| ObjectiveThe aim of the present study was to identify the potential IncRNA and its molecular mechanisms involved in regulating radiosensitivity of ESCC as to assess whether it could be a novel biomarker for prognosis and prediction of response to radiotherapy on ESCC patients.Methods(1)The patient's tumor tissue and blood samples were collected prospectively according to the "National 863 Tumor Molecular Typing and Individualized Diagnosis and Treatment" project group,"Tumor Genome" major project team,the Chinese Association of Pharmaceutical Biotechnology Association.(2)All the patients were treated with IMRT.After the complete of radiotherapy,the short-term effect of radiotherapy was evaluated according to RECIST criteria.The patients were divided into radiosensitive(CR + PR)and radioresistance group(SD + PD)based on the short-term effect.Survival data(overall survival,local fail-free survival and distant fail-free survival time)were analyzed by SPSS software.Survival curves were established by Kaplan-Meier and compared by log rank test.A multivariable analysis by gender,age,ECOG score,tumor location,clinical T and N stages,the radiotherapy dose of GTV and CTV,regimens and cycles of concurrent chemotherapy,and tumor response to treatment was performed using the Cox proportional hazards model.A two-tailed p value of less than 0.05 was considered statistically significant.(2)Each three patients in radiosensitive and radioresistant group were candidate and the lncRNAs were detected by Agilent human IncRNA.The candidate lncRNAs potentially associated with radiosensitivity were selected by bioinformatics analysis.(3)The candidate lncRNAs expression were detected by PCR in more esophageal cancer tissue samples to identify the potential relationship between candidate-lncRNAs and tumor radiosensitivity(short-term effect),prognosis of patients(OS).The IncRNA with the most obvious difference in expression,the highest in group expression was considered as the lncRNA that was most potential correlation with ESCC radiosensitivity.The overexpression and interference RNA sequences of the candidate-lncRNA were constructed according to its origin gene for constructing the plasmids.(4)The radiation-sensitive ESCC Eca109 cell line was candidate to construct the radioresistant cell(Eca109R)after 3 cycles of 10Gy/F irradiating by the X-ray radiation device.Both the Eca109 and Ecal09R cell were transfected the overexpress/interference RNA of candidate IncRNA(overexpress-/si-RNA lncRNA).The MTT was used to check the survival of the transfected cell lines and PCR was used to validate the transfected efficacy.(5)The Eca109/R cell lines transfected by the overexpression/RNAi lncRNA were irradiated by X-ray with 0,2,4,6,8,10Gy.The candidate-lncRNA expression was detected by PCR at various dose points,cell viability was detected by MTT assay,Annexin-V flow cytometry was used to detect apoptosis.The overexpress/RNAi IncRNA efficacy and evaluate the effect of overexpression/siRNA-candidate lncRNA on ionizing radiation sensitivity of ECa109/R cells.(6)The potential target genes downstream of lncRNAs were predicted by using the combination of PiTAR,MIRDB and BLAST databases.Luciferase reporter was used to detect the relationship between lncRNA and potential downstream target genes in wild type and mutant to identify the potential downstream target genes.PCR was used to validate the prediction.(7)Western blotting was used to detect the expression of the potential protein of the potential downstream target gene of the candidate lncRNA in the both Eca 109 and Eca109R cell lines.(8)Overexpression/siRNA-candidate IncRNA lentiviral plasmid was constructed and transfected into Eca109/R cells.The transfection results were detected by PCR and the survival of cells was determined by MTT.The transfection was successful and cell survival was good.Nude mouse model of xenograft tumors was established.Tumor volume was measured and the tumorigenic curve recorded.The tumor growth(volume and weight)was recorded by X-ray 10Gy/F tumor three days after the tumor formation.The growth curve of tumor radiosensitivity was made and the effect of overexpression/RNAi candidate lncRNA on the tumorigenicity of the nude mice was analyzed.Results(1)A total of 41 patients were enrolled in the study,including 23 radiosensitive patients and 18 radiosensitive patients.There was no significant difference between the two groups in terms of gender,age,ECOG score,tumor location and clinical stage.Four patients(9.7%)had CR after radiotherapy,19(46.3%)were PR,9(22%)were SD and 9(22%)were PD.The last follow-up date was December 2017,with 26 surviving and 15 deaths,with a median survival of 15 months.ly-OS,LFFS,DFFS were 74.9%,84.8%and 62.7%respectively.The ly-OS of patients with radiosensitivity and radioresistance were 68.4%vs.49.3%,respectively(p?0.021);the rates of 1y-LFFS were 75.4%vs.75.4%(p=0.181);ly-DFFS were 80,5%vs,67.1%,respectively(p?0.919).(2)Six fresh tumor tissues from radiation-sensitive and-resistant patients was used to detect the expression of the IncRNAs by the Agilent human IncRNA expression microarray chip.A total of 113 differentially expressed lncRNAs were found with 71 up and 42 down.There were 20 lncRNAs with significant difference between radiosensitivity and radioresistance groups(>2 folds,p<0.05).Starbase online software was used to select CASC2,FAM201A,DLEU2,DLX6-AS1 and MCF2L-AS1 as the potentially lncRNAs related to radiosensitivity.(3)Expand the sample size of 41 cases and detect the expression of lncRNAs-CASC2,FAM201A,DLEU2,DLX6-AS1 and MCF2L-AS1 in esophageal cancer patients by PCR showing that the FAM201A was the most significant difference between the two groups(resistance:1=0.312,p<0.05),the highest homogeneity within the group(SE=0.1068,p<0.01),and the highest correlation with radiosensitivity IncRNA.Overexpression of FAM201A had a poor sensitivity to radiotherapy,and the patients had short survival time(1,2y-OS was 68.4%vs.49.3%,37.5%vs.32.4%,p?0.021,respectively).(4)Overexpression/siRNA-FAM201A transfected Eca109 cells,PCR detected the FAM201A expression in both groups of cells.Compared with Ctrl cells,overexpression-RNA FAM201A was highly expressed(1:3.291,p<0.001);si-RNA FAM201A was significantly low expression(1:0.312,p<0.001),indicating that overexpression/si-RNA FAM201 A transfected Eca109 cells successfully.(5)Compared with the control group,the survival rate of overexpression/si-RNA FAM201A Eca109/R cells decreased with the increase of X-ray dose.However,in Eca109 cells,the function of overexpressing FAM201A to promote the survival of Eca109 cells showed a significant difference(0.834 vs.0.913,p=0.024)when the cells were DT6Gy;while siRNA-FAM201A promoted the function of Eca109 cells in DT1OGy(0.603 vs.0.364,p?0.001),indicating that FAM201A is more likely to promote radioresistance to Eca109 cells.However,in Eca109R cells,siRNA-FAM201A could significantly promote the death of Eca109R cells at DT8Gy compared with control cells(0.913 vs.0.641,p?0.02),but overexpression of FAM201A did not significantly promote cell survival,indicating the expression of FAM201A in radioresistant cells was already high.Transfection of overexpression FAM201A did not continue to increase the radiation tolerance of Eca109R cells,but interfering with the expression of FAM201A could significantly increase the ionizing radiation sensitivity of Eca109 and Eca109R cells.Annexin-V flow cytometry apoptosis,in support of the above conclusion.(6)The downstream target gene of FAM201 was predicted by PiTAR,MIRDB and BLAST databases.MiR590/101 were potential targets of FAM201A.And the miR101 was considered as the downstream target of FAM201A by the Luciferase reporter.(7)According to the literature,ATM and mTOR proteins are the downstream targets of miR101.In Eca109/R cells,the expression of ATM and mTOR increased and the expression of miRNA101 decreased in FAM201A-overexpressing cells compared with the control cells.Interfering FAM201A,the expression of ATM and mTOR was down-regulated and the expression of miRNA101 was increased.Compared with non-irradiated cells,the expression of ATM and mTOR increased after X-ray irradiation.WB confirmed the results of PCR.(8)The overexpression/siRNA-FAM201A lentiviral plasmid was successfully transfected into Eca109 cells(PCR test,sh-NC,sh-FAM201A were 1,0.01,0.33,respectively,p<0.001).Compared with NC and Ctrl,the tumor volume of Eca109 transfected with sh-FAM201 A significantly decreased after X-ray irradiation.Although the number of nude mice too few to reach statistical significance,but it was suggested that interference with FAM201A Improve radiosensitivity and thus confirm that FAM201A regulates radiosensitivity at animal level.ConclusionFAM201A is a novel lncRNA that is related to the radiosensitivity of esophageal squamous cell cancer.Overexpression of FAM201A led to radiation resistance which predicted poor survival in esophageal squamous cell cancer patients.Interference FAM201A expression increase the sensitivity of esophageal cancer cells to ionizing radiation.The lncRNA FAM201A,which mediated the radiosensitivity of ESCC by regulating ATM and mTOR expression via miR-101 in the present study,may be a potential biomarker for predicting radiosensitivity and patient prognosis,and may be a therapeutic target for enhancing cancer radiosensitivity in ESCC. |
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