Exosome-transmitted MiR-1290 Mediates The Activation Of Lung Cancer-related CAFs And Its Mechanism

Lung cancer is the most common malignant tumor with the highest morbidity and mortality in the world.Five-year survival rate of advanced lung cancer is only 20%-25%.Clinically,Metastasis of lung cancer is one of the main causes of death.Therefore,it is of great significance to study the metastasis of lung cancer in depth.Recent studies have shown that tumor metastasis is not only related to the tumor cell itself,but also to the tumor microenvironment.Cancer associated fibroblasts(CAFs)are important interstitial cells in TME.They are mainly activated by normal fibroblasts(NFs)in a variety of ways.Exosome is the main mediators of cell communication in TME.It can carry proteins,lipids and nucleic acids from one cell to another.In order to investigate whether tumor cells can transmit miRNAs to NFs via exosome in TME and mediate the activation of NFs to CAFs,and participate in the remodeling of TME and promote tumor metastasis.We have carried out the following work:1.To screen miRNAs secreted by exosome which may be involved in tumor metastasis and activation of CAFs.The materials ued in this study were 95 C,95D,NFs and CAFs.The differentially expressed miRNAs were screened by gene chip,q-PCR,electron microscope,particle size analysis and western blot.There were three miRNAs seleted: miR-652,miR-222 and miR-1290.Their expression was much higher in 95 D cells and exosome than in 95 C cells and exosome,and higher in CAFs cells than in NFs cells.It indicates that these three miRNAs are likely to be involved in the activation of NFs and related to the metastasis of lung cancer.2.To determine the mechanism of exosome transmitting miR-1290-mediated activation of lung cancer-related CAFs.The results of exosome uptake and mixed culture experiment showed tha t lung cancer cells could activate NFs to CAFs by secreting exosome,and then,through q-PCR,western blot and immunofluorescence assay,the activated miRNA could be selected.The results show that when NFs overexpressed miR-1290,the expression levels of FAP and α-SMA in NFs cells were higher than those in the control group,when the 95 D exosome which inhibited miR-1290 was mixed culture with NFs,the expression levels of FAP and α-SMA in NFs were down-regulated,indicating that miR-1290 transmitted by exosome can activate NFs to CAFs.Then,through bioinformatics analysis,q-PCR,western blot and double luciferase reporter gene detection found that: miR-1290 could target the 3’UTR sequence of MT1 G gene,down-regulate its expression,and then up-regulate the expression of p-AKT,thus,the AKT signaling pathway is activated.These results suggest that lung cancer cells can transmit miR-1290 through exosome to NFs,directly targeting MT1 G in NFs,inhibiting its expression,thus activating AKT signaling pathway and transmitting NFs to CAFs.3.To explore the effect of activated CAFs on tumor metastasis.The materials were A549,NFs and CAFs.Transwell and scratch test showed that activated NFs could promote the migration ability of lung cancer cells.In conclusion,it is clear that in TME,lung cancer cells can trans mit miR-1290 to NFs by secreting exosome;it is confirmed that miR-1290 can target MT1 G,and then up-regulate p-AKT.It is also found that activated CAFs can promote the metastasis ability of lung cancer cells.Therefore,in tumor microenvironment,lung cancer cells can transmit miR-1290,down-regulate MT1 G and activate AKT signal pathway through exosome,so that NFs can be activated into CAFs.The new mechanism of lung cancer metastasis was deeply discussed in this paper from the perspective of tumor microenvironment.