The Role And Mechanism Of Long Non-coding RNA G077640 In Esophageal Squamous Cell Carcinoma

Objective:To detect the differential expression of long non-coding RNAs in esophageal squamous cell carcinoma tissues and their adjacent tissues,and to explore its pathogenesis in esophageal squamous cell carcinoma.Methods : 1.Screening for lncRNA: First,we collected 3 pairs of esophageal squamous cell carcinoma tissues,paracancerous tissues,and cancer metastasis-positive lymph node tissues without chemoradiotherapy,and pathological HE staining confirmed their pathological characteristics,were used for lncRNA microarray experiment.There are 64 paired esophageal squamous cell carcinoma tissues,paracancerous tissues,and cancer metastasis-positive lymph nodes were collected,and total RNA was extracted by trizol method and reverse transcribed into cDNA.According to the results of lncRNA microarray,the target lncRNA was screened,and the expression of the target lncRNA was detected by qRT-PCR in esophageal squamous cell carcinoma tissues and their adjacent tissues.According to the expression of the target lncRNA G077640,the clinical information of the collected samples was statistically analyzed.2.To detect the effect of lncRNA G077640 on the biological behavior of esophageal squamous cell carcinoma cells: firstly,qRT-PCR was used to detect the expression of lncRNA G077640 in immortalization esophageal squamous cell line(Het-1A)and esophageal squamous cell carcinoma cell lines(TE-1,TE-10,TE-11,KYSE140,KYSE150,EC109,EC9706).The esophageal squamous cell carcinoma cell lines TE-11 and KYSE150 with high expression of lncRNA G077640 were used as cell models,and lncRNA G077640 in the two cell lines was interfered by siRNA interference technology,and compared the change of cell proliferation,cell cycle,cell apoptosis,cell migration according to lncRNA G077640 expression.Constructed stable interference lncRNA G077640 cell line,and performed nude mice tumor formation experiment,measured tumor volume in nude mice and immunohistochemical staining for expression of Ki67 were used to test the results of in vitro experiments.3.To explore the mechanism of lncRNA G077640 in promote esophageal squamous cell carcinoma: identified the localization of lncRNA G077640 by fluorescence in situ hybridization assay;obtained the end sequences of lncRNA G077640 gene by terminal rapid amplification experiments,thus got the full length of lncRNA G077640;RNA pull down and mass spectrometry experiments detected the possible binding proteins of lncRNA G077640;genome-wide expression profiling microarray detected the change of downstream target genes after interference with lncRNA G077640,and verified by qRT-PCR.Results: 1.LncRNA microarray showed there are 374 lncRNAs elevated and 476 lncRNAs down regulated in cancer tissues compared with adjacent tissues.We chose lncRNA G077640 as our research object and lncRNA G077640 was elevated in esophageal squamous cell carcinoma tissues in a large number of matched paracancerous samples(n=64)(P<0.05).There was no statistical difference in age,gender,tumor size,grade,stage,and lymph node metastasis(P>0.05).2.The expression of lncRNA G077640 in esophageal squamous cell carcinoma cell lines(TE-1,TE-10,TE-11,KYSE140,KYSE150,EC109,EC9706)were all higher than in normal esophageal squamous cell line(Het-1A)(P <0.05).Knock down lncRNA G077640 inhibited the growth and proliferation of esophageal squamous cell carcinoma cells TE-11 and KYSE150,lead to cell cycle arrest,promoted apoptosis and impaired migration ability.The KYSE150 cell line stably interfered with lncRNA G077640 grew slowly in the nude mice compared with the control group,and also has weaker expression of proliferation index Ki67.3.Fluorescence in situ hybridization experiments confirmed that lncRNA G077640 almost localized in the nucleus;and the full-length sequences of lncRNA G077640 was obtained by rapid amplification experiment.After designed the synthetic probe,lncRNA G077640 pull down followed by mass spectrometry confirmed that lncRNA G077640 related to protein H2 AX through bind to each other;after interfered lncRNA G077640,the whole gene expression profile microarray and qRT-PCR assay showed that genes which involved in proliferation,apoptosis and migration are down-regulated,and GO analysis showed that knock down lncRNA G077640 mainly affects the expression of genes which involved in metabolic processes.Conclusion:LncRNA G077640 is elevated in esophageal squamous cell carcinoma,but there is no difference between age,gender,tumor size,grade,stage,degree of differentiation,and metastasis.LncRNA G077640 is high expressed in esophageal squamous cell carcinoma cell lines,and can promote tumor cell growth,migration,regulate cell cycle and inhibit cell apoptosis.LncRNA G077640 mostly located at nucleus may cause metabolic reprogramming by interacting with H2 AX protein,thereby affecting downstream proliferation,apoptosis and other related genes,leading to change in cell biological behaviors and promoting tumor development.