| BackgroundOSCC has a high incidence rate in oral tumors,which can easily recur and metastasize with a low five-year survival rate.Although significant progress has been made in the treatment of OSCC,its long-term treatment effect is still not ideal.Therefore,to find the intervention sites and molecular markers during the occurrence of OSCC was important and of great significance for the early diagnosis,personalized treatment and prognosis for those patients.Although long non-coding RNA(lncRNA)are not translated to generate proteins,they participate in the occurrence and development of diseases by altering chromatin conformation,mRNA transcription,post-transcriptional RNA splicing,and protein degradation and transport.LncRNA regarded as tumor biomarker and therapeutic target,was with huge potential.Further research to reveal the mechanism of specific lncRNA in the occurrence and progression of OSCC,could greatly help patients with OSCC to diagnose the disease in the early stage and accurately predict the prognosis,and develop more effective individualized treatments.Therefore,to investigate the potential relationship between lncRNA and OSCC and explore its role in the development and prognosis is of great significance.PurposeIn this study,we selected the lncRNA with further research value in oral squamous cell carcinoma through bioinformatics analysis,and further explored its role and molecular mechanism in the progression,so as to provide new ideas for clinical auxiliary diagnosis and targeted treatment for oral squamous cell carcinoma.Part 1.Expression and clinical value of IGF2BP2-AS1 in OSCCMethods1.Bioinformatics analysisWe screened out the data of OSCC In the TCGA database and analyzed the relevant data of cancer tissues and normal oral tissues.Among the differences of lncRNAs,survival analysis was used for the significantly up regulated lncRNAs to select the ones with further research value.2.Analysis of clinicopathological dataThe correlations of the expression level of IG2BP2-AS1 in OSCC and clinicopathological parameters of those patients was analyzed through clinicopathological data.3.Real-time quantitative PCR detectionAfter RNA was extracted from OSCC and normal tissues around the tumor edge collected in our hospital,we detected the expression level of IGF2BP2-AS1 in tumor and adjacent tissues using qPCR technology.4.Fluorescence in situ hybridization(FISH)The localization of IGF2BP2-AS1 in OSCC cell lines Cal27 and SCC9 was detected by fluorescence in situ hybridization.Results1.Bioinformatics analysisThe results of TCGA database analysis showed that the expression of IGF2BP2AS1 in OSCC was significantly higher than that in normal oral tissues.The survival analysis showed that patients with high expression of IGF2BP2-AS1 live significantly shorter than those with low expression.2.Analysis of clinicopathological dataThe analysis of clinical and pathological data showed that the expression of IGF2BP2-AS1 was significantly correlated with the age of patients and tumor T stage.3.Real-time quantitative PCR detectionCompared with normal tissues adjacent to cancer,the expression of IGF2BP2AS1 in OSCC was significantly increased.4.Fluorescence in situ hybridizationIn OSCC cell lines Cal27 and SCC9,IGF2BP2-AS1 was mainly expressed in the cytoplasm.ConclusionThe expression of IGF2BP2-AS1 was increased in OSCC,and the prognosis of patients with high expression of IGF2BP2-AS1 was poor.Part 2.Effect of IGF2BP2-AS1 on biological behavior of OSCCMethods1.Cell transfectionThe OSCC cell line Cal27 and SCC9 were transfected with IGF2BP2-AS1 small interfering RNA to construct the OSCC cell model with IGF2BP2-AS1 instantaneous knockdown.Transfect IGF2BP2-AS1 knockdown virus into Cal27 OSCC cells to construct a stable knockdown OSCC cell model of IGF2BP2-AS1.2.colony formation assays and cell cycle experimentThe effects of IGF2BP2-AS1 on the proliferation and cell cycle of OSCC cells in vitro were detected by colony formation assays and flow cytometry.3.Wound healing assay and Transwell migration assayThe effect of IGF2BP2-AS1 on the migration ability of OSCC cells in vitro was detected by wound healing assay and Transwell migration assay.4.Subcutaneous tumorigenesis experiment in nude miceSubcutaneous tumorigenesis test was injected to nude mice to check the effect of IGF2BP2-AS1 on OSCC tumor cell proliferation.5.Hematoxylin-eosin staining(H&E staining)The xenograft tumor model of OSCC was constructed in nude mice.The effect of IGF2BP2-AS1 on the invasive ability of OSCC cells in vivo was observed by hematoxylin-eosin staining after the isolated tumor body was fixed.6.Immunohistochemistry(IHC)Immunohistochemical staining was used to observe the effect of IGF2BP2-AS1 on the proliferation of OSCC cells in vivo.Results1.Down-regulating the expression of IGF2BP2-AS1 inhibited the proliferation of OSCC cells and promoted cell apoptosis in vitroThe results of colony formation assays showed that the decreased expression of IGF2BP2-AS1 could significantly inhibit the proliferation of OSCC cells in vitro.The results of cell cycle experiment showed that IGF2BP2-AS1 knockdown could inhibit cell proliferation by inducing cell cycle arrest.2.Down-regulating the expression of IGF2BP2-AS1 inhibited the migration of OSCC cells in vitroThe results of wound healing assay and Transwell migration assay showed that the reduced expression of IGF2BP2-AS1 could inhibit the migration ability of OSCC cells in vitro.3.Down-regulating the expression of IGF2BP2-AS1 inhibited the proliferation and invasion of OSCC cells in vivoThe results of subcutaneous tumorigenesis in nude mice showed that the size and weight of the subcutaneous tumor in the experimental group were significantly lower,indicating that the decreased expression of IGF2BP2-AS1 could inhibit the tumorigenesis of OSCC in vivo.The results of H&E staining and immunohistochemical staining showed that the decreased expression of IGF2BP2AS1 could significantly inhibit the proliferation and invasion of OSCC in vivo.ConclusionThe decreased expression of IGF2BP2-AS1 could inhibit the proliferation,migration,and invasion of OSCC cells.Part 3.Study on the mechanism of IGF2BP2-AS1 affecting the progression of OSCCMethods1.RNA sequencing(RNA-seq)The changes of gene expression after IGF2BP2-AS1 knockdown were detected by transcriptome sequencing,and the pathway enrichment analysis was performed.2.Immunohistochemical stainingThe xenograft tumor model of OSCC was constructed in nude mice.After the isolated tumor was fixed,immunohistochemical staining was performed to observe the effect of IGF2BP2-AS1 knockdown on β-catenin in vivo,the key protein of the Wnt/β-catenin signaling pathway.3.Western blot analysisAfter IGF2BP2-AS1 was knock down in CAL27 and SCC9 cells,western blot was used to detect the effect of that on β-catenin and downstream target proteins Cyclin D1,MMP2,Bcl-2 and Bax.4.Signal pathway rescue experimentIGF2BP2-AS1 was knocked down in CAL27 and SCC9 cells and pathway activator LiCl was added,to verify whether IGF2BP2-AS1 could affects the malignant progression of OSCC through Wnt/β-Catenin signaling pathway by western blot and Transwell migration assay.Results1.Analysis of pathway enrichment after down-regulation of IGF2BP2-AS1 expressionRNA-seq results showed that Wnt signal pathway and other metabolic and signal pathways could be changed after IGF2BP2-AS1 expression was down regulated.2.Down-regulation of IGF2BP2-AS1 expression could inhibit expression of βcatenin protein in vivo.Immunohistochemistry staining results showed that the expression of β-Catenin was significantly lower than that of the control group,indicating that the decreased expression of IGF2BP2-AS1 could inhibit expression of β-catenin protein in vivo.3.Down-regulation of IGF2BP2-AS1 expression could inhibit Wnt/β-Catenin signal pathway and influence the expression of downstream related proteinsWestern blot results showed that in CAL27 and SCC9 cells,after IGF2BP2-AS1 was down-regulated,the expression of β-Catenin was significantly down-regulated.Compared with the control group,the expression of proliferation-related target protein Cyclin D1 and Bcl-2 was significantly down-regulated,the expression of migration and invasion related protein MMP2 was significantly down-regulated,and the expression of apoptosis-related protein Bax was significantly up-regulated in the experimental group.These results suggest that the downregulation of IGF2BP2 AS1 expression could inhibit Wnt/β-Catenin signal pathway and affect the expression of downstream related proteins.4.IGF2BP2-AS1 affects the malignant progression of OSCC through Wnt/βCatenin signal pathwayWestern blot results showed that the expression of β-catenin was significantly enhanced after LiCl was added,and LiCl could partially reverse the inhibitory effect of IGF2BP2-AS1 on β-Catenin.The results of Transwell migration assay showed that the cell migration ability was significantly enhanced after LiCl treatment,and the inhibitory effect on cell migration after IGF2BP2-AS1 knockdown could be partially reversed by LiCl.These results showed that IGF2BP2-AS1 could affects cell malignant progression through Wnt/β-Catenin pathway.ConclusionThe decreased expression of IGF2BP2-AS1 could play a biological role in OSCC through Wnt/β-Catenin pathway. |
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