| BACKGROUNDTumor metastasis is a special and complex process.It is an important factor that causes tumor treatment failure,leads to patient death,and results in a high tumor mortality rate.Tumor metastasis is divided into Oligometastatic(the number of systemic cumulative metastases or no more than 5 organs)and Polymetastatic(the number of systemic cumulative metastases or more than 5 organs).Oligometastatic and Polymetastatic are the second stage of tumor metastasis-two forms of distant metastasis.At this stage,a new microenvironment for tumor growth must be formed.There are many factors that affect the microenvironment of tumor growth.Among them,cancer cells affect the establishment of tumor microenvironment through their own secretion.However,the research on the mechanism of this aspect is still very vague.miRNA is an endogenous non-coding single-stranded small RNA molecule,which has a variety of important regulatory effects on tumor metastasis.miRNA can change the microenvironment of tumor growth by affecting the secretion of cancer cells,and affect the clonal proliferation of distantly metastatic tumors and the formation of tumor metastases.But RNA molecules are easily degraded outside the cell.Exosomes are secreted by various types of cells,are widely present and distributed in various body fluids,can carry various RNAs and protect them from degradation,and are closely related to the occurrence and progression of many diseases.Current studies have shown that exosomes secreted by tumor cells participate in the regulation of the occurrence and development of tumors,play the physiological functions of material transport and information transmission between cells,and reshape the tumor microenvironment.However,the specific effects of exosomes on tumors and their regulatory mechanisms are still unclear and still require in-depth research.PURPOSE1.To explore the influence of exosomes on the evolution of Oligometastatic to Polymetastatic.2.To verify the regulatory effect of miRNA carried by exosomes on tumor metastasis.3.To study the molecular mechanism of miRNA carried by exosomes in the process of tumor clone proliferation.METHOD1.Co-culture is used to simulate the effect of Polymetastatic in vivo on Oligometastatic.2.Extract exosomes by ultracentrifugation.3.Exosomes were detected by TEM,NTA,WB.4.The target gene is knocked down transiently by si RNA transfection.5.Transient or stable overexpression of the target gene by miRNA mimics transfection or lentiviral infection.6.Detect the target gene expression level by RNA extraction and RT-qPCR methods.7.Cell migration experiments,cell invasion experiments and clonal plaque formation experiments were used to detect the ability of tumor cells to migrate and proliferate in vitro.8.The changes in the uptake of exosomes by cells were observed by confocal fluorescence microscope.9.High-throughput sequencing is used to detect changes in the expression level of miRNA in exosomes.10.Bioinformatics analysis is performed through KEGG database,TCGA database,Target Scan Human database,GEPIA database,Onco Lnc database and BGI’s multi-omics system.11.Analyze cell cycle changes by flow cytometry.RESULT1.Through the established in vitro co-culture model of Oligometastatic tumor cells and Polymetastatic tumor cells,Polymetastatic tumor cells mediate the evolution of Oligometastatic to Polymetastatic by secreting exosomes in vitro process.2.In the stage of distant metastasis,exosomes secreted by Polymetastatic tumor cells can enhance the in vitro migration,invasion,and clonal proliferation capabilities of Oligometastatic tumors.3.Exosomes can regulate the evolution of Oligometastatic to Polymetastatic by transporting miRNA.High-throughput sequencing analysis showed that the miRNAs carried by exosomes secreted by Oligometastatic tumor cells and Polymetastatic tumor cells are different.4.The miR-199a-1-5p carried by exosomes can obviously promote the in vitro migration,invasion and clonal proliferation of Oligometastatic tumor cells.5.CDKN1B gene is the target gene of miR-199a-1-5p.miR-199a-1-5p can inhibit the expression level of CDKN1B gene,thereby enhancing the in vitro migration,invasion and clonal proliferation ability of Oligometastatic tumor cells,and induce the transition from G1 phase to S phase.CONCLUSION1.This study verified that during the stage of distal metastasis,Polymetastatic tumor cells can regulate the evolution of Oligometastatic to Polymetastatic in vitro by secreting exosomes,and enhance the ability of migration,invasion,cloning and proliferation of Oligometastatic tumor cells.It was demonstrated for the first time that exosome secretion can mediate the progression of Oligometastatic to Polymetastatic by regulating cell cycle2.This study proved and verified for the first time the molecular mechanism of exosomes inhibiting CDKN1B and promoting tumor metastasis through the transport of miR-199a-1-5p.Through the transportation of miR-199a-1-5p,exosomes enhance the gene expression of miR-199a-1-5p in Oligometastatic tumor cells,thereby inhibiting the expression level of CDKN1B gene,and finally enhancing the in vitro migration,invasion,cloning and proliferation ability of Oligometastatic tumor cells,inducing the transition from G1 phase to S phase.This molecular mechanism is completely different from the previous mechanism by which exosomes inhibit tumor metastasis through the transport of miR-199a-1-5p.It was first proved that exosomes can inhibit the gene expression of CDKN1B through the transport of miR-199a-1-5p,induce the transition from G1 phase to S phase,and promote tumor metastasis and proliferation.It was first demonstrated that CDKN1B is an inhibitory factor in the evolution of Oligometastatic to Polymetastatic.This provides a new research idea for the study of tumor metastasis,especially exosome-mediated tumor metastasis.3.The Oligometastatic and Polymetastatic models used in this study were derived from M14 melanoma cells,so this study proved for the first time that miR-199a-1-5p can promote the metastasis and proliferation of melanoma by inhibiting CDKN1B. |
