Study On The Mechanism Of MSC-mirna-198-exos Regulating MAPK1 In Malignant Pleural Effusion Associated With Non-small Cell Lung Cancer

Objective In this experiment,an animal model of MPE was established for the first time.Exosomes were used as the therapeutic vector.A high content of human umbilical cord derived msc-mirna-198-exos was constructed by retroviral transfection to study its effect on pleural invasion and the mechanism of msc-mirna-198-exos in NSCLC related MPE.Materials and methods 1.High content msc-mirna-198 was constructed by retroviral transfection of mirna-198 into MSC.The exosomes of mscmirna-198(msc-mirna-198 exos)and MSc exos(MSc exos)were extracted by differential centrifugation.The morphology of exosomes was observed by transmission electron microscope,and the concentration,particle size and phenotype of exosomes were determined by nanoparticle tracking analysis.2.The pleural effusion animal model of BALB / C nude mice was established.Based on the animal model,the effects of intrathoracic injection of MSC exos,msc-mirna-198 and phosphate buffer(PBS)on the volume of pleural effusion and the size of intrathoracic tumor were discussed.3.Based on the malignant pleural effusion model,the tumor tissues of MSC exos treatment group,msc-mirna-198-exos treatment group and PBS treatment group were collected.The preliminary mechanism of mirna-198 in inhibiting the formation of pleural effusion was clarified by immunohistochemical detection,microvessel density counting and ELISA detection of pleural supernatant,so as to verify the therapeutic effect of exosomes on NSCLC related MPE 4.Bioinformatics analysis and Western blot were used to explore whether overexpression of mir-198 can inhibit the activity of MAPK downstream of HGF / cMet pathway by inhibiting the expression of c-Met,so as to inhibit the invasion and migration of NSCLC and partially inhibit the occurrence of MPE.Experimental result 1.Compared with PBS group,the tumor tissue in MSC-miRNA-198-Exos group decreased significantly(MSC-miRNA-198-Exos group 138 ± 14.13,PBS group 326 ± 38.39;P = 0.0008),and the pleural fluid volume decreased(MSCmiRNA-198-Exos group 183 ± 26.11,PBS group 340 ± 38.70;P = 0.0087).The content of Evans blue in pleural fluid of MSC-miRNA-198-Exos group and MSC Exos group was lower than that of PBS group(MSC-miRNA-198-Exos group 32.67± 3.144,PBS group 69.05 ± 0.6188;P = 0.0033,MSC Exos group 38.23 ± 7.276,PBS group 69.05 ± 0.6188;P = 0.0076).MSC-miRNA-198-Exos could reduce the permeability of pleural blood vessels in nude mice.2.Immunohistochemistry and immunofluorescence of CD31 in tumor tissue of nude mice showed that the number of blood vessels in MSC Exos group was significantly lower than that in PBS group(MSC Exos group 14.39 ± 0.7066,PBS group 19.67 ± 1.501;P = 0.0087),and the number of blood vessels in MSC-miRNA-198-Exos group was less than that in PBS group(MSC-miRNA-198-Exos group 11.28 ± 0.8231,PBS group 19.67 ± 1.501;P =0.0002).The expression of vascular endothelial cell growth factor(VEGF)in mscMSC-miRNA-198-Exos group was significantly lower than that in PBS group(MSCmiRNA-198-Exos group 137.5 ± 4.554,PBS group 252 ± 39.01;P = 0.0317).3.Bioinformatics analysis showed that there was a correlation between miR-198 and MAPK1.The protein was extracted from the tumor tissue of nude mice after grinding.c-MET and ERK were detected by Western blot.It was found that the activities of cmet and p-ERK in MSC-miRNA-198-Exos group and MSC Exos group were significantly inhibited compared with PBS group.Conclusion 1.Intrapleural injection of msc-mirna-198-exos can significantly inhibit the growth of intrathoracic tumors and the formation of malignant pleural effusion.2.Exosomes are therapeutic vectors.Overexpression of mir-198 can inhibit the expression of c-met gene,inhibit the MAPK activity downstream of HGF / c-Met signaling pathway,inhibit the migration and invasion of NSCLC,reduce vascular permeability and inhibit the occurrence of MPE.3.Overexpression of mir-198 can inhibit the formation of MPE by inhibiting the proliferation,migration and invasion of A549 cells and reducing vascular permeability.The treatment of NSCLC is related to the secretion of NSCLC.