Long Non-coding RNA91H Contributes To The Occurrence And Progression Of Esophageal Squamous Cell Carcinoma By Inhibiting IGF2Expression

Objective Exploring the role of lncRNA91H in the regulation oIGF2expression and the association between91H and ESCC progression.Method1. Collected232cases of ESCC patients’tumor tissues and adjacent normal tissues. Buy cultured ESCC cell lines TE-1and Eca-109, normal human gastric epithelial cell line GES-1;2. Making construction and identification of91H carrier interference. Transfecting ESCC cell lines by the use of liposomes Lip2000transiently and screening stably transfected cell lines by G418;3. Adjusting demethylation agent5-aza-CdR to2.5,5,10mol/L by DMSO. Then the agent were used to demethylate the ESCC cell lines. Bisulfite sequencing PCR was used to test the methylation of theses demethylated cell lines;4. Real-time PCR was used to explore the difference of91H RNA expression in tumor and normal cell lines;5. Real-time PCR was used to explore the difference of91H and IGF2mRNA expression after being transfected in ESCC cell lines. Immunofluorescence technique was also used to detected the expression of IGF2protein in ESCC cell lines after being transfected;6. Extracting the DNA from tumor tissues. Then the association between the methylation status of H19and the ESCC progression was explored by BSP;7. Extracting the DNA from tumor tissues and adjacent normal tissues. Then the association between the91H and IGF2mRNA expression and the ESCC progression were explored by real-time PCR.Results 1. After detecting the expression of91H RNA in tumor and normal cell lines, the expression of91H RNA in TE-1and Eca-109was0.75±0.14and0.46±0.12which was both significantly higher than normal cell lines(p<0.05);2. The expression of IGF2mRNA in TE-1after being transfected was4.91±1.68,which was higher than the negative control(1.38±0.61, p<0.05); The expression of91H RNA in Eca-109after being transfected was3.62±1.44,which was higher than the negative control(1.75±1.00, p<0.05). Then the expression of IGF2protein was detected by IF, the expression of IGF2in TE-1was7.60±0.66×104, which was higher than the negative control(2.01±0.43×104, p<0.05); the expression of IGF2in Eca-109was8.64±1.74×104, which was higher than the negative control(3.7±0.68×104, p<0.05);3. The expression of91H RNA was tested by real-time PCR which showed that the expression of91H usually decreased in TE-1and Eca-109when treated with demethylation agent(p<0.05);4. The methylation status of H19DMD of ESCC patients’ tumor tissues were tested by BSP which were also compared with patients’ lymph nodes involvement, depth of invasion, neoplastic grading and TNM. However none of them was found to be correlated to patients’methylation status(p>0.05);5.91H expression was significantly lower in patients with higher depth of invasion, neoplastic grading and TNM which usually leads to the overexpression of IGF2in tumor progression after being tested by real-time PCR.Conclusion1. The lncRNA91H was associated with H19DMD methylation status;2. The lncRNA91H inhibited IGF2expression of ESCC patients;3.91H expression potentially represent a novel clinically relevant event to identify individuals at increased risk for the occurrence of ESCC.