Expression And Mechanism Of Long Non Coding RNA THOR In Esophageal Squamous Cell Carcinoma

Background:Esophageal squamous cell carcinoma(ESCC)is one of the most common malignant tumors in the world.There are obvious regional and histological differences in the incidence of ESCC,which is closely related to race,genetics,geographical location and eating habits.At present,esophagectomy is mainly aimed at ESCC cases with limited invasion and distant metastasis in the early stage,but recurrence often occurs and the prognosis is poor;for advanced patients,multimodal therapy of systemic chemotherapy and radiotherapy can only bring limited clinical benefits.At present,there is no molecular targeted therapy for patients with advanced metastasis.In recent years,despite the progress in the diagnosis and treatment of ESCC,due to the lack of specific and sensitive early tumor diagnostic markers,ESCC patients are often in advanced stage.Therefore,screening more effective tumor markers and therapeutic targets for diagnosis and prognosis has become the main direction of basic and clinical research of ESCC.A very conserved long noncoding RNA,testis associated highly conserved oncogenic long non coding RNA(THOR),was newly discovered in human genome sequencing.The gene is located on chromosome 2 and has a total length of 631 bp,It is composed of three exons.It was initially found that it is specifically expressed in testicular tissue.Now it has been confirmed that it also exists widely in a variety of human tumor tissues.Its high expression can significantly promote the occurrence and development of malignant tumors.Moreover,THOR can interact with insulin-like growth factor 2 mRNA binding protein 1(IGF2BP1)to promote the occurrence and development of a variety of human tumors.In this project,we studied the expression of THOR in esophageal squamous cell carcinoma,and explored its mechanism of promoting proliferation,cloning,migration and a series of cell biological functions,which opened up a new field of vision for the future research of new targets for the diagnosis and treatment of esophageal cancer.Objective:1.To explore the expression of THOR in ESCC and analyze the correlation between THOR and pathological information of patients;2.Using siRNA to knock down THOR in ESCC,to explore the possible role and molecular mechanism of THOR in promoting the occurrence and development of ESCC.In order to open up a new field of vision for the future research of new targets for the diagnosis,prognosis evaluation and treatment of esophageal cancer.Methods:1.The expression of THOR in esophageal cancer was analyzed by using ualcan database,and the correlation between THOR and pathological characteristics of esophageal cancer such as sample type,race,gender,race,weight,smoking,tumor stage,tumor grade and tumor subtype was analyzed;2.Tissue samples of 37 patients with ESCC were collected from the Department of thoracic surgery of North Sichuan Medical College from 2018 to 2019.qRT-PCR was used to quantitatively detect the expression level of THOR in 37 pairs of ESCC cancer tissues and paired normal esophageal epithelial tissues adjacent to cancer,and the correlation between the expression level of THOR and clinicopathological information of the patient was analyzed;3.Normal esophageal epithelial cell lines HET-1A and ESCC cell lines TE1,EC 109 and KYSE150 were cultured in vitro.The expression of Thor was detected by qRT-PCR,and the high expression cell lines of THOR were selected as the experimental subjects for the follow-up study;4.For ESCC cell line te1 with significantly high expression of THOR,siRNA was used to knock down the expression of THOR at two sites(si-THOR-1,si-THOR-2).CCK-8 test,plate clone formation test,scratch test and cell cycle test were used to detect the effects of knocking down the expression of THOR on the proliferation,colony formation,migration and cell cycle of TE1;5.For TE1 cell line,siRNA was used to knock down the expression of THOR.The mRNA and protein expressions of IGF2BP1 and the dependent genes GLI-1 and MYC andwere detected by qRT-PCR and Western blotting,respectively.Results:1.The results of online analysis of ualcan database showed that the expression of Thor in EC patients was significantly higher than that in normal people,and the expression of THOR was also significantly higher in male patients with esophageal adenocarcinoma(EAC)and ESCC,whose tumor stage was Ⅱ and Ⅲ,race was Caucasian and Asian,age was 40-80 years,and tumor grade was Ⅱ and Ⅲ.2.qRT-PCR was used to detect the expression of THOR in 37 pairs of ESCC cancer tissues/adjacent tissues.It was found that the expression level of Thor in ESCC cancer tissues was significantly higher than that in adjacent normal esophageal epithelial tissues(P<0.01).At the same time,the correlation between the tumor location,differentiation grade,tumor diameter and lymph node metastasis was analyzed.The results were consistent with the data of ualcan,which confirmed the high expression of THOR in ESCC,suggesting that THOR may be a oncogene of ESCC.THOR may play an important role in the occurrence and development of ESCC.3.Through the detection of THOR in ESCC cell lines TE1,EC 109,KYSE150 and normal esophageal epithelial cell line HET-1A,we found that the expression level of THOR in TE1 and EC 109 was significantly higher than that in normal esophageal epithelial cell line HET-1A.TE1 can be used as an experimental object for further study of cell biological function.4.In cell line TE1,after siRNA knockdown of THOR at two sites(si-THOR-1,si-THOR-2),THOR was significantly lower than that in the control group(si-NC)(P<0.01).After si-THOR-1 and si-THOR-2 were transfected into TE1 cells,the results of CCK8 experiment confirmed that the cell proliferation of the experimental group was inhibited,and the difference was statistically significant.Compared with the control group(si-NC),the colony forming ability of the experimental group(si-THOR-1,si-THOR-2)was significantly decreased.Scratch test showed that compared with the meaningless control group(si-NC),the migration ability of TE1 cells transfected with two targets(si-THOR-1,si-THOR-2)was significantly inhibited at 12hours,24hours and 48 hours.Flow cytometry showed that the cells in the two experimental groups(si-THOR-1,si-THOR-2)had obvious G1 phase arrest.5.In TE1 cell line,the expression of IGF2BP1 and its dependent genes GLI-1,MYC mRNA and protein were significantly down regulated by siRNA targeting at two sites(si-THOR-1,si-THOR-2)compared with the control group(si-NC)by qRT-PCR and Western blot(P<0.05).Conclusion:Knockdown of the expression of THOR in ESCC cell line TE1 significantly inhibited the proliferation,cloning,migration and cell cycle of TE1.The molecular mechanism of IGf2BP1 may be that it mediates the expression of GLI-1 and MYC,thus promoting the proliferation,clone formation,migration and cell cycle of tumor cells.It opens up a new field of vision for better diagnosis and treatment of ESCC in the future.