Comparison of two in vitro angiogenesis assays for evaluating the effects of netrin-1 on tube formation.

Netrin-1 is a neural guidance cue that also regulates vascular development. Controversial results, however, have been obtained concerning the roles of netrin-1 in vascular development both in vivo and in vitro. In the present study, two in vitro angiogenesis assays were compared to evaluate the effects of netrin-1 secreted by retrovirally transduced melanoma cells (Mel2a-netrin1) on tube formation. The results showed that there was no obvious difference in tube formation induced by conditioned media (CM) from the control, Mel2a-netrin1 and Mel2a cells in a matrigel assay. The results of another in vitro assay, in which endothelial cells were co-cultured with human fibroblasts, however, showed that Mel2a-netrin1 CM inhibited the tube formation, supposedly through blocking the elongation and coalescence of human umbilical vein endothelial cells (HUVECs). These results confirmed that the matrigel assay is not able to demonstrate the anti-angiogenic roles of netrin-1.

Multi-omics joint analysis revealed the metabolic profile of retroperitoneal liposarcoma.

Retroperitoneal liposarcoma (RLPS) is the main subtype of retroperitoneal soft sarcoma (RSTS) and has a poor prognosis and few treatment options, except for surgery. The proteomic and metabolic profiles of RLPS have remained unclear. The aim of our study was to reveal the metabolic profile of RLPS. Here, we performed proteomic analysis (n = 10), metabolomic analysis (n = 51), and lipidomic analysis (n = 50) of retroperitoneal dedifferentiated liposarcoma (RDDLPS) and retroperitoneal well-differentiated liposarcoma (RWDLPS) tissue and paired adjacent adipose tissue obtained during surgery. Data analysis mainly revealed that glycolysis, purine metabolism, pyrimidine metabolism and phospholipid formation were upregulated in both RDDLPS and RWDLPS tissue compared with the adjacent adipose tissue, whereas the tricarboxylic acid (TCA) cycle, lipid absorption and synthesis, fatty acid degradation and biosynthesis, as well as glycine, serine, and threonine metabolism were downregulated. Of particular importance, the glycolytic inhibitor 2-deoxy-D-glucose and pentose phosphate pathway (PPP) inhibitor RRX-001 significantly promoted the antitumor effects of the MDM2 inhibitor RG7112 and CDK4 inhibitor abemaciclib. Our study not only describes the metabolic profiles of RDDLPS and RWDLPS, but also offers potential therapeutic targets and strategies for RLPS.

JMJD2D stabilises and cooperates with HBx protein to promote HBV transcription and replication.

Background & Aims: HBV infection is a global health burden. Covalently closed circular DNA (cccDNA) transcriptional regulation is a major cause of poor cure rates of chronic hepatitis B (CHB) infection. Herein, we evaluated whether targeting host factors to achieve functional silencing of cccDNA may represent a novel strategy for the treatment of HBV infection. Methods: To evaluate the effects of Jumonji C domain-containing (JMJD2) protein subfamily JMJD2A-2D proteins on HBV replication, we used lentivirus-based RNA interference to suppress the expression of isoforms JMJD2A-2D in HBV-infected cells. JMJD2D-knockout mice were generated to obtain an HBV-injected model for in vivo experiments. Co-immunoprecipitation and ubiquitylation assays were used to detect JMJD2D-HBx interactions and HBx stability modulated by JMJD2D. Chromatin immunoprecipitation assays were performed to investigate JMJD2D-cccDNA and HBx-cccDNA interactions. Results: Among the JMJD2 family members, JMJD2D was significantly upregulated in mouse livers and human hepatoma cells. Downregulation of JMJD2D inhibited cccDNA transcription and HBV replication. Molecularly, JMJD2D sustained HBx stability by suppressing the TRIM14-mediated ubiquitin-proteasome degradation pathway and acted as a key co-activator of HBx to augment HBV replication. The JMJD2D-targeting inhibitor, 5C-8-HQ, suppressed cccDNA transcription and HBV replication. Conclusion: Our study clarified the mechanism by which JMJD2D regulates HBV transcription and replication and identified JMJD2D as a potential diagnostic biomarker and promising drug target against CHB, and HBV-associated hepatocarcinoma. Impact and implications: HBV cccDNA is central to persistent infection and is a major obstacle to healing CHB. In this study, using cellular and animal HBV models, JMJD2D was found to stabilise and cooperate with HBx to augment HBV transcription and replication. This study reveals a potential novel translational target for intervention in the treatment of chronic hepatitis B infection.

[Establishment of esophageal squamous carcinoma cell lines stably over-expressing LLGL2].

Objective To prepare a lentiviral vector expressing LLGL2 and establish KYSE450 and TE-1 cell lines for the stable expression of LLGL2. Methods The full-length LLGL2 sequence was amplified by high-fidelity PCR, and then it was inserted into pCDH-CMV-IRES-GFP-EF1-Puro vectors. The recombinant plasmid was confirmed by double enzyme digestion and sequencing. After co-infection of pCDH-CMV-LLGL2-IRES- GFP-EF1-Puro with vesicular stomatitis virus glycoprotein (VSVG) and PHR into HEK293T cells, the lentivirus was harvested and used for infecting esophageal squamous cell carcinoma cell lines including KYSE450 and TE-1 cells. These two cell lines infected with the lentivirus were screened with puromycin, and the stable cell lines were further confirmed with green fluoresence and Western blotting. Results Dual-enzyme digestion and sequencing confirmed that the pCDH-CMV-LLGL2-IRES-GFP-EF1-Puro vector, a lentiviral expression vector for the overexpression of LLGL2, was successfully constructed through high-fidelity PCR and ligation. Western blotting showed the increased expression level of LLGL2 protein in KYSE450 and TE-1 stable cell lines compared with the controls. Conclusion The experiment successfully established KYSE450 and TE-1 stable cell lines for the overexpression of LLGL2.

Effects of Slit3 silencing on the invasive ability of lung carcinoma A549 cells.

Slit proteins function as chemorepellents in axon guidance and neuronal migration by binding to cognate Robo receptors. The Slit/Robo signaling pathway is also involved in the regulation of tumor cell metastasis. However, whether the Slit/Robo signaling pathway exerts prometastatic or antimetastasis functions remains controversial. To date, most of the research on Slit/Robo has focused on Slit2, and the effects of Slit3 on metastasis remain largely unknown. Based on the Oncomine database, overall expression of Slit3 is low in tumor tissues compared to its level in normal tissues. The underlying mechanism for slit3 silencing in tumor tissues is likely related to hypermethylation of the slit3 promoter. However, lung carcinomas appear to be an exception. Several studies have reported that the frequency of Slit3 methylation in lung cancers is far lower than the frequency of Slit2. In the present study, high Slit3 expression at the mRNA level, yet not at the protein level, was detected in lung adenocarcinoma A549 cells. The function of Slit3 in tumor migration and invasion was examined by silencing of Slit3 expression in A549 cells. Silencing of Slit3 promoted proliferation, migration and invasion of A549 cells and induced epithelial-mesenchymal transition by downregulation of E-cadherin and upregulation of vimentin. The inhibitory effects of Slit3 on tumor migration and invasion are likely related to matrix metalloproteinases (MMPs). Silencing of Slit3 in the A549 cells enhanced MMP2 and MMP9 expression. These results indicate that Slit3 is a potential tumor suppressor in lung adenocarcinoma.

[The development and clinical applications of automatic negative pressure gun for biopsy].

Using the new negative pressure biopsy technology, the automatic negative pressure gun and the specific puncture needle for the biopsy have been developed. Renal biopsies were conducted in 1136 cases, with the success rate being 99.9%, and 29 cases gross hematuria while the hepatic biopsies were conducted in 16 cases, mass biopsies in 3 cases with the success rates being 100% respectively. The biopsy gun has the advantages of easy manipulation, higher success rate and lower incidence of complications.