Subcellular trafficking of tubular MDM2 implicates in acute kidney injury to chronic kidney disease transition during multiple low-dose cisplatin exposure.

Acute kidney injury (AKI) is the leading cause of renal failure, and quite a few patients will advance to chronic kidney disease (CKD) in the long term. Here, we explore the roles and mechanisms of tubular epithelial cells (TECs) during repeated cisplatin (CP) induced AKI to CKD transition (AKI-CKD). Previously, we reported that murine double minute 2 (MDM2), an E3-ubiquitin ligase, is involved in tubulointerstitial fibrosis. However, whether tubular MDM2 is implicated in AKI-CKD is undefined. Currently, we confirmed that during AKI-CKD, MDM2 shifts from nucleus to cell membrane in TECs both in vivo and in vitro. Whereas regulating MDM2 distribution chemically or genetically has a prominent impact on tubular disorders. And then we investigated the mechanisms of the above findings. First, in the nucleus, repeated CP administration leads to MDM2 reduction with escalated p53 and cell cycle G2/M arrest. On the other hand, multiple CP treatment increases the level of membranous MDM2 with ensuing integrin ß8 degradation and TGF-ß1 activation. More interestingly, anchoring MDM2 on cell membranes can mimic the reduction of integrin ß8 arousing by repeated CP exposure. Collectively, our findings provided the evidence that tubular MDM2 subcellular shuttling is involved in AKI-CKD through p53-G2/M arrest and integrin ß8 mediated TGF-ß1 activation.

Integrin ß8 facilitates tumor growth and drug resistance through a Y-box binding protein 1-dependent signaling pathway in bladder cancer.

The transmembrane receptors integrins are the bridges for cell-cell or cell-ECM interaction, which is strictly correlated to cancer development in several tumor types. Here, we revealed that integrin ß8 serves as a driver to mediate sustained growth of bladder cancer and development of drug resistance. The elevated expression of integrin ß8 was observed in highly malignant bladder tumor tissues from patients. The in vitro and in vivo results further indicated that integrin ß8 overexpression in Biu87/T24 bladder cancer could mediate and strengthen cell proliferation and resistance to mitomycin C and hydroxycamptothecin. Mechanistically, integrin ß8 on the cellular surface might recruit phosphorylated Y-box binding protein 1, leading to the activation of c-Myc and nuclear factor-κB signals. Pharmacological targeting of integrin ß8 by Arg-Gly-Asp-Ser efficiently suppressed sustained growth and drug resistance in bladder cancer cells. Our findings identified integrin ß8 as a marker of bladder cancer diagnosis and development, and provides an innovative approach for clinical bladder cancer therapy.

[Investigation of Lentiviral Vectors Based Integrin ß8 RNAi System in Neonatal Rats' Brain].

OBJECTIVE: To construct lentiviral vectors expressing pSicoR-ß8 shRNA and evaluate its efficiency of RNA interference in neonatal rats' brain. METHODS: Plasmid vectors pSicoR-ß8 shRNA and pSicoR-control,as well as lentiviral packaging system pDM2G,g/p RRE and pRSV Rev were amplified respectively and plasmid DNA was identified by restriction enzyme digestion. Lentiviral packaging system and expressing vector pSicoR-ß8 shRNA/pSicoR-control were co-transfected into packaging cell line 293T. Lentiviral particles expressing ß8-shRNA or control sequence packaged and secreted by 293T were collected,concentrated by PEG-it,and viral titers were assayed by 50% tissue culture infective dose (TCID50). RNAi for integrin ß8 in neonatal rats' brain was performed by intraventricular injection of lentivirus expressing ß8-shRNA and rats received lentivirus expressing ß8-shRNA were served as control. Green fluorescent protein (GFP) expression after intraventricular injection of GFP-Lentivirus was observed under fluorescence microscope,ß8 mRNA and ß8 protein expression were detected by RT-PCR and Western blot respectively,all of which were performed to evaluate the RNAi efficiency and to choose the optimal time for intervention. RESULTS: Restrictive endonuclease digestion and agarose gel electrophoresis showed plasmids as same as the expected size. Lentiviral titers for LV-control after concentration was 1.0×108 PFU/mL,and for LV-ß8 shRNA 5.0×108 PFU/mL.One day after intraventricular injection of lentiviral vectors containing GFP sequence,lenticivirus genome was integrated into host cells and emitted green fluorescence. A relatively strong green fluorescence could be observed in brain slides 2 d,3 d and 5 d after intraventricular injection. Western blot and RT-PCR demonstrated a maximum inhibition happened 3 d after intraventricular injection of LV-ß8 shRNA,the inhibitory rate for ß8 mRNA and ß8 protein were 56% and 51%,respectively. CONCLUSION: Lentiviral vectors expressing ß8-shRNA are successfully constructed and lentiviral mediated ß8-RNAi is successfully applied for in vivo use.

Integrin ß8 prevents pericyte-myofibroblast transition and renal fibrosis through inhibiting the TGF-ß1/TGFBR1/Smad3 pathway in diabetic kidney disease.

Diabetic kidney disease (DKD) is one of the leading causes to develop end-stage kidney disease worldwide. Pericytes are implicated in the development of tissue fibrosis. However, the underlying mechanisms of pericytes in DKD remain largely unknown. We isolated and cultured primary pericytes and rat mesangial cells (HBZY-1). Western blot and qRT-PCR analysis were used to explore the role and regulatory mechanism of Integrin ß8/transforming growth factor beta 1 (TGF-ß1) pathway. We also constructed pericyte-specific Integrin ß8 knock-in mice as the research objects to determine the role of Integrin ß8 in vivo. We discovered that reduced Integrin ß8 expression was closely associated with pericyte transition in DKD. Overexpressed Integrin ß8 in pericytes dramatically suppressed TGF-ß1/TGF beta receptor 1 (TGFBR1)/Smad3 signaling pathway and protected glomerular endothelial cells (GECs) in vitro. In vivo, pericyte-specific Integrin ß8 knock-in ameliorated pericyte transition, endothelium injury and renal fibrosis in STZ-induced diabetic mice. Mechanistically, Murine double minute 2 (MDM2) was found to increase the degradation of Integrin ß8 and caused TGF-ß1 release and activation. Knockdown MDM2 could partly reverse the decline of Integrin ß8 and suppress pericytes transition. In conclusion, the present findings suggested that upregulated MDM2 expression contributes to the degradation of Integrin ß8 and activation of TGF-ß1/TGFBR1/Smad3 signaling pathway, which ultimately leads to pericyte transition during DKD progression. These results indicate MDM2/Integrin ß8 might be considered as therapeutic targets for DKD.

Baicalein Inhibits Metastatic Phenotypes in Nasopharyngeal Carcinoma Cells via a Focal Adhesion Protein Integrin ß8.

Baicalein, a prominent flavonoid from the indigenous herbal plant Scutellaria baicalensis Georgi, possesses broad-spectrum anticancer activities. However, the biological effects of baicalein on nasopharyngeal carcinoma (NPC) and its underlying mechanisms remain unclarified. Thus, in this study, we examined the effects of baicalein on NPC cell lines and investigated the corresponding molecular mechanism through transcriptome profiling. In the study, four NPC cell lines were treated with various concentrations of baicalein at different time points. Cellular toxicity and proliferative inhibition of baicalein were examined by MTT assay. Metastatic phenotypes of NPC cells were investigated by wound healing, transwell, and adhesion assays. Additionally, microarray experiments were performed to determine the cellular pathways affected by baicalein. The expression and localization of the integrin ß8 were validated by western immunoblotting and immunofluorescence. Our results revealed that baicalein exhibited its cytotoxicity and antiproliferative activity on all tested NPC cell lines. It also significantly inhibited metastatic phenotypes at sub-lethal concentrations. Transcriptomic analysis showed that baicalein significantly affected the focal adhesion pathway in NPC, where integrin ß8 was greatly diminished. Thus, the present study results suggested that baicalein inhibits the metastatic phenotypes of NPC cells by modulating integrin ß8, one of the major molecules in a focal adhesion pathway.

LncRNA TCF7 Promotes Epithelial Ovarian Cancer Viability, Mobility and Stemness via Regulating ITGB8.

This study aimed to investigate the carcinogenic role of long non-coding RNA T-cell factor 7 (lnc-TCF7) in epithelial ovarian cancer (EOC). Lnc-TCF7 overexpression and shRNA plasmids were transfected into SKOV3 and OVCAR3 cells, followed by measurement of cell proliferation, migration, invasion, apoptosis, stemness, and mRNA profile (via microarray). Besides, lnc-TCF7 expression was measured in tumor and adjacent tissues from 76 EOC patients. Lnc-TCF7 was upregulated in EOC cell lines; its overexpression increased cell proliferation, migration, invasion, but decreased apoptosis and promoted CD44, CD133 expressions, CD44+CD133+ cell proportion, spheres formation efficiency and drug resistance to cisplatin in SKOV3 and OVCAR3 cells. Besides, lnc-TCF7 ShRNA exhibited opposite effects comparing with its overexpression. Microarray analysis revealed 267 mRNAs were modulated by lnc-TCF7 dysregulation, among which ITGB8 was the most dysregulated one, which was validated by subsequent western blot and RT-qPCR. Furthermore, ITGB8 overexpression not only induced proliferation, migration, invasion and stemness, but also attenuated the effect of lnc-TCF7 ShRNA on these functions in SKOV3 and OVCAR3 cells. In addition, lnc-TCF7 was upregulated in tumor tissues and correlated with higher pathological grade, tumor size, International Federation of Gynecology and Obstetrics (FIGO) stage and worse overall survival in EOC patients. Conclusively, lnc-TCF7 regulates multiple oncogenic pathways, promotes proliferation, migration, invasion, stemness via upregulating ITGB8. It also correlates with advanced tumor features and poor prognosis in EOC, implying its potential as a target for EOC treatment.

Integrin ß8 Deletion Enhances Vascular Dysplasia and Hemorrhage in the Brain of Adult Alk1 Heterozygous Mice.

Brain arteriovenous malformation (bAVM), characterized by tangled dysplastic vessels, is an important cause of intracranial hemorrhage in young adults, and its pathogenesis and progression are not fully understood. Patients with haploinsufficiency of transforming growth factor-ß (TGF-ß) receptors, activin receptor-like kinase 1 (ALK1) or endoglin (ENG) have a higher incidence of bAVM than the general population. However, bAVM does not develop effectively in mice with the same haploinsufficiency. The expression of integrin ß8 subunit (ITGB8), another member in the TGF-ß superfamily, is reduced in sporadic human bAVM. Brain angiogenic stimulation results at the capillary level of vascular malformation in adult Alk1 haploinsufficient (Alk1 +/- ) mice. We hypothesized that deletion of Itgb8 enhances bAVM development in adult Alk1 +/- mice. An adenoviral vector expressing Cre recombinase (Ad-Cre) was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brain of Alk1 +/-;Itgb8-floxed mice to induce focal Itgb8 gene deletion and angiogenesis. We showed that compared with Alk +/- mice (4.75 ± 1.38/mm2), the Alk1 +/-;Itgb8-deficient mice had more dysplastic vessels in the angiogenic foci (7.14 ± 0.68/mm2, P = 0.003). More severe hemorrhage was associated with dysplastic vessels in the brain of Itgb8-deleted Alk1 +/- , as evidenced by larger Prussian blue-positive areas (1278 ± 373 pixels/mm2 vs. Alk1 +/-  : 320 ± 104 pixels/mm2; P = 0.028). These data indicate that both Itgb8 and Alk1 are important in maintaining normal cerebral angiogenesis in response to VEGF. Itgb8 deficiency enhances the formation of dysplastic vessels and hemorrhage in Alk1 +/- mice.

MicroRNA-17/20a functions to inhibit cell migration and can be used a prognostic marker in oral squamous cell carcinoma.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) accounts for>90% oral cancer which is a leading cause of cancer death worldwide. Early diagnosis may well offer an opportunity to increase survival to this neoplasm. Micro(mi)RNA-interfered cancer progression is crucial, yet its migration machinery of OSCC is still unknown. To access whether the possible miRNA prognostic markers and underlying mechanisms, we developed a highly migratory TW2.6 MS-10 cells from TW2.6 cells to investigate the issue. MATERIALS AND METHODS: miRNA profiling was performed on TW2.6 and TW2.6 MS-10. Target miRNA was correlated to pathological status in OSCC patients by real-time RT-PCR. A downstream effector was identified using a bioinformatics analysis, and a 3'-untranslated region (UTR) reporter assay was used. RESULTS: An miRNA cluster, miR-17-92, including miR-17, miR-19b, miR-20a, and miR-92a, was found to be significantly down-regulated in TW2.6 MS-10 compared to TW2.6 cells. Overexpression of this cluster decreased the migratory ability of OSCC cell lines. We further demonstrated that miR-17 and miR-20a are the main miRNAs of miR-17-92 cluster which modulate OSCC migration. Clinically, miR-17/20a showed negative correlation with TNM stage and lymphatic metastasis. Through a bioinformatics screening analysis and 3'UTR reporter assay, we confirmed the integrin (ITG) ß8 as a direct target of miR-17/20a, and knockdown of ITGß8 reduced cell migratory capability of OSCC. CONCLUSIONS: miR-17/20a acts as a prognostic predictor of OSCC patients' outcome and a tumor migration suppressor miRNA.