IGF2BP2 Acts As Proliferation And Invasion Regulator In Laryngeal Squamous Cell Carcinoma Through Facilitating CDK6

Chapter 1 IGF2BP2 regulates the proliferation and invasion of laryngeal squamous cell carcinoma Objective: To identify key genes affecting the progression of laryngeal squamous cell carcinoma(LSCC)by bioinformatics analysis,and to target N6-adenylate methylation(m6A)protein for validation.Methods:(1)Two sets of public chip data were integrated to analyze the differentially expressed genes(DEGs)between LSCCS and normal samples,and the function and signaling pathways of differentially expressed genes were explored through enrichment analysis.(2)The relative expression of insulin-like growth factor 2 m RNA binding protein1(IGF2BP1)and insulin-like growth factor 2 m RNA binding protein 2(IGF2BP2)was measured by q PCR in cancer and paracancer paired clinical samples from 15 LSCC patients.(3)Hematoxylin and eosin(HE)and immunohistochemical(IHC)staining were used to analyze the histopathological features and IGF2BP2 levels of LSCC and paracancer control specimens.(4)The m RNA and protein expression levels of IGF2BP2 in LSCC cells and normal bronchial epithelial cells were detected by quantitative fluorescence PCR(q PCR)and western blotting(Wb).Results:(1)In the cancer and para-cancer samples of GSE59102 and GSE143224 chips,it was found that IGF2BP1 and IGF2BP2 of the m6 A protein IGF2 BP family were differentially expressed in the above two chips.(2)Increased expression of IGF2BP1 and IGF2BP2 was found in cancer and paracancer matched clinical samples from 15 LSCC patients.(3)The survival prognosis of IGF2BP2 was analyzed in conjunction with the plots of head and neck squamous cell carcinoma cases in the KMPLOTS database,and it was found that the lower the expression of IGF2BP2,the higher the survival rate of head and neck squamous cell carcinoma patients.(4)IHC,q PCR and Wb verified that IGF2BP2 was up-regulated in LSCC.Conclusion:(1)The differential expression gene profile of LSCC was constructed by analyzing the difference between LSCC and paracancer samples of GEO data.(2)IGF2BP2 was an up-regulated regulator of m6 A in LSCC public data,clinical samples and cell lines.(3)In patients with LSCCS and other head and neck squamous cell carcinomas,lower IGF2BP2 is associated with better survival.Chapter 2 In vitro and in vivo studies of Ig F2BP2-mediated CDK6 regulation of LSCC proliferation and invasion Objective: In order to further explore the relationship between IGF2BP2 and LSCC,this part of the experiment will study IGF2BP2 at two levels in vitro and in vivo.Methods:(1)IGF2BP2 was knocked down in human laryngeal carcinoma cell lines TU686 and FD-LSC-1 in vitro,and the effects of IGF2BP2 on the cells were investigated by CCK-8,colony formation,transwell and flow cytometry.(2)In vivo,a subcutaneous transplantation tumor model of nude mice was constructed using human laryngeal carcinoma cell TU686 with IGF2BP2 expression down-regulated.The model was divided into three groups: 5 mice in group Lv-sh-NC,5 mice in group Lv-sh-IGF2BP2#1,and 5 mice in group Lv-sh-IGF2BP2#2,with a total of 15 nude mice.Solid tumor volume was observed and recorded on a regular basis,growth curves were drawn,and the expression of IGF2BP2 and Ki67 were detected histopathologically in the removed tumor.Results:(1)CKK8 and plate cloning experiments showed that the growth of LSCC cells was inhibited after IGF2BP2 silencing.(2)Flow cytometry showed that IGF2BP2 deletion led to G0/G1 phase cell cycle arrest.(3)histopathological analysis of the three groups of tumors showed that the knockout of IGF2BP2 significantly reduced the levels of Ki67 and IGF2BP2 in the transplanted tumor tissues.Conclusions:(1)The effective construction of IGF2BP2 interference model in laryngeal cancer cells can effectively silence the expression of IGF2BP2 in TU686 and FD-LSC-1 cells,which provides an experimental basis for the study of the role of IGF2BP2 in laryngeal cancer cells.(2)The expression level of IGF2BP2 in LSCC cells is correlated with the proliferation and invasion ability of laryngeal carcinoma cells in vitro.(3)IGF2BP2 can affect the cell cycle and its associated proteins,namely,silencing IGF2BP2 blocks the cell cycle in G0/G1 phase,thereby reducing the invasiveness of LSCC cells by regulating cell cycle arrest.(4)The expression level of IGF2BP2 in LSCC-transplanted nude mice was positively correlated with the tumor,that is,low expression of IGF2BP2 would reduce the growth of the transplanted tumor.Chapter 3 IGF2BP2 promotes invasiveness of LSCC cells by affecting CDK6 expression and stability Objective: To study IGF2BP2 targeting molecules and verify their causal relationship,and to evaluate the effect of IGF2BP2 expression on cell cycle-related proteins at the mechanistic verification level Methods:(1)Public databases were used to explore the high correlation between IGF2BP2 and IGF2BP2 molecules affecting the cell cycle and to verify their correlation in expression.(2)Determine the half-life to understand the stability.(3)The response experiment finally clarified the interaction between the two.Results:(1)The expression of CDK6 was high in LSCCS,and the two were positively correlated,that is,the expression of CDK6 decreased with the down-regulation of IGF2BP2.(2)IGF2BP2 mediates the stabilization of CDK6,that is,the half-life of CDK6 m RNA is shortened after IGF2BP2 knockout.(3)IGF2BP2 promoted the invasivity of LSCC cells through CDK6,and the parts with enhanced cell proliferation and invasivity induced by IGF2BP2 overexpression were neutralized by CDK6 knockdown.Conclusion: IGF2BP2 regulates the expression of CDK6 by promoting the stabilization of CDK6 m RNA.Response tests showed that down-regulation of CDK6 did not cause changes in IGF2BP2 expression,but partially eliminated the promoting effect of IGF2BP2 overexpression on the invasion of LSCC cells.