LTCONS₀0014107-miR-29b-2-3p-GAS7 Axis Promotes Esophageal Precancerous Lesions In A Heat Stimulation-induced Rat Model

Background and purposeEsophageal cancer is a common malignant tumor of the digestive system and ranks sixth leading cause of cancer-related death worldwide.It caused about 509,000 deaths in 2018 accounting for about 5%of all cancer-related deaths.Esophageal squamous cell carcinoma?ESCC?is the major histological subtype of esophageal cancer,and it mainly occurs in northern China,north-eastern Iran and South Africa.Multiple epidemiological evidence shows that consumption of hot temperature food and beverages is an important risk factor for developing ESCC.For example,innorthern China,particularly people living around the Taihang Mountains,including Linzhou City at the Henan province,have a habit of eating very hot soup,which has led to one of the highest incidence areas of ESCC in the whole country.Although association between consumption of hot food and beverages and ESCC has long been speculated,its underlying mechanism still remains unclear.Long non-coding RNA?lncRNA?is a non-coding RNA of greater than 200 nucleotides in length.Studies show that lncRNA plays an important role in many life activities such as epigenetic regulation,cell cycle transition and cell differentiation.MicroRNA?miRNA?is a small,highly conserved,single-stranded,non-coding RNA that typically consists of 20 to 24 nucleotides.It can bind to the 3'-untranslated region?3'-UTR?of target mRNA and induce their degradation or inhibit their translation.This leads to changes in biological functions,such as cell proliferation,differentiation and.turnover.It has been reported that cellular non-coding RNA expression profile changes in response to different stress conditions including heat shock.Therefore,identifying non-coding RNAs,both lncRNA and miRNA,related to heat induction and elucidating their molecular mechanism in the development of ESCC are of great significance in ESCC prevention and treatment.In this study,we simulated the habit of drinking hot beverages to generate an animal model of heat-induced rat esophageal cancer.Both lncRNA and miRNA expression of esophageal tissues were profiled by RNA-seq.Differentially expressed lncRNA LTCONS₀0014107 and miRNA miR-29b-2-3p were screened out since it is speculated that LTCONS₀0014107 could be a precursor of miR-29b-2-3p.Both play an important role in heat-induced ESCC and then verified by cell heat induction assays and cell phenotypic assays.Finally,data mining from the publicly available databases followed by validation using qRT-PCR,it was found GAS 7 may be a potential target gene of miR-29b-2-3p.Through the study of the role of GAS7 in ESCC,we further clarified the mechanism of LTCONS₀0014107-miR-29b-2-3p-GAS7 axis in heat-induced ESCC.Taken together,this study provides a theoretical and experimental basis of miR-29b-2-3p as one of the biomarkers and therapeutic targets in ESCC.Methods1.A heat-induced rat esophageal cancer animal model was established,and HE staining was used to detect the effect of heat induction on rat esophageal mucosa.2.RNA-seq was performed on the rat esophageal mucosa tissues of each experimental group to screen differentially expressed lncRNAs and miRNAs related to heat induction.3.To validate the RNA-seq data by designing specific qRT-PCR primers of a differentially expressed lncRNA,LTCONS₀0014107,and determine its relationship to miR-29b-2-3p in rat skin cells.4.In rat skin cells,used siRNA to knock down the expression of LTCONS₀0014107,and studied its biological function by MTT and Soft agar colony formation assays.5.Detected the expression of miR-29b-2-3p in ESCC by qRT-PCR technology.6.Constructed miR-29b-2-3p sponge vector to down-regulate miR-29b-2-3p expression,used lentivirus to up-regulate miR-29b-2-3p expression,and the biological function of miR-29b-2-3p in ESCC was studied by MTT,plate cloning and Soft agar colony formation assays.7.Using the bioinformatics website,combined with relevant literature reports,to screen target genes regulated by miR-29b-2-3p,and verify the relationship between miR-29b-2-3p and target genes by qRT-PCR technology.Results1.Long-term consumption of 65? hot water induced precancerous lesions of esophageal mucosa in rats.2.RNA-seq analysis showed that LTCONS₀0014107 expression is downregulated whereas and up-regulate the expression of miR-29b-2-3p in rat esophagus tissue.3.Sequence alignment of LTCONS₀0014107 and miR-29b-2-3p and heat-induced assay suggested that LTCONS₀0014107 could be a precursor of miR-29b-2-3p.4.Knockdown of LTCONS₀0014107 significantly slowed down rat skin cell proliferation and colony formation ability.5.Majority of ESCC cell lines exhibited an up-regulation of miR-29b-2-3p level comparing to normal esophageal epithelial cell lines.6.Silencing of miR-29b-2-3p inhibited ESCC cell proliferation and colony formation,whereas over-regulation of miR-29b-2-3p promoted ESCC cell proliferation and colony formation.7.GAS7 is a potential target gene of miR-29b-2-3p due to the fact that their expression is negatively correlated.Conclusions1.Long-term heat induction can induce precancerous lesions of the esophageal mucosa,leading to degradation of LTCONS₀0014107 and up-regulation of miR-29b-2-3p expression.2.miR-29b-2-3p promotes cell proliferation and colony formation of ESCC cell lines by inhibiting the target gene GAS 7.3.Heat induction promotes the occurrence and development of ESCC through the LTCONS 00014107-miR-29b-2-3p-GAS7 axis.