The aim of this study was to clone the chicken zp1 gene encoding zona pellucida 1 (Zp1) and investigate its tissues expression profile and its effect on osteoblast mineralization. The expression level of zp1 was quantified in various tissues of laying hens and in the tibia of the pre- and post-sexual maturity by RT-qPCR. Zp1 overexpressed vector was transfected into chicken calvarial osteoblasts which were induced differentiation for 8 days, and the extracellular mineral and the expression of mineralization-related genes were detected. The full-length chicken zp1 gene is 3 045 bp, encoding 958 amino acids residuals, and has two N-glycosylation sites. The highest expression level of the zp1 gene was found in the liver, followed by the tibia and yolk membrane, while no expression was detected in the heart and eggshell gland. Compared with the pre-sexual maturity hens, the concentration of estrogen (E2) in plasma, the content of glycosaminoglycan (GAG) and the expression level of the zp1 gene in the tibia with post-sexual maturity were higher. The extracellular matrix and the level of osteoblast mineralization-related genes showed a significantly upregulated expression in chicken calvarial osteoblasts with Zp1 overexpressed and addition of estrogen. The expression of the zp1 gene is tissue-specific and positively regulated osteoblast mineralization under the action of estrogen, laying the foundation for elucidating the functional properties of Zp1 in chicken bones during the egg production period.
Female ; Animals ; Zona Pellucida Glycoproteins ; Membrane Glycoproteins/metabolism* ; Chickens/genetics* ; Egg Proteins/metabolism* ; Receptors, Cell Surface ; Estrogens

OBJECTIVE: To investigate the clinical significance of SFRP1 gene and its methylation in childhood acute lymphoblastic leukemia (ALL) . METHODS: Methylation-specific PCR (MSP) was used to detect the methylation status of SFRP1 gene in bone marrow mononuclear cells of 43 children with newly diagnosed ALL before chemotherapy (primary group) and when the bone marrow reached complete remission d 46 after induction of remission chemotherapy (remission group), the expression of SFRP1 mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR), the expression of SFRP1 protein was detected by Western blot, and clinical data of children were collected, the clinical significance of SFRP1 gene methylation in children with ALL was analyze. RESULTS: The positive rate of SFRP1 gene promoter methylation in the primary group (44.19%) was significantly higher than that in the remission group (11.63%) (χ2=11.328, P<0.05). The relative expression levels of SFRP1 mRNA and protein in bone marrow mononuclear cells of children in the primary group were significantly lower than those in the remission group (P<0.05). Promoter methylation of SFRP1 gene was associated with risk level (χ2=15.613, P=0.000) and survival of children (χ2=6.561, P=0.010) in the primary group, children with SFRP1 hypermethylation had significantly increased risk and shortened event-free survival time, but no significant difference in other clinical data. CONCLUSION Hypermethylation of SFRP1 gene promoter may be involved in the development of childhood ALL, and its hypermethylation may be associated with poor prognosis.
Child ; Humans ; Clinical Relevance ; DNA Methylation ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; Bone Marrow/metabolism* ; RNA, Messenger/metabolism* ; Membrane Proteins/genetics* ; Intercellular Signaling Peptides and Proteins/metabolism*

OBJECTIVE: To investigate the mechanism of drug reversing resistance of Agaricus blazei extract FA-2-b-β on T cell acute lymphoblastic leukemia (T-ALL) cell lines. METHODS: Cell proliferation was detected by CCK-8 assay; the apoptosis, cell cycle mitochondrial membrane potential, and intracellular rhodamine accumulation were detected by flow cytometry, and apoptosis-related gene and protein expression were detected by qPCR and Western blot; the membrane surface protein MDR1 was observed by immunofluorescence microscopy. RESULTS: Different concentrations of FA-2-b-β significantly inhibited proliferation and induced apoptosis of CCRF-CEM and CEM/C1 (P<0.05), and CCRF-CEM cell cycle were arrested at S phase, and CEM/C1 cells were arrested at G₀/G₁ phase. Western blot and qPCR results show that FA-2-b-β inhibited ABCB1、ABCG2、CTNNB、MYC and BCL-2 expression, but upregulated Bax expression. In addition, FA-2-b-β reversed the resistance characteristics of CEM/C1 drug-resistance cells, which decreased mitochondrial membrane potential, and significantly increased the intracellular rhodamine accumulation, and weakening of the expression of the membrane surface protein MDR1. With the Wnt/β-catenin inhibitor (ICG001), the process was further intensified. CONCLUSION Agaricus Blazei Extract FA-2-b-β inhibits cell proliferation, promotes apoptosis, regulates the cell cycle, reduces mitochondrial energy supply, and down-regulate MDR1 expression to reverse the resistance of CEM/C1, which all suggest it is through regulating the Wnt signaling pathway in T-ALL.
Humans ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; Wnt Signaling Pathway ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism* ; Apoptosis ; Drug Resistance, Multiple ; Membrane Proteins ; Cell Line, Tumor ; Cell Proliferation

Cancer stem cell-like cells (CSCs) play an integral role in the heterogeneity, metastasis, and treatment resistance of head and neck squamous cell carcinoma (HNSCC) due to their high tumor initiation capacity and plasticity. Here, we identified a candidate gene named LIMP-2 as a novel therapeutic target regulating HNSCC progression and CSC properties. The high expression of LIMP-2 in HNSCC patients suggested a poor prognosis and potential immunotherapy resistance. Functionally, LIMP-2 can facilitate autolysosome formation to promote autophagic flux. LIMP-2 knockdown inhibits autophagic flux and reduces the tumorigenic ability of HNSCC. Further mechanistic studies suggest that enhanced autophagy helps HNSCC maintain stemness and promotes degradation of GSK3β, which in turn facilitates nuclear translocation of β-catenin and transcription of downstream target genes. In conclusion, this study reveals LIMP-2 as a novel prospective therapeutic target for HNSCC and provides evidence for a link between autophagy, CSC, and immunotherapy resistance.
Humans ; Autophagy ; Carcinoma, Squamous Cell/pathology* ; Cell Line, Tumor ; Glycogen Synthase Kinase 3 beta/metabolism* ; Head and Neck Neoplasms/pathology* ; Neoplastic Stem Cells/pathology* ; Squamous Cell Carcinoma of Head and Neck/pathology* ; Lysosome-Associated Membrane Glycoproteins

Melanoma is the most aggressive cutaneous tumor. Neuropilin and tolloid-like 2 (NETO2) is closely related to tumorigenesis. However, the functional significance of NETO2 in melanoma progression remains unclear. Herein, we found that NETO2 expression was augmented in melanoma clinical tissues and associated with poor prognosis in melanoma patients. Disrupting NETO2 expression markedly inhibited melanoma proliferation, malignant growth, migration, and invasion by downregulating the levels of calcium ions (Ca²⁺) and the expression of key genes involved in the calcium signaling pathway. By contrast, NETO2 overexpression had the opposite effects. Importantly, pharmacological inhibition of CaMKII/CREB activity with the CaMKII inhibitor KN93 suppressed NETO2-induced proliferation and melanoma metastasis. Overall, this study uncovered the crucial role of NETO2-mediated regulation in melanoma progression, indicating that targeting NETO2 may effectively improve melanoma treatment.
Humans ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism* ; Cell Line, Tumor ; Cell Proliferation ; Melanoma/genetics* ; Membrane Proteins/genetics* ; Phosphorylation ; Signal Transduction

Objective: To investigate the expression of glycoprotein non metastatic melanoma protein B (GPNMB) in renal eosinophilic tumors and to compare the value of GPNMB with CK20, CK7 and CD117 in the differential diagnosis of renal eosinophilic tumors. Methods: Traditional renal tumor eosinophil subtypes, including 22 cases of renal clear cell carcinoma eosinophil subtype (e-ccRCC), 19 cases of renal papillary cell carcinoma eosinophil subtype (e-papRCC), 17 cases of renal chromophobe cell carcinoma eosinophil subtype (e-chRCC), 12 cases of renal oncocytoma (RO) and emerging renal tumor types with eosinophil characteristics [3 cases of eosinophilic solid cystic renal cell carcinoma (ESC RCC), 3 cases of renal low-grade eosinophil tumor (LOT), 4 cases of fumarate hydratase-deficient renal cell carcinoma (FH-dRCC) and 5 cases of renal epithelioid angiomyolipoma (E-AML)], were collected at the Affiliated Drum Tower Hospital of Nanjing University Medical School from January 2017 to March 2022. The expression of GPNMB, CK20, CK7 and CD117 was detected by immunohistochemistry and statistically analyzed. Results: GPNMB was expressed in all emerging renal tumor types with eosinophil characteristics (ESC RCC, LOT, FH-dRCC) and E-AML, while the expression rates in traditional renal eosinophil subtypes e-papRCC, e-chRCC, e-ccRCC and RO were very low or zero (1/19, 1/17, 0/22 and 0/12, respectively); the expression rate of CK7 in LOT (3/3), e-chRCC (15/17), e-ccRCC (4/22), e-papRCC (2/19), ESC RCC (0/3), RO (4/12), E-AML(1/5), and FH-dRCC (2/4) variedly; the expression of CK20 was different in ESC RCC (3/3), LOT(3/3), e-chRCC(1/17), RO(9/12), e-papRCC(4/19), FH-dRCC(1/4), e-ccRCC(0/22) and E-AML(0/5), and so did that of CD117 in e-ccRCC(2/22), e-papRCC(1/19), e-chRCC(16/17), RO(10/12), ESC RCC(0/3), LOT(1/3), E-AML(2/5) and FH-dRCC(1/4). GPNMB had 100% sensitivity and 97.1% specificity in distinguishing E-AML and emerging renal tumor types (such as ESC RCC, LOT, FH-dRCC) from traditional renal tumor types (such as e-ccRCC, e-papRCC, e-chRCC, RO),respectively. Compared with CK7, CK20 and CD117 antibodies, GPNMB was more effective in the differential diagnosis (P<0.05). Conclusion: As a new renal tumor marker, GPNMB can effectively distinguish E-AML and emerging renal tumor types with eosinophil characteristics such as ESC RCC, LOT, FH-dRCC from traditional renal tumor eosinophil subtypes such as e-ccRCC, e-papRCC, e-chRCC and RO, which is helpful for the differential diagnosis of renal eosinophilic tumors.
Humans ; Kidney Neoplasms/pathology* ; Carcinoma, Renal Cell/pathology* ; Diagnosis, Differential ; Angiomyolipoma/diagnosis* ; Biomarkers, Tumor/metabolism* ; Leukemia, Myeloid, Acute/diagnosis* ; Membrane Glycoproteins

OBJECTIVE: To investigate the effects of LASS2/TMSG1 gene overexpression on proliferation and apoptosis of human lung cancer A549 cells and explore the possible mechanism. METHODS: We examined LASS2/TMSG1 expression level in a previously constructed A549 cell line overexpressing LASS2/TMSG1 using Western blotting. The proliferation and apoptosis of the cells were detected using colony-forming assay, CCK-8 assay, Hoechst/PI double staining and flow cytometry. Fourteen nude mice were randomized into 2 groups (n=7) to receive subcutaneous injection of A549 cells with or without LASS2/TMSG1 overexpression on the back of the neck, and the cell proliferation in vivo was observed. The expression levels of p38 MAPK protein and p-p38 MAPK protein in the xenografts were detected with Western blotting. ELISA was used to detect the levels of ceramide and p38 MAPK protein in cultured A549 cell supernatants and the xenografts in nude mice. RESULTS: Compared with the negative control cells, A549 cells with LASS2/TMSG1 overexpression had significantly lowered proliferation ability in vitro with increased early apoptosis rate (P < 0.05), and showed obvious growth inhibition after inoculation in nude mice(P < 0.05). Western blotting showed that in both cultured A549 cells and the xenografts in nude mice, LASS2/TMSG1 gene overexpression significantly increased the expression levels of p38 MAPK protein and p-p38 MAPK protein (P < 0.05); the results of ELISA also revealed significantly increased levels of ceramide and p38 MAPK protein in the cell supernatant andxenografts as well (P < 0.05). CONCLUSION Overexpression of LASS2/TMSG1 gene can significantly inhibit the proliferation and promote early apoptosis of human lung cancer A549 cells both in vitro and in vivo possibly by upregulating the expressions of ceramide and p38 MAPK protein to activate a signal transduction cascade.
Animals ; Humans ; Mice ; A549 Cells ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Lung Neoplasms ; Membrane Proteins/metabolism* ; Mice, Nude ; p38 Mitogen-Activated Protein Kinases/metabolism* ; Signal Transduction ; Tumor Suppressor Proteins/metabolism*

OBJECTIVE: To screen the differentially expressed long non-coding RNAs (lncRNAs) in non-small cell lung cancer (NSCLC) cells with acquired resistance to osimertinib and explore their roles in drug resistance of the cells. METHODS: The cell lines H1975_OR and HCC827_OR with acquired osimertinib resistance were derived from their osimertinib-sensitive parental NSCLC cell lines H1975 and HCC827, respectively, and their sensitivity to osimertinib was assessed with CCK-8 assay, clone formation assay and flow cytometry. RNA sequencing (RNA-seq) and real-time quantitative PCR (qPCR) were used to screen the differentially expressed lncRNAs in osimertinib-resistant cells. The role of the identified lncRNA in osimertinib resistance was explored using CCK-8, clone formation and Transwell assays, and its subcellular localization and downstream targets were analyzed by nucleoplasmic separation, bioinformatics analysis and qPCR. RESULTS: The resistance index of H1975_OR and HCC827_OR cells to osimertinib was 598.70 and 428.82, respectively (P < 0.001), and the two cell lines showed significantly increased proliferation and colony-forming abilities with decreased apoptosis (P < 0.01). RNA-seq identified 34 differentially expressed lncRNAs in osimertinib-resistant cells, and among them lnc-TMEM132D-AS1 showed the highest increase of expression after acquired osimertinib resistance (P < 0.01). Analysis of the TCGA database suggested that the level of lnc-TMEM132D-AS1 was significantly higher in NSCLC than in adjacent tissues (P < 0.001), and its high expression was associated with a poor prognosis of the patients. In osimertinib-sensitive cells, overexpression of Lnc-TMEM132D-AS1 obviously promoted cell proliferation, colony formation and migration (P < 0.05), while Lnc-TMEM132D-AS1 knockdown partially restored osimertinib sensitivity of the resistant cells (P < 0.01). Lnc-TMEM132D-AS1 was localized mainly in the cytoplasm, and bioinformatics analysis suggested that hsa-miR-766-5p was its candidate target, and their expression levels were inversely correlated. The target mRNAs of hsa-miR-766-5p were mainly enriched in the Ras signaling pathway. CONCLUSION The expression of lnc-TMEM132D-AS1 is significantly upregulated in NSCLC cells with acquired osimertinib resistance, and may serve as a potential biomarker and therapeutic target for osimertinibresistant NSCLC.
Humans ; Carcinoma, Non-Small-Cell Lung/metabolism* ; Lung Neoplasms/genetics* ; RNA, Long Noncoding/metabolism* ; Sincalide/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Cell Movement ; MicroRNAs/genetics* ; Gene Expression Regulation, Neoplastic ; Membrane Proteins/metabolism*

PiT2 is an inorganic phosphate (Pi) transporter whose mutations are linked to primary familial brain calcification (PFBC). PiT2 mainly consists of two ProDom (PD) domains and a large intracellular loop region (loop7). The PD domains are crucial for the Pi transport, but the role of PiT2-loop7 remains unclear. In PFBC patients, mutations in PiT2-loop7 are mainly nonsense or frameshift mutations that probably cause PFBC due to C-PD1131 deletion. To date, six missense mutations have been identified in PiT2-loop7; however, the mechanisms by which these mutations cause PFBC are poorly understood. Here, we found that the p.T390A and p.S434W mutations in PiT2-loop7 decreased the Pi transport activity and cell surface levels of PiT2. Furthermore, we showed that these two mutations attenuated its membrane localization by affecting adenosine monophosphate-activated protein kinase (AMPK)- or protein kinase B (AKT)-mediated PiT2 phosphorylation. In contrast, the p.S121C and p.S601W mutations in the PD domains did not affect PiT2 phosphorylation but rather impaired its substrate-binding abilities. These results suggested that missense mutations in PiT2-loop7 can cause Pi dyshomeostasis by affecting the phosphorylation-regulated cell-surface localization of PiT2. This study helps understand the pathogenesis of PFBC caused by PiT2-loop7 missense mutations and indicates that increasing the phosphorylation levels of PiT2-loop7 could be a promising strategy for developing PFBC therapies.
Humans ; Cell Membrane ; Mutation, Missense ; Phosphates/metabolism* ; Sodium-Phosphate Cotransporter Proteins, Type III/genetics*

NDFIP1 has been previously reported as a tumor suppressor in multiple solid tumors, but the function of NDFIP1 in NSCLC and the underlying mechanism are still unknown. Besides, the WW domain containing proteins can be recognized by NDFIP1, resulted in the loading of the target proteins into exosomes. However, whether WW domain-containing transcription regulator 1 (WWTR1, also known as TAZ) can be packaged into exosomes by NDFIP1 and if so, whether the release of this oncogenic protein via exosomes has an effect on tumor development has not been investigated to any extent. Here, we first found that NDFIP1 was low expressed in NSCLC samples and cell lines, which is associated with shorter OS. Then, we confirmed the interaction between TAZ and NDFIP1, and the existence of TAZ in exosomes, which requires NDFIP1. Critically, knockout of NDFIP1 led to TAZ accumulation with no change in its mRNA level and degradation rate. And the cellular TAZ level could be altered by exosome secretion. Furthermore, NDFIP1 inhibited proliferation in vitro and in vivo, and silencing TAZ eliminated the increase of proliferation caused by NDFIP1 knockout. Moreover, TAZ was negatively correlated with NDFIP1 in subcutaneous xenograft model and clinical samples, and the serum exosomal TAZ level was lower in NSCLC patients. In summary, our data uncover a new tumor suppressor, NDFIP1 in NSCLC, and a new exosome-related regulatory mechanism of TAZ.
Humans ; Carcinoma, Non-Small-Cell Lung/metabolism* ; Carrier Proteins/metabolism* ; Cell Line ; Cell Proliferation ; Exosomes/metabolism* ; Lung Neoplasms/genetics* ; Membrane Proteins/metabolism* ; Transcriptional Coactivator with PDZ-Binding Motif Proteins/metabolism*