[Hydnocarpin inhibits malignant progression of triple negative breast cancer via CNOT4-mediated ubiquitination and degradation of YAP].

This study aims to investigate the effect and mechanism of hydnocarpin(HC) in treating triple negative breast cancer(TNBC). Cell counting kit-8(CCK-8), xCELLigence real-time cellular analysis(RTCA), and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines: MDA-MB-231 and MDA-MB-436. The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis, wound-healing assay, and Transwell assay. The changes in the epithelial-mesenchymal transition(EMT) and the expression of invasion-and migration-associated proteins [E-cadherin, vimentin, Snail, matrix metalloproteinase-2(MMP-2), and MMP-9] were detected by Western blot. Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP) and downstream targets(CTGF and Cyr61). TNBC cells were transfected with Flag-YAP for the overexpression of YAP, and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay, Transwell assay, and wound-healing assay. The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay. The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4) was detected by microscale thermophoresis(MST) assay and drug affinity responsive target stability(DARTS) assay. The results showed that HC significantly inhibited the proliferation, colony formation, invasion, and EMT of TNBC cells. HC down-regulated the protein and mRNA levels of CTGF and Cyr61. HC down-regulated the total protein level of YAP, while it had no effect on the mRNA level of YAP. The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation, migration, and invasion of TNBC cells. HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP. The results of MST and DARTS demonstrated direct binding between HC, YAP, and CNOT4. The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.

Silencing of circDPP4 suppresses cell progression of human prostate cancer and enhances docetaxel cytotoxicity through regulating the miR-564/ZIC2 axis.

BACKGROUND: Circular RNA derived from dipeptidyl peptidase 4 (circDPP4; ID: hsa_circ_0056881) is one of the top increased circRNAs in prostate cancer (PC) and docetaxel (DTX)-based chemotherapy is the primary therapeutic choice for PC. However, its repertoire in PC development and chemoresistance remains to be documented. METHODS: Expression of circDPP4, microRNA (miR)-564, and zinc finger of the cerebellum 2 (ZIC2) was detected by a real-time quantitative polymerase chain reaction and western blotting; the direct interaction was validated by an RNA pull-down assay, a dual-luciferase reporter assay, and RNA immunoprecipitation. Cell progression was measured by a cell-counting kit-8 assay, colony formation assay, flow cytometry, a transwell assay, a xenograft experiment, and immunohistochemistry. DTX cytotoxicity was confirmed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. RESULTS: Expression of circDPP4 is upregulated in PC tumors from 60 patients and PC cell lines, and a higher circDPP4 might predict poor overall survival. Decreasing circDPP4 suppresses cell proliferation, colony formation, migration/invasion, and 50% inhibitory concentration of DTX in PC cells, and promotes the apoptosis rate. Both overexpressing miR-564 and inhibiting ZIC2 could imitate those effects, whereas inhibiting miR-564 and restoring ZIC2 could separately counteract them. Mechanistically, circDPP4 functions as a miR-564 sponge and regulates the expression of ZIC2, a target gene for miR-564. Tumor growth is retarded by silencing circDPP4, accompanied by elevated miR-564 and attenuated Ki-67 and ZIC2. CONCLUSIONS: Blocking circDPP4 antagonizes cell progression of PC and contributes to in vitro DTX cytotoxicity via regulating the miR-564/ZIC2 axis, at least. The present study suggests that circDPP4 is a potential biomarker and target for PC.

Rs6265 polymorphism in brain-derived neurotrophic factor (Val/Val and Val/Met) promotes proliferation of bladder cancer cells by suppressing microRNA-205 and enhancing expression of cyclin J.

BACKGROUND: In this study, we evaluated the effect of rs6265 polymorphism on the expression of brain-derived neurotrophic factor (BDNF) and relevant downstream targets, as well as the involvement of this polymorphism in bladder cancer. METHOD: A computational analysis and luciferase assays were used to explore the interaction among BDNF, miR-205, and cyclin J (CCNJ). Real-time polymerase chain reaction (RT-PCR) and Western blot analysis were carried out to determine the effect of rs6265 polymorphism on the expression of BDNF and relevant downstream genes. RESULT: BDNF directly inhibited miR-205 expression but enhanced the expression of CCNJ, which was identified as a virtual target gene of miR-205. Furthermore, the inhibitory effect of BDNF carrying the Val genotype, defined as BDNF (Val), on miR-205 expression was much stronger than that of BDNF (Met), while the inductive effect of BDNF (Val) on CCNJ expression was much weaker than that of BDNF (Met). miR-205 and CCNJ small interfering RNA (siRNA) were found to reduce cell proliferation and arrest the cells in G0/G1 phase. In addition, miR-205 expression in patients carrying BDNF genotyped as Met/Met (defined as Met/Met group) was much higher than patients carrying BDNF genotyped as Val/Val and Val/Met (defined as Val/Val group and Val/Met group). As an inhibitor of CCNJ expression, the inhibitory effect of miR-205 was much higher in the Met/Met group than that in the Val/Val and Val/Met groups. CONCLUSION: In summary, we suggested that the rs6265 polymorphism in BDNF upregulates the expression of CCNJ in bladder cancer via the inhibition of miR-205 expression, which leads to the promoted proliferation of bladder cancer cells.