MiR-346 Mediates Tumor-derived Exosomes To Promote The Growth And Metastasis Of Cervical Cancer

[Objective]Exosomes are tiny vesicles with a diameter of 30-150 nm that are secreted by various cells.Exosomes contain various nucleic acids,proteins and enzymes.Exosome miRNAs are currently a hot spot in tumor research,and more and more studies have shown that exosome miRNAs also play an important role in the occurrence and development of tumors.Our previous experiments found that miR-346 can promote the malignant behavior and affect the occurrence and development of cervical cancer.This paper aims to explore the effects of exosomes with high expression of miR-346 on tumor cells themselves and Tumor associated stromal cells,the specific mechanism of exosomes entering receptor target cells,and how miR-346 exerts its cancer-promoting effect after entering target cells.[Methods] Firstly,exosomes of tumor cells were extracted and identified through various experimental methods.Then,we constructed cell line with stable expression of miR-346,and the expression of miR-346 in exosomes was detected by RT-q PCR.We established the co-culture model,and through the cell biology assays and in vivo animal assays to explore the functional role of exosomes with high expression of miR-346 in vivo and in vitro.Further,possible target genes of miR-346 were predicted by bioinformatics software analysis,and its targeting effect was verified by EGFP fluorescence reporting system.The regulatory effect of miR-346 on target genes was detected by Western blotting and RT-q PCR.The function of the target gene of miR-346 was explored by MTT assay,clone formation assay,Transwell migration and invasion assay,Western blot assay,RT-q PCR assay and ELISA assay.Then,we constructed cell line with stable expression of EGFP and CD63 fusion protein.Fluorescence localization assay verified that exosomes successfully entered receptor target cells.Furthermore,proteins from exosome with high expression of miR-346 were extracted for silver staining and mass spectrometry analysis to screen the protein molecules related to the exosome entry into the recipient cells.A large number of literature review and binding experiments were conducted to confirm that this molecule was CD44.Then Flag affinity purification and mass spectrometry were performed to screen the interaction proteins of CD44,and the detailed mechanism of exosome entry into recipient cells was finally determined by immunoprecipitation,immunofluorescence,Western blot,q PCR and ELISA assay.[Results] Exosomes with dual membrane structure,particle size of 30-150 nm in diameter and expression of CD63,CD9,TSG101 were successfully extracted.And it was determined that exosomes could successfully enter receptor target cells.Stable cell lines with overexpression of miR-346 were constructed,and the expression of miR-346 in exosomes was detected to be higher than that of the control group.Exosomes with high expression of miR-346 can promote the proliferation,migration and invasion of tumor cells,and promote the adhesion of tumor-related fibroblasts and the transformation of macrophages,which can promote the growth of heterotopic tumor and lung metastasis of tumor in mice.Then,we found that miR-346 can directly target and regulate SMAD4,SMYD3 and Bcl-6,In addition,we found that transmembrane protein CD44 on exosomes can interacted with HYAL2 and down-regulating its expression,thereby promoting receptor-ligand interaction between CD44 and HA and ultimately promoting exosome entry into receptor target cells.[Conclusions]We found that cervical cancer cells-derived exosomes were rich in miR-346,exosomes that overexpress miR-346 can promote exosomes to enter receptor target cells through the ligand action of CD44 and HA.Exosomes entering receptor target cells can release miR-346 to increase the expression of miR-346 in cells,and the highly expressed miR-346 can down-regulate the expression of SMAD4 in tumor cells or up-regulate the expression of SMYD3 and Bcl-6 in tumor-related stromal cells,ultimately promoting the growth and metastasis of tumors.In addition,in vivo animal studies have shown that exosomes with high expression of miR-346 can promote tumor growth and metastasis.In a word,exosomes,as carriers of various signaling molecules,play a key role in the occurrence,development and metastasis of tumors,and provide a new opportunity for the diagnosis and treatment of malignant tumors.