Visualization Of Microrna Therapy In Gastric Cancer Delivered By Engineered Small Extracellular Vesicles

Objective:Globally,Gastric cancer(GC)has been one of the most common cancers and the third most common causes of cancer death.At present,anticancer treatment represented by surgery is the cornerstone of clinical treatment.For patients with no chance of radical surgery or metastatic gastric cancer,radiotherapy and chemotherapy are usually used as adjuvant treatment.Although it has significant anticancer effect,long-term use of radiotherapy and chemotherapy leads to systemic side effects and drug resistance of patients.Therefore,the introduction of gene therapy is hopeful to solve the bottleneck of current clinical cancer treatment.At the same time,it is of great significance to innovate drug delivery methods to improve the efficacy and organ targeting of gene drugs.Small extracellular vesicles(sEVs)are a new type of drug delivery system with great potential that has emerged in recent years.Compared with delivery systems such as nanoparticles or viruses,sEVs are natural nanoscale membrane vesicles with high stability and biocompatibility,low immunogenicity,and no cytotoxicity.sEVs have provided a new drug delivery strategy for the treatment of diseases.Therefore,this study aims to build a kind of delivery platforms based on sEVs for tumor targeted delivery of small nucleic acid drugs.In order to deeply explore the feasibility of the project’s transformation and application,GQDs/Cy5-miR@sEVs has been carried out in the GC animal model(subcutaneous tumor model),the biosafety and anticancer effect of this sEVs-based drug delivery system will be evaluated.Methods:MiR-193a-3p has a clear inhibitory effect on gastric cancer.We selected miR-193a-3p as the gene drug for the treatment of gastric cancer,and the miRNA target gene prediction database Targetscan Human8.0(http://www.targetscan.org),miRDB(http://www.mirdb.org)and Pic Tar(http://www.pictar.org)were used to predict its target mRNA.The possible target genes regulated by miR-193a-3p were detected and named as the target gene prediction data set.TCGA database(https://portal.gdc.cancer.gov/)and GTEx database(https://www.genome.gov/Funded-Programs-Projects/Genotype-TissueExpression-Project)were used to analyze the expression distribution of the genes in the above target gene prediction data set in gastric cancer patients’ tissues and healthy controls,and the mRNAs that significantly highly expressed in GC tissues were selected as gene sets.Then the data sets in the GEO gene expression database(GSE77229)and the KEGG signal pathway enrichment analysis were carried out to obtain the relevant information analysis on the microarray data results of mRNAs in gastric cancer tissues and healthy control mRNAs.The set of genes related to cell cycle signal pathway significantly regulated by miR-193a-3p was outputed.The gene set was intersected with the cell cyclerelated gene set,and the obtained gene was finally used as the target gene of the research object.Quantitative real time polymerase chain reaction(q RT-PCR)was applied to validate the differential expression of candidate genes in gastric cancer cell lines and normal gastric mucosal epithelial cell lines,and the gene with the largest difference was selected as our research target gene.Subsequently,Cy5-miRNA or FAM-miRNA were co-incubated with graphene quantum dots(GQDs)to prepare miRNA-loaded nanosensor GQDs/miR,and their particle size and surface potential were identified and analyzed.In order to load GQDs/Cy5-miR nano-sensors into sEVs,the optimizing conditions for preparing engineering sEVs by ultrasonic method were selected by observing the morphology and particle size distribution of sEVs under different sonication conditions.Successfully prepared GQDs/Cy5-miR@sEVs were detected by q RT-PCR.The actual encapsulation efficiency of miRNA in GQDs/Cy5-miR and GQDs/Cy5-miR@sEVs were detected.We studied the process of GQDs/Cy5-miR@sEVs regulating tumor promoting target genes in vitro and its inhibitory effect on the proliferation,migration,and invasion of gastric cancer cells.After that,we injected Cy5-miR,GQDs/Cy5-miR,GQDs/Cy5-miR@sEVs into tumor-bearing animals through the tail vein,and observed GQDs/Cy5-miR@sEVs in vivo distribution through small animal live imaging system.We also explored the action time of GQDs/Cy5-miR@sEVs by the fluorescent “OFF-ON effect”in vivo.To study the tumor inhibition ability of GQDs/Cy5-miR@sEVs,we recorded the tumor growth curve of GQDs/Cy5-miR@sEVs after the different groups’ intervention of gastric cancer bearing animals.The antitumor effect was evaluated by TUNEL fluorescence staining and immune histochemistry of Ki-67.The toxic side effects of GQDs/Cy5-miR@sEVs were evaluated by HE staining of tissue sections of the heart,liver,spleen,lung and kidney of the experimental animals,as well as detection of liver and kidney function by serum samples.Results:A series of public database screening and bioinformatics analysis were conducted to screen the target gene of miR-193a-3p.We selected cyclin D 1(CCND1)as the final target gene of miR-193a-3p for follow-up research.We successfully constructed GQDs/Cy5-miR and GQDs /FAM-miR,with an average particle size of 3～4 nm.Every 20 μg GQDs carried about 100 pmol of Cy5-miR and every 60 μg GQDs carried about 100 pmol of FAM-miR.The surface potential of GQDs/Cy5-miR was(11.3 ± 0.2)m V.The target gene sequence fragment could rapidly combine with the miRNA in the nano-sensor through base complementary pairing after it added into the GQDs/Cy5-miR aqueous solution.When miRNA was hybridized with its corresponding target gene to form a duplex,the duplex detaches from the surface of GQDs and leads to the recovery of fluorescence for detection of CCND1 mRNA.We have successfully prepared GQDs/Cy5-miR@sEVs and GQDs/FAM-miR@sEVs by optimizing the experimental conditions of the "sonicationincubation" method and selecting the ultrasonic power of 250 J as the sonication condition.Every 100 μg EVs contain about 100 pmol of Cy5-miRNA or FAM-miRNA.Our study showed the stability of GQDs/Cy5-miR@sEVs was 1.5-fold than that of GQDs/Cy5-miR in animal serum.WB analysis showed that the surface of GQDs/Cy5-miR@sEVs also expressed the positive markers,including CD63,CD9 and Alix,and did not express the negative marker Calnexin.QRT-PCR results showed that the encapsulation efficiency of miRNA for the GQDs/Cy5-miR@sEVs was about 19%.Our immunofluorescence results demonstrated that intracellular CCND1 mRNA triggered the Cy5-miR released in cytoplasm.Our results confirmed that GQDs/Cy5-miR@sEVs can finally induce the apoptosis of gastric cancer cells,inhibit the migration and invasion of gastric cancer cells.In vivo experiments,GQDs/Cy5-miR@sEVs could be enriched in the gastric cancer subcutaneous tumor site and demonstrated the fluorescent “OFF-ON” effect.Antitumor efficacy evaluation findings showed GQDs/Cy5-miR@sEVs could significantly inhibit tumor growth and promote tumor cell apoptosis.In addition,GQDs/Cy5-miR@sEVs had no obvious organ toxicity to nude mice after treatment.Conclusion:In this study,we successfully construct GQDs/Cy5-miR@sEVs delivery system based on huc MSC-sEVs.The results confirm GQDs/Cy5-miR@sEVs can deliver the anti-cancer miR-193a-3p and regular the CCND1 mRNA in GC cells.As results,GQDs/Cy5-miR@sEVs inhibit the proliferation and promote the apoptosis of GC cells.Compared with the free form of GQDs /Cy5-miR,sEVs can significantly improve the stability of GQDs/Cy5-miR and protect the miRNA from degradation under the RNase.Our in vivo studies validate GQDs/Cy5-miR@sEVs can realize visualized gene delivery and inhibit the tumor growth effectively.Our research elaborates the effect and prospect of sEVs as a natural drug delivery carrier in the precise visual treatment of tumors,providing new theoretical and experimental basis for the transformation and application of this treatment strategy.