Study On Exosomal MiRNAs As Biomarkers Of Non-small Cell Lung Cancer And The Mechanism Involved In Invasion And Metastasis

BackgroundAccording to the 2020 global cancer statistics analysis,lung cancer ranks second in the world for new cancers(accounting for 11.4%of the total cases),but it is the leading cause of cancer-related deaths(accounting for 18%).Non-small cell lung cancer(NSCLC)accounts for about 80%of all lung cancer cases and is mainly composed of squamous cell carcinoma and adenocarcinoma.Although great advances have been made in the current diagnostic methods and treatments,the overall 5-year survival rate of patients with NSCLC remains low,at 10-15%.The main reason for poor prognosis is that most patients have already been in local advanced stage or distant metastasis at the time of diagnosis,thus missing the best opportunity for treatment.Therefore,the early diagnosis and metastasis warning of NSCLC is one of the main challenges in the field of lung cancer research.More importantly,exploring the metastasis and its potential molecular mechanism of NSCLC is useful for finding therapeutic targets to inhibit NSCLC metastasis and improving the survival rate of patients.In recent years,extracellular vectors have attracted renewed interest because of their numerous functions in physiology and pathology.Exosomes are extracellular vesicles with lipid bilayer structure,about 30nm-100nm in size.They originate from the endosomal system and are formed by the invagination of lysosomal particles,and contain DNA,mRNA,non-coding RNA,proteins and lipids in the vesicles.Cells can use exosomes as a medium to transmit biomolecules to neighboring or distant cells and influence their functions.All cell types,including tumor cells,are capable of producing exosomes under specific physiological or pathological conditions.It is worth noting that RNA molecules(such as miRNAs)encapsulated in exosomes are more resistant to RNase than free ones and can directly reflect the physiological and pathological state of source cells,thus serving as novel biomarkers.In addition,tumor-derived exosomes have been identified as mediators of tumor microenvironment,playing an important role in coordinating tumor formation and progression,promoting angiogenesis,invasion,metastasis and immune escape.More and more studies have shown that exosomes produced by tumors are involved in the process of tumor metastasis,mainly in the following aspects:(1)The exosomes produced by tumor can endow the recipient cells with mesenchymal characteristics and make them undergo epithelial mesenchymal transformation,which can activate the initial stage of metastasis by improving the migration activity of cells;(2)Tumor derived exosomes can transform mesenchymal stem cells into cancer associated fibroblasts,which cross fusion with cancer cells to change the tumor microenvironment and further induce EMT while reducing the production of normal epithelial cells;(3)Exosomes derived from tumor cells could further form tubular structures by stimulating endothelial progenitor cells planted by tumor cells,so as to induce the formation of neovascularization,which could provide oxygen for metastatic lesions and provide an entrance for blood borne dissemination;(4)Exosomes produced by tumor cells play a decisive role in organ-specific metastasis,that is to say,the metastasis of tumor cells will occur only when the seeds are compatible with the soil,in other words,when the tumor cells are matched with the host organ.MiRNA is an endogenous small non-coding RNA with a length of 19-22 nucleotides.In some cases,miRNA can target specific regions of messenger RNA(mRNA),such as the 3'untranslated region(3'UTR),resulting in degradation of mRNA or inhibition of protein translation.It can be seen that miRNA plays key functions in various biological processes by targeting mRNA.In addition,because MiRNA is usually located in the "fragile regions"of chromosomes,the frequency of deletion,rearrangement and amplification of these regions is very high,and is related to malignant tumors,including tumor occurrence and development,cell proliferation,migration,invasion and apoptosis.Therefore,MiRNA has attracted the attention of scholars in the field of tumor research.Exosomes in the serum are secreted into the blood by cells,which can truly reflect the state of the source cells.Since miRNAs in exosomes have the ability to regulate gene expression and exist stably in the extracellular environment,they are widely used as biomarkers for non-invasive diagnosis or prognosis of cancer and other diseases.Exosomal miRNAs have also been found to be signal molecules that regulate tumor proliferation,angiogenesis,metastasis,immune-regulation,and inhibit drug resistance.They are involved in multiple links of tumor progression,and more importantly,play an important role in the process of tumor metastasis.In addition,some scholars have studied how highly metastatic liver cancer cell lines and highly metastatic breast cancer cell lines edutated low metastatic cell lines by transferring exosomal miRNAs.However,in the field of lung cancer research,this part is still blank.Based on the above research background and theoretical basis,we designed and conducted this research.The purpose is to find serum exosomal miRNAs biomarkers that can be applied to the early diagnosis and early warning of metastasis of NSCLC,and further study the molecular mechanism of exosomal miRNAs involved in its invasion and metastasis.It mainly includes two parts:Part 1:Research on serum exosomal miRNAs as biomarkers of non-small cell lung cancerPurposeTo screen differentially expressed miRNAs in serum exosomes of NSCLC patients and evaluate their diagnostic efficacy as biomarkers for early diagnosis and metastasis warning of NSCLC.Method1.Serum exosomes from healthy volunteers and NSCLC patients were obtained by ultracentrifugation at low temperature.The size,morphology and surface marker proteins(TSG101 and CD9)of exosomes were detected by qNano,transmission electron microscope and western blot respectively;2.Screening the differentially expressed exosomal miRNAs(ExmiRNAs)between healthy volunteers and NSCLC patients,and between non-metastatic and metastatic non-small cell lung cancer patients by miRNAs chip technology;3.Total serum exosomal RNAs were extracted from large sample cohort studies,and the differentially expressed miRNAs were quantitatively analyzed by real-time quantitative PCR(RT-PCR)to screen out biomarkers for early diagnosis and early predicting of metastasis of NSCLC.At the same time,the commonly used clinical tumor markers(CEA,CYFRA21-1)were combined and ROC curve was calculated to evaluate their diagnostic efficacy;4.Inpatient information and clinicopathological parameters of NSCLC patients were reviewed and recorded,such as age,sex,smoking history,drinking history,lymph node metastasis,distant metastasis and TNM stage,etc.NSCLC was staged according to AJCC clinical tumor staging criteria.The correlation between target miRNAs expression level and common clinicopathological features of NSCLC patients was analyzed;5.RNAs in serum exosome(EXO)and exosome-depleted supernatant(EDS)were extracted to detect the location of miRNAs;After exosomes were treated with RNA enzymes or placed at room temperature for different periods,RNAs were extracted to detect the expression changes of target miRNAs and to test the stability of miRNAs in exosomes;6.Patients receiving first-line chemotherapy were followed up to evaluate the role of exosomal miRNAs in predicting the efficacy of chemotherapy in NSCLC according to their treatment status and expression level of exosomal miRNAs.Results1.The results of qNano and electron microscopy analysis showed that exosomes were round vesicles with diameters ranging from 60nm to 160nm.The exosome surface marker proteins TSG101 and CD9 can be detected by western blot.2.Screening of early diagnosis markers:According to the criterion of Fold change? 2,twenty-eight differentially expressed miRNAs between healthy volunteers and NSCLC patients screened by miRNAs chip technology(nine up-regulated in NSCLC patients,nineteen down-regulated)were selected as the research objects,and these targets were detected by the expression of miRNAs in serum exosomes of 282 healthy volunteers and 276 NSCLC patients found that the expression levels of exosomes miR-20b-5p and miR-3187-5p in NSCLC patients were significantly down-regulated(p<0.0001).The diagnostic capabilities of these two ExmiRNAs were evaluated by the ROC curve,and the results showed that for miR-20b-5p and miR-3187-5p,the AUC were 0.818(95%CI:0.784-0.853)and 0.690(95%CI:0.646-0.733).Combining miR-20b-5p and miR-3187-5p can increase AUC to 0.848(95%CI:0.817-0.880).The AUC of CEA combined with ExmiR-20b-5p and ExmiR-3187-5p in the diagnosis of NSCLC increased to 0.905(95%CI:0.880-0.929).Similarly,the combined application of CYFRA21-1 with ExmiR-20b-5p and ExmiR-3187-5p also increased the diagnostic ability of NSCLC to 0.894(95%CI:0.868-0.920).In addition,the expression levels of ExmiR-20b-5p and miR-3187-5p were signifycantly correlated with early NSCLC(stage 0 and I,n=104)(p<0.0001)compared with healthy controls.The AUC of miR-20b-5p and miR-3187-5p were 0.810(95%CI:0.764-0.865)and 0.673(95%CI:0.617-0.728),respectively.The AUC increased to 0.838(95%CI:0.794-0.881)after the combination of the two drugs.Using CEA and CYFRA211 in combination with exosomes miR-20b-5p and miR-3187-5p,the results showed that the AUC increased to 0.930(95%CI:0.900-0.959)and 0.928(95%Cl:0.896-0.959),respectively.After analyzing the clinical characteristics data,it was found that the two early diagnosis markers have no correlation with the patient's age,gender,pathological type and other factors.3.Screening of early predicting markers for metastasis:Similarly,according to the criterion of Fold change>2,30 miRNAs that were differentially expressed between non-metastatic and metastatic NSCLC patients screened by miRNAs chip technology(15 were up-regulated in patients with metastatic NSCLC and 15 were down-regulated)were selected as analysis objects.By detecting the expression levels of these target miRNAs in serum exosomes of 173 non-metastatic and 132 metastatic NSCLC patients,it was found that exosomes miR-212-5p and miR-651-3p were significantly down-regulated in metastatic NSCLC patients(p<0.0001).The diagnostic capabilities of these two ExmiRNAs were evaluated by the ROC curve.The results showed that for ExmiR-212-5p and ExmiR-651-3p,the AUC were 0.746(95%CI:0.691-0.802)and 0.688(95%CI:0.628-0.749).Combining miR-212-5p and miR651-3p can increase AUC to 0.751(95%CI:0.697-0.806).The AUC of CEA combined with ExmiR-212-5p and ExmiR-651-3p in the diagnosis of NSCLC increased to 0.787(95%CI:0.736-0.838).The expression levels of ExmiR-212-5p and ExmiR-651-3p are closely related to tumor stage.In the analysis of the relationship between clinical features,they have no correlation with age,gender,smoking and other factors,but they are closely related to lymph node metastasis and distant metastasis.4.Compared with the supernatant without exosomes,the expression level of miRNAs in exosomes were significantly higher;The expression levels of miRNAs contained in exosomes treated with RNA enzymes or placed at room temperature for different periods of time did not change significantly.5.In the follow-up of 64 patients receiving first-line chemotherapy,the high expression level of exMir-212-5p was found to be correlated with better chemotherapy efficacy(P=0.024).Conclusion1.Serum exosomal miR-20b-5p and miR-3187-5p can be used as biomarkers for early diagnosis of NSCLC,and serum exosomal miR-212-5p and miR-651-3p can be used as biomarkers for metastasis warning.The combination of CEA or CYFRA21-1 can improve diagnostic efficiency;2.Serum exosomal miR-212-5p can be used as a potential predictor of chemotherapy efficacy.Part 2:Study on the mechanism of exosomal miRNA-632 involved in the metastasis of non-small cell lung cancerPurpose1.To study the effects of miRNA-632 on NSCLC cell proliferation,migration and invasion,EMT and other biological behaviors,as well as the regulation of human umbilical vein endothelial cells(Human Umbilical Vein Endothelial Cells,HUVEC)angiogenesis;2.Verify that exosomes can carry miRNA-632 from high-metastatic lung cancer cells to low-metastatic lung cancer cells and HUVEC,thereby affecting the biological behavior of recipient cells,further confirming the regulatory function of the target miRNA-632 molecule;3.To find the target gene of mirNA-632 and explore the molecular mechanism of exosomal miRNA promoting proliferation and metastasis of NSCLC.Method1.Thirty candidate miRNAs with differential expression in serum exosomes between non-metastatic and metastatic NSCLC patients(all of which are up-regulated in metastatic patients)were selected and simultaneously analyze them in NSCLC cell lines(high metastatic cell line 95-D,low metastatic cell line A549 and SPCA-1)by QRT-PCR to screen out the target miRNA;then detect the expression levels of target miRNA in serum exosomes of patients with non-metastatic and metastatic NSCLC;2.Target miRNA was overexpressed in low metastatic NSCLC cell lines A549,SPCA-1 and HUVEC.CCK8 assay,clone formation assay,transwell assay,scratch assay and Western blot assay were used to study the proliferation,clonogenesis,migration and invasion of the cells.And the effect on epithelial mesenchymal transformation ability;Angiogenesis assay measures regulation of angiogenesis ability;3.Fluorescent dyes PHK67 and DAPI label the nuclei of 95-D cell line exosomes and A549 cell line respectively.After the two are incubated together,observe and evaluate the uptake of exosomes by recipient cells;4.Exosomes of high metastatic NSCLC cell line 95-D were extracted,and low metastatic NSCLC cells A549,SPCA-1 cells and human umbilical vein endothelial cells were treated with exosomes.Continue to use proliferation,migration and invasion experiments,EMT experiments and angiogenesis experiments to study the changes in the biological behavior of recipient cells after exosomes are incubated;5.After the subcutaneous tumor implantation model of nude mice was constructed,PBS,95-d exosomes and 95-d exosomes+ inhibitor miRNA were injected simultaneously in the tumor of the control group and the experimental group in multiple directions to verify the effect of exosomal miRNA on the proliferation of tumor cells in the in vivo;6.Using bioinformatics analysis and dual luciferase reporter gene assay to find the target gene of the target miRNA,by synthesizing a plasmid containing the target gene,and transfecting it into the cell to observe its effect on the function of NSCLC cells and HUVEC.Result1.Through qRT-PCR,we found that compared with low metastatic NSCLC cell lines A549 and SPCA-1,miRNA-632 is highly expressed in the highly metastatic cell line 95-D and the expression level in 95-D exsomes is also significant increase;further analysis found that the expression level of miRNA-632 in the serum exosomes samples of patients with metastatic NSCLC(n=83)was significantly higher than that of patients with nonmetastatic NSCLC(n=199),and the difference was statistically significant(P=0.0006);2.Overexpression of miRNA-632 can improve the proliferation,clonogenesis,migration and invasion ability of A549 and SPCA-1 cells,promote mesenchymal transformation of tumor cells,and improve the neovascularization ability of HUVEC cells;3.Fluorescence microscope observed that 95-D cell derived exosomes can be internalized by recipient cells(A549 cells);4.Exosomes from high metastatic cell line 95-D can carry miRNA-632 and transfer to low metastatic NSCLC cell line A549,SPCA-1 and HUVEC,which can not only improve the proliferation,clonal formation,migration and invasion ability of recipient cells,promote EMT,but also promote vascular formation.When the expression level of miRNA632 in exosomes was inhibited,the positive promoting effect of exosomes was inhibited(the ability of migration and invasion,EMT and angiogenesis were reduced);5.In animal experiments,exosomal miRNA-632 can promote tumor growth in nude mice,and inhibition of miRNA-632 reduces the tumor volume.6.The results of the dual luciferase report experiment show that miRNA-632 can directly and specifically bind to the 3'UTR of Sec23A and affect gene expression at the post-transcriptional level.Compared with the control group,overexpression of Sec23A could inhibit the clonal formation,invasion migration and EMT of NSCLC cells,and inhibit the angiogenesis of endothelial cells;The results of showed that overexpression of Sec23A could reverse the positive promoting effect of miRNA-632.Conclusion1.Exosomes from high metastatic cell line 95-D can enter low metastatic NSCLC cell line,promote proliferation,clone formation,migration and invasion ability and epithelial mesenchymal transformation,and also enter human umbilical vein endothelial cells to promote angiogenesis.The above effects are achieved by down-regulating the expression of Sec23A by miRNA-632.2.Exosomal miRNA-632 is a potential biomarker for predicting NSCLC metastasis.