DEPDC1B is a Novel Direct Target of B-Myb and Contributes to Malignant Progression and Immune Infiltration in Lung Adenocarcinoma.

BACKGROUND: Lung cancer is the primary cause of cancer-related deaths, with one of the highest incidence and mortality rates of all malignant tumors. Dysregulated expression of DEPDC1B has been reported to occur in various tumor types. However, the functional implications of this alteration in lung adenocarcinoma (LUAD) and the underlying molecular mechanism remains unclear. In this study, we investigated the role and clinical significance of DEPDC1B in LUAD. METHODS: The expression of DEPDC1B in LUAD and its relationship with prognosis were systematically evaluated in several publically available datasets. The effects of DEPDC1B knockdown on the proliferation and motility of LUAD cells were assessed using the JULI Stage Real-time Cell History Recorder, while the effect of knockdown on the cell cycle was studied by flow cytometry. Furthermore, RNA-Sequencing (RNA-Seq) analysis was conducted to identify the downstream target genes and pathways regulated by DEPDC1B. Correlations between the expression of DEPDC1B and immune cell infiltration, immunotherapy resistance, and chemoresistance were also examined. Additionally, molecular biological methods were used to explore the regulatory mechanism of B-Myb on DEPDC1B expression. RESULTS: DEPDC1B was found to be upregulated in LUAD patients and this was associated with poor clinical outcomes. Knockdown of DEPDC1B inhibited cell growth, migration and motility, as well as cell cycle progression. Knockdown also resulted in the down-regulation of several downstream genes, including NID1, FN1, and EGFR, as well as the inactivation of multiple critical pathways, such as the ERK and PI3K-AKT pathways. Analysis of the tumor immuno-environment in LUAD revealed that high DEPDC1B expression was associated with an abundance of activated CD4+ memory T cells, M0 macrophages, M1 macrophages, and CD8+ T cells. Moreover, these tumors responded poorly to immunotherapy. Analysis of chemo-drug sensitivity showed that LUADs with high DEPDC1B expression were more responsive to frontline chemotherapeutic drugs such as Vinorelbine, Cisplatin, and Etoposide. Additionally, mechanistic investigations revealed that DEPDC1B is a direct target gene of B-Myb, and that its knockdown attenuated the proliferation and motility effects of B-Myb. CONCLUSIONS: In summary, our findings indicate that DEPDC1B is a critical regulator during the malignant progression of LUAD. DEPDC1B could therefore be a promising prognostic marker and therapeutic target in LUAD diagnosis and treatment.

[Inhibition of proliferation in MCF-7 breast cancer cells by plasmid-based siRNA targeting to oncogene c-myc].

OBJECTIVE: To investigate the effects of plasmid-based siRNA targeting to oncogene c-myc on c-myc/ c-Myc expressions and cells proliferation in MCF-7 breast cancer cells. METHODS: siRNA eukaryotic expression plasmid p-Mat01-1 targeting to the sequence 589-609 of oncogene c-myc and its mismatch plasmid p-Mis09-1 were constructed, and transiently transfected MCF-7 cells using Lipo2000. Semi-quantitative RT-PCR and Western blot were used to analyze the expressions of c-myc/c-Myc in MCF-7 cells, and cells proliferation was detected by MTT assay. RESULTS: p-Mat01-1 inhibited the expressions of c-myc mRNA (24 h: P < 0.01) and c-Myc protein (5 d. P < 0.01) in MCF-7 cells as compared with pEGFP-C1 and p-Mis09-1 controls, and suppressed the proliferation of MCF-7 cells significantly (3 d: P < 0.05, 5, 7 d: P < 0. 01). CONCLUSION: Plasmid-based siRNA targeting to oncogene c-myc could inhibit the expressions of c-myc/c-Myc in MCF-7 breast cancer cells efficiently, suggesting that the downregulation of c-myc/c-Myc could suppress the proliferation of MCF-7 cells in vitro.

[Construction and expression of anti-human AFP ScFv gene in BL-21 (DE3) E. coli].

OBJECTIVE: To construct anti-human AFP single chain fragment variable (ScFv) gene, transform it into BL-21 (DE3) E. coli for expression, and identify its bioactivity. METHODS: VH and VL genes of anti-human AFP monoclonal antibody were cloned by RT-PCR from hybridoma. The ScFv gene was spliced by sequence overlap extending (SOE) PCR, and then it was ligated into pGEM-T vector to be identified by endonuclease digestion, PCR and sequencing. ScFv gene was cloned into pET32 (a+) vector and transformed into BL-21 (DE3) E. coli. The positive clones were screened out by IPTG induction, and the ScFv antibody was purified to be identified by SDS-PAGE and competitive inhibition ELISA test. RESULTS: The VH DNA consisted of 339 bases, coming from the mouse IgG gamma chain. The VL DNA consisted of 312 bases, coming from the mouse IgG kappa chain. The VH and VL genes were spliced by 45 bases coding a (G4S)3 flexible linker. The ScFv gene consisted of 696 bases. The ScFv antibody expressed by BL-21 (DE3) fused with TrxA tag protein and formed inclusions. The relative molecular mass of TrxA-ScFv fusion protein is about 40 x 10(3) and that of ScFv is about 24 x 10(3). The ScFv antibody has excellent activity tested by competitive inhibition ELISA, the TrxA-ScFv could inhibit about 41% of the McAb to bind antigen and ScFv could inhibit about 53%. CONCLUSION: We have successfully constructed an anti-human AFP ScFv gene with 696 bases; it can express in BL-21 with high activity.

[A change in antigen presentation ability of spleen macrophage from Balb/c mouse during late pregnancy].

OBJECTIVE: To study the antigen presentation ability of spleen macrophage from Balb/c mouse during late gestation. METHODS: The spleen of Balb/c mouse during late gestation was collected aseptically and macrophage was separated; the mice in estrus were used as control. The ability of macrophage to prime original T cells to human gamma globulin antigen (HGG-Ag) or placenta antigen (Pla-Ag) was evaluated by a delayed-type hyper sensitivity (DTH) response induced by the same antigen, and the ability of macrophage to induce the proliferation of primed normal T cells to HGG-Ag or Pla-Ag was assessed by an antigen special lymphocyte transformation in vitro. RESULTS: After being sensitized previously by spleen macrophage from Balb/c mice during late gestation that has been pulsed by HGG-Ag or Pla-Ag, the DTH intensity of mice response to the same antigen was significantly weaker than that being sensitized by macrophage from mice in estrus (P < 0.05). When spleen macrophage from Balb/c mice during late gestation was used as antigen presentation cell (APC), the proliferability of primed T cell induced by HGG-Ag or Pla-ag was significantly lower than that when macrophage from estrous Balb/c mouse was used in vitro (P < 0.05). CONCLUSION: The antigen presentation ability of spleen macrophage from Balb/c mouse is inhibited during late pregnancy. This may be the important mechanism by which maternal immune system tolerates embryo antigen and may be responsible for the down-regulation of maternal cell mediated immunity during pregnancy.

[Inhibition of constitutively activated Jak3 and induction of apoptosis in NALM-6 cell line].

OBJECTIVE: To investigate the relation of Jak3 constitutive activation and acute lymphoblastic leukemia(ALL). METHODS: NALM-6 cells were treated with varying concentrations of AG490, a Jak3 inhibitor. Apoptosis and proliferation of NALM-6 cells were tested by flow cytometry (FCM) analysis and MTT assay. RESULTS: With the exception of AG490 5 mumol/L, the AG490 10, 15, 20 mumol/L induced a strong apoptotic response in NALM-6 cells by FCM analysis(P < 0.05) and significantly inhibited the proliferation of NALM-6 cells by MTT assay(P < 0.05). All of the effects were dose-dependent. CONCLUSION: Jak3 inhibitor AG490 can inhibit proliferation and induce apoptosis in NALM-6 cells, and Jak3 activation is associated with pre-B ALL.