Long Noncoding RNA AC003092.1 Regulate Temozolomide Resistance Through MiR-195/TFPI2 Signaling Pathway In Glioblastoma

BackgroundGlioblastoma(GBM)is one of the most aggressive primary brain tumors in adults.Currently,temozolomide(TMZ)-based chemotherapy after surgical excision is one of the most frequently used therapeutic strategies for GBM patients.Unfortunately,a large proportion of patients developing resistance to TMZ becomes the major barrier to the efficacy of GBM treatment.Studies have shown that lncRNA was correlated with development of tumor and drug resistance.Exploring the role of lncRNA in glioma TMZ resistance could provide new stratedgy to overcome TMZ chemoresistance.lncRNA microarray analysis indicidated that IncRNA AC003092.1 and its nearby gene,Tissue factor pathway inhibitor-2(TFPI-2),showed remarkably down-regulated in U87TR cells when compared with its parental U87 cells,which meaned TFPI-2 might be regulated by IncRNA AC003092.1.Theory of CeRNA could explain the regulation of IncRNAs to its targeted gene.Studies have shown that IncRNAs can sponge miRNA,which attentuates the inhibition of miRNA on its targeted gene.Bioinformation analysis predicated that miR-195 has potential target site with IncRNA AC003092.1 and TFPI-2.Also,miR-195 has been reported that correlated with glioma TMZ resistance.Collectively,we hypothesized that lncRNA AC003092.1 participated in the enhancement of TMZ sensitivity by competitively sponging and then inhibiting miR-195 to augment TFPI-2 expression in GBM cells.ObjectiveFirstly,we evaluated expression of IncRNA AC003092.1 in GBM cell lines,glioma tissues and estimated its clinical relevance.Next,we explored the role of IncRNA AC003092.1 and its genomic neighboring gene TFPI-2 on GBM cell proliferation,cell apoptosis and TMZ chemo-resistance.Finally,we test the hypothesis that lncRNA AC003092.1 participated in the enhancement of TMZ sensitivity by competitively sponging and then inhibiting miR-195 to augment TFPI-2 expression in GBM cells.MethodsThe expression of lncRNA AC003092.1 in GBM cell lines and glioma tissues were detected by qRT-PCR.CCK-8 assay was conducted to explore the role of IncRNA AC003092.1 and TFPI-2 on glioma TMZ chemo-sensitivity.Flow cytometry assay and TUNEL staining were applied to detected cell apoptosis after intervention.While cell proliferation was investigated by EdU assay.The relationship between IncRNA AC003092.1,miR-195 and TFPI-2 were investigated by CNC analysis,qRT-PCR,Western blot,bioinformatic analysis,FISH assay,Dual-Luciferase reporter assay and RIP assay et al.Results1.IncRNA AC003092.1 remarkably down-regulated in U87TR cells when compared with its parental U87 cells.Low IncRNA AC003092.1 expression was negatively correlated to TMZ chemo-resistance and glioma grades.Also,lncRNA AC003092.1 downregulation predicated poor prognosis.2.IncRNA AC003092.1 overexpression could enhance TMZ chemo-sensitivity in TR cells.IncRNA AC003092.1 overexpression facilitates cell apoptosis and inhibits cell proliferation in TMZ resistant glioma cells.In contrast,IncRNA AC003092.1 knockdown decreased TMZ chemo-sensitivity in U87 and U251 cells.3.Co-expression analyses between protein-coding RNAs and IncRNA AC003092.1 indicated that TFPI-2 expression was highly correlated to lncRNA AC003092.1 expression.IncRNA AC003092.1 overexpression significantly increased TFPI-2 mRNA and protein expression in TR cells.4.TFPI-2 interference significantly decreased cell viability and reversed the lncRNA AC003092.1 mediated TMZ sensitivity.In addition,the flow cytometric analysis and TUNEL assays showed that TFPI-2 inhibition decreased cell apoptosis.TFPI-2 upregulation significantly decreased the cell viability of TR cells.Moreover,TFPI-2 overexpression dramatically increased the pro-apoptotic protein.5.IncRNA AC003092.1 was mainly located in cytoplasm.Bioinformatic prediction showed that miR-195-5p had putative binding sites with IncRNA AC003092.1 and TFPI-2.qRT-PCR assay analysis showed that expression of miR-195 was significantly downregulated after IncRNA AC003092.1 overexpression.Dual-Luciferase reporter assay indicated IncRNA AC003092.1 is targeted by miR-195.In addition,lncRNA AC003092.1 functions as ceRNA and negatively modulates miR-195 expression.6.The expression of miR-195 were drastically higher in both TR cells and Relapsed GBM tissues.Dual-luciferase Reporter Assay showed miR-195 can directly target TFPI-2.IncRNA AC003092.1 could abolish miR-195's inhibition on TFPI-2;MiR-195 could reverse IncRNA AC003092.1's positive regulation of TFPI-2.Conclusion1.LncRNA AC003092.1 downregulation correlates to TMZ resistance and poor prognosis in glioma.2.LncRNA AC003092.1 overexpression enhances TMZ chemosensitivity,facilitates cell apoptosis and inhibits cell proliferation in TR cells.3.LncRNA AC003092.1 regulates TMZ chemosensitivity through TFPI-2 mediated cell apoptosis.4.miR-195/TFPI-2 mediates IncRNA AC003092.1 function in glioma TMZ chemo-sensitivity.