Study On Exosomal LncRNAs As Biomarkers Of Colorectal Cancer

Purpose（1）To screen differentially expressed lncRNAs in serum exosomes of CRC patients and evaluate their diagnostic efficacy as biomarkers for CRC and early-stage CRC.（2）To study the effects of FOXD2-AS1on malignant biological behavior such as CRC cell proliferation,cytokinetic formation,cell scratches and migration.（3）To verify exosomes can carry and deliver FOXD2-AS1 to EA.hy926 cells,and promote the migration,invasion and angiogenesis of EA.hy926 cells.Method（1）Serum exosomes from healthy volunteers and CRC patients were obtained by ultracentrifugation.The morphology,size and surface marker proteins（TSG101 and CD9）of exosomes were verified by transmission electron microscope（TEM）,qNano and immunoblotting;（2）Screening the differentially expressed exosomal lncRNAs（ExlncRNAs）between 4 CRC primary patients and 2 healthy donors by lncRNA microarray analysis,16 lncRNAs with the most significance were selected for primer design and specificity verification and 8 of them were excluded due to poor primer specificity,and others were subjected to a cohort with 48 healthy people and 48 CRC patients,and 5 of them were excluded due to no difference.Finally,FOXD2-AS1 and NRIR and XLOC₀09459 were identified and then subjected to a more expanded cohort for future validation;（3）The isolated exosomes were incubated with RNase A and stored at room temperature.RNA were extracted to detect the expression changes of FOXD2-AS1 and NRIR and XLOC₀09459 and to prove the stability of exosomal lncRNAs;（4）Total serum exosomal RNAs were extracted from 203 CRC patients,203 healthy donors and 20 patients with benign intestinal diseases（BD）and the differentially expressed lncRNAs were quantitatively analyzed by real-time quantitative PCR（RT-PCR）to screen out biomarkers for diagnosis of CRC and early-stage CRC;The receiver operating characteristic curve（ROC）was used to assess the single and joint diagnostic efficiency of serum exosomal lncRNAs;（5）Inpatient information and clinicopathological parameters of CRC patients were reviewed and recorded,such as age,sex,drinking history,diabetes,histological,tumor position and size,lymph node metastasis,distant metastasis and TNM stage,etc.CRC was staged according to AJCC clinical tumor staging criteria.The correlation between the expression level of FOXD2-AS1 and NRIR and XLOC₀09459 and common clinicopathological features of CRC patients was analyzed;（6）Constructing the overexpress plasmid and small interference RNA of FOXD2-AS1,which expressed and knocked FOXD2-AS1 in CRC cells,and uses CCK8 cell proliferation experiments,cytokines,cell scratches experiment,cell migration and invasive experiment.Research on the impact of the proliferation,cloning of CRC cells,the ability to form and migration;（7）SW480 cells have expressed FOXD2-AS1 to extract the exosomes in the supernatant in the cells,incubate EA.hy926 cells,and investigate their impact on EA.hy926 migration,invasion,and angiogenesis.Results（1）Exosomal FOXD2-AS1,NRIR,and XLOC₀09459（TCONS₀0020073）levels were significantly upregulated in 203 CRC patients and 80 early-stage CRC patients compared to 201 healthy donors,possessing the area under the curve（AUC）of 0.728,0.660 and 0.682 for CRC,as well as 0.743,0.660 and 0.689 with 73.1%for early-stage CRC,respectively.Notably,their combination demonstrated the markedly elevated AUC of 0.736 for CRC and 0.758 for early-stage CRC,indicating their potential as diagnostic biomarkers for CRC;（2）Knocking of FOXD2-AS1 can suppress the proliferation,clonogenesis andmigration ability of CRC cells,overexpression of FOXD2-AS1 can improve the proliferation,clonogenesis and migration ability of CRC cells;（3）SW480 cells have expressed FOXD2-AS1 to extract the exosomes in the supernatant in the cells,incubate EA.hy926 cells,which can improve the migration,invasion and angiogenesis of EA.hy926.Conclusion（1）Our data suggested that exosomal lncRNAs FOXD2-AS1,NRIR and XLOC₀09459 act as the promising biomarkers for the diagnostics of CRC and early-stage CRC;（2）FOXD2-AS1 can promote proliferation,cytokinetic formation,cell scratches and migration of CRC cells;（3）Exosomes can carry and deliver FOXD2-AS1 to EA.hy926 cells,and promote the migration,invasion and angiogenesis of EA.hy926 cells.