Proteomics identifies EGF-like domain multiple 7 as a potential therapeutic target for epidermal growth factor receptor-positive glioma

Background:Glioma,the most frequent primary tumor of the central nervous system,has poor prognosis.The epidermal growth factor receptor(EGFR)pathway and angiogenesis play important roles in glioma growth,invasion,and recurrence.The present study aimed to use proteomic methods to probe into the role of the EGF-EGFR-angiogenesis axis in the tumorigenesis of glioma and access the therapeutic efficacy of selumetinib on glioma.Methods:Proteomic profiling was used to characterize 200 paired EGFRpositive and EGFR-negative glioma tissues of all pathological types.The quantitative mass spectrometry data were used for systematic analysis of the proteomic profiles of 10 EGFR-positive and 10 EGFR-negative glioma cases.Consensusclustering analysis was used to screen target proteins.Immunofluorescence analysis,cell growth assay,and intracranial xenograft experiments were used to verify and test the therapeutic effect of selumetinib on glioma.Results:Advanced proteomic screening demonstrated that the expression of EGF-like domain multiple 7(EGFL7)was higher in EGFR-positive tumor tissues than in EGFR-negative tumor tissues.In addition,EGFL7 could act as an activatorin vitro and in vivo to promote glioma cell proliferation.EGFL7 was associated strongly with EGFR and prognosis.EGFL7 knockdown effectively suppressed glioma cell proliferation.Selumetinib treatment showed tumor reduction effect in EGFR-positive glioblastoma xenograft mouse model.Conclusions:EGFL7 is a potential diagnostic biomarker and therapeutic target of glioma.Selumetinib could target the EGFR pathway and possibly improve the prognosis of EGFR-positive glioma.

The expression of TGF-β_1,ADAM12 and HB-EGF in primary hepatic carcinoma

Objective: To detect the expression and location of TGF-β1, ADAM12 and HB-EGF in primary hepatic carcinoma and study their effect on the growth and metastasis of hepatoma carcinoma cell. Methods: TGF-β1, ADAM12 and HB-EGF were detected by RT-PCR and immunohistochemistry in 30 cases of hepatic carcinoma tissues, 30 cases of adjacent carci- noma tissues and 5 cases of normal hepatic tissues. Results: RT-PCR analyses showed that the mRNA expression of TGF-β1, ADAM12 and HB-EGF were markedly increased in each hepatic carcinoma tissue compared with its adjacent tissue (P < 0.01), but no signal was detected in normal hepatic tissue. Immunohistochemistry showed the same outcome on the expression of above three factors in hepatic tissues as RT-PCR. Proteins location analyses showed the proteins of TGF-β1, ADAM12 and HB-EGF all distributed in the stroma of hepatic carcinoma tissues. The positive correlation was found between TGF-β1 and ADAM12 (r = 0.6137, P < 0.05), as well as ADAM12 and HB-EGF (r = 0.5763, P < 0.05). The protein expression of TGF-β1, ADAM12 and HB-EGF were correlated with the size of tumors, degree of differentiation of hepatoma carcinoma cells, portal vein thrombus and the metastasis of absorbent glands, especially with hepatic cirrhosis caused by hepatitis B virus. Conclu- sion: TGF-β1, ADAM12 and HB-EGF possibly play an important role in the process of growth, invasion and metastasis of hepatoma carcinoma cell, meanwhile, the above three factors may collectively participate in the transition from hepatic cirrhosis caused by hepatitis B virus to hepatocellular carcinoma.

The expression of Hoxa10 and its regulation by sex steroids andHB-EGF at the time of implantation in the human endometrium

Objective:The aim of this study was to determine the expression of Hoxa 10 in endometrium during the menstrual cycle and their response to sex steroids and HB-EGF.Methods:Forty endometrial samples from regularly cycling women were studied.The endometrial epithelial(EEC)and stromal(ESC)cells isolated by collagenase Ⅰ digestion and mechanical dissociation was cultured from every sample.The endometrial stromal cells(ESC)were incubated with 17-beta estradiol(10-8mol/L),medroxyprogesterone acetate(MPA)(10-6mol/L),RU486(10-8mol/L)or HB-EGF(10ng/ml)for 48 hours respectively.The expression of Hoxa10 mRNA was demonstrated by reverse transcription-polymerase chain reaction(RT-PCR).Result:Hoxa10 showed a regulated cycle phase-dependent expression pattern in stromal cells and epithelial cells.Hoxa10 expression dramatically increased during the midsecretory phase of the menstrual cycle,at the time of implantation.The expression of Hoxa10 in cultured endometrial stromal cells was stimulated by estrogen,progesterone and HB-EGF.Conclusion:The cycle phase-specific expression of Hoxa10 and up-regulated by sex steroids and HB-EGF suggests a tight regulation and establishing conditions necessary for implantation.

Low-Molecular-Weight Heparin and Protamine-Based Polyelectrolyte Nano Complexes for Protein Delivery (A Review Articles)

We produced low-molecular-weight heparin/protamine micro (nano) particles (LMW-H/P MPs·NPs) as a carrier for heparin-binding growth factors (GFs), such as fibroblast growth factor (FGF)-2 and various GFs in platelet-rich plasma (PRP). A mixture of LMW-H (MW: approximately 5000 Da, 6.4 mg/ml) and protamine (MW: approximately 3000 Da, 10 mg/ml) at a ratio of 7:3 (vol:vol) yields a dispersion of micro (nano) particles (200 nm - 3 μm in diameter). The diluted LMW-H solution in saline (0.32 mg/ml) mixed with diluted protamine (0.5 mg/ml) at a ratio at 7:3 (vol:vol) resulted in soluble nanoparticles (approximately 100 nm in diameter). The generated NPs could be then stabilized by adding 2 mg/ml dextran (MW: 178-217 kDa) and remained soluble after lyophilization of dialyzed LMW-H /P NPs solution. The LMW-H/P MPs·NPs adsorb GFs, control their release, protect GFs and activate their biological activities. Furthermore, administration of GFs-containing F/P MPs·NPs exhibited significantly higher inductions of vascularization and fibrous tissue formation in vivo than GFs alone. LMW-H/P MPs·NPs can also efficiently bind to tissue culture plates and retain the binding of GFs. The LMW-H/P MPs·NP-coated matrix with various GFs or cytokines provided novel biomaterials that could control cellular activity such as proliferation and differentiation. Thus, LMW-H/P MPs·NPs are an excellent carrier for GFs and are a functional coating matrix for various kinds of cell cultures.

Hydroxysafflor Yellow A Promotes HaCaT Cell Proliferation and Migration by Regulating HBEGF/EGFR and PI3K/AKT Pathways and Circ_0084443

Objective:To investigate the effect and possible mechanism of hydroxysafflor yellow A(HSYA) on human immortalized keratinocyte cell proliferation and migration.Methods:HaCaT cells were treated with HSYA.Cell proliferation was detected by the cell counting kit-8 assay,and cell migration was measured using wound healing assay and Transwell migration assay.The mRNA and protein expression levels of heparin-binding epidermal growth factor(EGF)-like growth factor(HBEGF),EGF receptor(EGFR),phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT),mammalian target of rapamycin(mTOR),and hypoxia-inducible factor-1α(HIF-1α) were detected by quantitative real-time polymerase chain reaction(qRT-PCR) and Western blot,respectively.Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA.The expression of circ_0084443 was detected by qRT-PCR.Results:HSYA(800 μmol/L) significantly promoted HaCaT cell proliferation and migration(P<0.05or P<0.01).It also increased the mRNA and protein expression levels of HBEGF,EGFR,PI3K,AKT,mTOR and HIF-1α,and increased the phosphorylation levels of PI3K and AKT(P<0.05 or P<0.01).Furthermore,HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/m TOR signaling pathways(P<0.01).Circ_0084443 attenuated the mRNA expression levels of HBEGF,EGFR,PI3K,AKT,mTOR and HIF-1α(P<0.05).HSYA inhibited the circ_0084443 expression,further antagonized the inhibition of circ_0084443on HBEGF,EGFR,PI3K,AKT,m TOR and HIF-1α,and promoted the proliferation of circ_0084443-overexpressing HaCaT cells(P<0.05 or P<0.01).However,HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration(P>0.05).Conclusion:HSYA played an accelerative role in HaCaT cell proliferation and migration,which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways,and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.