The Role And Mechanism Study Of Long Non-coding RNA Inhibits The Proliferation Of Residual Cancer Cells After Radiofrequency Ablation Of Hepatocellular Carcinoma

Purpose and significanceHepatocellular carcinoma(HCC)is the third leading cause of cancer-related death.Radiofrequency ablation(RFA)is a radical and minimally invasive treatment technique commonly used in clinic that has been included in the guidelines for the diagnosis and treatment of HCC.The tumor-free survival rate of small HCC treated with RFA is lower than that treated with surgical resection,and tumors are prone to early recurrence after RFA treatment.The mechanism of recurrence has not been fully elucidated.However,there is a significant relationship between the micro-residual tumors caused by insufficient RFA or Incomplete RFA(IRFA).At present,it is believed that the research of micro-residual cancer cells should focus on cell proliferation or apoptosis.Inhibiting proliferation or promoting apoptosis of residual cancer cells is the key to improve the efficacy of RFA,and also the key to improve the prognosis of patients.Long non-coding RNA(lncRNA)is abnormally expressed in many tumors,plays an important role in the occurrence and development of HCC and is involved in the regulation of cell proliferation.Therefore,to study the role and molecular mechanism of lncRNA in regulating the proliferation of residual cancer cells after IRFA is expected to provide therapeutic strategies for delaying the recurrence of HCC.The microscopic residual cancer cells after IRFA as the research object in this study,and we established a 50? IRFA cell and animal model,and used gene chip technology and RNA-protein interaction technology to analyze the role and mechanism of lncRNA in the proliferation of residual cancer cells after RFA treatment for HCC.These studies will provide new ideas and methods for promoting apoptosis of residual cancer cells and improving the efficacy of RFA for HCC.Materials and methodsSMMC-7721 hepatoma cells were treated in water bath at 50? for 10 minutes to simulate IRFA and extracted cellular RNA after recovery for 24 hours.The lncRNA and mRNA expression profiles were detected by Agilent human lncRNA + mRNA chip V4.0.The differentially expressed lncRNA and mRNA was analyzed by bioinformatics methods.Then,some differentially expressed lncRNA and mRNA were screened for qRT-PCR validation and identify the lncRNA to be studied.lncRNA pulldown assay was used to detect the binding proteins of target lncRNA.The full length sequence of lncRNA was determined by RACE.Double luciferase gene reporting method and ChIP assay was used to detect the transcripts binding to the target lncRNA gene.Co-IP assay was used to detect protein-protein interaction.The localization of target lncRNA and co-localization of target lncRNA and protein was detected by FISH assay.CCK-8 assay was used to detect the proliferation of HCC cells in each group.Lentivirus transfection technique was used to detect the biological function of target lncRNA.Animal experiments were conducted to verify the biological function of target lncRNA in vivo.The expression of target lncRNA in human HCC tissues was detected by qRT-PCR.Results(1)We obtained the data of lncRNA and mRNA expression profiles,differentially expressed lncRNA and mRNA and bioinformatics analysis data in HCC cells that normal cultured or treated at 50? for 10 minutes.(2)Screening and validating 23 differentially expressed lncRNA and 17 differentially expressed mRNA,identifying the target lncRNA ENST00000570843.1,and named it as lncRNA SHILA(Sub-lethal Heat Inducing Long noncoding RNA).(3)lncRNA SHILA was highly expressed in HCC cells after 50? treated for 10 minutes and lasted for 1 week.(4)Transcription factor IRF9 promotes the expression of lncRNA SHILA.(5)lncRNA SHILA located in cytoplasm.(6)lncRNA SHILA binds to ENO1 protein and Glucose Regulated Protein 78 kD(GRP78)protein and down-regulates ENO1 expression.(7)There is protein-protein interaction between ENO1 protein and HSC70(heat shock 70 kDa protein 8)and HSP90(heat shock protein 90).(8)The proliferative capacity and expression of ENO1 was decreased in HCC cells after 50? treated for 10 minutes.(9)lncRNA SHILA inhibits the proliferation of HCC cells treated at 50? for 10 minutes and the growth of HCC tissues in nude mice of IRFA HCC model.(10)The expression of lncRNA SHILA in human HCC tissues was lower than that in adjacent normal tissues.Conclusions(1)The expression profiles of lncRNA and mRNA in HCC cells were significantly different between the treated at 50? for 10 minutes group and the normal cultured group.Bioinformatics analysis showed that the differentially expressed mRNA were mainly related to metabolic pathways.(2)The results of lncRNA+RNA expression microarray were consistent with those of qRT-PCR.(3)The expression of lncRNA SHILA in HCC cells was up-regulated by 50? treatment for 10 minutes,and IRF9 transcription factor played an important role.(4)lncRNA SHILA negatively regulates the expression of ENO1 in HCC cells treated at 50? for 10 minutes(5)lncRNA SHILA inhibits the proliferation of HCC cells treated at 50? for 10 minutes.