The role of WNT1 mutant variant (WNT1c.677C>T ) in osteogenesis imperfecta.

Osteogenesis imperfecta (OI), also known as "brittle bone disease," is a rare inherited genetic disorder characterized by bone fragility and often associated with short stature. The mutation in WNT1 causes autosomal recessive OI (AR-OI) due to the key role of WNT/ß-catenin signaling in bone formation. WNT1 mutations cause phenotypes in OI of varying degrees of clinical severity, ranging from moderate to progressively deforming forms. The nucleotide change c.677C > T is one of the recurrent variants in the WNT1 alleles in Chinese AR-OI patients. To explore the effects of mutation c.677C > T on WNT1 function, we evaluated the activation of WNT/ß-catenin signaling, cell proliferation, osteoblast differentiation, and osteoclast differentiation in WNT1c.677C>T , WNT1c.884C>A , and wild type WNT1 transfected into MC3T3-E1 preosteoblasts. Plasmids containing wild type WNT1, WNT1c.677C>T , and WNT1c.884C>A cDNAs were constructed. Protein levels of phosphorylation at serine 9 of GSK-3ß (p-GSK-3ß), GSK-3ß, nonphosphorylated ß-catenin (non-p-ß-catenin), and ß-catenin were detected with western blot. Cell proliferation was determined using MTS. BMP-2 and RANKL mRNA and protein levels were detected by qPCR and western blot. Our results showed that WNT1c.677C>T failed to activate WNT/ß-catenin signaling and impaired the proliferation of preosteoblasts. Moreover, compared to wild type WNT1, WNT1c.677C>T downregulated BMP-2 protein expression and was exhibited a diminished capacity to suppress the RANKL protein level. In conclusion, mutation c.677C > T hindered the ability of WNT1 to induce the WNT/ß-catenin signaling pathway and it affected the WNT/ß-catenin pathway which might potentially contribute to hampered bone homeostasis.

[Sequencing of 100 common variation sites of 18 deafness susceptibility genes among 16 182 individuals from Dongguan].

OBJECTIVE: To determine the type and carrier rate of deafness-related variants in Dongguan, China. METHODS: A total of 16 182 subjects were screened. Heel blood samples were collected from newborns, while peripheral venous blood samples were collected from the remainders. For each individual, 100 variations of 18 deafness susceptibility genes were detected. RESULTS: In total 1631 deafness-related variants (including 5 homozygous mutations) were detected, which gave a detection rate of 10.08%. The detection rate of SLC26A4 gene variants was the highest (845 cases, 5.22%), which was followed by GJB2 (673 cases, 4.16%), GJB3 (100 cases, 0.62%), TMC1 (12 cases, 0.07%), and MYO15A (1 case, 0.01%). The detection rate for GJB2 c.235delC variant was the highest (524 cases, 3.24%), which was followed by SLC26A4 IVS7-2A>G variant (270 cases, 1.67%). Thirty three individuals (0.20%) carried two variants at the same time, 7 of them (0.04%) carried compound heterozygous variants of the same gene. CONCLUSION: To expand the range of screening can help with determination of the carrier status and provision of early intervention and genetic counseling for the examinees.

Inhibition of the proliferation, invasion, migration, and epithelial-mesenchymal transition of prostate cancer cells through the action of ATP1A2 on the TGF-ß/Smad pathway.

BACKGROUND: Prostate cancer (PC) is one of the major male malignancies worldwide. Because Na+-K+-ATPase is widely involved in various pathological processes, but the action of its α2 subtype (ATP1A2) in PC is unclear, we investigated the role of ATP1A2 in the invasion and migration of PC cells. METHODS: We measured the expression levels of ATP1A2 in human normal prostate epithelial cell line (RWPE-1) and PC cell lines (PC-3 and DU145) by quantitative real-time PCR (qRT-PCR) and western blot. Cell proliferation, apoptosis, migration, and invasion of PC-3 and DU145 cells were investigated through clone formation assay, EdU assay, flow cytometry and transwell assay, respectively. The effect of ATP1A2 on a tumor-inhibitory pathway [transforming growth factor-ß (TGF-ß)/Smad] was assessed using western blot. In addition, tumor formation was detected using in vivo xenograft model in male BALB/c nude mice. RESULTS: The Cancer Genome Atlas (TCGA) analysis showed that ATP1A2 expression was reduced in PC patients (P<0.05), and patients with low ATP1A2 expression had a lower survival rate (P<0.05). ATP1A2 levels were significantly reduced in PC-3 and DU145 cells, compared with RWPE-1 cells (P<0.01). We also demonstrated that overexpression of ATP1A2 significantly inhibited the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of PC-3 and DU145 cells (P<0.01) and promoted apoptosis (P<0.01). However, silencing ATP1A2 had the opposite effect (P<0.01). In addition, overexpression of ATP1A2 significantly inhibited the TGF-ß/Smad pathway (P<0.01), whereas silencing ATP1A2 activated the TGF-ß/Smad pathway (P<0.01). Meanwhile, the effect of ATP1A2 silencing on the proliferation, apoptosis, migration and invasion was reversed by TGF-ß/Smad pathway inhibitor (LY364947). Furthermore, ATP1A2 inhibited tumor growth in vivo. CONCLUSIONS: ATP1A2 inhibited proliferation, apoptosis, migration, invasion, and EMT in PC by inhibiting the TGF-ß/Smad pathway.

Rapid and sensitive detection of hepatitis B virus by lateral flow recombinase polymerase amplification assay.

Hepatitis B virus (HBV) infection is a major public health priority. In the present study, a lateral flow strip combined with the recombinase polymerase amplification (LF-RPA) assay was developed and evaluated for rapid HBV detection. A primer/probe pair targeting the conserved region of the HBV genome was designed and applied to the LF-RPA. TheRPA was achieved at the isothermal temperature of 39℃ for 30 min, and the RPA products were detected using the LF test. DNA extraction, RPA reaction and endpoint detection will take about 70 min. The LF-RPA assay could detect HBV at as low as 10 copies/reaction, with no cross-reactions with other common pathogens. The LF-RPA assay was performed on 85 samples. Of these, 36 samples tested HBV positive, whereas 49 were negative. Similar results were obtained using the conventional polymerase chain reaction method. Thus, the newly developed LF-RPA assay can be an improved diagnostic tool for rapid and simple HBV detection.

Effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast differentiation via the WNT1/ß-catenin signaling pathway.

BACKGROUND: WNT1 c.110 T>C and c.505G>T missense mutations have been identified in patients with osteogenesis imperfecta (OI). Whether these mutations affect osteoblast differentiation remains to be determined. This study aimed to investigate the effects of WNT1 c.110 T>C and c.505G>T mutations on osteoblast function, gene expression, and pathways involved in OI. METHODS: Empty vector (negative control), wild-type WNT1, WNT1 c.110 T>C, WNT1 c.505G>T, and WNT1 c.884C>A (positive control) mutant plasmids were constructed and transfected into preosteoblast (MC3T3-E1) cells to investigate their effect on osteoblast differentiation. The expressions of osteoblast markers, including BMP2, RANKL, osteocalcin, and alkaline phosphatase (ALP), were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), enzyme-linked immunosorbent assay, and ALP staining assay, respectively. The mRNA and protein expression levels of WNT1 or the expression levels of the relevant proteins involved in the WNT1/ß-catenin signaling pathway were also determined using RT-qPCR, WB, and immunofluorescence (IF) assays after the different plasmids were transfected into MC3T3-E1 cells. RESULTS: Compared with those in the wild-type group, in the mutation groups, the mRNA and protein expression levels of BMP2 were suppressed, the expressions of osteocalcin and ALP were inhibited, and the mRNA and protein expression levels of RANKL were enhanced in MC3T3-E1 cells. WB and IF assays revealed that the protein expression levels of WNT1 in MC3T3-E1 cells were downregulated in the mutation groups compared with those in the wild-type WNT1 group. Furthermore, the expression levels of nonphosphorylated ß-catenin (non-p-ß-catenin) and phosphorylated GSK-3ß (p-GSK-3ß) were downregulated in the mutation groups compared with those in the wild-type group. However, no significant changes in the expression level of non-p-ß-catenin or p-GSK-3ß were observed in the mutation groups. CONCLUSIONS: WNT1 c.110 T>C and c.505G>T mutations may alter the proliferation and osteogenic phenotype of MC3T3-E1 linked to the progression of OI via the inhibition of the WNT1/ß-catenin signaling pathway. This is the first study to confirm the effect of WNT1 c.110 T>C and c.505G>T missense mutations on osteoblast differentiation and propose a new molecular mechanism for OI development.

CircRNA CDR1as/miR-1287/Raf1 Axis Modulates Hepatocellular Carcinoma Progression Through MEK/ERK Pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common lethal malignant tumor worldwide. Circular RNAs (circRNAs) have been reported to affect the development of human cancers, including HCC. In this project, we aim to clarify the functional effect of circular CDR1as (circ_CDR1as) on HCC progression. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot is implemented to detect the expression of circ_CDR1as, microRNA (miR)-1287 and Raf-1 proto-oncogene, serine/threonine kinase (Raf1). Cell proliferation is assessed via colony formation and 3-(4, 5)-dimethylthiazole-2-y1)-2, 5-biphenyl tetrazolium bromide (MTT) assays. Cell migration and invasion are measured by Transwell assay. The target relationship between miR-1287 and circ_CDR1as or Raf1 is validated through dual-luciferase reporter assay. The levels of epithelia-mesenchymal transition (EMT) markers and the MEK/ERK signal pathway-related proteins are examined by Western blot. Model in nude mice is constructed to determine the role of circ_CDR1as in vivo. RESULTS: Expression of circ_CDR1as and Raf1 is elevated, while miR-1287 expression is decreased in HCC. Depletion of circ_CDR1as or Raf1 could inhibit proliferation and metastasis of HCC cells. Besides, circ_CDR1as regulates Raf1 expression by targeting miR-1287. MiR-1287 upregulation or Raf1 depletion could partially counterbalance circ_CDR1as depletion-mediated inhibitory effects on HCC cell behaviors. Moreover, circ_CDR1as depletion represses HCC progression through inactivating MEK/ERK pathway. In addition, circ_CDR1as depletion suppresses tumor growth in vivo via regulating miR-1287/Raf1 pathway. CONCLUSION: Circ_CDR1as depletion inhibits HCC cell proliferation and metastasis by miR-1287/Raf1 and MEK/ERK pathways, highlighting a promising molecular target for HCC treatment.

[Analysis of hepatitis B surface antigen levels in patients with chronic hepatitis B in Guangzhou].

OBJECTIVE: To assess the value of quantitative analysis of hepatitis B surface antigen (HBsAg) levels in the diagnosis and therapeutic evaluations in patients with chronic hepatitis B (CHB). METHODS: According to the staging criteria defined by the American Association of Liver Diseases, 96 patients with CHB admitted in Zhujiang Hospital were classified in immune-tolerant (IT), HBeAg-positive hepatitis (EPH), inactive carrier (IC) and HBeAg-negative hepatitis (ENH) phases. Serum HBsAg, HBV-DNA and ALT levels were quantified and their correlations were evaluated in each phase of infection. RESULTS: The mean HBsAg titers (measured in log10U/L) differed significantly between the phases of CHB (4.12 in IT, 4.02 in EPH, 2.85 in EPH, and 3.29 in ENH). The correlation coefficient of HBsAg with HBV-DNA was 0.6828 in IT, 0.5759 in EPH, 0.3280 in IC, and 0.1083 in ENH. Serum HBsAg titers were significantly higher in HBeAg-positive patients than in HBeAg-negative patients. No correlation was found between HBsAg level and ALT in each phase of CHB. CONCLUSION: The median baseline serum HBsAg levels vary between different phases of CHB in Guangzhou, suggesting the value of HBsAg in accurate classification of hepatitis B patients and evaluation of the therapeutic effect and outcomes of the patients.