LncRNA-SNHG26 Activates Akt/mTOR Pathway By Inhibiting PGK1 Ubiquitination To Promote The Proliferation,Migration,Invasion And Cisplatin Resistance Of Tongue Squamous Cell Carcinoma

Background:Tongue squamous cell carcinoma(TSCC)is a epidermal malignant tumor occurring in the anterior 2/3 of the tongue.It is one of the main categories of head and neck cancer(the sixth largest cancer in the world),with high invasiveness and metastasis.With the development of medical research,the treatment of TSCC has developed from simple surgical treatment to comprehensive sequential therapy including preoperative induction chemotherapy,surgery and postoperative radiotherapy and chemotherapy,but the 5-year survival rate of patients is still about50%.Existing evidence shows that the main reason for poor prognosis of TSCC patients is the poor clinical chemotherapy efficacy caused by cisplatin based chemotherapy resistance.Therefore,it is urgent to explore the internal mechanism of TSCC chemotherapy resistance and provide solutions for improving the clinical chemotherapy efficacy of TSCC.In recent years,with the deepening of the research in the field of tumor genes,long non coding RNA(lncRNA)is considered to be an important determinant of tumor occurrence,development and chemoresistance.However,the effect and mechanism of lncRNA on TSCC are still relatively lacking.Objective:The purpose of this study was to explore the role and mechanism of lncRNA in the occurrence,development and chemoresistance of TSCC.Methods:(1)The normal TSCC cell line(CAL27)and cisplatin-resistant TSCC cell line(CAL27/CDDP)were compared and detected by next-generation transcriptomic sequencing technology,and the differential lncRNAs related to drug resistance of TSCC were screened and the target lncRNA was identified for further study.The basic characteristics of lncRNA was analyzed in public databases,and the subcellular localization of target lncRNA was detected by fluorescence in situ hybridization(FISH)and nucleoplasmic separation assay.(2)Through The Cancer Genome Atlas(TCGA)public database and clinical tissues,bioinformatics analysis techniques,quantitative real-time PCR(q RT-PCR)and tissue FISH techniques,The relationship between target lncRNA and the occurrence and development of TSCC was analyzed from the clinical tissue level.Through the public database of the Cancer Genome Atlas(TCGA)and the collected clinical case tissues,the relationship between the target lncRNA and the occurrence and development of TSCC was analyzed at the tissue level with the help of bioinformatics analysis technology,quantitative real-time PCR(q RT-PCR)and tissue FISH technology.(3)In TSCC cell lines(CAL27 and SCC15),the target lncRNA was knocked down or overexpressed,the cisplatin sensitivity of the cells was detected by CCK-8assay,the proliferation ability of the cells was detected by CCK-8 and Ed U assay,and the migration and invasion ability of the cells were detected by Scratch and Transwell chamber assay.While expression levels of proteins related to proliferation,migration and invasion is assessed by Western blot(WB)and Immunohistochemistry(IHC).A xenograft nude mouse model was constructed to verify whether the growth of TSCC cells in vivo was consistent with the results of cell experiments in vitro.(4)RNA pull down and mass spectrometry were used to screen targeted lncRNA binding proteins in TSCC cells.It was verified by RNA Binding Protein Immunoprecipitation(RIP),WB and q RT-PCR assays.Co-immunoprecipitation(co-IP)and WB assays were used to detect the specific regulatory mechanism of lncRNA on target protein.Downstream signaling pathway of target protein were screened by literature review and public database analysis.The feasibility of target lncRNA to target protein and downstream signaling pathway was verified by gene knockdown overexpression technique,q RT-PCR,WB and IHC assays.Results:(1)SNHG26 is a highly expressed lncRNA in cisplatin-resistant TSCC cells,which is significantly different from that in normal TSCC cells.It meets the basic characteristics of lncRNA and is mainly located in TSCC cell cytoplasm.(2)In TCGA-HSNC database,SNHG26 was highly expressed in head and neck cancer relative to paracancer tissues,and had good cancer diagnosis ability and prognosis prediction ability;In clinical tissues,SNHG26 is highly expressed in TSCC compared with paracancer tissues,and is closely related to T stage and pathological grade of patients.(3)The high expression of SNHG26 could improve the cisplatin resistance,proliferation,migration and invasion of TSCC cells,and increase the expression of proliferation and epithelial-mesenchymal transition(EMT)related proteins;On the contrary,the low expression of SNHG26 could reduce the cisplatin resistance,proliferation,migration and invasion of TSCC cells,and reduce the expression of proliferation and EMT related proteins.The results of experiments in vivo were consistent with the aboved results.(4)In the cytoplasm of TSCC,SNHG26 could specifically bind to PGK1 protein,inhibit the ubiquitination level of PGK1 and prevent its degradation;The increase of PGK1 protein expression level will increase the phosphorylation level of Akt / mTOR pathway,and then increase the expression of proliferation and EMT related proteins.Conclusions:(1)SNHG26 is a specific high expression lncRNA associated with cisplatin resistance in TSCC cells.(2)SNHG26 is positively correlated with the occurrence,development and poor prognosis of TSCC.(3)SNHG26 could promote the proliferation,migration,invasion and cisplatin resistance of TSCC cells.(4)At the mechanism level,SNHG26 binds to PGK1 in TSCC cells,inhibits its ubiquitination,and then activates Akt / mTOR pathway,so as to enhance the expression of proliferation and EMT related proteins.Thus,lncRNA-SNHG26 can be used as a potential target to inhibit the progression of TSCC and improve its cisplatin chemosensitivity.