Construction Of MiR-34a-loaded Nano-drug Delivery System And Its Regulation Of TAM For Ovarian Cancer By Sensitization And Targeting

Objective:Ovarian cancer is currently one of the most common clinical causes of death from malignancies in the female reproductive system,with a low 5-year survival rate.The tumor microenvironment plays an integral role in tumor development,in which tumor-associated macrophages have tumor-supporting properties.We aimed to prepare micro RNA-34a（miR-34a）-loaded nano-microspheres through a nano-drug delivery system and investigate their stability and mechanism of action on ovarian cancer and its tumor-associated macrophages in ovarian cancer to provide a new perspective for selective immunotherapy of ovarian cancer.Methods:Liposome-modified magnetic triiron tetroxide microspheres（CLs@Fe₃O₄）were synthesized chemically and characterized using UV spectrophotometer and Fourier transform infrared spectrometer.Agarose gel experiments were performed to explore the optimal binding ratio of miR-34a to CLs@Fe₃O₄.The uptake of miR-34a-loaded CLs@Fe₃O₄by human ovarian cancer SKOV3 and A2780 cells was observed by fluorescence microscopy,and the transfection efficiency of CLs@Fe₃O₄was determined by RT-q PCR.The expression levels of miR-34a in different ovarian cancer cells as well as in human normal ovarian epithelial cells ISOE-80 were quantified and clarified.The CCK-8 kit was applied to detect the toxicity of empty magnetic nanomicrospheres on ovarian cancer cells.Overexpression of miR-34a in ovarian cancer cells was detected by CCK-8 and plate clone formation assay on the proliferation ability of ovarian cancer cells;cell scratching and Transwell assay on the migration and invasion ability of ovarian cancer cells;flow cytometry to detect changes in apoptosis of ovarian cancer cells.CCK-8 assay to detect the toxicity of cisplatin and the effect of the combination with miR-34a on the survivability of ovarian cancer cells.A model of THP-1 differentiation into M1 and M2subtypes of macrophages was established.q RT-PCR was performed to detect the molecular markers of macrophages and their subtypes and the expression of miR-34a;fluorescence uptake assay and transfection efficiency assay were performed to verify the transfection of miR-34a into the cells.After co-culture of M2-type macrophages transfected with miR-34a with ovarian cancer cells,the effects on the biological functions of ovarian cancer cells were again determined by CCK-8,plate clone formation assay,cell scratch assay,Transwell assay,and flow apoptosis assay.In M2-type macrophages,miR-34a was overexpressed,and the expression levels of M2-type molecular markers as well as miR-34a were detected.miR-34a was transfected in SKOV3 and M2-type macrophages,and CCL5 and NF-κB levels were detected by PCR,respectively.Macrophages were stimulated with CCL5 stimulating factor,or transfected with si RNA CCR1,si RNA CCR5 followed by stimulation with CCL5,and NF-κB and p-NF-κB levels in macrophages were detected by Western blot.Results:1.Construction and Characterization of the Nanodrug Delivery SystemThe liposome-modified magnetic triiron tetroxide microspheres were successfully prepared.the average particle size of CLs@Fe₃O₄was305.50 nm,the average zeta potential was+23.33 m V,and the optimal mass ratio of miR-34a to CLs@Fe₃O₄binding was 1:3.2.The miR-34a-Containing Nanodelivery System Inhibits Ovarian Cancer ProgressionmiR-34a is extremely low in human ovarian cancer,while it is highly expressed in normal ovarian epithelial tissue.In ovarian cancer cells transfected with miR-34a,it was determined that CLs@Fe₃O₄as a nanocarrier could bring miR-34a into the cells well compared with the free drug by fluorescence uptake experiments and transfection efficiency experiments.The CCK-8 results showed that empty CLs@Fe₃O₄had a toxic effect on human ovarian cancer SKOV3 and A2780 cells and showed concentration-dependent and time-dependent effects,therefore,the CLs@Fe₃O₄concentration of 4μg/ml was selected to exclude the effect of the material on ovarian cancer cells.CCK-8 experiments showed that the effect of miR-34a on the proliferation of ovarian cancer cells did not significant changes,while the proliferation ability of ovarian cancer cells was significantly inhibited in the plate clone formation assay.The cell scratch assay and Transwell assay demonstrated that miR-34a could inhibit the migration and invasion ability of ovarian cancer cells.Flow cytometry assay detected that miR-34a promoted apoptosis in ovarian cancer cells,with late apoptosis occurring mainly in SKOV3 cells,while in A2780 cells apoptosis occurred mainly in the early stage.3.The miR-34a-Containing Nanodelivery System Increases Ovarian Cancer Sensitivity to Cisplatin In VitroWhether miR-34a affects the proliferative capacity of ovarian cancer cells was further determined by increasing cisplatin（DDP）using the CCK-8 method when selecting the appropriate DDP concentration alone or in combination with miR-34a.The toxicity of cisplatin to ovarian cancer increased with concentration and time in a concentration-dependent and time-dependent manner.The combination of the two inhibited the proliferative ability of ovarian cancer cells more potently,indirectly demonstrating the role of miR-34a.4.the Ability of the miR-34a-Containing Nanodelivery System to Inhibit Ovarian Cancer Progression by Modulating the TAM PhenotypeIn the exploration of ovarian cancer tumor microenvironment,human monocytes THP-1 could differentiate into different macrophage subtypes induced by different stimuli.In M2 macrophages,CD163 and CD206 expression levels were high,and in M1 macrophages,CD80expression was more.miR-34a expression was higher in M1 than in M2.CLs@Fe₃O₄could likewise be used as a vector to bring miR-34a into macrophages,and the transfection efficiency was high.When the transfected macrophages were co-cultured with ovarian cancer cells,the survival rate of ovarian cancer cells remained meaningless by CCK-8method,but the inhibition of proliferation ability was significant in the plate clone formation assay;cell scratching and Transwell assays confirmed that miR-34a inhibited ovarian cancer by affecting tumor-associated macrophages in terms of lateral migration,longitudinal migration and invasion ability,respectively.The flow cytometry assay showed that miR-34a had no significant effect on the apoptosis of ovarian cancer cells after co-culture;therefore,miR-34a mainly inhibited the migration and invasion of ovarian cancer by regulating tumor-associated macrophages.5.The Ability of the miR-34a-Containing Nanodelivery System to Regulate the TAM Phenotype by Targeting CCL5/NF-κB to Inhibit Ovarian Cancer ProgressionIn M2-type macrophages transfected with miR-34a,miR-34a expression increased and CD163 and CD206 expression levels decreased,indicating that miR-34a overexpression could promote the polarization of M2-type macrophages to M1-type macrophages.The levels of CCL5 and NF-κB were detected in human SKOV3 cells and M2-type macrophages transfected with miR-34a,respectively,and the levels of CCL5 were increased in SKOV3 and NF-κB levels were increased in M2.After direct stimulation of M0 macrophages with CCL5 stimulating factor for a certain time,NF-κB and p-NF-κB expression levels were increased,while CCR1 and CCR5 were silenced,and NF-κB and p-NF-κB expression levels were lower after CCL5 stimulation,especially si CCR5 was the most obvious.Thus,miR-34a promotes increased M1 polarization and decreased M2 polarization in macrophages through activation of intracellular NF-κB signaling pathway by CCL5-CCR1 or CCL5-CCR5.Conclusion:Liposome-modified magnetic triiron tetroxide microspheres are used as carriers with good deliverability.miR-34a is lowly expressed in human ovarian cancer cells,and increasing miR-34a expression can inhibit ovarian cancer proliferation,migration,and invasion,promote apoptosis,and exert anti-cancer effects.miR-34a can also act synergistically with cisplatin to inhibit the proliferative ability of ovarian cancer.miR-34a could inhibit the migration and invasive ability of ovarian cancer by regulating the tumor-associated macrophage phenotype,with less effect on proliferation and apoptosis.miR-34a could promote the transformation of tumor-associated macrophage（TAM）phenotype by targeting and regulating the CCL5/NF-κB pathway.These results conclusively suggest that miR-34a can significantly inhibit ovarian cancer progression by targeting CCL5 and activating the NF-κB signaling pathway in macrophages,directly or indirectly regulating the phenotypic changes of tumor-associated macrophages in the ovarian cancer microenvironment,providing a new idea for immunotherapy of ovarian cancer.