional TSHß splice variant was predominately expressed by plasma cells infiltrated around follicles and germinal center in injured thyroid of patient with HT. Of particular interest, the TSHß splice variant was expressed at significantly higher levels in the thyroid tissues of patients with HT than that in the normal thyroid tissues, furthermore, expression level of TSHß splice variant was positive related with the degree of follicles damage in thyroid of patient with HT. These findings defined the immune-derived functional TSHß splice variant that resided in the thyroid of patient with HT, which exerted the unique effects on the pathogenesis of HT, meanwhile, we considered these findings to have significant implications for understanding immune-endocrine interactions in a number of ways.

[Study on reversion of malignant phenotype of glioma by siRNA targeting p75 neurotrophin receptor].

OBJECTIVE: To study the therapeutic efficacy of siRNA fragments silencing p75 neurotrophin receptor (p75(NTR)), which may be a key regulator of glioma cell apoptosis and invasion. METHODS: The siRNA sequence fragments targeting p75(NTR) were designed and transferred into human glioma cell line U251. RT-PCR and immunocytochemistry method were used to explore the expression of p75(NTR) mRNA and protein. Cell adhesion assay was employed to detect cellular adhesion ability, and soft agar clone formation assay was adopted to identify oncogenicity, and a U251 glioma model was established in nude mice. The intracranial tumor volume was detected by MRI. The expression of p75(NTR), NGF and cyclin D2 were identified using immunohistochemistry. Cell apoptosis was detected by apoptosis kit in situ. RESULTS: The siRNA fragments targeting p75(NTR) were capable of decreasing mRNA and protein expression of p75(NTR) in U251 glioma cell line. Both the cellular adhesion ability and oncogenicity were weakly relevant. The p75(NTR) expression level was negatively correlated with cyclin D2 and apoptosis, and positively correlated with NGF expression. The siRNA sequence fragments targeting p75(NTR) were effective in decreasing the gross volume of tumor; prolonged the survival time of mice, and the edge of tumor was much sharper than that of the control group. CONCLUSIONS: The gene silencing technique by siRNA targeting p75(NTR) is capable of decreasing tumor invasion and cell proliferation as well as inducing cell apoptosis. It is expected to be a new choice for glioma gene therapy.

[The study in primary cultured astrocytes following fluid percussion injury].

OBJECTIVE: To investigate the morphology alterations and proteomics changes in primary astrocytes following fluid percussion injury. METHODS: Primary cultures of astrocytes were prepared from cerebral hemispheres of 1-3 d-old SD rats, then, astrocytes were randomly divided into control group and injury group which were subjected to (0.2 +/- 0.01) MPa fluid percussion injury. The changes of protein expression pattern in astrocytes between injury and control groups were monitored with two dimensional gel electrophoresis. RESULTS: Astrocytes' s abnonmalities of morphology after injury were apparent. The fluid percussion injury caused astrocytes edema, shrinkage, cell junction disconnection and necrosis at 2 h after injury. 24 h and 48 h after injury, most part of astrocytes's dendrites and soma became hypertrophy and showed a higher rate of cell proliferation. The dynamic proteomics changes were identified and total different 13 spots were detected in this study from the 2DE gels. The different displayed 5 spots were identified via MALDI-TOF: cofilin 1, destrin, phosphoglycerate mutase 1, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10, annexin 1. CONCLUSION: The obvious alteration of morphology and protein expression pattern in primary cultured astrocytes could be induced after fluid percussion injury. The differential proteins detected were probably related to stress responses.

[Differential proteomics in glioblastoma].

OBJECTIVE: To establish differential proteomics profiles of glioblastoma cell lines from Chinese, and to provide reference for future basic studies. METHODS: Total protein was extracted from 3 glioblastoma cell lines, CHG-5, TJ899 and TJ905. After normalization, the total protein was presented by two-dimensional (2D) electrophoresis, scanned and analyzed. Some of the identified protein spots were verified by immunocytochemistry of cell lines and immunohistochemistry of solid tumors. The glia cells were used as the control throughout the study. RESULTS: A total of 13 differential protein spots were selected, and eventually 10 were identified as unique proteins. These 10 proteins were involved in cytoskeleton forming, cellular metabolism, tumor migration, stress and inflammatory reaction. Immunocytochemistry and immunohistochemistry further confirmed these proteins present in the solid tumors. CONCLUSION: Distinct differential proteomics profiles exist in glioblastoma cell lines and normal glia cells, likely related to the transformation of normal glia to glioma.