LncRNA LURAP1L-AS1 Promotes Fibroblasts Activating And Oral Cancer Epithelial-mesenchymal Transition

[Objective]Tumor metastasis is a multi-step and complicated process regulated by tumor cells interact with mesenchymal cells in the microenvironment.However,the underlying mechanism of how the cancer-associated fibroblasts promote the oncogenesis and progression of tumors when they are in an activated state is unclear.Our previous faresearch found that platelet-derived factor-BB(PDGF-BB)can induce the activation of oral mucosal fibroblasts and secrete the chemokine CCL25.The present study explored the role and molecular process of PDGF-BB inducing fibroblast activation from the perspective of long non-coding RNA regulating gene expression.We elaborated on the molecular mechanism by which the cross-talk between oral squamous carcinoma cells and fibroblasts can promote tumor progression.Our study revealed the internal relationship between cancer cells and cancer-related fibroblasts,and provide new ideas and theoretical basis for the treatment of oral cancer.[Method]1.We routinely culture normal human oral mucosal fibroblasts(hOMF),human oral tongue squamous cell carcinoma cells CAL-27,TCA8113 and TSCCA.The Transwell chamber is used co-cultivation of fibroblasts and oral cancer cells.2.30ng/ml PDGF-BB factor wfor as used to induce activation of hOMF fibroblasts(Activated Fibroblast,AF).Western blotting was utilized to detect the expression of fibroblast activation marker ?-SMA and related signal pathway protein molecules.Q-PCR and ELISA were used to detect the expression and secretion of activated fibroblasts CCL25.Western blotting was used to detect the activation of fibroblasts in co-culture.3.CCK8 method was used to detect cancer cell proliferation.Annexin V-FITC/PI double staining method was utilized to detect cancer cell apoptosis.Transwell and scratch test was carried to investigate cancer cell invasion/migration.Cancer cell epithelial-mesenchymal transition Related protein expression was confirmed by immunofluorescence.4.The expression levels of lncRNAs and mRNAs before and after activation of fibroblasts was detected through Arraystar human lncRNA chip,combined analysis and screening of relevant differential lncRNAs,and verified by parallel qPCR and Western blotting.Gene Ontology(GO)analysis performs functional annotation and enrichment scores on genes,clarifies the correlation between differential genes and their enriched functional items.We inferred the signal pathways involved in differential genes by Kyoto encyclopedia of genes and genomes(KEGG)analysis.The function of lncRNA and target gene protein is predicted by amino acid sequence alignment,protein homology analysis and RNA binding proteins(RBPs).Actinomycin D assay was used to detect the stability of LURAP1L mRNA in fibroblasts.The dual luciferase gene report and FISH experimental analysis verified the binding of LURAP1L-AS1 and LURAP1L in fibroblasts.5.Stable knockdown LURAP1L-AS1 fibroblasts(named hOMF-siLURAP1L-AS1,control cells named hOMF-siNC)were established by lentiviral plasmids with small interfering RNA.The expression levels of LURAP1L-AS1 and LURAP1L in fiber cells after the transfection were detected through qPCR and Western blot methods.6.The LURAP1L-interacting proteins and downstream signaling pathways were clarified through co-immunoprecipitation and immunoblotting.[Result]1.The expression of the fibroblast activation marker ?-SMA in hOMF cells after PDGF-BB induction increased,and the addition of inhibitors of its receptor PDGFR-?can block this change(p<0.05).2.Co-culture of activated fibroblasts with oral cancer cells CAL-27,TCA8113 and TSCCA respectively can significantly reduce the expression of E-cadherin,significantly up-regulated the expression of N-cadherin in oral cancer cell lines(p<0.05).The secretion of chemokine CCL25 in the supernatant of activated fibroblasts increased,and the expression of CCL25 in activated fibroblasts increased.The scratch test and transwell test showed that CCL25 can promote the invasion/migration of oral cancer cells CAL-27.3.After chip sequencing,bioinformatics screened out the lncRNAs involved in regulating the activation of oral mucosal fibroblasts:LOC102724467,LURAP1L-AS1,G048372,G054461,G089447,LINC01054,RP1-193H18.2,XLOC₀02805,RP11-193M21.1 and G018999.Among them,the main signal pathways involved are chemokine signal pathway and NF-?B signal pathway.The expression of IncRNA LURAP1L-AS1 and LURAP1L increased after PDGF-BB inducing oral mucosal fibroblasts.4.Actinomycin D experiment showed that knocking down LURAP1L-AS1 can decrease the stability of LURAP1L mRNA in fibroblasts.Dual luciferase experiment confirmed that IncRNA LURAP1L-AS1 binds to the 5'UTR region of LURAP1L.FISH experiment showed that IncRNA LURAP1L-AS1 and LURAP1L co-localized in fibroblasts.5.After knocking down LURAP1L-AS1,the expression of LURAP1L in fibroblasts decreased.Compared with control cells,the expression levels of LURAP1L-AS1 and LURAP1L in fibroblasts were significantly increased after PDGF-BB induction,while PDGF-BB induced stable knockdown LURAP1L-AS1 fibroblast lines,the expression levels of LURAP1L-AS1 and LURAP1L has no difference(p>0.05).6.After PDGF-BB induction,the expression of IKK?,?-SMA and p-NF-?B in fibroblasts increased significantly,and the difference was statistically significant(p<0.01).However,after knockdown the expression of LURAP1L-AS1,PDGF-BB induced the expression of IKK?,?-SMA and p-NF-?B in fibroblasts has no difference(p>0.05).CO-IP showed that LURAP1L and IKKa could interact in fibroblasts.7.Compared with the control group,we found that the proliferation level of CAL-27 cell was significantly increased,its apoptosis level was significantly reduced,and its migration level was significantly increased in CAL-27 cells and activated fibroblasts co-cultured model(p<0.05),while in CAL-27 cells co-cultured with hOMF-siLURAP1L-AS1 cells induced by PDGF-BB,the above effects were weakened.8.Compared with hOMF-siNC cells,the CCL25 secretion of hOMF-siRURAP1L-AS1 cells was significantly reduced after PDGF-BB induction(p<0.05).In the co-culture model with CAL-27,hOMF-siLURAP1L-AS1 cells induced by PDGF-BB secreted CCL25 lower than hOMF-siNC cells induced by PDGF-BB.Immunofluorescence showed that the co-culture of activated fibroblasts and CAL-27 cells can significantly increase the expression levels of occludin and N-cadherin in CAL-27 cells,and significantly reduce the expression levels of E-cadherin.The above effect was weakened in the co-culture model of PDGF-BB induced hOMF-siLURAPIL-AS1 cells with CAL-27.9.Compared with hOMF cells cultured separately,the expressions of p-IKKa and p-NF-?B both increased significantly in hOMF cells co-cultured with CAL-27 and PDGF-BB-induced hOMF cells,while knockdown the expression of LURAP1L-AS1 can reduce this effect.[Conclusion]1.PDGF-BB induces the activation of oral mucosal fibroblasts by binding to its receptor PDGFR-?,secretes chemokine CCL25,and promotes the invasion and migration of oral squamous cell carcinoma cells2.LncRNA LURAP1L-AS1 plays a key role in the activation of oral mucosal fibroblasts induced by PDGF-BB.It may stabilize LURAP1L mRNA in cells and promote LURAP1L protein expression,thereby enhancing the interaction with IKKa and activating NF-?B signaling pathways to induce the expression and secretion of chemokines to feed oral cancer cells.