Screening of m6A gene-related lncRNAs in colon adenocarcinoma and construction of a prognostic prediction model.

OBJECTIVE: We aimed to screen the long non-coding RNAs (lncRNAs) related to N6-methyladenosine (m6A) gene and build the prognostic prediction model of colon adenocarcinoma (COAD). MATERIALS AND METHODS: The RNA sequencing data of 435 COAD cases with clinical survival and prognosis information and the GSE39582 dataset were obtained from TCGA and GEO, respectively. The lncRNAs related to the m6A gene with significant independent prognosis were identified. We used Cox regression analyses to acquire the lncRNAs associated with prognosis. Moreover, we built a prognostic prediction model of COAD. The Cox regression analyses were applied to obtain the independent prognostic clinical factors. Furthermore, we built the ceRNA regulation network of COAD, and the gene ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) enrichment analysis for the lncRNAs was applied. RESULTS: Overall, 5 lncRNAs (MAGI1-IT1, CSNK1G2-AS1, ALMS1-IT1, LINC01341, LOXL1-AS1) related to m6A gene with significant independent prognosis were acquired. A prognostic prediction model of COAD was built, and 4 correlation-independent prognostic factors were found. In addition, the ceRNA regulation network of COAD was built, and mRNAs were significantly enriched in the 15 GO biological processes (such as regulation of transcription) and in 14 KEGG pathways (such as taurine). CONCLUSIONS: We identified 5 lncRNAs related to the m6A gene with significant independent prognosis. The ceRNA regulation network of COAD was built, which has great significance for identifying the biomarkers associated with m6A in COAD.

Fibulin-5 protects the extracellular matrix of chondrocytes by inhibiting the Wnt/ß-catenin signaling pathway and relieves osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a common disease in the elderly and seriously affects the quality of life of patients. The purpose of this study was to explore the protective effect of Fibulin-5 on articular chondrocytes and its mechanism of action. PATIENTS AND METHODS: Articular cartilage tissues from patients with OA and normal people were selected and tested for differences in Fibulin-5 expression. In addition, human chondrocytes were cultured, and the effects of Fibulin-5 on the extracellular matrix (ECM) of chondrocytes and the level of inflammation were examined by means of cell transfection and cytokine intervention. SKL2001, an agonist of the Wnt/ß-catenin signaling pathway, was used to validate the mechanism of action of Fibulin-5 to protect chondrocytes. RESULTS: Fibulin-5 was lowly expressed in the cartilage tissue of patients with OA. Overexpression of Fibulin-5 significantly increased the expressions of ECM collagen II and aggrecan in chondrocytes, while decreasing the expressions of MMP-3 and MMP-13. In addition, Fibulin-5 reduced IL-1ß-induced inflammation of chondrocytes, as well as expressions of IL-6, IL-8, and TNF-α. Overexpression of Fibulin-5 also reduced the activity of Wnt/ß-catenin signaling pathway, and activation of Wnt/ß-catenin signaling pathway attenuated the protective effects of Fibulin-5 on the ECM of chondrocytes. CONCLUSIONS: Fibulin-5 can protect the ECM of chondrocytes and reduce the inflammatory response of chondrocytes by inhibiting the Wnt/ß-catenin signaling pathway.

[The characteristic of hereditary spherocytosis related gene mutation in 37 Chinese hereditary spherocytisis patients].

Objective: To reveal the genetic characteristics of erythrocyte membrane protein in hereditary spherocytosis (HS) in China. Methods: Next-generation sequencing technology was used to detect mutations in genes of erythrocyte membrane proteins in 51 clinically diagnosed HS patients. The relationship between gene mutations and clinical phenotypes was analyzed. Results: Mutations in erythrocyte membrane protein genes were detected in 37 patients, including 17 with ANK1 mutations (17/37, 45.9%), 14 with SPTB mutations (14/37, 37.8%), and 5 with SLC4A1 mutations (5/37, 13.5%). One patient carried both heterozygous ANK1 mutation and SPTB mutation (1/37, 2.7%). SPTA1 and EPB42 mutation was not fou nd in any patient. Nonsense mutations (36.8%) and missense mutations (31.6%) were most common. Of the 38 mutations detected, 34 were novel mutations and have not been reported elsewhere (89.5%). Sixteen HS patients underwent parental genetic validation, 6 patients (37.5%) inherited gene mutation from parents and 10 (62.5%) were de novo. The peripheral blood cell parameters of HS patients were not related to the mutant genes and gene mutation types. However, it seems that HS patients with mild clinical status are prone to carry SPTB mutations while more patients with severe clinical status have ANK1 mutations. Conclusions: ANK1 and SPTB are the most common mutant genes in Chinese HS patients, mainly with missense mutations and nonsense mutations. There was no significant correlation between the mutation of HS related genes and the severity of HS.

CD4+Foxp3+ regulatory T cell differentiation mediated by endometrial stromal cell-derived TECK promotes the growth and invasion of endometriotic lesions.

Endometriosis is associated with an abnormal immune response to endometrial cells, which can facilitate the implantation and proliferation of ectopic endometrial tissue. The proportion of CD4(+)Foxp3(+) regulatory T cells (Tregs) is significantly increased in the peritoneal fluid of women with endometriosis. The thymus-expressed chemokine TECK/CCL25 directly promotes the invasiveness of endometrial stromal cells (ESCs). The aim of this study was to investigate the effects of ESC-derived TECK on the crosstalk between Tregs and ESCs in the progress of endometriosis. We determined that the percentage of Tregs and the concentration of TECK increased in the peritoneal fluid with the progression of endometriosis. The supernatant from co-cultured human ESCs and macrophages not only induced Treg differentiation and increased Treg expression of interleukin-10 (IL-10), transforming growth factor-ß (TGF-ß) and CD73 by activating the AKT/STAT3 signaling pathway but also repressed Treg apoptosis by downregulating Fas and FasL expression and enhanced the Treg-mediated suppression of CD4(+)CD25(-) T cells. In addition, in vitro and in vivo trials confirmed that these effects could be inhibited by anti-TECK neutralizing Abs. The secretion of IL-10 and TGF-ß by Tregs increased MMP2 expression and decreased TIMP1 expression and further stimulated the proliferation and invasion of ESCs and the growth of ectopic lesions. These results indicate that TECK derived from ESCs and macrophages upregulates the number and function of Tregs in the ectopic milieu, which contributes to endometriotic immunotolerance and high levels of ESC proliferation and invasion, thereby facilitating the progression of endometriosis.

Closure of skin incision after thyroidectomy through a supraclavicular approach: a comparison between tissue adhesive and staples.

BACKGROUND: The objective of this study was to compare the effectiveness and cosmetic results of tissue adhesive or surgical staples in thyroidectomy through a supraclavicular incision. METHODS: This was a prospective, randomized study of consecutive patients undergoing thyroidectomy by a supraclavicular approach. Eligible patients were randomized into two groups: one group had the incision closed with tissue adhesive (the experimental group) and the other with surgical staples (the control group). The main outcomes included operative time, early postoperative pain measured by Visual Analog Scale, incidence of wound dehiscence and infection, perceived cosmetic outcome, and overall patient satisfaction by using Patient Satisfaction Assessment Form. RESULTS: There were 151 consecutive patients assessed for eligibility, and 132 patients were enrolled over 22 months. The clinical characteristics of the patients in the two groups were similar. Main outcomes were assessed in the first 24 h postoperatively, the first month, and the third month postoperatively. Operation time was longer in the experimental group (P = 0.027). Mean Visual Analog Scale scores for pain were lower in the experimental group in the early postoperative period (P < 0.001). No patients developed surgical site infections or wound dehiscence. Lower scores for scar assessment and higher overall satisfaction levels at the first month after surgery were found in the experimental group compared to the control group (P < 0.001). There was no significant difference between the two groups at the third month postoperatively in perceived cosmetic result (P = 0.052) or overall satisfaction (P = 0.059). CONCLUSIONS: Tissue adhesive is effective and reliable in skin closure for thyroid surgery. While this closure may take somewhat longer to perform, it leads to less postoperative pain, more acceptable wound cosmesis, and higher patient satisfaction levels in short postoperative follow-up.

RANKL promotes the growth of decidual stromal cells in an autocrine manner via CCL2/CCR2 interaction in human early pregnancy.

OBJECTIVE: Receptor-activator of NF-κB ligand (TNFSF11, also known as RANKL) and its receptor RANK are essential regulators on bone remodeling, mammary gland development and hormone-associated breast cancer development. However, the expression pattern and role of RANKL/RANK axis in decidual stromal cells (DSCs) are unclear in human early pregnancy. STUDY DESIGN: We analyzed RANKL/RANK expression in DSCs by real-time PCR, immunhistochemistry, enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively. Then BrdU cell proliferation assay, flow cytometry assay and ELISA were performed to investigate the effect of recombinant human RANKL and DSCs-derived RANKL on the proliferation, apoptosis, chemokine (C-C motif) ligand 2 (CCL2) secretion, C-C chemokine receptor type 2 (CCR2) and other target proteins expression in DSCs in vitro, respectively. RESULTS: Here we show that DSCs co-express RANKL/RANK. Not only recombinant human (rh) RANKL but also the DSC-secreted RANKL stimulate proliferation and anti-apoptosis, and elevate CCL2 secretion and CCR2 expression of DSCs. Furthermore, the stimulatory effects on the proliferation, anti-apoptosis and the expression of Bcl-2 and Ki67 and inhibitory signaling on Fas ligand (FasL) in DSCs induced by RANKL can be partly reversed by the way of blocking CCL2 and or CCR2. CONCLUSIONS: Our results have revealed that RANKL/RANK signal promotes Bcl-2 and Ki67 and decreases FasL expression, and further as a positive regulator for stimulating the proliferation and growth of DSCs through up-regulating CCL2/CCR2 signal, which finally contributes to the establishment and maintenance of physiological pregnancy.

Thymic stromal lymphopoietin promotes the proliferation of human trophoblasts via phosphorylated STAT3-mediated c-Myc upregulation.

Our previous study has demonstrated that thymic stromal lymphopoietin (TSLP) stimulates trophoblast proliferation and invasion, suggesting TSLP plays an important role in the placentation in early human pregnancy, but the intracellular molecular mechanism is currently unknown. The present study is undertaken to investigate whether the STAT3-c-Myc signaling pathway is involved in TSLP-mediated trophoblast proliferation. Primary human first-trimester trophoblasts were treated with TSLP only, or TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, and ERK). The levels of STAT3 tyrosine phosphorylation and c-Myc expression were determined by using Western blot analysis, and the proliferation of trophoblasts was analyzed by BrdU cell proliferation assay. JEG-3 cells were transfected with siRNA targeting to c-Myc, and the proliferation was determined in JEG-3 cells treated with TSLP only, or TSLP combined with c-Myc silencing. It was revealed that treatment with TSLP significantly enhanced STAT3 phosphorylation and c-Myc expression in human trophoblasts. The effect of TSLP upregulation on trophoblast proliferation was abrogated completely by either STAT3 inhibitor or c-Myc siRNA silence. We further found that the upregulation of TSLP on c-Myc expression was abrogated completely by the STAT3 inhibitor, which suggests that the intracellular STAT3 phosphorylation is an upstream signal of c-Myc expression in the TSLP-stimulated trophoblast proliferation. These results suggest that TSLP may upregulate c-Myc expression through activation of STAT3 pathway, thereby inducing trophoblast proliferation.

[Changes in plasma von Willebrand factor and nitric oxide levels in patients with pregnancy induced hypertension].

OBJECTIVE: To study the changes and clinical significance of plasma von Willebrand factor (vWF) and nitric oxide (NO) levels in patients with pregnancy induced hypertension (PIH). METHODS: Plasma vWF and NO were detected by ELISA and Griess assay respectively in 36 patients with PIH and 18 healthy pregnant women. RESULTS: The levels of plasma vWF in patients with moderate and severe PIH increased significantly. than those in healthy pregnancy. The levels of plasma NO in patients with PIH were significantly lower than those in healthy pregnancy. The higher the vWF levels or the lower the NO levels, the more severe in PIH. There was a negative correlation between the plasma vWF and NO levels. CONCLUSION: The results suggest that plasma vWF and NO concentration can be used as indicators in the judgement of the severity of PIH.

Monoclonal anti-androgen receptor antibodies: production, characterization and potential diagnostic applications.

Several monoclonal antibodies (mAbs) and novel mAb-based assays for the androgen receptors (AR) have been developed. Large amounts of the recombinant human AR protein produced by a baculovirus expression system were used as an antigen to produce mAbs. Twenty-nine AR-specific mAbs were first confirmed by Western blot analysis and were then characterized for their immunoglobulin isotypes, epitopes, and epitope localization in AR. Novel assays using flow cytometry and sandwich enzyme-linked immunosorbent assays (ELISA) were established to detect AR-expressing cells and to quantify soluble AR protein, respectively. Using immunostaining, we identified several anti-AR mAbs exclusively recognizing AR within the nuclei of the prostate cancer cell line LNCaP and of prostate tissues in both frozen and paraffin-embedded sections, whereas other mAbs could detect AR in both nuclear and cytoplasmic compartments. Interestingly, certain mAbs, such as G122-25 and G122-77, could distinguish the androgen-bound AR from the unoccupied AR. In sum, many purified AR protein and anti-AR mAbs, together with the assays developed, could be powerful tools for the study of functional AR and for the diagnosis of prostatic cancers.

The expression of prostatic acid phosphatase is transcriptionally regulated in human prostate carcinoma cells.

The expression of prostatic acid phosphatase (PAcP) in three human prostate carcinoma cell lines including LNCaP, DU 145 and PC-3, was studied to explore its potential role as a marker in the progression of prostate cancer. Although Southern blot analysis suggested the presence of PAcP gene in all three prostate carcinoma cell lines, the Northern blot analysis and the reverse transcriptase-polymerase chain reaction (RT-PCR) assay showed that PAcP mRNA can be detected only in LNCaP cells. As one of the major differences between LNCaP cells and PC-3 as well as DU 145 cells is the androgen-sensitivity of LNCaP cells, we then focused on the influence of PAcP expression by the presence of androgen receptor (AR) in human AR cDNA-transfected PC-3 cells and high passages of LNCaP cells. The results demonstrated that the transfection of human AR cDNA into PC-3 cells did not have any detectable effect on the expression of PAcP. Further, in LNCaP cells, while the level of PAcP mRNA diminished upon passage, the AR mRNA level remained approximately the same. Together, these data suggested that the differential expression of PAcP in different prostate carcinoma cells including high passages of LNCaP cells may occur at the transcriptional level and may have little linkage to the expression of AR.