The T-box transcription factor 18 (Tbx18) plays an essential role in normal development of the urinary system. It has been identified that Tbx18-positive cells contribute to multiple components of the mature urogenital system. However, it remains unclear whether Tbx18-positive progenitors play a specific role in the development of urologic fat tissues. Here we applied the Tbx18 lineage tracing model of Tbx18Cre/Rosa26EYFP and Tbx18Cre/Rosa26LacZ mice to identify the differentiation capacity of Tbx18-expressing progenitor cells in the urologic system. X-gal staining ofthe urinary tissue revealed that the X-gal protein is detected in the renal capsule, the ureter and perirenal fat tissue and that Tbx18-expressing precursors contribute to the renal capsule, ureter and perirenal adipocytes in Tbx18Cre/Rosa26LacZ fate-mapping mice. Immunostainingofthe kidney and perirenal tissue revealedsome yellowfluorescentprotein-positive(YFP+)cells express Perilipin, the marker for adipocytes and that Tbx18-positive progenitors contribute to perirenal adipocytes in Tbx18Cre/Rosa26EYFP fate-mapping mice. Thus, our analyses identified the multi-differentiation capacity of Tbx18-expressing progenitors in the urinary system. Further researches are needed to study the capacity of Tbx18-expressing progenitors to differentiate into adipocytes and other components of the urogenital system in models of kidney injury.
EGFR-TKI targeted therapy has been playing an important role in the treatment of non-small cell lung cancer (NSCLC). However, unavoidable therapeutic resistance significantly limits the clinical efficacy of TKI. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a member of the type I receptor tyrosine kinase family and plays an important role in cancer development and progression. The aim of this work is to investigate the effect and molecular mechanism of ROR1 on gefitinib resistance in NSCLC. EGFR-TKI-resistant HCC827/GR cells were established from parental HCC827 cells by continuous exposure to gefitinib. ROR1 expression on the mRNA and protein levels was detected by qRT-PCR and Western blot respectively. Influence of ROR1 to gefitinib resistance in HCC827/GR cells was assayed by ROR1 shRNA in vitro. Influence of ROR1 to gefitinib resistance was assayed by ROR1 overexpression in HCC827 cells in vitro or by ROR1 shRNA in HCC827/GR cells in xenograft mouse model. ROR1 downstream signal molecules were detected by Western blot. We found that the mRNA and protein levels of ROR1 are significantly increased in HCC827/GR. Knockdown of ROR1 significantly increased gefitinib sensitivity of HCC827/GR cells and gefitinib-induced cell apoptosis in vitro. Overexpression of ROR1 significantly increased gefitinib resistance of HCC827 cells in vitro. Moreover, in vivo assays also showed that knockdown of ROR1 significantly increased gefitinib sensitivity (P<0.05). Furthermore, we found that abnormal activation of AKT/FOXO1 signaling was detected in HCC827/GR cells, and knockdown of ROR1 significantly inhibited the phosphorylation of AKT, but increased the expression of FOXO1. Taken together, these results indicated that ROR1 could increase gefitinib resistance in HCC827 cells, by which mechanism is related to activation of AKT/FOXO1 signaling.
MicroRNAs (miRNAs) have emerged as critical regulators for target genes expression, and thus are involved in almost all physiological and pathological processes. SIRT1 exerts critical roles in diverse biological processes through deacetylation of substrates. Nonetheless, the relationship between SIRT1 and non-coding RNAs such as miRNAs is still unknown. In this study, we initially silenced SIRT1 in HEK293 cells, and noted that miR-221 and miR-222 were significantly down-regulated using quantitative RT-PCR. Meanwhile, ectopic expression of SIRT1 enhanced miR-221/222 expression levels in cells suggesting a the positive correlation between SIRT1 and miR-221/222. We further constructed a dual luciferase reporter vector containing the promoter of miR-221/222 cluster, which was co-transfected with SIRT1 over-expression plasmid or interference sequences of SIRT1 into the HEK293 cells. The results uncovered that SIRT1 significantly increased the miR-221/222 promoter activity, indicating that SIRT1 mightbe regulate miR-221/222 at the transcriptional level. Moreover, the Western blot analysis showed that forced expression of miR-221/222 promoted the autophagy of HEK293 cells, while inhibition of miR-221/222 decreased autophagy; Furthermore,inhibition of miR-221/222 significantly reversed the SIRT1-induced autophagy. In conclusion,SIRT1 induced autophagy in HEK293 cells by upregulating the expression of miR-221/222 to significantly promote the autophagy in HEK293 cells. Its mechanism needs to be further studied.
Many people in the modern society are inflicted with depression and psychological suboptimal health. Here we analyzed the metabolic biomarkers associated with these two conditions, aiming to provide a reference for the clinical diagnosis and identification as well as the early therapeutic prevention and treatment. 18 patients with depression and 23 suboptimal health subjects were screened, and their venous blood was collected. We used the method of 1H-NMR metabolomics, combined with the univariate analysis, multivariate statistics, correlation and Fold Change (FC), and screened the endogenous differential metabolites as potential metabolic biomarkers by 1r1>0.6 and FC>1.5 as critical indicators. Metabolites including 3-OH-butyrate, acetate, alanine, betaine and carnitine and so on showed significant differences between the two groups (P<0.05, 0.01). Then the receiver operating characteristic curve (ROC) was used to assess the ability of identification and diagnosis. The area under the receiver operating characteristic curve (AUC) showed that carnitine, choline, histidine, and lipid (AUC> 0.85) had a high diagnostic value and predictive ability for associating depression and psychological suboptimal health. In summary, the present research could set up new measures for improving the accuracy and reliability of clinical diagnosis of depression. It will play a critical role in the early identification of psychological suboptimal health and prevent it from turning into mental diseases.
Interaction of Aβ42 with the cell membrane is an important event in the pathogenesis of Alzheimer disease (AD). However, very little work has been done to examine the interaction of different aggregation states of Aβ42 with the cell membranes in combination with their toxicity. Transmission electron microscopy (TEM), membrane balance, ThT assay and cytotoxicity test showed that: (1) Aβ42 monomers can be inserted into the phospholipid monolayer whereas ADDLs do not have the membrane insertion ability; (2) Transmission electron microscopy and ThT fluorescence analysis showed that different Aβ42 aggregation states have different abilities to form fibrils. The Aβ42 monomers had the strongest fibril formation propensity, whereas the Aβ42 protofibrils also had some propensity to form fibrils. Fibril formation from Aβ42 ADDLs was difficult. (3) Aβ42 monomer cytotoxicity was weak, and the cytotoxicity of Aβ42 ADDLs and protofibrils was very strong. This result suggests that the monomer rapidly inserts into the membrane to form fibrils, thus reducing its cytotoxicity. ADDLs and protofibrils do not have the ability to insert into the membranes therefore are cytotoxic. These toxic effects are probably caused by binding to the membrane receptors. These results will provide some reference for the study of the mechanism of Aβ42 toxicity in AD. However, the mechanism of endocytosis of various Aβ oligomers still needs further study.
The Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) plays its anti-tumor role by activating and amplifying tumor-specific or non-specific killer cells in vitro. It has a good prospect in tumor immunotherapy. In this study, a recombinant lentiviral vector encoding the antiEGFRⅢ chimeric antigen receptor (CAR) was constructed. Then a stable CAR-modified Jurkat cell line was generated using the lentivirus. When stimulated with EGFRvⅢ or U87MG, the cell line was activated, suggesting that a recombinant plasmid pCDH-EGFRvⅢ-scFv-CAR-copGFP-T2A-puro was successfully constructed and an EGFRⅢ-CAR-expressing Jurkat cell line was successfully generated. After EGFRvⅢ stimulation for 12 h, the CCK8 assay showed that the proliferation rate of CAR-Jurkat cells was about 1.36 times faster than the control group (P<0.05). After co-incubation with U87MG cells, the ELISA assay showed that the concentration of IL-2 in the cell supernatant was 1.625 times more than the control group (P <0.01). The results above showed that the EGFRvⅢ-CARexpressing Jurkat cell line specifically targets the EGFRvⅢ molecule and EGFRvⅢ-positive cells and releases IL-2 cytokine, which provides a theoretical basis for the subsequent clinical application of the cell-based immunotherapy.
