MiR-326@SPION Inhibiting Endometrial Cancer Stemc Ells Tumor Formation Under Magnetic Resonance Monitor

Aim:To investigate the feasibility of superparamagnetic nanoparticles(SPION)loaded with miR-326 as gene carrier to inhibit the growth of endometrial cancer stem cells in nude mice and to detect the tumor size in vivo by magnetic resonance imaging.Material and methods:The physical and chemical characteristics of SPION were measured by transmission electron microscope and particle size analyzer.The potential regulatory target gene of miR-326 were predicted and confirmed by bioinformatics tools and luciferase report assay,respectively.The expression vector for miR-326 and its mutation sequence vector(miR-mut)were loaded on SPION in vitro and transfected into human endometrial carcinoma type II tumor stem cells(HuECSCs).Two groups of cells were inoculated subcutaneously in nude mice10 weeks after injection mice were sacrificed.The weight and volume of the tumor tissues were measured.The aggregation of superparamagnetic ferrite nanoparticles and the ultrastructure of tumor cells were examined by transmission electron microscopy.H&E and immunohistochemical staining were used to evaluate the tumor characteristics and the activity of tumor cells.The mRNA and protein expression levels of Gpr91/STAT3/VEGF signal pathway were detected by real-time fluorescence quantitative PCR,Western blot and immunofluorescence staining,respectively.Results:The size of SPION core was between 2nm and 20nm.When the SPION surface was modified by PEI,it could efficiently combine with the plasmid DNA expressing miR-326 to form "nucleic acid/nanoparticles" complex.Through the analysis of bioinformatics software,we found that the mature miR-326 with seven bases on the specific site(+121bp～+127bp)of succinic acid receptor 1(SUCNR1/GPR91)could completely complement each other,suggesting that GPR91 may be one of the targets of miR-326.Luciferase Report assay analysis showed that wild-type mature miR-326 could inhibit the expression of luciferase protein by targeting the specific site of wild-type GPR91 gene 3’UTR,indicating that SUCNR1/GPR91 was one of the potential targets of miR-326.The results of MRI showed that the tumor tissue image of nude mice could be traced clearly,and the tumor tissues formed by miR-326@SPION-HuECSCs were significantly smaller than that formed by miR-Mut@SPION-HuECSCs.The results of H&E staining showed that the tumors formed by the two groups of cells were consistent with the pathological characteristics of human type II endometrial carcinoma.The results of immunohistochemistry showed that the positive rate of Ki67 protein in miR-326@SPION-HuECSCs was significantly lower than that in miR-Mut@SPION-HuECSCs.The results of transmission electron microscopy showed that in the tumor tissues formed by the two groups of cells,there were high-density electron clouds with round particles,and most of the high-density particles were in the phagocytic vesicles of the cells,suggesting that there were metal nanoparticles in the cells.Both the results of qPCR and Western blot showed that overexpression of miR-326 could significantly reduce the expression of GPR91/STAT3/VEGF pathway in HuECSCs,and also could reduce the activity of key factors in the pathway(phosphorylation modification level).Conclusion:SPION is an efficient nucleic acid nano carrier.It can inhibit the activation of GPR91/STAT3/VEGF signal pathway and significantly weaken the activity of endometrial cancer stem cells by loading miR-326.At the same time,MRI can be used to trace and evaluate tumor state in vivo.