| BackgroundOvarian cancer(OC)is a common malignant tumor of the female reproductive system.Its morbidity ranks seventh among female malignant tumors worldwide,its mortality ranks eighth among cancer-related deaths,and it ranks first among gynecological tumor-related deaths.The 5-year survival rate is about 46 %,the recurrence rate is about 80%,and the prognosis is extremely poor.At present,the main plan of OC treatment is still surgery combined with chemotherapy.However,the resistance of chemotherapeutic drugs due to various factors during the chemotherapy process reduces the therapeutic effect of the drug,resulting in recurrence,metastasis and mortality of OC.Therefore,exploring the mechanisms and strategies to improve the sensitivity of OC chemotherapy is very important to improve the therapeutic effect of OC.Although angiogenesis of tumor tissue can maintain tumor growth,the incomplete structure,pressure occlusion and disorderly arrangement of tumor blood vessels lead to vascular dysfunction such as increased blood vessel wall permeability,insufficient blood perfusion,and increased interstitial hydraulic pressure.This affects the delivery and sensitivity of chemotherapeutic drugs and reduces the therapeutic effect of tumors.The normalization of tumor blood vessels can improve vascular dysfunction,make tumor blood vessels mature and stable,reduce the permeability,increase blood flow perfusion,increase the accumulation of chemotherapeutic drugs in tumor tissue,and increase the sensitivity of tumors to chemotherapeutic drugs,then improve the therapeutic effect of tumors.Therefore,the discovery of key regulators of tumor vessel normalization related to OC resistance is significant for the treatment of OC.MicroRNA is an important post-transcriptional regulatory molecule in the body.Existing research shows that micro RNA is closely related to tumor angiogenesis and chemotherapy resistance.Mi RNAs regulate angiogenesis at the post-transcriptional level by targeting m RNA degradation or translation inhibition,and its imbalanced expression may lead to abnormal angiogenesis.Therefore,micro RNA is an important source of therapeutic molecules related to OC vascular normalization.In this study,we took the search for key micro RNA molecules related to OC vascular normalization as the entry point,and used the ideal carrier for therapeutic nucleic acid delivery-exosomes,the establishment of an exosomal targeted drug delivery system carrying the gene of interest and its effectiveness was preliminary verified.It is significant for improving the sensitivity and prognosis of patients with OC.ObjectiveLooking for key molecules that regulate vascular normalization related to OC chemotherapy sensitivity,establish an exosomal targeted delivery strategy,establish a large-scale exosome production method,animal experiments explore its effect on improving the sensitivity of OC.Methods1.The RGD-Lamp2 b vector was constructed and transfected into HEK293 T cells.Exosomes were collected by Exo-quick kit,and identified by Western-blot,particle size analysis,and transmission electron microscopy(TEM).Observe the yield of exosomes by changing the time and intensity of ultrasound irradiation.QRT-PCR was used to detect the expression of genes related to exosomes synthesis and secretion,and to explore the mechanism of increased yield.2.Bioinformatics analysis of OC cell lines and normal ovarian epithelial cell lines and q RT-PCR were used to detect the expression of 10 miRNAs related to tumor angiogenesis,and to screen candidate miRNAs based on micro RNA expression cluster analysis,q RT-PCR was performed in OC and normal ovarian tissues for further verification.Screened miR484 may be an important candidate for OC angiogenesis.Transfection of miR-484 mimics directly and exosomes loaded with miR-484 mimics into HUVEC cells to increase the expression of miR-484,q RT-PCR was used to determine the transfection efficiency and the optimal electroporation parameters for miR-484 loading.Angiogenesis experiments were performed to analyze its effect on angiogenesis.3.Establish a nude mouse tumor-bearing model of OC,and injecting modified exosomes by RGD(Arginine-Glycine-Aspartic Acid)peptide that targeting tumor vessels loading with miR-484.Through blood vessel staining,α-SMA staining,and tail vein injection of FITC-Lectin,systematic observation of angiogenesis and vascular normalization-related indicators,establishing the effects of miR-484 targeted delivery by exosomes on blood vessels.The effect of the treatment of tumor growth and survival time of tumor-bearing mice was measured by combined therapy of exosomal targeted delivery and cisplatin.Results1.Targeting remodeling of exosomes by RGD peptides does not affect the morphology,particle size distribution,and expression of characteristic markers of exosomes.The tracer experiment found that compared with the control group,the remodeling of RGD peptide in HEK293 T cell-derived exosomes significantly enhanced the tumor tissue targeting of exosomes.However,the modification did not significantly enhance the targeting effect on exosomes derived from cells such as A2780 and SKOV3.Compared with the control,ultrasound irradiation can enhance the yield of exosomes,the increase of which is related to time and intensity,0.5W / cm2 irradiation for 60 min is the best,and its yield increased by 1.74 times.Increasing the yield of exosomes by ultrasound irradiation is closely related to changing the expression of exosomes synthesis and secretion-related genes.2.Differential analysis of angiogenesis-related micro RNAs of A2780 and IOSE-80 revealed the abnormal expression of multiple miRNAs,among which miR-484 expression was down-regulated most significantly,and OC tissue also verified that miR-484 expression was significantly down-regulated;Bioinformatics and dual luciferase reporter gene experiments confirmed that the target gene of miR-484 is VEGF-A.In vitro experiments on HUVEC cells found that VEGF-A expression was down-regulated after transfection with miR-484,and tubule formation experiments found that tubule formation was significantly reduced compared to the control group.3.After exosomes delivered miR-484 to OC tumor-bearing mice,qRT-PCR detected that miR-484 expression in tumor tissues increased.With the increase of miR-484 expression,the expression of its target gene VEGF-A decreased significantly,vascular density decreased,pericytes increased,blood flow perfusion increased,and local drug concentration increased;Chemosensitivity increased,tumor volume and weight decreased significantly,and prolonged survival.ConclusionmiR-484 is an important gene that inhibits angiogenesis and induces vascular normalization.RGD peptide engineered exosomes can effectively target tumor tissue blood vessels.Ultrasound irradiation can improve the yield of exosomes and produce exosomes on a large scale.The targeted delivery strategy of miR-484 based on reconstituted exosomes can improve the chemotherapeutic sensitivity of OC.In this study,while discovering new targets for tumor treatment,created new methods to increase targeting and yield of exosomes,that provides new ideas for the treatment of OC resistance and has potential clinical application value. |
