Expression Of Insulin-like Growth FactorⅡmRNA Binding Protein 3 And Its Clinical Significance In The Development Of Gastroenteropancreatic Neuroendocrine Neoplasm

The incidence of gastroenteropancreatic neuroendocrine neoplasms(GEP-NEN) has substantially increased over the last decades.The clinical course of the disease is almost indolent.Although up to two-thirds of patients are diagnosed as GEP-NEN with distant metastasis,the 5-years survival exceeds 60%.For this reason,the incidence of GEP-NEN is higher than that of pancreatic,gastric and esophageal adenocarcinomas,making it the second most prevalent cancer type of the gastrointestinal(GI) tract.The early detection and subsequent effective therapy might improve the prognosis of GEP-NEN. Diagnosis of these tumors has been impoved by advances in pathological diagnosis,classification and endoscopic ultrasound and somatostatin receptor fusion.Genetic and molecular advances have identified molecular targets in the treatment of these tumors.Surgery remains the mainstay of treatment,amply supported by interventional radiological techniques,including embolization. Treatment of metastatic disease has significantly improved with the addition of several new agents,Despite significant advances in the understanding and management of GEP-NETs,the survival of patients remains unchanged and there remains a need for the development of national and international research collaborations to spearhead future efforts.IMP3,a member of insulin-like growth factor 2(IGF-2) m RNA- binding proteins(IMPs) family,consists of IMP1,IMP2 and IMP3.IMPs bind to and influence the transportation,localization and stability of target m RNA, especially during early stages of both human and mouse embryogenesis.IMP3 is located on chromosome 7p11.5 and encodes a 4350 bp m RNA.IMP3 is expressed in the developing epithelium,muscle and placenta during the early stages of human and mouse embryogenesis, and low or undetectable levels of IMP3 are present in adult tissues.IMP3 has been shown to be overexpressed in gastric cancer,colon cancer and adenocarcinoma of the lung.The expression of IMP3 was identified to correlate with tumor aggressive progression,suggesting that IMP3 may play a role in tumor invasion and metastasis.Previous studies reported that the oncogenic effects of IMP3 have been suggested to be mediated through IGF-Ⅱ,the m RNA for which is activated by Imp3(11,12).IGF2 ligands activate a common receptor,the IGF1 receptor(IGF1R),which signals mitogenic,anti-apoptotic,and transforming activities.The IGF1 R is a cell-surface tyrosine kinase receptor coupled to several intracellular secondary messenger pathways,including theras-raf-MAPK and PI3K-AKT signaling cascades.The IGF1 R is vital for cell survival,as illustrated by the lethal phenotype of mice in which the IGF1 R gene is disrupted.A recent research reports mouse IMPS family gene deletion leads to CD44 m RNA down-regulation.CD44,a cell adhesion molecule,can promote the degradation of type IV collagen fibers and combine with the cytoskeletal protein,down-regulation of CD44 can reduce the adhesion between cell and matrix.Up to now,no independent prognostic value of IMP3 in GEP-NEN has been reported.To confirm the involvement of IMP3 in the prognosis of GEP-NEN we collected clinical samples of GEP-NEN tissue and cultured Gastroenteropancreatic neuroendocrine neoplasm cell line.The methods including pathology,immunohistochemistry,western blot,quantitative real-time PCR(q RT-PCR),MTT colorimetric method,Immunohistochemical staining and Transwell were used to investigate the relationship of the expression of IMP3 and biological behaviours of GEP-NEN.The expression of IMP3 was examined by immunohistochemistry,western blot and q RT-PCR.The effects of IMP3 on proliferation,and invasion of GEP-NEN cell were detected after silencing of endogenous IMP3 using si RNA technique,for clarifying the important roles of IMP3 in the process of development of GEP-NEN.All the four parts of studies were aimed to investigate the effects of IMP3 on the generation,migration and invasion of GEP-NEN,and to provide evidence as well as target for diagnosis and treatment of GEP-NEN.The detailed methods and results of this study are as follows: Part I Expr ession of IMP3 and its clinical significance r elated to the differentiation of Gastr oentero-pancreatic neuroendocrine neoplasmObjectives:To investigate the expression and clinical significance of Oncofetal Protein IMP3 in the differentiation gastroenteropancreatic neuroendocrine neoplasm(GEP-NEN).Methods: A total of 162 patients who were diagnosed as GEP-NEN and underwent surgery or endoscopy resection from January 2006 to March 2013 were enrolled in this study.including 85 cases of neuroendocrine tumor G1,40 cases of neuroendocrine tumor G2,28 cases of neuroendocrine carcinomas G3 and 9 cases of mixed adenoneuroendocrine carcinomas,(MANEC).The clinical and pathological data were recorded for analysis.The expression of IMP3,CD44,IGF1 R,and MMP2 were determined by immunohistochemistry,SPSS 16.0 software was used for data processing and analyses, and significance was determined when P<0.05.Result: Oncofetal Protein IMP3 showed a considerably high expression rate of 74.69% in GEP-NEN.IMP3 positive cases showed significantly decreased overall and disease-free survival time as compared to IMP3-negative cases(P=0.012).Overexpression of IMP3 was correlated with tumor grade,clinical stage,tumor size and poor prognosis(all Ps<0.05).Patients with overexpression of IMP3 had a poorer prognosis(P<0.01),The COX regression showed that the overexpression of IMP3,tumor grade,tumor size and metastasis of GEP-NEN were associated with clinical outcomes.Our results also showed that the expression of CD44,IGF1 R,and MMP2 in GEP-NEN were 19.75%,53.7% and 55.56%,respectively.While it was negatively correlated with the expressions of CD44(r=-0.131;P=0.096),the expression of IMP3 was positively correlated with the expression of IGF1 R and MMP2(r=0.288;P<0.01 and r=0.208;P=0.008).In addition,the expression of IGF1 R and MMP2 were positively correlated(r=0.687; P<0.01).Summary:1 The positive expression rate of IMP3 protein in NEC G3 was significantly higher than that in NET G1 and G2 tissues;2 The survival rate of patients with positive expression of IMP3 is low;3 High IMP3 expression is associated with poor differentiation and tumor sizes in GEP-NEN patients;4 Positive expression of IMP3 was positively correlated with IGFR1 and MMP2. Part II Expression of IMP3 and its endoscopic and pathological featur es related to the low-grade gastrointestinal neuroendocrine neoplasmObjectives: To investigate the expression of insulin-like growth factor 2 m RNA binding protein 3(IMP3) in the low-proliferation neuroendocrine tumor tissue and its pathological features of endoscope.Methods: the endoscopic diagnosis and pathology of neuroendocrine tumor endoscopic ultrasonography for assessment of lesions,select resectable cases underwent endoscopic resection and in measuring the size of the lesion,combined with endoscopic findings and clinical data of follow-up analysis,collected the 18 cases of neuroendocrine tumor patients underwent endoscopic resection and 3 cases of biopsy specimens.All the specimens were placed in sterile EP tubes,and quickly stored in liquid nitrogen transport to 80℃ refrigerator.All patients did not receive radiotherapy and chemotherapy before and after surgery,they were confirmed by pathological examination.Western-blot method was used to detect tissues in 21 cases of neuroendocrine tumors.Result:1 21 cases of patients were examined by endoscopic ultrasound to determine the depth of lesions,18 cases subjected to endoscopic resection,The diagnostic accuracy of endoscopic ultrasonography(EUS) was 88.9%(16/18), EUS has high accuracy for evaluating the feasibility of resection on lesions of low-grade gastrointestinal neuroendocrine tumor.2 18 cases of resection patients were followed up for 12 to 24 months,no recurrence and metastasis,it is an effective and safe method to treat the smaller lesions(less than 1cm) of low-grade(G1 or G2)gastrointestinal neuroendocrine tumors by endoscopic ultrasound.3 the expression of IMP3 protein was detected in issues of patients with low-proliferation neuroendocrine tumor by Westem-blot,and the expression of IMP3 protein was positive in 21 cases.The results showed that IMP3 protein over-expression was significantly associated with proliferation grade,tumor size and invasion(1.4176+0.1168 vs 0.8187+0.3169,P<0.001;1.3806+0.1286 vs 0.8372+0.3491,P<0.05;1.3801+0.1268 vs 0.7470+0.2814,P<0.05),however, no correlation was observed between IMP3 positive expression and age, gender,and numbers of tumor.Summary:1 The endoscopic resection for low proliferation gastrointestinal neuroendocrine tumor less than or equal to 1cm is safe and effective.2 The IMP3 expression was significantly higher in neuroendocrine tumor Grade G2 compared to that of Grade G1 patients.3 The expression of IMP3 protein was associated with tumor size and invasion depth in low proliferative neuroendocrine tumor. Part III The Effect of IMP3 on the biological characteristics of Gastroenteropancreatic neuroendocrine neoplasm STC-1 cell lineObjective: To investigate the effect of si RNA on the proliferation and invasion of neuroendocrine carcinoma STC-1 cells by inhibiting the expression of insulin-like growth factor m RNA binding protein 3(IMP3) gene.Method: to construct three si RNA sequences for IMP3 and one nonspecific si RNA sequence,the best transfection efficiency of IMP3-si RNA was selected to transfect the mouse neuroendocrine carcinoma cell STC-1. Immunocytochemical staining to observe IMP3 expression in cells.MTT assay was used to detect the cell growth curve;Brd U labeling method to detect cell DNA synthesis,migration and invasion of STC-1 cells were detected by Transwell model.Results: the successful construction of IMP3-si RNA transfection system and STC-1 cells transfected with IMP3-si RNA-1.Immunocytochemical staining results showed that, compared with the con group and NS-si RNA group,shallow IMP3-si RNA group cell lines IMP3 staining,MTT assay and Brd U incorporation into the experiment found that,compared with the con group and NS-si RNA group and weaken the IMP3-si RNA group DNA synthesis,cell proliferation decreased significantly;Transwell model analysis found compared with con group and NS-si RNA group,IMP3-si RNA group of cells migration and transmembrane invasion was significantly inhibited(P< 0.05).Summary:1 Three IMP3-si RNA sequences were successfully constructed,the highest transfection efficiency was IMP3-si RNA-1,which is the best inhibition of IMP3;2 inhibition of IMP3 expression made STC-1 cell proliferation activity decreased;3 inhibition of IMP3 expression significantly inhibited STC-1 cell migration and invasion. Part IV The role and mechanism of IMP3 in proliferation,migration and invasion of Gastr oentero-pancreatic neuroendocrine neoplasm STC-1 cell lineObjective: To investigate the effect of si RNA on the expression of insulin-like growth factor m RNA binding protein 3(IMP3) gene on the proliferation,invasion and migration of neuroendocrine carcinoma STC-1 cells in mice.Methods:IMP3-si RNA-1were transfected into the mouse neuroendocrine carcinoma cell strains of STC-1.Western blot was used to detect the interference of IMP3 expression and detection NS-si RNA group tumor proliferation of growth(p27,Cyclin D1,EGFR and Ki67) and angiogenesis(VEGFA) and invasion and metastasis(IGF1R, MMP9 and MMP2) related indicators of change.Results: STC-1 cells in the con group and NS-si RNA group transfected by IMP3-si RNA-1 compared to the IMP3-si RNA group,tumor proliferation protein, EGFRA and Ki67 protein expression levels were significantly reduced, and Cyclin D1 and p27 protein expression showed no obvious change;as well as the invasion metastasis associated protein IGF1 R,VEGFA,MMP2,MMP9 protein was decreased.Summary:1 IMP3 silencing may inhibit the proliferation of neuroendocrine cancer cells by downregulation of the expression of protein Ki67 and EGFR associated with tumor proliferation.2 IMP3 silencing may down regulate VEGFA,matrix metalloproteinases MMP2,MMP9 and other related proteins through the IGF1 R signal transduction pathway,then affect the invasion and migration of neuroendocrine tumor cells, and promote the development of tumor.Conclusion:1 IMP3 expression is related to the biological characteristics of gastroenteropancreatic neuroendocrine tumors;the IMP3 expression in neuroendocrine carcinoma G3 and MANEC tissue was higher than that of neuroendocrine tumors(G1, G2);The survival rate of GEP-NEN patients with positive expression of IMP3 is low;IMP3 is one of predictive factors of GEP-NEN with distant metastasis.2 Endoscopic resection of low-proliferation neuroendocrine tumors(less than or equal to 1cm) is safe and effective.In the lowg-rade neuroendocrine tumors, IMP3 expression was different,the tumor size and depth of invasion were also different in IMP3 expression,the higher the GEP-NET proliferation activity,the deeper the depth of invasion,the higher the IMP3 expression.3 We constructed three IMP3-si RNA sequences successfully,the IMP3-si RNA-1,which is the highest transfection efficiency,the best inhibition of IMP3,decreased IMP3 expression of STC-1 cells,inhibited cell proliferation;decreased the activity of the planar cell migration;cell transmembrane migration activity and cell migration rate were all significantly inhibited,too.4 IMP3 silencing may inhibit the proliferation of neuroendocrine cancer cells by downregulation of the expression of protein Ki67 and EGFR associated with tumor proliferation.IMP3 silencing may down regulate VEGFA,matrix metalloproteinases MMP2,MMP9 and other related proteins through the IGF1 R signal transduction pathway,then affect the invasion and migration of neuroendocrine tumor cells,and promote the development of tumor.5 IMP3 may be a key molecular target in diagnosis and treatment of Gastroenteropancreatic neuroendocrine neoplasm.