Clinical Significance And Mechanism Of NIPAL1 And GRHL1 In Esophageal Squamous Cell Carcinoma

Chapterl Clinical Significance and Mechanism of NIPAL1 in Esophageal Squamous Cell CarcinomaBackgroundEsophageal cancer is the eighth most common type of cancer and also the sixth leading cause of cancer-related death in the world[1].About 50%of esophageal cancers in the United States and other western countries are adenocarcinomas.However,in Asian countries,about 90%of esophageal carcinomas are squamous cell carcinomas(SCC)and SCC is a major histological subtype of esophageal cancer,accounting for 80%of all esophageal cancers in the world[2].At present,the molecular mechanisms of the invasion and metastasis about esophageal squamous cell carcinoma are not cdear.But,researchers are working to dig deeper into the mechanisms by which these functional biomarkers develop in esophageal squamous cell carcinoma,as these markers can make esophageal squamous cell carcinoma(ESCC)patients more accurately delaminate and help clinicians make the best treatment decisions to improve patient outcomes.Three ESCC patients’ tumors and normal esophageal epithelial tissues adjacent to cancer were sequenced by the second generation deep sequencing technique.Sequencing results showed that NIPAL1 was one of the significantly down-regulated genes.In addition to oral squamous cell carcinoma,the expression and function of NIPAL1 in other tumors have not been reported.Therefore,it is necessary to study the role and mechanism of NIPALI in esophageal squamous cell carcinoma..Materials and methods1.Patients and tissue specimentsWe collected 60 fresh tissues and 299 paraffin-embedded tissues from patients with esophageal squamous cell carcinoma.2.MethodsThe expression of NIPAL1 in 60 cases of esophageal squamous cell carcinoma and its adjacent normal esophageal epithelium was detected by qRT-PCR.The expression level of NIPAL1 in 299 cases of ESCC tissue microarray was detected by immunohistochemistry,and the correlation among the expression level of NIPAL1,clinicopathological characteristics and prognosis was analyzed.Esophageal cancer cell lines EC 109 and EC9706 stably expressing NIPAL1 were successfully constructed by Lentivirus.At the same time,a stable knockdown NIPAL1(shRNA)in KYSE510 was successfully constructed.The effects of NIPAL1 on cell growth and migration in vitro and in vivo were studied by CCK-8 experiment,foci formation,subcutaneous tumorigenesis experiment in nude mice,invasion and migration Experiment and tail vein pulmonary metastasis model in nude mice.In the study of molecular mechanism of NIPAL1,we used Western blotting,flow cytometry,F-actin,immunofluorescence,JC-1 and live cell imaging to analyze and explore the mechanism of NIPAL1 inhibiting the growth and metastasis of tumors.ResultsThe results showed that the expression of NIPAL1 in ESCC tissues and most cell lines decreased.At the same time,the low expression of NIPAL1 was significantly correlated with clinical stage(TNM),lymph node metastasis and prognosis of ESCC.In vivo and in vitro functional tests showed that NIPAL1 could effectively inhibit the proliferation and migration of esophageal cancer cells.The results of mechanism study showed that:1.NIPAL1 can block G2/M phase by negative regulation of p-CDC2;2.NIPAL1 can promote cell apoptosis by down-regulating Bcl-2;3.NIPAL1 can inhibit ESCC cell migration by negative regulation of slug expression;ConclusionsNIPAL1 has the function of inhibiting the proliferation,invasion and metastasis of ESCC.NIPAL1 plays an anti-oncogene role by blocking G2/M phase,inhibiting cell mitosis and reducing the formation of filamentous pseudopods and plate pseudopods in the cytoskeleton,.Chapter2 Suppressor gene GRHL1 is related to the clinical prognosis in patients with Esophageal Squamous Cell CarcinomaBackgroundGRHL1 is one of family genes encoding transcription factors that assumes a part in restraining tumor cell clone development,proliferation,and development of embedded tumor cells.This study aims to assess the expression and prognostic estimation of GRHL1 in esophageal squamous cell carcinoma(ESCC).MethodsGRHL1 mRNA and protein level were detected in ESCC cell lines and clinical ESCC tissues by quantitative RT-PCR,agarose gel electrophoresis,and western blotting.The expression of GRHL1 was stained by immunohistochemical staining in 266 paraffin-implanted ESCC samples with clinical methodologies,and cox regression was utilized to distinguish autonomous prognostic components.The practical part of GRHL1 in ESCC cell lines was assessed by lentiviral construct containing GRHL1 overexpressed of the protein revealed by the cell growth and foci formation assays.ResultsThe expression of GRHL1 was downregulated in most ESCC cell lines and clinical tissues at both the mRNA and protein levels.Low expression of GRHL1 was essentially connected with tumor invasion(P=0.008),clinical stage(P=0.004)and patient death(P<0.001).Furthermore,Kaplan-Meier demonstrated that the low expression of GRHL1 was notably related to poorer overall survival(OS)(log-rank test,P<0.001,HR,2.073;95%Cl,1.491-2.881).Moreover,Cox regression analysis demonstrated that the low expression of GRHL1 and bad differentiation were autonomous prognostic pointers for poor survival of patients with ESCC(both P<0.05).Besides,the GRHL 1-overexpressing cells restrained the invasive capacity of ESCC cells in vitro.ConclusionsThe low expression of GRHL1 is related with altogether poorer OS in ESCC and overexpressing of GRHL1 restrained ESCC cell intrusion.GRHL1 can be filled in as another autonomous prognostic marker and additionally as a novel potential restorative focus for ESCC.