The Biologicalroles Of LncRNA ECIR-1 In Esophageal Cancer And Its Correlation With Radiosensitivity

Esophageal cancer is one of the most frequently diagnosed malignant tumor derived from esophageal epithelium and glands,and China is one of the countries with the highest incidence and mortality rates of esophageal cancer.In 2014,the incidence rate of esophageal cancer in China was 18.85/100,000,and the mortality rate was 14.11/100,000.The majority of patients are in middle or advanced stage when diagnosed.Radiotherapy is one of primary treatments for esophageal cancer,which has obvious advantages in palliative care involving treating symptoms such as obstruction and pain,and improving the quality of life of patients.However,recurrence and metastases are still the main cause of death.With the development of equipment and techniques in radiotherapy,the application of new technology,including three dimensional conformal intensity modulated radiotherapy and helical tomotherapy etc,have improved conformal dose distributions and target dose uniformity,and reduced the incidence of adverse reactions,such as radiation pneumonia,radioactive myocardial injury etc.But overall survival have not been significantly improved,with 5-year survival rate only 10%.Long non-coding RNA(lncRNA)is a class of RNA molecules with a length of over 200 nucleotides(nt)that does not encode proteins.LncRNA was initially considered to be the "background noise" of genome transcription without biological functions.Recent studies have shown that lncRNA is widely involved in the biological process of cell development,differentiation,growth and apoptosis both in and out of the nucleus through chromatin modification,transcription and post-transcriptional regulation,RNA sponge and other mechanisms.Genome-wide studies have shown that lncRNAs regulate the malignant transformation of cells by interacting with DNA,RNA,proteins and other biological macromolecules.In lung,stomach,breast,colorectal,pancreatic and kidney cancers,lncRNA expression levels were disregulated and correlated with poor prognosis of malignant tumors.Targeted disruption of lncRNA-macromolecules interactions can modulate gene expression,and provides a new strategy for tumor treatment.In conclusion,IncRNA is an important regulator of tumor aggressiveness and a potential therapeutic target.There were a large number of differentially expressed lncRNA between esophageal cancer and normal paracancerous tissues.Comprehensive differential expression analysis of RNA sequencing(RNA-seq)data showed that compared with normal tissues,127 lncRNAs were more than fourfold change,suggesting that abnormal lncRNA expressions may be closely related to malignant phenotype of esophageal cancer cells.POU3F3,Linc00152 and other long non-coding RNA in blood can be used for early diagnosis,and the high expression of MALAT1 is closely related to poor prognosis of esophageal cancer.Further studies have shown that lncRNA can promote proliferation,differentiation and invasion of esophageal cancer cells through epigenetic modification,transcriptional and post-transcriptional regulation,single nucleotide polymorphism and other mechanisms.However,studies about the roles of lncRNA in esophageal cancer are relatively less,and its function and mechanism need to be further elucidated.Radiotherapy is an important treatment option for esophageal cancer.Individual differences in radiosensitivity are common,and some patients are resistant to radiotherapy.Therefore,it is important to clarify the molecular mechanism of radiosensitivity and identify new molecular targets to improve the efficacy of radiotherapy for esophageal cancer.For example,suppressor of cytokine signaling 1(SOCS1)could suppress the STAT3-Mcl-1 signaling pathway and improve the radiosensitivity of esophageal cancer,by inducing apoptosis and enhancing DNA damage of radiotherapy,which provided a basis for new therapeutic strategies for treating esophageal cancer.High-throughput sequencing or microarray raise the level of research from single gene to whole genome.Gene expression profiles could be obtained from literature.A radiation classifier based on gene expression profiles was used to predict the inherent radiosensitivity of tumor cell lines,which successfully predicted survival fraction at 2 Gy(SF2)of 22 cell lines and determined three new genes associated with radiosensitivity(RbAp48,RGS19,R5PIA).Further study found that G2/M phase arest was more common in cells overexpressed RbAp48,suggesting that RbAp48 may induce radiosensitization through antagonizing the Ras pathway.High-throughout sequencing or microarray has become a powerful tool for studying the molecular mechanisms of radioresistance or tumorigenesis.This study was based on the analysis of high-throughput sequencing data for esophageal cancer cell line,and verified by real-time fluorescence quantitative PCR(RT-PCR).A new lncRNAECIR-1,was found to be differentially expressed before and after radiotherapy.This study aimed to characterize the roles and mechanism of ECIR-1 on biological behavior of esophageal cancer.1.Screening of differentially expressed IncRNA before and after radiotherapy for esophageal cancerAfter radiation,cells have complex responses at the gene level.Radiosensitivity was heterogeneous in different cell lines,and many factors could affect the changes in molecular levels after radiation,such as radiation dose,dose rate,detection time,etc.There may be a dose-effect relationship between radiation dose,dose rate and geneexpression level,and different detection times may result in different expression profiles.Therefore,to identify the differentially expressed non-coding genes before and after radiation,the radiation dose and time point for detection should be determined first.We selected 50%concentration of inhibition(IC50)as radiationdose,and the highest peak of DNA double strand breakage(DNA DSB)within 24h was used as the time point for post-radiation detection.Inhibition rates of esophageal cancer cell lines EC-109,TE-1 and KYSE150 were detected by CCK-8 assay on the seventh day after radiationwith different doses,and IC50 was calculated.After irradiation with IC50 dose(dose rate 2Gy/min),Western blot was used to detect the changes in protein expression levels of yH2AX in esophageal cancer cell lines at 0,0.25,0.5,1,3,5,8,12 and 24h after radiation.yH2AX was used as the sensitive molecular marker for DNA DSB,whoseexpression levels reflected DNA DSB effect after radiation.The IC50 values of esophageal cancer cell lines EC-109,TE-1 and KYSE150 were 5.7Gy,3.7Gy and 4.3Gy respectivelyon the seventh day after radiation.The protein expression levels of yH2AX in the three cell lines all arrived peak value at 1h after radiation.Esophageal cancer cell lines EC-109 and TE-1 were irradiated with 5.7Gy and 3.7Gy seperately.1h after radiation,total RNA were extracted from cells by Trizol reagent.Total RNA of EC-109 and TE-1 without radiation were taken as negative control,and RNA-seq was conducted to analyze differentially expressed genes before and after radiation.lncRNAs that were co-upregulated or co-downregulated in the two cell lines with differential expression level more than 2-fold were selected,and the results of RNA-seq were confirmed by RT-PCR.RNA-seq data analysis indicated that after radiation,EC-109 and TE-1 had 121 and 129 co-downregulated and co-upregulated genes seperately.Genes with differential expression level more than 2-fold were 13 and 12 seperately.25 candidate genes were subjected to RT-PCR verification.The results indicated that 4 IncRNAswere up-regulated and 4 lncRNAs were down-regulated after radiation.LncRNA ECIR-1 was down-regulated in both cell lines and selected to conduct further research.The above results indicated that the expression levels of many IncRNAs changed in esophageal cancer after radiation,and the unknown lncRNA ECIR-1 was screened out by RNA-seq and RT-PCR,and further studies were needed to verify whether ECIR-1was asociated with malignant phenotype of esophageal cancer.2.The role and mechanism of lncRNA ECIR-1 in the development of esophageal cancerTo investigate the association between lncRNA ECIR-1 and development of esophageal cancer cell,we adopt nonsense small interfering RNA sequences(siRNA-nc)and siRNAECIR-1(97#,195#,351#)to transfect esophageal cell EC-109,TE-1 respectively,to induce an effcient silencing of ECIR-1.RT-PCR was used to verify transfection efficiency;pcDNA3.1 null vector and pcDNA3.1 ECIR-1 were adopted to transfect esophageal cancer cell,to overexpress ECIR-1 and the overexpression level of the targeted gene was also validated by RT-PCR.The effect of ECIR-1 on the proliferation of esophageal cancer cells were measured by cell counting method at 24h,48h and 72h after transfection with siRNA or pcDNA3.1.The ability of esophageal cancer cells to form colonies was determined by plate clonal formation assay.Cell migration capacity was assessed by wound healing assay.Cell cycle was detected by flow cytometry after propidium iodide(PI)staining,and apoptosis was detected by Annexin-FITC/PI double staining.Knockdown of ECIR-1 in EC-109 and TE-1 obviously inhibited the capability of cell proliferation,clone formation,migration,and inuced apoptosis.Oppositely,overexpression of ECIR-1 could increased the capacity of cell proliferation,clone formation,and decreased apoptosis in esophageal cancer cells.There was no significant change in cell cycle distribution by knockdown or overexpression.The above studies showed that IncRNA ECIR-1 was closely related to the malignant proliferation,clone formation,migration and apoptosis of esophageal cancer cell EC-109 and TE-1.ECIR-1 is an antisense lncRNA.Studies have shown that antisense lncRNA have a significant site-specific effect,which can form RNA-RNA dimer with messenger RNA(mRNA)of adjacent genes in justice chain,so to improve the stability of mRNA,and then upregulate expression of adjacent genes.Theadjacent genes of ECIR-1 gene is interleukin 6 signal transducers(IL-6ST),and IL-6ST encoded protein gp130 which is the signal transduction chain ofinterleukin-6 receptor(IL-6R).Only when IL-6 binds to its receptor and form IL-6/IL-6R/gp130 complex,can gp130activate downstream of IL-6 pathway.So gp130 plays a key role in activating downstream of IL-6 signaling pathway.To evaluate whether ECIR-1 can affect the expression of IL-6ST,lncRNA ECIR-1 of EC-109 was downregulated or overexpressed,and changes in the expression levels of IL-6ST mRNA and proteins were detected by RT-PCR and Western blot respectively.Knockdown of ECIR-1 signifcantly suppressed the mRNA and protein level of IL-6ST in EC-109 cells.Overexpression of ECIR-1 improved protein level of IL-6ST,while there was no significant change in IL-6ST mRNA level.To determine whether ECIR-1 further activated downstream signaling pathway of gp130(IL-6ST-encoded protein),Western Blot was used to detect phosphorylation of downstream Janus kinase(phosphorylated Janus kinase,pJAK).The background protein expression of pJAK in EC-109 was extremely low,which was not activated even when IL-6 was added into culture medium.This suggested that the effect of ECIR-1 on phenotype of esophageal cancer may be JAK-independent.Further studies showed that expressions of ECIR-1 and phosphorylated extracellular signal regulating kinase(pERK)were positively correlated.Knockdown of ECIR-1 reduced downstream pERK;on the contrary,pERK was activatedbyoverexpression of ECIR-1.Meanwhile,the total ERK level remained unchanged regardless of expression level of ECIR-1 The above results suggested that lncECIR-1 may promote the developments of esophageal cancer by activating ERK/mitogen-activated protein kinase(MAPK)pathway.In summary,lncRNA ECIR-1 regulated the expression of IL-6ST at transcriptional and post-transcriptional levels,and may partly affect the malignant phenotype of tumor cells and promote the malignant proliferation and migration of esophageal cancer by activating ERK/MAPK pathway.3.Expression of lncRNA ECIR-1 in esophageal cancer and its relationship with radiosensitivityIn order to clarify the expression status of ECIR-1 in tissues,RT-PCR was conducted in cancer and adjacent tissues of 20 patients with esophageal squamous cell carcinoma.The results showed that the expression level of ECIR-1in esophageal cancer tissues was higher than that of adjacent tissues.To determine the effect of ECIR-1 on radiosensitivity of esophageal cancer cells,small interfering RNA nonsense sequences and siRNA(97#,195#,351#)were used to transfect EC-109 cells respectively,to silence the expression of targeted gene.Plate clonal formation assay and MTT assay were performed to verify changes in colony-forming ability and cell viability of cells after radiotherapy.The colony-forming ability and cell viability gradually decreased with the increase of radiotherapy dose,and ECIR-1 had obvious synergistic effect with radiotherapy.In order to verify the effect of ECIR-1 on objective response rate(ORR)further,expressions of gp130 protein were detected by immunohistochemistry in 53 patients with esophageal cancer.The correlations between radiotherapy and gp130 expressions in patients were retrospectively analyzed,indirectly reflecting the relationship between ECIR-1 and radiosensitivity of esophageal cancer.The results showed that patients with low gp130 expressions had better radiologic response rates than patients with high gp130 expressions.In the univarirate analysis,the significant prognostic factors for progression-free survival(PFS)were ORR and radiotherapy dose,and multivariate analysis suggested that ORR was the only prognostic factor for PFS.In conclusion,lncRNA ECIR-1 was highly expressed in esophageal cancer and had obvious synergistic effect with radiotherapy.Patients with low gp130 expressions indicated higher ORR.This study is the first to sequence differentially expressed genes before and after radiotherapy for esophageal cancer to identify genes or core pathways associated with tumorigenesis or prognosis.A new long non-coding RNA ECIR-1 was selected for stable down-regulation after radiotherapy.This study confirmed the effect of ECIR-1 on biological behavior of esophageal cancer and preliminarily discussed the effect and mechanism of ECIR-1 on phenotype of esophageal cancer.The differential expressions of ECIR-1 in cancer and adjacent tissues were detected and the effects of ECIR-1knockdown on radiosensitivity of esophageal cancer cells were verified.Further,the correlations between IL-6ST-encoded protein gp130 and patients radiosensitivity were investigated.These results provided experimental basis for understanding the mechanism of esophageal cancer and potential therapeutic targets.