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.

Clinicopathological and prognostic value of long noncoding RNA SNHG7 in cancers: a meta-analysis and bioinformatics.

The long intergenic non-coding RNA SNHG7 has been reported to be abnormally expressed in many types of cancer, the results remain controversial. In this study, a meta-analysis was performed to evaluate the clinicopathologic and prognostic value of SNHG7 in cancers. Electronic databases of PubMed, Web of Science, Cochrane Library and Embase were used to search relevant studies. A combined hazard ratio (HR) and its corresponding 95% confidence interval (CI) were used to assess the association between SNHG7 expression and prognosis in cancer patients. Pooled odds ratio (OR) and 95% CI were calculated to elaborate the association between SNHG7 expression and clinicopathological features in cancers. Besides, the data from The Cancer Genome Atlas (TCGA) dataset was used to validate the results. In total, eighteen studies compromising 1303 participants were enrolled in this analysis. The pooled results showed increased SNHG7 expression could predict unfavorable overall survival (OS) (HR = 1.75, 95%CI = 1.52-2.02, P = 0.000). Analysis stratified by follow-up time, cancer types, analysis types, sample sizes and cut off further verified the prognostic value of SNHG7. Additionally, elevated SNHG7 expression was correlated with TNM stage (OR: 3.31, 95%CI = 2.29-4.80, P = 0.000), lymph node metastasis (OR = 3.32, 95%CI = 1.61-6.83, P = 0.004), and tumor differentiation (OR = 1.92, 95%CI = 1.22-3.03, P =0.005) in patients with cancers. Excavation of TCGA dataset valuated that SNHG7 was upregulated in some cancers and predicted worse OS, which partially confirmed our results in this meta-analysis.

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.

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.

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.

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.