| Background and ObjectivesEsophageal cancer is a common digestive tract tumor with high incidence in China.The main pathological type is squamous cell carcinoma(ESCC).The development of endoscopy and imaging technology promotes the early diagnosis of esophageal cancer.However,due to the lack of early diagnostic markers at the molecular level,the screening methods are very limited and the positive detection rate is low.At present,surgery,adjuvant chemotherapy and radiotherapy are the mainly treatments of esophageal cancer.Although a breakthrough in the theory of immunotherapy had been made for ESCC,the exploration of new immune-checkpoint is under research.Therefore,in a long run,scientific exploration at the molecular level will be helpful to establish new targets for early diagnosis and treatment in ESCC.Long non coding RNA(lncRNA)is generally longer than 200 nucleotides,and it’s mainly produced by RNA polymerase II.In the past,it was thought that most of lncRNAdid not have the ability to encode protein,and the research value was low.However,it has been found that lncRNAhas played many important functions at the molecular level in recent years.Whether it is molecular signal,bait function,or molecular support and guidance function,the basis of its biological function lies in its position and structure..Although a large number of reports have confirmed that many lncRNAs play an extremely critical role in the growth of esophageal cancer,and the cancer promoting molecules include hotair,casc9,H19,etc.,the research content has revealed the correlation between lncRNAand tumor biological function,but the direct mechanism has not been elaborated in detail.The reports of lncRNArelated to esophageal cancer are relatively more Less.In this study,we sequenced the lncRNAof clinical case tissue samples,selected MIR22HG,a key tumor suppressor with different expression,as the research object,and revealed its association with esophageal squamous cell carcinoma from clinical correlation and biological function.In further experiments,we further verified its combination with HNRNPH1 to regulate downstream target by interfering MIR22HG transcriptome sequencing and pulldown experiment Because of KLB,it can realize the negative regulation of tumor.MethodsPart Ⅰ:RNA sequencing and clinical information statistics of tumor samples.(1)Four pairs of cancer and paracancerous tissues were selected,and the clinical information,such as age,sex,smoking and alcohol history,esophageal tumor size,TNM stage,etc.,were statistically analyzed.(2)The key molecules were screened,and the results of sequencing were verified by qPCR of cancer and paracancerous tissue samples.(3)The expression of MIR22HGqpcr was measured in tumor tissue samples.According to the expression level of MIR22HG,MIR22HG was divided into high expression group and low expression group.The clinical characteristics(age,tobacco and alcohol history,TNM stage)were statistically analyzed.The correlation between MIR22HG expression and prognosis was analyzed by Kaplan Meier survival analysis.Part Ⅱ:Study on the biological function of MIR22HG in esophageal cancer.(1)Culture esophageal cancer cell line.(2)Overexpression and interference of MIR22HG were used in CCK-8 proliferation experiment.(3)Overexpression and interference of MIR22HG in Transwell invasion and migration experiments.(4)Overexpression and interference of MIR22HG in flow cytometry and MIR22HG in vivo were used to observe the tumorigenic effect of nude mice.Part Ⅲ:To study the regulatory mechanism of MIR22HG,to find the downstream target genes,and to interfere with MIR22HG posttranscriptome sequencing.(1)Transcriptome sequencing was performed after interfering MIR22HG expression in TE-1 cells.(2)Based on transcriptome sequencing,differentially expressed mRNA was selected.(3)The results of qPCR and Western blotting were verified.Part Ⅳ:continue to explore the mechanism of MIR22HG regulating downstream target genes,and analyze its possible binding proteins by mass spectrometry.(1)Immunofluorescence localization(FISH)of MIR22HG(2)pulldown mass spectrometry experiment of MIR22HG(3)in vitro biological function experiment:CCK-8 proliferation function experiment,Transwell invasion and migration function experiment.ResultsPart Ⅰ:lncRNAsequencing was used to screen the differentially expressed lncRNA,qPCR was used to verify the expression difference of MIR22HG in clinical samples,and the key molecule MIR22HG was screened.The expression of MIR22HG was relatively low in esophageal squamous cell carcinoma,and its expression was not related to TNM stage,age,gender and smoking history.The prognosis of MIR22HG with low expression was poor.Part Ⅱ:using siRNA technology and overexpression vector plasmids to construct interference and overexpression MIR22HG,to carry out the biological experiments of proliferation,migration,invasion and apoptosis,and the tumorigenesis experiments of nude mice after interference.The results show that overexpression MIR22HG can significantly inhibit the proliferation,migration and invasion of esophageal cancer cells,and promote the apoptosis of cells,while interference MIR22HG can promote the growth of esophageal cancer cells The proliferation,migration and invasion of esophageal cancer cells inhibited the apoptosis,suggesting that they regulate the proliferation,migration and apoptosis of esophageal cancer cells at the cellular level.In vivo animal experiment-nude mouse tumor formation experiment further rises from the cell level to the animal level.The experiment proves that the tumor formation ability is enhanced after the interference of in vivo state with the expression of MIR22HG,which is mutually supported by the results at the cell level.Part Ⅲ:after interfering with MIR22HG,the transcriptome sequencing was continued.Six possible target genes were screened out from the sequencing results.After overexpression and interfering with MIR22HGqpcr,the key target gene KLB was screened out.It was proved that MIR22HG inhibited the proliferation,migration and invasion of ESCC cells and promoted apoptosis by regulating the expression of downstream target gene KLB.Part Ⅳ:at the mechanism level,pulldown experiment screened out the binding protein molecule HNRNPH1,qPCR,Western blotting experiment and recovery function experiment proved that MIR22HG may regulate the downstream target gene KLB to play an anti-cancer role through the binding protein HNRNPH1.ConclusionsMIR22HG is located in the nucleus of esophageal squamous cell carcinoma(ESCC).It can inhibit the proliferation,migration,invasion,apoptosis and other biological functions of ESCC by affecting the downstream target gene KLB through binding protein hnrnph... |
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