| ObjectiveIn this study,we aim to investigate the expression of FOXP4-AS1 in papillary thyroid carcinoma(PTC)tissue and its relationship with various clinicopathological characteristics of patients.Furthermore,we intend to clarify the specific biological function of FOXP4-AS1 in PTC,and explore its mechanism of action preliminarily.Methods(1)Bioinformatics screening of the correlation between FOXP4-AS1 and prognosis in thyroid cancer:GSE66783 dataset has been downloaded from GEO database(GENE EXPRESSION OMNIBUS),and the expression data,clinical data,somatic mutation and other data related to 33 cancer tissues and paracancerous tissues were retrieved from The Cancer Genome Atlas(TCGA).Differential analysis and survival analysis were performed through Limma and Survival packages of R studio software;ggplot2 package was deployed for the visualization and screening for differential expression of long noncoding RNAs(lnc RNAs).Analysis of FOXP4-AS1expression differences,clinical relevance,and survival prognosis in 33 cancers and GSEA analysis of FOXP4-AS1 enrichment pathways in thyroid cancer.(2)Validation of human PTC cancer tissue specimens:The expression levels of FOXP4-AS1 in 72 PTC tissues and adjacent tissues were detected by real-time quantitative PCR(RT-q PCR),then the relationship between the expression of FOXP4-AS1 and the clinicopathological characteristics of patients was statistically analyzed.(3)Cell experiments:FOXP4-AS1 expression levels in TPC-1,K1,BCPAP and Nthy-ori3-1 cell lines was detected by RT-q PCR.Then apply lentivirus infection and si RNA interference technology to induce overexpression and interference of FOXP4-AS1,the efficiency was detected by RT-q PCR.Afterwards,the effects of FOXP4-AS1 on the proliferation,migration and apoptosis of PTC cells were detected by CCK8,clone formation,Transwell,as well as flow cytometry.Furthermore,utilize RT-q PCR to detect the changes on BAX,MMP2,MMP9 gene expression after overexpression and interference of FOXP4-AS1 to verify whether they are involved in the apoptosis and migration process above.The subcellular localization of FOXP4-AS1 in PTC cells was detected by RNA fluorescence in situ hybridization(FISH).The Illumina next-generation high-throughput sequencing platform was used to detect the changes of the whole-genome m RNA expression profile of the thyroid cancer cell line TPC1 before and after FOXP4-AS1overexpression by using the involved transcriptome sequencing technology.The differentially expressed genes were clustered by GO analysis,meanwhile,the differentially expressed genes caused by overexpression of FOXP4-AS1 were analyzed by KEGG to seek for potential candidate genes and regulatory mechanisms.Western blot was used to detect the expression levels of PI3K,p-AKT protein and total AKT protein in thyroid cancer cell lines overexpressing and interfering with FOXP4-AS1 expression,and to verify the altered PI3K/AKT signaling pathway caused by changes in FOXP4-AS1 expression in bioinformatics and transcriptome sequencing.(4)Animal experiment:The expression of FOXP4-AS1 in K1 cells was overexpressed by lentivirus infection technology.The concentrations of cell suspension in the experimental group and the control group were both 1×10~6cells/ml,and 100μl/nude mice were injected into the double armpits of nude mice respectively.After the subcutaneous transplanted tumor model established successfully,the tumor growth indicators were collected every 3 days until the 48th day of transplantation.Immunohistochemistry was adopted for detecting tumor proliferation index Ki67 and p AKT protein to identify the signaling pathway in vitro.Results(1)Bioinformatics:Through bioinformatics analysis and screening of differential lnc RNAs in thyroid cancer,it implies that FOXP4-AS1 is low expressed in thyroid cancer and is in close relation to PI3K-AKT-MTOR,cell cycle,apoptosis and other pathways.The expression of FOXP4-AS1 has statistical differences in some clinical features,for instance,T stage,N stage,Clinical stage,and extraglandular invasion(p<0.01),moreover,and patients with low expression have a poor prognosis.(2)Human PTC tissue specimens:FOXP4-AS1 exhibits low expression in papillary thyroid carcinoma,furthermore,the degree of low expression shows significantly positively correlated with tumor T1/T2 and T3/T4 stages,N0 and N1stage,Stage I and stage II+III,and extraglandular invasion in PTC patients(p<0.05),while,there was no statistical correlation with gender,age,and distant metastasis(all p>0.05).(3)Cellular experiments:Overexpression of FOXP4-AS1 inhibited the proliferation and migration of PTC cells,induced apoptosis(all p<0.05);decreased the expression of the anti-apoptotic gene BCL-2,increased the expression of the pro-apoptotic gene BAX,meanwhile,it significantly decreased the expression of the pro-migration genes MMP2 and MMP9(p<0.01);Interference of FOXP4-AS1promoted the proliferation and migration of PTC cells,inhibited apoptosis(all p<0.01)as well as the increase in the expression of the anti-apoptotic gene BCL-2,decreased the expression of pro-apoptotic gene BAX,and significantly increased the expression of the pro-migration genes MMP2 and MMP9(p<0.05).FOXP4-AS1 distributed in both the nucleus and cytoplasm of thyroid cancer cells,and mainly expressed in the cytoplasm.Transcriptome sequencing GSEA analysis showed that FOXP4-AS1 was enriched in some remarkable tumor signaling pathways PI3K-AKT-MTOR,KRAS,cell cycle and other signaling pathways.Western blot results showed that the expression levels of PI3K and p-AKT protein in PTC cells were significantly decreased after overexpression of FOXP4-AS1(both p<0.001);the expression levels of PI3K and p-AKT protein in PTC cells were significantly increased after interfering with FOXP4-AS1(both p<0.001).While,the total AKT protein expression level had no significant change(p>0.05).FOXP4-AS1 is an antisense long non-coding RNA(ln c RNA)of the transcription factor FOXP4,which functions by inhibiting the expression of host m RNA and is one of the potential mechanisms of action of lnc RNA.After overexpression of FOXP4-AS1,the expression of transcription factor FOXP4 m RNA in PTC cells was significantly decreased(p<0.01);the expression of transcription factor FOXP4 m RNA in PTC cells interfered with FOXP4-AS1 was significantly increased(p<0.001).MK-2206,the AKT inhibitor,can attenuate the effect of interfered FOXP4-AS1 on promoting the proliferation,migration and inhibiting apoptosis of PTC cells(both p<0.05).(4)Animal experiments:Subcutaneous tumor proliferation in nude mice overexpressing FOXP4-AS1 group was significantly inhibited,and the activation levels of tumor proliferation indexes Ki67 and p-AKT significantly declined(both p<0.05).Conclusions(1)FOXP4-AS1 is generally differentially expressed in tumor and non-tumor tissues,and it low expressed in PTC,in which of the degree are positively correlated with the malignant progression of PTC.(2)Overexpression of FOXP4-AS1 can inhibit the proliferation and migration of PTC cells and promote apoptosis;while interfering with FOXP4-AS1 can promote the proliferation and migration of PTC cells and inhibit apoptosis.(3)FOXP4-AS1 inhibits the proliferation and migration of PTC cells and induces apoptosis by inhibiting activating PI3K/AKT signaling pathway.(4)FOXP4-AS1 can negatively regulate FOXP4,which might be a tumor suppressor target gene of FOXP4-AS1 in PTC cells. |
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