The Function And Related Mechanism Study Of IGF2BP2 Induces Metastasis Of Esophageal Cancer

ObjectiveEsophageal cancer is one of the most common malignant tumors in digestive system,the morbidity and mortality of which have been top in the world for a long time with a 5-year survival rate<30%.The metastasis and recurrence are the principal causes of death for esophageal cancer patients.Therefore,the research on the mechanism of esophageal cancer metastasis is of particular importance.Insulin-like growth factor 2 messenger RNA binding protein 2(IGF2BP2)has a wide range of pathophysiological functions in embryonic development,neuronal differentiation,metabolism,insulin resistance and tumorigenesis.IGF2BP2 is over-expressed in esophageal adenocarcinoma tissue and Barret’s esophagus tissue,closely correlated with metastasis and poor survival in patients,relevant mechanism of which is unknown.The genetic polymorphisms of the IGF2BP2 rs1470579 gene could be associated with the genetic susceptibility to esophagogastric junction adenocarcinoma in eastern Chinese Han population.The correlation between IGF2BP2 and metastasis of esophageal squamous cell carcinoma(ESCC)and its molecular mechanism remain to be explained.MethodIn this study,in vivo and in vitro experiments were performed to investigate the role of IGF2BP2 in esophageal squamous cell carcinoma and the potential metastasis-promoting mechanism of the gene.(1)The expression of IGF2BP2 in esophageal squamous cell carcinoma tissues and adjacent normal tissues were detected.Then,the relationship between the expression of IGF2BP2 and clinicopathological parameters was analyzed.(2)The stable ESCC cell lines for knocking down and over-expressing IGF2BP2 were constructed through lentiviral stable transfection experiments.Effects of IGF2BP2 on the clonal formation,proliferation,migration and invasion of esophageal squamous cell carcinoma cells were studied.(3)Western blot and real-time fluorescent quantitative PCR were used to explore the correlation between the expression levels of IGF2BP2 and IGF2 and EMT(Epithelial to mesenchymal transition)markers(E-cadherin,N-cadherin,etc.).We analyzed the possible related pathways of metastasis by bioinformatics and verified it by western blot.(4)Experiment in vivo:through nude mouse tumor formation experiment,the effect of IGF2BP2 on transplanted tumor growth in nude mice was clarified.We explored the role of IGF2BP2 in potential metastasis-promoting mechanism in esophageal squamous cell carcinoma by analyzing the results.The mechanism provides a new candidate markers for the prognostic judgment and treatment of esophageal squamous cell carcinoma.Results(1)The expressions of IGF2BP2,IGF2,phospho-p38MAPK and EMT markers were detected in 203 cases of esophageal cancer tissues and adjacent normal tissues.The results showed that the expression of IGF2BP2,IGF2,phospho-p38MAPK,Vimentin,Twist,Snail,and N-cadherin was significantly up-regulated and the expression of E-cadherin was significantly down-regulated in tissue samples of esophageal carcinoma.IGF2BP2 expression was detected in cell lines.Compared with normal esophageal epithelial cells,Het,the expression of IGF2BP2 protein in ESCC cells lines was significantly up-regulated.(2)After knocking down IGF2BP2,the proliferation inhibition rate of esophageal squamous cell carcinoma was over 15%,the colony formation rate decreased by 4.8%,the wound healing rate decreased by 39.2%,and the invasive ability decreased.After over-expressing IGF2BP2,the proliferation promotion rate of ESCC cells was over35%,the colony formation rate increased by 13.0%,the wound healing rate increased by 19.8%,and the invasive ability increased too.(3)The m RNA expression of IGF2 gene was down-regulated by 52.1%after knocking down IGF2BP2(P<0.01),and the m RNA expression of IGF2 gene was up-regulated by 69.3%when over-expressed IGF2BP2(P<0.001).When IGF2BP2knocked down the expression level of epithelial marker E-cadherin increased,the expression level of mesenchymal marker N-cadherin and Slug protein decreased,and the expression level of IGF2、p38MAPK and phospho-p38MAPK were down-regulated.When IGF2BP2 over-expressed,the expression of epithelial marker E-cadherin declined,while the expression level of mesenchymal marker N-cadherin and Slug increased,in meanwhile the expression level of IGF2、p38MAPK and phospho-p38MAPK were up-regulated.(4)The model of subcutaneous transplanted tumor in nude mice was successfully established.After knocking down IGF2BP2,the tumor volume and mass were significantly smaller than before and the metastasis rate of transplanted tumors reduced.After over-expressing IGF2BP2,the tumor volume and mass were significantly larger than before and the metastasis rate of transplanted tumors increased.Conclusion(1)IGF2BP2 highly expresses in human ESCC tissues,and the high expression of)IGF2BP2 is a risk factor for metastasis of ESCC.(2)IGF2BP2 can promote the proliferation,clone formation,invasion,and metastasis of ESCC cells.The metastasis may be associated with EMT in ESCC cells.(3)IGF2BP2 may affect the EMT process of ESCC cells through the IGF2/p38MAPK pathway.