The Effect Of Exosome-derived MiR221-3p On Angiogenesis In Cervical Squamous Cell Carcinoma

Cervical cancer is the third most common type of cancer in women worldwide and a leading cause of mortality among females in developing countries.While early-stage and locally advanced cancers can often be cured with the current standard treatment,metastatic or recurrent cervical cancer has limited treatment options and a high rate of mortality.Angiogenesis is essential for tumour progression and has shown great promise as a therapeutic target for the treatment of advanced cervical cancer.Currently,the addition of bevacizumab to combine chemotherapy improved median overall survival in patients with recurrent,persistent or metastatic cervical cancer.However,in unselected patient populations,the treatment with angiogenesis-targeted combination regimens is marred by variable responses,non-negligible toxicity and questionable economy.Therefore,the identification of alternative anti-angiogenetic factors or predictive biomarkers will be critical for future drug development.Within the tumour microenvironment(TME),angiogenesis is a complex process governed by a balance between pro-angiogenic factors and anti-angiogenic factors.During angiogenesis,transferring angiogenic factors and intercellular communication between the malignant and nonmalignant cells of the host are necessary.Exosomes,a group of extracellular 40-to 100-nm membrane vesicles secreted by multiple cells.including cancer cells,contain abundant genetic materials,such as mRNA and miRNA,and thus carry genetic messages.Cancer cell-derived exosomal miRNAs can be transferred into recipient cells to mediate intercellular communication and instruct biological processes,including angiogenesis.microRNAs(miRNAs)are a class of endogenous 22-to 25-nt non-coding single-stranded RNA molecules that function as major players of posttranscriptional gene regulation in diverse biological processes.Therefore,it is critical to understand the role of exosomal miRNAs in tumour angiogenesis due to their diagnostic and therapeutic potential to improve outcomes for cancer patients.Our previous studies have indicated that miR-221-3p was overexpressed in cervical squamous cell carcinoma(CSCC).And we also found that miR-221-3p was positively correlated with the vascular density.Even so,the mechanisms of miR-221-3p in cervical cancer progression and angiogenesis remain unknown and thus warrant further study.Here,we investigated the mechanisms of cancer cell-derived exosomal miR-221-3p in angiogenesis in CSCC and explore the potential clinical application in diagnosis and therapy.There are four parts as follow:Part ?:The relationship between the expression level of miR-221-3p and the density of blood vessels in cervical cancer.In situ hybridisation was used to detect the expression level of miR-221-3p in the samples of cervical cancer using the specific primers of microRNAs.IHC was used to mark the blood vessels by CD31 and accessed the density of blood vessels in the samples.We found that miR-221-3p was overexpressed in CSCC and there was a positive correlation between the vascular density and the H-score of miR-221-3p.These results suggest the likelihood that miR-221-3p is an angiogenesis-promoting miRNA in CSCC.Part ?:The effects of exosome-derived miR-221-3p on regulating angiogenesis in vitro.Firstly,exosomes were isolated from two typical CSCC cell lines,SiHa and C33a,and characterised by electron microscope analysis and western blot analysis.We found that miR-221-3p was enriched in exosomes,and exosomes were carriers transferred miR-221-3p into HUVECs.The exosomal miR-221-3p promoted the migration of HUVECs.Exosomal miR-221-3p induced more tube formation than the control groups.Exosomal miR-221-3p significantly facilitated HUVEC sprouting compared to the controls.Part III:The effects of exosome-derived miR-221-3p on regulating angiogenesis and tumour growth.The administration of SiHa-221-Exo and C33a-221-Exo for intra-tumour injection significantly promoted tumour growth.SiHa-221-Exo and C33a-221-Exo significantly promoted peritumoural vessels.Collectively,these results indicate that CSCC-secreted exosomal miR-221-3p significantly promoted tumour angiogenesis and facilitated tumour growth in return.Part IV:The downstream target of miR-221-3p.The miRWalk and PicTar databases were used to predict targets.We took advantage of Gene Ontology analyses to point out that 401 genes negatively regulated angiogenesis genes.And miRanda(http://www.microrna.org)was used to further identify the target sites of PDCD10.It was found that PDCD10 contains seed sequences and regions of potential base-pairing with miR-22 1-3p in their 3'UTRs.To confirm this speculation,a 1187-bp fragment of the 3'UTR region of PDCD10 mRNA that include the predicted miR-221-3p recognition site was subcloned and then inserted into a luciferase reporter plasmid.miR-221-3p mimic decreased the expression level of PDCD10 in mRNA and the protein level.In conclusion,the above results verify that miR-221-3p decreased the expression level of PDCD10 by directly binding to the 3'UTR region of PDCD10 and validate that PDCD10 is a direct downstream target of miR-221-3p.