The involvement of translationally controlled tumor protein during lamb rumen epithelium development.

Early weaning is usually applied to improve the reproductive efficiency of sheep in mutton production, while the development of rumen is of vital importance for sheep weaning age. Translationally controlled tumor protein (TCTP) is a highly conserved protein which participates in multiple tissue and organ development. Thus, we hypothesized that TCTP was involved in sheep rumen development. Histological analyses of sheep rumen epithelium showed that the epithelium formed tough shaped papillae without growing from birth to day 15 of age, after which it rapidly developed to functional epithelia on day 45 of age. We then found TCTP expressed in stratum basale, stratum spinosum and stratum granulosum of rumen epithelium. TCTP protein expression remained at a relative low level from day 0 to day 15 of age, it then significantly increased on day 30 (p < 0.05) and gradually decreased until day 60. Furthermore, to explore the role of TCTP in sheep rumen and its regulation, we found the ratio of Ki67 positive cell in stratum basale cells followed the similar pattern as the expression of TCTP. We also found the ratio of acetate:propionate in rumen fluid decreased from day 30 to day 60 of age (p < 0.05). To conclude, our data indicated that TCTP participated in rumen papillae growth by promoting rumen stratum basale cell proliferation.

MicroRNA-376b-3p targets RGS1 mRNA to inhibit proliferation, metastasis, and apoptosis in osteosarcoma.

BACKGROUND: To investigate the role of microRNA-376b-3p (miR-376b-3p) and regulator of G protein signaling 1 (RGS1) in the proliferation, metastasis, and apoptosis of osteosarcoma. METHODS: Differentially expressed genes (DEGs) between tumor and normal tissues from GSE14359 and GSE33382 in the cancer genome atlas (TCGA) dataset were analyzed with GEO2R online. Similarly, differentially expressed miRNAs from GSE70367 were also analyzed with GEO2R. The interaction between the differentially expressed miRNAs and the shared distal metastasis-related DEGs from the two datasets were analyzed using miRWalk and Cytoscape. RGS1 and miR-376-3p were chosen to verify the prediction. RGS1 stably expressing and silencing cells were established based on the MG63 and U2OS cell lines. The targeting of RGS1 with miR-376b-3p was confirmed with Starbase prediction and luciferase reporter assay. Cell proliferation, metastasis, and apoptosis were characterized in vitro and in xenograft mice. RESULTS: A total of 10 up-regulated and 8 down-regulated DEGs were characterized as shared metastasis-related DEGs for GSE14359 and GSE33382. Among these DEGs, RGS1 was targeted with miR-376b-3p, a predicted down-regulated miRNA in GSE70367. High expression of RGS1 predicted proliferation, invasion, metastases, and poor prognosis in osteosarcoma. Overexpression of RGS1 promoted proliferation, invasion, mobility, and stemness in MG63 and U2OS cells, while silencing of RGS1 had the opposite effect in both cell lines. High expression of RGS1 promoted tumor growth in xenograft nude mice. RGS1 was targeted with miR-376b-3p; the addition of miR-376b-3p down-regulated RGS1, and suppressed cell proliferation, invasion, and metastasis. Meanwhile, sponging of miR-376b-3p had the opposite effect. The suppressive effects of miR-376b-3p could be abolished with RGS1, as cell proliferation, stemness, metastasis, and invasion were all promoted with RGS1 co-transfection in both cell lines. CONCLUSIONS: Our study indicated that RGS1 is a tumor-promoting gene in osteosarcoma, which could be inhibited with miR-376b-3p.

LncRNA-SARCC sensitizes osteosarcoma to cisplatin through the miR-143-mediated glycolysis inhibition by targeting Hexokinase 2.

Chemotherapy is one of the primary treatments used against cancer. Cisplatin is a conventional chemotherapy drug used to treat osteosarcoma; however, due to the development of cisplatin resistance, advantageous therapeutic outcomes and prognosis of osteosarcoma remain low. Thus, investigation of the specific targeted therapies to circumvent the anti-chemoresistance of osteosarcoma depends on understanding the molecular mechanisms underlying cisplatin resistance. Tumor cells display an increased utilization of glycolysis rather than oxidative phosphorylation. This phenomenon is called the "Warburg effect," which presents a survival advantage for tumor cells, leading to chemoresistance. To date, the molecular mechanism underlying osteosarcoma cisplatin resistance remains to be fully elucidated. In this study, we reported the significant down-regulation of the long noncoding RNA-Suppressing Androgen Receptor in Renal Cell Carcinoma (lncRNA-SARCC) in the cells of osteosarcoma and in the specimens from osteosarcoma patients. Moreover, we observed a negative correlation between the lncRNA-SARCC and cisplatin resistance in the osteosarcoma tissues. Overexpression of the lncRNA-SARCC sensitizes osteosarcoma cells to cisplatin. From microarray analysis, we screened several miRNAs, which are significantly regulated by the lncRNA-SARCC in osteosarcoma cells, and revealed that lncRNA-SARCC promoted microRNA-43 (miR-143) expression in osteosarcoma. Interestingly, miR-143 showed the same expression pattern with the lncRNA-SARCC in osteosarcoma patient specimens. By establishing a cisplatin-resistant cell line from Sarcoma Osteogenic-2 (Saos-2), we found the cisplatin-resistant cells with down-regulated expressions of the lncRNA-SARCC and miR-143, but with a higher glycolysis rate compared to that in parental cells. We identified the glycolysis key enzyme, Hexokinase 2 (HK2), as a direct target for miR-143 in osteosarcoma. Restoration of the HK2 expression in the lncRNA-SARCC-overexpressing osteosarcoma cells reversed cisplatin resistance, suggesting that lncRNA-SARCC-mediated cisplatin sensitivity may be via glycolysis in the miR-143-inhibited osteosarcoma cells. Finally, results from both in vitro and in vivo xenograft models demonstrated that the lncRNA-SARCC was an effective therapeutic agent for overcoming cisplatin resistance in osteosarcoma. Our findings suggest an essential axis of the lncRNA-SARCC-miR-143-HK2 in regulation of osteosarcoma chemosensitivity, presenting the lncRNA-SARCC as a new therapeutic target against cisplatin-resistant osteosarcoma.

LncRNA-XIST promotes the oxidative stress-induced migration, invasion, and epithelial-to-mesenchymal transition of osteosarcoma cancer cells through miR-153-SNAI1 axis.

Osteosarcoma (OS) is the most common type of primary bone tumor that exhibits invasive growth and long-distance organ metastasis. Thus, investigating the specifically targeted therapeutic agents against metastatic osteosarcoma depends on understanding the molecular mechanisms. The long noncoding RNAs (lncRNA) XIST (X-inactive specific transcript) has been reported to have oncogenic roles in various malignant tumors including OS. However, its molecular mechanisms in OS migration and invasion are still under investigation. In the current study, we demonstrate that XIST is significantly upregulated in 30 pairs of OS tissues compared with their matched adjacent nontumor tissues by the quantitative reverse transcription polymerase chain reaction. Overexpression of XIST significantly induced the invasion, migration, and the epithelial-to-mesenchymal transition (EMT) phenotype. The epithelial marker, E-cadherin was effectively suppressed by XIST overexpression. On the other way, the mesenchymal marker, Fibronectin, Snail, and Vimentin were significantly activated by exogenous XIST overexpression. Furthermore, we observed XIST was upregulated by the oxidative stress-induced EMT. Bioinformatical analysis indicated that miR-153 has multiple biding sites for XIST and miR-153 was inversely suppressed by oxidative stress. XIST was verified to directly downregulate miR-153 via sponging. We identified the mesenchymal marker, SNAI1 was a direct messenger RNA target of miR-153. Importantly, inhibiting XIST successfully blocked the H2 O2 -induced EMT of OS cells. In conclusion, this work demonstrates that lncRNA-XIST promotes the oxidative stress-induced OS cell invasion, migration, and EMT through the miR-153/SNAI1 pathway, presenting lncRNA-XIST as a promising therapeutic target for treating metastatic OS.

MAD2 Combined with Mitotic Spindle Apparatus (MSA) and Anticentromere Antibody (ACA) for Diagnosis of Small Cell Lung Cancer (SCLC).

BACKGROUND MAD2 is the gene controlling mitosis. Many studies have assessed MAD2 in various types of carcinoma. Antinuclear mitotic spindle apparatus antibody (MSA) and anticentromere antibody (ACA) are related mitotic antibodies, playing roles in autoimmune diseases and carcinomas, but the expression of MAD2, MSA, and ACA in SCLC is unclear. MATERIAL AND METHODS We enrolled 70 SCLC patients, 72 non-small cell lung cancer (NSCLC) patients, and 65 pulmonary nodule (PN) patients. MAD2 expression was measured through agarose electrophoresis and qt-PCR. Antinuclear mitotic spindle apparatus antibody (MSA) and anticentromere antibody (ACA) were detected by indirect immunofluorescence (IIF). RESULTS MAD2 was found both in SCLC and NSCLC. Interestingly, there was a significant difference found between SCLC and NSCLC using qt-PCR (P<0.05). The area under the ROC curve of MAD2 expression was 0.799, with medium diagnostic value. MAD2 expression was related to age, lymphatic metastasis, and survival time, but not with sex. The positivity for MSA and ACA by IIF assay were 37.20% and 34.00%, respectively, in the SCLC group, which were higher than in the NSCLC and pulmonary nodule groups (P<0.05). The kappa values of MSA and ACA with MAD2 expression were 0.73 and 0.65, respectively, with moderate consistency. Combining MAD2 with MSA and ACA enhanced the sensitivity and specificity for diagnosing SCLC. CONCLUSIONS MAD2 expression was found to be involved in carcinogenesis and prognosis of SCLC. The combination of MAD2 with MSA and ACA is useful for early diagnosis and shows promise in treatment of SCLC.

The clinical significance of anti-mitotic spindle apparatus antibody (MSA) and anti-centromere antibody (ACA) detected in patients with small cell lung cancer (SCLC).

PURPOSE: The project is aimed to detect anti-mitotic spindle apparatus antibody (MSA) and anti-centromere antibody (ACA) and explore the clinical value for the diagnosis of small cell lung cancer (SCLC), providing clinical evidence for molecular studies of SCLC. METHODS: 93 SCLC patients, 208 patients with other cancers and 50 healthy controls were enrolled in this study. MSA antibodies were detected by enzyme linked immunosorbent assay (ELISA). MSA, ACA and anti nuclear antibodies (ANA) were examined by indirect immuno-fluorescence (IIF). And the results were retrospectively analyzed. RESULTS: ① the positivity for MSA and ACA by IIF assay was respectively 36.56% and 30.11% in SCLC group, higher than in other tumor groups (P<0.01), ② in correlative analysis, the RR (Relative Ratio) value between MSA and SCLC was as high as 12.93, 12.74, and the RR value of ACA and ANA with SCLC was respectively 4.31 and 3.48. ③ the area under ROC (Receiver operating characteristic) curve (AUC) of MSA detection for SCLC was 0.778, with medium diagnostic value. CONCLUSION: MSA and ACA might serve as a new marker for SCLC because of its high detection rate. These two markers may participate in the occurrence and development of SCLC, resulting from the highly strong risk. So, the study have some application value for early detection, clinical diagnosis and potential treatments of SCLC.

Comparison of biomarkers between PLA2RAb+ and PLA2RAb- in patients with idiopathic membranous nephropathy.

OBJECTIVE: The presence of antibodies against the M-type phospholipase A2 receptor (PLA2RAb) is considered to be a promising serological diagnostic biomarker of idiopathic membranous nephropathy (IMN). We compare the change in serum Cystain C (CysC), urea, creatinine (CREA), uric acid (UA), total protein (TP), albumin (ALB), IgG4 and 24-h urinary protein (proteinuria, PRO) between PLA2RAb+ and PLA2RAb- of IMN patients. MATERIALS AND METHODS: The serum and urine samples were collected from 120 patients with IMN. The presence of circulating PLA2RAb was determined by indirect immunofluorescence, and their titer was quantified by ELISA. CysC, urea, CREA, UA, TP, ALB and 24-h PRO were examined by automatic biochemical analyzer. RESULTS: Serum IgG4 level was determined by specific protein analyzer. PLA2RAb-positive percentage by ELISA was higher than that by IIF, but no significant difference was found by McNemar's Chi-square test. Serum IgG4 level and 24-h PRO level were significantly higher in PLA2RAb+ than in PLA2RAb- (P < 0.05). In PLA2RAb+ group, PLA2RAb is positively related to serum IgG4 and 24-h PRO and negatively related to serum TP and ALB (P < 0.01). CONCLUSION: This is the first study to show that combined detection of IgG4 concentration and PLA2RAb was usable for IMN patients.

Validation study of urinary metabolites as potential biomarkers for prostate cancer detection.

BACKGROUND: Urinary metabolomic profiles have recently drawn a lot of attention owing to a debate regarding their possible role as potential clinical markers for prostate cancer. In this study, levels of proline, kynurenine, uracil and glycerol-3-phosphate in 126 patients with genitourinary malignancies were analyzed using a validated method and compared with no evidence of malignancy. RESULTS: The statistical results showed that these biomarkers cannot differentiate prostate cancer from no evidence of malignancy or from other related cancer types, such as bladder cancer. In addition, there was no significant difference in biomarker levels for T1 stages, T2 stages and Gleason scores <7, ≥7. From the correlation study, results showed/demonstrated that age or serum prostate-specific antigen levels do not influence these metabolite concentrations in urine. However, the strong correlation between these metabolites and urinary creatinine concentrations implies that their occurrence is mainly due to renal excretion. CONCLUSION: This detailed study shows that the aforementioned urinary metabolites are not reliable biomarkers for prostate cancer detection or for differentiating the aggressiveness of prostate cancer.

Quantitative determination of sarcosine and related compounds in urinary samples by liquid chromatography with tandem mass spectrometry.

The current prostate cancer (PCa) diagnosis, based on the blood prostate-specific antigen (PSA) level measurement, is not a precise science. The widely used PSA biomarker for PCa has poor sensitivity and specificity and often leads to false-negative and false-positive test results. Recently, sarcosine, proline, kynurenine, uracil, and glycerol 3-phosphate were found in higher concentrations in metastatic prostate cancer urine samples. By measuring all five of these metabolites, doctors may be better able to diagnose prostate cancer with high accuracy. However, there is no method reported for simultaneous detection of these compounds in urine samples. In this study, a novel method was developed to separate and quantify six urinary metabolites including creatinine in urine samples by using liquid chromatography/tandem mass spectrometry. Chromatographic separations of the analytes were carried out using a phenyl-hexyl column with 0.1% formic acid in water and acetonitrile, respectively, under a gradient program. The six metabolites were detected in the multiple reaction monitoring modes with the ESI-positive mode. The linear range of the analytes was from 0.003 to 40 µmol/L. The limit of detection was from 0.05 to 4 nmol/L, and the limit of quantification ranged from 3 to 20 nmol/L. The factors affecting the separation and quantification of the six metabolites, such as mobile-phase and MS conditions, were also investigated. The technique developed in this study is simple, fast, sensitive, and selective. It can be used for quantifying these six metabolites in urine samples for potential early cancer screening.

A fast capillary electrophoresis method for separation and quantification of modified nucleosides in urinary samples.

Modified nucleosides are formed at the post-transcriptional stage by chemical modification of normal nucleosides within the ribonucleic acid (RNA). These modified nucleosides cannot be reutilized or further degraded, but they are excreted in the urine as intact molecules. The elevated levels of modified nucleosides in the urine samples have served as potential cancer biomarkers in many studies. Although different analytical techniques have been reported for determining nucleosides levels, they are practically difficult to use as a routine tool for early cancer screening. In this paper, a novel method was developed to separate and quantify 10 nucleosides--adenosine, cytidine, guanosine, uridine, inosine, xanthosine, pseudouridine, N(2)-methylguanosine, 1-methyladenosine, and N(2),N(2)-dimethylguanosine--in urine samples using capillary electrophoresis with an ultraviolet (UV) detector (abbreviated as CE-UV) at a wavelength of 254 nm. A 50 microm (i.d.) x 38 cm (effective length) fused silica capillary was used for the separation, and a borate-phosphate buffer containing 25 mM cetyltrimethylammonium bromide (CTAB) at pH 9.50 was used as a background electrolyte. The separation was performed at 15 kV under reverse polarity and completed within 10 min. The linear range of the analytes was 5.0-500 micromol/L, and the limit of detection was <2.0 micromol/L. The effects of pH, buffer concentrations, CTAB concentration, and the operation voltages on the separation and quantification of the modified nucleosides were also investigated. The technique developed in this study is much simpler and faster, compared to previous studies, and can be used to quantify modified nucleosides in urine samples.

[Treatment of multilevel cervical intervertebral disc protrusion using titanium mesh cage with self-locked titanium plate].

OBJECTIVE: To observe the effect of titanium mesh cage with self-locked titanium plate on the cervical inter-body fusion after anterior discectomy of multilevel cervical intervertebral disc protrusion. METHODS: The therapeutic effect of 18 patients who received the treatment of titanium mesh cage with self-locked titanium plate from September 2004 to June 2007 were retrospectively analyzed, including 11 males and 7 females (aged 32-75 years, 54 years on average). The course of disease was 6 months to 15 years (5.8 years on average). Sixteen patients suffered sensory dysfunction in limbs. Concerning the muscle strength of extremities, 3 cases were on the second level, 12 were on the third level, 1 was on the fourth level, and 2 were on the fifth level. Sixteen cases had pyramidal sign, 13 had retention of urine and feces, 8 had sexual dysfunction, and 7 combined with symptoms of nerve root type. Twelve cases were consecutive multilevel and 6 cases were discontinuous multilevel. The mean JOA scores were 8.30 +/- 1.21 preoperatively. X-ray films and MRI showed degeneration and protrusion of intervertebral disc and compression of cervical cord preoperatively. The height of involved intervertebral space was (6.40 +/- 0.87) mm on X-ray films preoperatively. RESULTS: All the incisions were healed by first intention. All the patients were followed up for 6-36 months (18 months on average). The recovery of extremities motor function was better than that of sensory function at the early stage after operation. Muscular tension decreased after operation and mobility of limbs was improved. The operated segments were instantly stable and solid fusion was observed at 3-6 months after operation. JOA scores at 6 months after operation was 12.60 +/- 0.78 with an improvement rate of 51.8%, indicating significant difference was evident when compared with the JOA scores before operation (P < 0.05). The height of involved intervertebral space increased significantly to (8.20 +/- 0.46) mm postoperatively, indicating the difference was significant (P < 0.05). According to Odom's evaluation scale, 17 patients were graded as excellent and 1 as good. No death and complications of spinal cord, nerve, trachea and esophagus were observed. The cage witnessed no translocation. CONCLUSION: Titanium mesh cage with self-locked titanium plate is more stable and effective for the treatment of multilevel cervical intervertebral disc protrusion.

Molecular mechanisms of cyclic nucleotide-gated ion channel gating.

Cyclic nucleotide-gated ion channels (CNGs) are distributed most widely in the neuronal cell. Great progress has been made in molecular mechanisms of CNG channel gating in the recent years. Results of many experiments have indicated that the stoichiometry and assembly of CNG channels affect their property and gating. Experiments of CNG mutants and analyses of cysteine accessibilities show that cyclic nucleotide-binding domains (CNBD) bind cyclic nucleotides and subsequently conformational changes occurred followed by the concerted or cooperative conformational change of all four subunits during CNG gating. In order to provide theoretical assistances for further investigation on CNG channels, especially regarding the disease pathogenesis of ion channels, this paper reviews the latest progress on mechanisms of CNG channels, functions of subunits, processes of subunit assembly, and conformational changes of subunit regions during gating.

Changes of cyclic AMP levels and phosphodiesterase activities in the rat ovary.

Cyclic AMP (cAMP) is a second messenger that plays a critical role in follicular recruitment, development and luteinization in the mammalian ovary. The cellular level of cAMP is largely dependent on the activity of phosphodiesterase (PDE), which degrades cAMP into 5'-AMP. The present study was conducted to investigate the level of cAMP and the activity of cAMP-PDE in postnatal rats; immature rats during gonadotropin-primed follicular development, ovulation and luteinization; adult rats during normal estrous cycling; and aged rats that spontaneously developed persistent estrous (PE) by radioimmunoassay (RIA). All four rat models were confirmed by histological examination of one ovary and assayed using the other ovary by RIA. In the postnatal rats, the ovarian cAMP level was high on day 10 after birth, while ovarian cAMP-PDE activity was highest at 21 days of age. In the immature female rats, both the ovarian cAMP level and cAMP-PDE activity increased remarkably after treatment with equine chorionic gonadotropin (eCG), increased continuously 24 h after injection of human chorionic gonadotropin (hCG) for induction of ovulation and luteinization, and then declined significantly. In the adult rats during the normal estrous cycle, the ovarian cAMP levels were low on the day of estrus, and there were no significant changes in ovarian cAMP-PDE activity throughout the estrous cycle. In the PE rats, the ovarian cAMP levels were similar to those of the adult rats on the day of estrus but were lower than those on the other days of the estrous cycle; ovarian cAMP-PDE activity was lower than that in the adult rats on any day of the estrous cycle. Together, these findings indicate that the ovarian cAMP level and cAMP-PDE activity were regulated in a stage-dependent manner during ovarian follicular development, atresia and luteinization and providing evidences that cAMP and cAMP-specific PDEs are involved in these physiological processes.

Regulation of FoxO1 transcription factor by nitric oxide and cyclic GMP in cultured rat granulosa cells.

FoxO1 is a transcription factor implicated in a multitude of physiological processes including cell cycle progression, apoptosis and insulin signaling. Recent findings indicate that FoxO1 is a key regulator during the proliferation and maturation of granulosa cells. Over the past several years, it has become evident that nitric oxide (NO) and cGMP modulate ovarian function. There has been no information, however, about whether NO-cGMP affects FoxO1 expression or about the relationship between NO-cGMP and FoxO1. In the present study, we used immunoblot analysis to determine whether NO and cGMP affect FoxO1 expression in cultured granulosa cells. Our results clearly showed that FSH suppressed FoxO1 expression in a time-dependent manner, and that NO-cGMP stimulated FoxO1 expression in cultured granulosa cells. In addition, this stimulatory effects of NO and cGMP can be blocked by FSH in cultured granulosa cells. These findings demonstrate that NO and cGMP influence FoxO1 expression possibly through antagonizing the action of FSH in cultured granulosa cells. Results of both immunoblot analysis and immunohistochemistry also show that estradiol implantation do not affect the expression of FoxO1 in rat granulosa cells as gonadotrophins do, indicating that mechanism of estradiol on granulosa cells is different from gonadotrophins. Together, our experiments suggest that expression of FoxO1 in rat granulosa cells can be regulated by gonadotrophins and the NO/cGMP signaling pathway.