The Mechanism Of IGF2BP2 Regulating Slug To Promote Lymphatic Metastasis Of Head And Neck Squamous Cell Carcinoma Through M6A Methylation Modification

Background:Head and neck squamous cell carcinoma(HNSCC)is the sixth most common cancer worldwide,resulting in approximately350,000 deaths evey year [1].In recent years,despite advances in multi-disciplinary comprehensive therapy and targeted immunotherapy,the prognosis of patients is still poor,and cervical lymph node metastasis is an important reason for the poor prognosis of patients with HNSCC.Therefore,elucidating the molecular mechanisms that underlie HNSCC initiation and metastasis is of great significance for the improvement in patient prognosis and development of targeted therapeutic strategies.N6-methyladenosine(m6A)modification is the most extensive reversible RNA epigenetic modification in eukaryotes discovered at this stage.RNA methyltransferase(writer)and demethylation transferase(eraser)can dynamically regulate the methylation modification of the nitrogen atom at the 6th position of adenine in RNA,and regulate the alternative splicing,positioning,translation and other processes of RNA under the regulation of m6 A binding proteins(readers).In this dynamic and reversible process,the fate and biological function of m6A-modified RNAs mainly depend on the reader.As a newly discovered reader protein,IGF2BP2 can recognize m6A-modified m RNA by targeting and enhance its stability.It has been reported that IGF2BP2 can regulate the progression of some tumors,but the functional role and mechanism of IGF2BP2 and its mediated m6A-related regulation in HNSCC have not been studied.Objective:To investigate the expression of IGF2BP2 in HNSCC and analyze its correlation with the clinical prognosis;to identify the biological role of IGF2BP2 in lymphatic metastasis of HNSCC,and the molecular mechanism of the interaction between IGF2BP2 and Slug;by constructing a nude mouse model of footpad tumorigenesis and popliteal lymph node metastasis,to investigated whether targeting IGF2BP2 could become a new therapeutic target for patients with head and neck squamous cell carcinoma with lymph node metastasis,providing new ideas for precise treatment.Methods : 1.Bioinformatics technologies such as TCGA database,GEPIA2 online website,and R language were used to analyze the expression and overall survival rate of m6A-related regulators in HNSCC;clinical tissue specimens were used to verify the expression pattern of IGF2BP2 in HNSCC,and the correlation between IGF2BP2 expression and clinicopathological characteristics was further analyzed.2.Knockdown or stably overexpress IGF2BP2 HNSCC cell lines were constructed by small interfering RNA or lentivirus infection;RT-q PCR and western blotting were used to verify the transfection efficiency;Wound-healing assay and Transwell assay were used to detect the effects of IGF2BP2 on the migration and invasion ability of HNSCC cells.3.Using nude mice to establish a popliteal lymph node metastasis model;Fa Du cells were transfected with lentiviral vector expressing sh RNA to stably silence IGF2BP2 expression,and the silencing efficiency of IGF2BP2 was verified by RT-q PCR and western blotting;in vivo bioluminescence imaging and immunohistochemical staining were used to further clarified the effect of IGF2BP2 on lymphatic metastasis and microlymphangiogenesis in HNSCC.4.The EMT cell model was established using TGF-β;to verify whether the EMT cell model was successfully constructed,the changes of cell morphology were observed by inverted microscope,immunofluorescence staining and confocal microscope;and the expression of E-Cadherin was detected by western blotting.5.To clarify the role of IGF2BP2 in EMT process,RT-q PCR and Western blotting were used to detect the expression of EMT markers in silencing-or overexpressing-HNSCC cells,such as E-Cadherin,N-Cadherin and Vimentin.6.To clarify the role of Slug in the regulation of EMT by IGF2BP2,TCGA database,HNSCC cell lines and clinical tissue samples were used to analyze the expression relationship between Slug and IGF2BP2 in HNSCC.7.RIP-q PCR and Me RIP-q PCR were conducted to verify the direct interaction between IGF2BP2 and Slug;SRAMP analysis software was used to predict m6 A modification sites of Slug;luciferase reporter assay and m RNA stability assay were used to further confirm the m6 A dependence between IGF2BP2 and Slug m RNA.Results:1.Twenty m6 A regulators are generally highly expressed in head and neck squamous cell carcinoma,of which only the higher expression of IGF2BP2 is associated with a lower survival probability in HNSCC patients;IGF2BP2 is overexpressed in HNSCC tumor tissues compared with adjacent normal tissues;Compared with tissues without lymphatic metastasis,IGF2BP2 is highly expressed in HNSCC tumor tissue with lymphatic metastasis;high expression of IGF2BP2 is significantly correlated with lymphatic metastasis and poor prognosis in HNSCC,and IGF2BP2 expression was an independent prognostic factor in patients with HNSCC.2.In vitro functional assays: compared with the si-NC group,the m RNA and protein levels of IGF2BP2 in the si-IGF2BP2#2 and si-IGF2BP2#3 groups were significantly decreased;compared with the vector group,the expression of IGF2BP2 in Fa Du and SCC15 cells was successfully up-regulated.The results of cell scratch and Transwell assays showed that compared with the si-NC group,the migration and invasion ability of HNSCC cells in the si-IGF2BP2#2 and si-IGF2BP2#3 groups was significantly inhibited;compared with the vector group,the migration and invasion ability of HNSCC cells in the IGF2BP2 group was significantly enhanced.3.In vivo animal experiments: Compared with the sh-NC group,the m RNA and protein levels of IGF2BP2 in the sh-IGF2BP2 group were significantly decreased;in vivo bioluminescence imaging showed that silencing IGF2BP2 significantly inhibited the lymphatic metastasis of HNSCC cells;volume and weight analyses showed that the footpad tumors and the popliteal lymph nodes were both smaller and lighter in the sh-IGF2BP2 group than in the sh-NC group;immunohistochemical staining analysis showed that silencing IGF2BP2 significantly decreased the levels of microlymphatic vessel density in both the intratumoral and peritumoral regions of mice tissues.4.After treating Fa Du and SCC15 cells with 10ng/ml TGF-β for 72 hours,the cell-to-cell contact was lost and the cell morphology changed from paving stone-like to spindle or spindle characterized by mesenchymal cells.Immunofluorescence staining and confocal imaging analysis showed that HNSCC cells treated with TGF-β formed extensive filopodia and lamellipodia;Western blotting analysis showed that the expression of E-Cadherin was significantly decreased in HNSCC cells treated with TGF-β-,indicating that EMT cells model was successfully established.5.RT-q PCR and Western blotting analyses showed that the expression of epithelial marker E-Cadherin was increased after silencing IGF2BP2,while the mesenchymal markers of N-Cadherin and Vimentin were down-regulated.After overexpression of IGF2BP2,E-Cadherin expression was down-regulated,while the expression levels of N-Cadherin and Vimentin were significantly up-regulated at both m RNA and protein levels.Immunofluorescence staining and confocal imaging analysis showed that knockdown of IGF2BP2 significantly inhibited the expression of Vimentin,while the expression of E-Cadherin was significantly up-regulated.6.Spearman correlation analysis revealed the strongest positive correlation between Slug and IGF2BP2 in HNSCC,and IGF2BP2 was significantly positive correlated with Slug expression;RT-q PCR and western blot analyses revealed that IGF2BP2 inhibition significantly decreased Slug expression at m RNA as well as protein levels in Fa Du and SCC15 cells,whereas overexpression of IGF2BP2 significantly enhanced Slug expression.The results of western blot analysis indicated that Slug knockdown antagonized the upregulation of vimentin and downregu-lation of E-Cadherin induced by IGF2BP2 overexpression in Fa Du cells,whereas IGF2BP2 overexpression partially reversed vimentin’s downregulation and E-Cadherin’s upregulation induced by Slug inhibition in Fa Du cells.Immunohistochemical staining showed that IGF2BP2 high expression specimens were characterized by high Slug expression,whereas IGF2BP2 low expression specimens displayed low to moderate expression of Slug.The quantitative score analysis showed that IGF2BP2 and Slug expression disclosed a robust positive correlation(r =0.752,p-value <0.001).7.The results of RIP-q PCR assays showed that compared with the Ig G negative control group,the IGF2BP2 group significantly enriched Slug m RNA in Fa Du and SCC15 cells;the results of Me RIP-q PCR assays showed that knockdown of IGF2BP2 significantly reduced the m6 A modification levels of Slug;SRAMP software analysis predicted a very high confidence m6 A single-base modification site in the CDS region of Slug m RNA;luciferase reporter assays showed that the luciferase activity was significantly attenuated in Slug-WT group when IGF2BP2 was silenced,while that of Slug-MT group seemed to be unaffected;m RNA stability assays revealed that the m RNA half-lives of Slug were continually reduced by silencing IGF2BP2.Conclusion : Our study reveals for the first time the clinical and biological function of IGF2BP2 in facilitating lymphatic metastasis in HNSCC,and demonstrated that IGF2BP2 promotes EMT and cell metastasis via stabi-lizing Slug m RNA in an m6 A dependent-manner.These outcomes indicate that IGF2BP2 could function as a predictive biomarker of lymphatic metastasis and potential target for anti-metastasis therapies for HNSCC patients.