A Study On The Effect And Mechanism Of Peptide-engineered Exosomes With Overexpressed MiR-92b-3p On Anti-angiogenesis In Ovarian Cancer

Ovarian cancer is a malignant tumor with the highest fatality rate among women,and most patients are already in the advanced stage(stage Ⅲ-Ⅳ)at the time of diagnosis.At present,the main treatment for ovarian cancer is still a combination of surgery and platinum-based chemotherapy,but most of patients have recurrence or metastasis after the initial treatment,and appear to be resistant to chemotherapy.Therefore,finding new chemotherapy methods is one of the research directions in the treatment of ovarian cancer.The tumor microenvironment(TME)is closely related to the occurrence and development of tumors.Tumor-associated angiogenesis has been recognized as an essential component in tumor microenvironment that can be controlled by cancer cells.In the last decade,anti-angiogenic agents,including antibody and small molecule showed the promise as therapeutics in solid tumors.Apatinib,as a novel receptor tyrosine kinase inhibitor of vascular endothelial growth factor receptor 2(VEGFR2)with a high specificity,has been clinically used in cancer anti-angiogenic therapy.Some clinical trials showed the effect of Apatinib in recurrent ovarian cancer,but the evidence that the application of Apatinib in newly diagnosed ovarian cancer is lacking.Exosome,as a vesicle between 30 nm to 100 nm in diameter,enriches in miRNAs,proteins,lipids,and others and acts as a mediator of intercellular communication.MicroRNA(miRNA)is small non-coding RNA molecule and typically comprised of approximately 20 nucleotides.MiRNAs occupy a large proportion in the exosomal components.Previous studies showed the role of exosomal miRNAs in promoting tumor angiogenesis,but the potential of exosomal miRNAs as anti-angiogenic agents in cancers is still completely unclear up to date.Exosomal miRNA as a mediator of intercellular communication plays a role in tumor-associated angiogenesis,and therapy against angiogenesis has been demonstrated to have a remarkable anti-tumor efficacy in various malignancies,but not as expected in ovarian cancer.Here,we found that ovarian cancer cells derived exosomes promoted the angiogenesis and migration viability of vascular endothelial cells in vitro and in zebrafish models.We further identified that miR-92b-3p expression was lower in ovarian cancer cell derived exosomes than that in immortalized ovarian epithelial cell derived exosomes through exosomal miRNA-sequencing and found by gene function experiment that exosomal miR-92b-3p modulated tumor-associated angiogenesis via targeting SOX4.Besides,we verified exosomes with overexpressed miR-92b-3p to produce antiangiogenic and anti-tumor effects and a synergetic inhibition with Apatinib in vitro and in zebrafish and nude mouse models.Furthermore,we generated a peptide-engineered exosome with overexpressed miR-92b-3p that specifically targets vascular endothelial cells,and found its stronger anti-angiogenic and anti-tumor effects,whether alone or combined with Apatinib,than that of parental exosomes in nude mouse models.Our findings demonstrate the effect and mechanism of exosomal miR-92b-3p from ovarian cancer cells on tumor-associated angiogenesis and the potential of artificially generated exosomes with overexpressed miR-92b-3p to be used as a anti-angiogenic agent,which may provide a new approach for anti-angiogenic therapy of ovarian cancer.Objective:To explore the different effects on tube formation and migration of vascular endothelial cells between different exosomes derived from normal ovarian epithelial cells or ovarian cancer cells,as well as the effects of angiogenesis in Tg(fli-1:EGFP)zebrafish.Methods:First,ultracentrifugation was used to extract the exosomes derived from normal ovarian epithelial cell line(IOSE-80)and ovarian cancer cells SKOV3 and A2780,and the exosomes were identified by transmission electron microscopy and western blotting.Second,a confocal microscope was used to observe the process of the uptake of PKH-67 green fluorescently labeled exosomes by human umbilical vein endothelial cells(HUVECs).Third,after co-culturing HUVECs with different exosomes for 48 hours,the tube formation assay and transwell assay were used to detect the effect of exosomes on the tube formation and migration abilities of HUVECs.Finally,through micro-injection,the changes of angiogenesis in Tg(fli-1:EGFP)zebrafish treated with exosomes were observed.Results:1.Ultracentrifugation could effectively extract exosomes secreted by immortal ovarian epithelial cells(IOSE-80)and ovarian cancer cells SKOV3 and A2780.2.Exosomes could be taken up by vascular endothelial cells.3.Compared with exosomes derived from normal ovarian epithelial cells(IOSE80/exo),exosomes derived from ovarian cancer cells(SKOV3/exo,A2780/exo)could significantly promote the tube formation and the migration abilities of HUVECs in vitro.4.Compared with exosomes derived from normal ovarian epithelial cells(IOSE80/exo),exosomes derived from ovarian cancer cells(SKOV3/exo,A2780/exo)can significantly promote angiogenesis in Tg(fli-1:EGFP)zebrafish.Conclusions:Compared with exosomes derived from immortalized ovarian epithelial cells,exosomes derived from ovarian cancer cells have a stronger ability to promote angiogenesis.Part Ⅱ Exosomal mir-92b-3p regulates angiogenesis via targeting SOX4Objective:To explore the differentially expressed and functional miRNAs in exosomes derived from normal ovarian epithelial cells and exosomes derived from ovarian cancer cells and determine the downstream genes and functions regulated by the miRNA and the effects of exosomes carrying the specific miRNA.Methods:First,exosomal miRNA-sequencing used to identify the differential expressed miRNAs in IOSE-80/exo vs SKOV3/exo or A2780/exo.The miRNAs with significant differences in expression(|Log2(Fold Change)|>1.5)and large expression levels(expression level>100)in the sequencing were verified by qRT-PCR.SKOV3 cell line that stably overexpressed miR-92b-3p were constructed by lentivirus to obtain exosomes with more miR-92b-3p(SKOV3-92b/exo).In vitro,the effects of miR-92b-3p and SKOV3-92b/exo on anti-angiogenesis were analyzed by tube formation assay and migration assay.The anti-angiogenic effects in vivo were assessed in Tg(fli-1:EGFP)zebrafish models.Secondly,after predicting the target genes of miR-92b-3p by Targetscan,miRTarbase,miRDB and miRWalk software,SRY-Box Transcription Factor 4(SOX4)was selected as a possible target gene of miR-92b-3p,and the regulatory relationship between miR-92b-3p and SOX4 was verified by qRT-PCR,western blotting,dual-luciferase reporter assay and rescue experiments.Finally,small RNA interference and plasmids overexpression were used to down-regulate or up-regulate the expression of SOX4 in vascular endothelial cells,and the effect of SOX4 on angiogenesis in vitro was also tested by the tube formation assay and migration assay.Results:1.Compared with exosomes derived from normal epithelial cells,the expression of miR-92b-3p in exosomes derived from ovarian cancer cells was significantly reduced.2.HUVECs with miR-92b-3p overexpression showed significantly the weakened abilities of tube formation and migration,whereas HUVECs with miR-92b-3p inhibition presented significantly the enhanced abilities of tube formation and migration.Fluorescence microscope showed remarkably decreased sprouting and vascular endothelial cell migration in 48 hpf zebrafish with miR-92b-3p mimics injection compared to those with NC mimics.3.Tube formation assay and migration assay showed that angiogenesis and migration of HUVECs treated with SKOV3-92b/exo were inhibited compared those with SKOV3/exo;zebrafish embryos injected with SKOV3-92b/exo also presented less newly formed blood vessels compared to those with SKOV3/exo.4.Predictions of bioinformatics software and results of dual luciferase report experiment proved that miR-92b-3p could regulate SOX4 by targeted the 3’-UTR region of SOX4 gene mRNA.qRT-PCR analysis illustrated the significantly decreased expression of SOX4 mRNA,and western blotting assay showed significantly decreased expression of SOX4,in HUVECs with miR-92b-3p mimics,vice versa.5.Tube formation and migration assays of HUVECs were inhibited after the overexpression of miR-92b-3p.This inhibition could be rescued by co-transfection with SOX4 overexpression plasmids.6.Overexpression of SOX4 could promote the tube formation and migration abilities of HUVECs in vitro,and further promote the expression of Endothelin-1 and the activation of p-Akt/Akt pathway;vice versa.Conclusions:1.Compared with exosomes derived from normal ovarian epithelial cells,the expression of miR-92b-3p is significant lower in exosomes derived from ovarian cancer cells.2.Exosomes with overexpressed miR-92b-3p can inhibit the angiogenesis of ovarian cancer.3.SOX4,as a direct target gene of miR-92b-3p,participates in miR-92b-3p’s regulation of ovarian cancer angiogenesis.Part Ⅲ Effects of DSPE-PEG2K-RGD engineered exosomes carrying miR-92b-3p on ovarian cancerObjective:To explore the possibility of combining exosomes carrying miR-92b-3p with antitumor angiogenesis drugs on anti-tumor and anti-angiogenesis effects,and construct exosomes modified by DSPE-PEG2K-RGD to enhance their ability of targeting to the vascular endothelial cells.Methods:First,CalcuSyn software was used to calculate the drug combination index(CI)of miR-92b-3p and Apatinib and zebrafish models were used to verify.Second,the antiangiogenetic effect of the combination of SKOV3-92b/exo and Apatinib was verified in vitro through tubule formation assay and migration assay.Next,we co-cultured DSPEPEG2K-RGD with SKOV3-92b cells that stably overexpressed miR-92b-3p,thereby obtaining a new kind of exosomes with DSPE-PEG2K-RGD modification on the exosomal membrane with miR-92b-3p(RGD-SKOV3-92b/exo).The difference in the uptake speed of PKH-67-labeled SKOV3-92b/exo and RGD-SKOV3-92b/exo was observed by a confocal microscope.Finally,SKOV3 cells(SKOV3-Luc cells)that stably transduced with luciferase plasmids were used to construct nude mice model with abdominal tumors.Mice were then injected with different exosomes through tail vein every 3 days,according to grouping,respectively and Apatinib were administrated orally.We evaluated the effects of different treatments on tumor growth by the in vivo imaging system(IVIS)Lumina LT system under anesthesia.Finally,the micro vessels density of abdominal tumors in nude mice was calculated by immunohistochemical staining of CD31.Results:1.In vitro,when the concentration of miR-92b-3p is 5 nM and Apatinib is 50 nM,the synergistic inhibition effect on the tube formation and migration ability of HUVECs was the strongest.In addition,zebrafish embryos treated with Apatinib(1 μM)plus miR92b-3p mimics for 48 hours demonstrated remarkably stronger angiogenetic inhibition compared to those treated with Apatinib or miR-92b-3p mimics alone.2.Tube formation assay and migration assay of HUVECs treated with combined SKOV3-92b/exo and Apatinb(50 nM)were significantly more inhibited compared with those with SKOV3-92b/exo and Apatinb alone.3.Confocal microscopy showed that the captured amount of RGD-SKOV392b/exo were more than that of SKOV3-92b/exo in HUVECs.4.IVIS showed that the fluorescence amount in SKOV3-92b/exo or RGD-SKOV392b/exo plus Apatinib group presented further significantly decreased than that in SKOV3-92b/exo or RGD-SKOV3-92b/exo alone group.Conclusions:1.MiR-92b-3p and exosomes loaded with miR-92b-3p both have a strong synergistic anti-angiogenesis effect with Apatinib.2.Modification of exosomes by DSPE-PEG2K-RGD can effectively improve the efficiency of HUVEC uptake of exosomes by vascular endothelial cells.3.DSPE-PEG2K-RGD modified engineered exosomes loaded with miR-92b-3p can produce a combined anti-tumor and anti-angiogenetic effect with Apatinib in nude mice abdominal tumor models.