The Role And Molecular Mechanism Of LncRNA-UCA1 In The Pathogenesis Of Esophageal Cancer Induced By NMBzA Combined MC-LR

Background and Objectives:Esophageal cancer(EC)is one of the most common malignancies which threaten the human health severely in China.The results show that EC is a complexity disease caused by the interaction between environmental factors and genetic factors as an environment relation tumor.Nitrosamines compounds is a strong carcinogen.Epidemiological studies have found that Nitrosamines compounds are an important pollutants which could induced EC.Recently,for the water environment were widely polluted in China,and the water eutrophication become more serious,this coursed large algae propagation.Microcystion were secondary metabolites produced by microcystis cell,it existed in the source water which were serious entrophication.Microcystion can enter in the terminal tap water,and people could exposure in microcystis by drinking water.Microcystion has a strong toxicity,and study has confirmed that it could promote the cinoma occurred in vivo or vitro.Our previous study has found that NMBA combined with MCLR could induce EC carcinoma.However,if the long nocoding RNA involved in the process,and whether the lncRNA could play important role in regulation of EC oncogenesis.The mechanism of EC were not clear.So,the objective of this study was through the microarray of lncRNA,and compared the expression profiles of lncRNA and mRNA in different group.We select the lncRNA related with EC by co-expression analysis,lncRNA subclass analysis,and adjacent coding genetic information analysis and so on.We measured the expression of lncRNA-UCA1 to validated the results of microarray.We disussed the relationship between the expression of lncRNA-UCA1 to the clinical pathologic correlation parmeter,and assessment the EC risk.To explore the effect of lncRNA-UCA1 on the cell phenotype and the regulation mechanism of lncRNA-UCA1.Objective to elucidate the molecular regulation mechanism of LncRNA-UCA1 in EC carcinoma.Also the marker of lncRNA-UCA1 are to developed as potential special target and a new type of genetic marker for early diagnosis and identify EC high risk populationMethods1.A high-throughput ChIP-Seq was applied to screen aberrantly expressed lncRNA and mRNA expression profile in the primary ESCC tumor compared to matched adjacent normal tissues(N=3?)We constructed lncRNA-mRNA co-expression networks based on lncRNA subclass analysis,GO and KEGG analysis of mRNA,and bioinformatics analysis,for further screening ESCC related lncRNAs.Quantitative polymerase chain reaction(RT-qPCR)was used to further validate the expression levels of the above candidate lncRNAs in expanding esophageal cancer samples Conditional logistic regression analysis was used to investigate the relationship between the above aberrantly expressed lncRNAs and the onset risk of esophageal cancer.The independent-samples t test was used to explore the clinical association between the above candidate lncRNAs expression and esophageal cancer pathological parameters2.LncRNA-UCA1 over-expression lentiviral vectors and interference vector system were constucted to transfect to EC 109,respectively,for developing stable lncRNA-UCA1 overexpression and interference cell lines.RT-qPCR was applied to detect the expression of lncRNA-UCA1 after stable transfection for the assessment of the efficiency of overexpression and interference of lentiviruses Subsequently,we used the stable cell lines including lncRNA-UCA1 overexpression and interference to analyze the influence on the cell proliferation,invasion and migration of the EC 109.Cell Counting Kit-8(CCK-8)assay and transwell assay were used for the evaluation of EC 109 proliferation and migration,respectively.Subcutaneous tumorigenesis and tail vein assay of cancer metastasis in mice were used to observe the influence of overexpression lncRNA-UCA1 on tumor formation and metastatic capacity in mice3.Eukaryotic expression vector containing lncRNA-UCA1 was constructed in EC 109 cells.RT qPCR,dual luciferase reporter(DLR)assay,western blot,RNA-pulldown,protein silver staining assay,mass spectrometry,RNA immunoprecipitation(RIP)were applied for exploring and validating the target proteins bonding to lncRNA-UCA1 directly and related signalling pathway moleculars4.The signal axis of lncRNA-UCA1/miR-18a-5p/SORBS2 was selected by DLR analysis,lncRNA-miRNA-mRNA coexpression net constructed by CeRNA theory combined with bio-infromatics prediction software,the datas were come from the miRNA and mRNA which are different from EC tissues and normal tissues,all the raw data were downlode from the TCGA database.Next,DLR,qRT-PCR,MS2-Rip,Western blot and so on were applied to validate the effect of proliferation of EC 109cells and molecule regulation mechisam of this signal axis5.We first construt the model of malignat Het-IA induced by NMBA and MCLR,then qRT-PCR techniques was applied to measure the expression levels of lncRNA-UCA1 in the ten or twenty generation exposed NMBA and MCLR on the Het-IA cells.And the levels of lncRNA-UCA1 were also measured at different time point on 12h,24h,48h,72h for exposed NMBA and MCLR by qRT-PCR.Western blot was used to measure the protein level of the target proteins like FGRR2 Ⅲb,Ⅲc,SORBS2,and the key proteins(PI3K,AKT,P-AKT,PTEN)in PI3K-AKT signals and EMT marker proteins(ZO-1,N-Cadherin,E-Cadherin,Snail)Results1.Screening of esophageal cancer-related lncRNA and study on the relationship between LncRNA-UCA1 and the risk of esophageal cancer1.1 Functional analysis of esophageal cancer related lncRNA expression profile and its adjacent genesLncRNA expression profile microarray identified 1360 lncRNAs significantly differences in esophageal cancer,of which 473 were up-regulated and 887 were down-regulated.According to the Fold Change(FC)more than 2 times,745 different lncRNAs were further screened out,among which 300 were up-regulated and 445 were down-regulated.A total of 1992 differentially expressed transcripts were screened out by adjacent mRNA expression spectrum analysis,of which 1026 were up-regulated and 896 were down-regulated.According to FC>2,1480 different transcripts were further screened,among which 801 were up-regulated and 679 were down-regulated.LncRNA subclass analysis,GO analysis and KEGG Pathway analysis of mRNA showed that these differentially expressed genes were mainly related to MAPK signaling pathway,p53 signaling pathway,cytokine-cytokine receptor interaction signaling pathway and extracellular matrix receptor interaction.According to the differentially expressed lncRNAs and miRNAs,the co-expression analysis of lncRNA-mRNA was performed.Based on the overall structure of the co-expression network,a core gene located in the network was found,namely UCA1(NR 015379),which was significantly down-regulated in the chip results(P<0.05),suggesting that UCA1 is closely related to esophageal cancer and may play an important biological function in esophageal cancer1.2 Epidemiological studies on the risk of LncRNA-UCA1 and esophageal cancerThe expression characteristics of LncRNA-UCA1 in human EC cells(EC109,EC9706,CaEs-17,KYSE150 and TE-1)and human normal esophageal epithelial cells(Het-IA)were detected by qRT-PCR,and the results showed that the expression of LncRNA-UCA1 in 5 EC cell lines was significantly lower than that of Het-1A(P<0.05).The results of qRT-PCR showed that LncRNA-UCA1 expression was significantly lower in 115 cases of esophageal squamous cell carcinoma than that in paracancerous normal tissue(P<0.05).Logistic regression analysis showed that the expression of LncRNA-UCA1 in esophageal cancer tissues was negatively correlated with the risk of esophageal cancer,that is,LncRNA-UCA1 with low expression significantly increased the risk of esophageal cancer(P<0.05).Single sample t test was performed to analyze the sequencing expression of LncRNA-UCA1 in 81 cases of esophageal cancer tissues from TCGA database and the clinical pathological parameters(Age,Gender,Race,Stage,T/N/M,Grade)of EC patients.The results showed that the expression level of LncRNA-UCA1 in cancer tissues with lymph node metastasis was significantly lower than that in patients without lymph node metastasis(P<0.05).These results indicated that the expression of LncRNA-UCA1 in esophageal cancer tissues and cells was significantly lower than that of the corresponding control group,and the low expression of LncRNA-UCA1 could promote lymph node metastasis and increase the risk of EC2.Functional of esophageal squamous cell carcinoma by long non-coding RNA UCA12.1.The localization of LncRNA-UCA1 in EC 109 cellFluorescence in situ hybridization(FISH)was used to detect the distribution of LncRNA-UCA1 in EC 109 cells.The results showed that the transcripts of LncRNA-UCA1 were distributed in cytoplasm and nucleus of esophageal squamous cell carcinoma EC 109 cells,but the fluorescence signal in cytoplasm was significantly higher than that in nucleus(P<0.05).It suggests that the different distribution of LncRNA-UCA1 in the cytoplasm and nucleus may be related to its involvement in different signal transduction pathways2.2.Effect of LncRNA-UCA1 on the biological founctions of EC 109After transfecting lentivirus with overexpression and interfering UCA1 into EC109 cells,qRT-PCR results showed that expression level of overexpression LncRNA-UCA1 group(LV-UCA1)was 274.9 times higher than that of negative control group(LV-NC).The expression level of LncRNA-UCA1 in interfering group(UCA1-shRNA#2)was knocked down to 32%compare with the control group.When LncRNA-UCA1 was overexpressed,the results of the CCK8 experiment,transwell invasion and migration experiment indicated that the proliferation,invasion and migration ability of EC109 in LV-UCA1 group was significantly reduced compared with the LV-NC group(P<0.05)After LncRNA-UCA1 interfered,the proliferation,invasion and migration ability of EC 109 in UCA1-shRNA#2 group was significantly increased(P<0.05).The results suggest that LncRNA-UCA1 may play a role in tumor inhibition in EC2.3.Effects of LncRNA-UCA1 on xenograft tumor and in vivo metastasis in nude miceThe effect of LncRNA-UCA1 on tumor growth and metastasis in vivo was observed in nude mice and tail vein metastasis.The tumor formation test results of nude mice showed that the tumor volume of the LV-UCA1 group was significantly lower than that of the LV-NC group(271.29±83.26 mm3 vs 676.39±56.80 mm3),showing statistically significant differences(P<0.05).The tumor weight of LV-UCA1 group was significantly lower than that of LV-NC group(152.37±78.32mg vs 384.64±68.08mg),and the difference was statistically significant(P<0.05).Experimental results of tail vein metastasis in nude mice showed that no obvious metastasis was found in liver of LV-UCA1 group,while relatively obvious metastasis was found in LV-NC group.These results further confirmed the ability of LncRNA-UCA1 to inhibit tumor cell growth and distal metastasis in vivo3.LncRNA-UCA1 is involved in the regulation of FGFR2 variable splicing by binding hnRNP F to affect the EMT process of esophageal squamous cell carcinoma3.1.LncRNA-UCA1 can bind hnRNP F in nucleotide anisotropyThe RNA-pulldown combined with NanoLC-ESI-MS/MS analysis results identified three proteins that could bind to LncRNA-UCA1,namely hnRNP F(UniProtKB-P52597),ACTA2(UniProtKB-P62736)and PRSS3(UniProtKB-P35030)),with a relative enrichment degree of 51.6%,19.9%and 28.5%,respectively,with a reliability of 99%,89.7%and 91.1%.Based on the result,the protein with the highest enrichment and reliability was selected,which were hnRNP F,RIP and Western blot further confirmed the specific binding of LncRNA-UCA1 to hnRNP F.Western blot showed that hnRNP F was mainly expressed in the nucleus,but not in the cytoplasm.The results of qRT-PCR and Western blot showed that the expression of hnRNP F in EC109,EC9706 cell lines and Het-IA cell lines showed no significant difference(P>0.05).Both qRT-PCR and Western blot results showed that overexpression or interference with LncRNA-UCA1 did not affect the mRNA and protein expression of hnRNP F(P>0.05).The above results showed that LncRNA-UCA1 could bind hnRNP F in nuclear anisotropic,and the expression of hnRNP F was not affected by LncRNA-UCA1.This suggests that LncRNA-UCA1 may participate in its signal transcription pathway in nucleus by binding hnRNPF3.2.LncRNA-UCA1 regulates the generation of FGFR2 variable splicing Ⅲb and Ⅲc by binding hnRNP FThe results of MiasDB database retrieval found that hnRNP F can be involved in regulating the generation of FGFR2 variable splicing Ⅲb and Ⅲc.The results of qRT-PCR and Western blot showed that overexpression of LncRNA-UCA1 could promote the expression of Ⅲb and increase the ratio ofⅢb/Ⅲc(P<0.01).Interference with LncRNA-UCA1 can promote the expression of Ⅲc,inhibit the expression of Ⅲb,and reduce the ratio of Ⅲb/Ⅲc(P<0.01).The total mRNA expression level of FGFR2 was not affected by LncRNA-UCA1 overexpression or interference(P>0.05).After the expression of hnRNP F was knocked down by siRNA technology,Western blot results showed that the generation of Ⅲc was significantly increased(P<0.01).Th results indicate that LncRNA-UCA1 can regulate the variable splicing of FGFR2 by binding hnRNP F,and thus affect the generation ofⅢb and Ⅲc and the change of their ratios3.3.LncRNA-UCA1/hnRNP F/FGFR2 Ⅲc signal axis participates in the regulation of esophageal cancer EMT process through PI3K-AKT signal pathwayWestern blot results showed that the expression of EMT marker zo-1 and e-cadherin was significantly higher in the LncRNA-UCA1 overexpression group(LV-UCA1)than in the negative control group(LV-NC),and significantly higher in the LncRNA-UCA1 interference group(ucal-shrna)and hnRNP F interference group(si-F)than in the corresponding control group(Ctrl-UCA1 and si-Ctrl).The expression of n-cadherin and Snail in the LV-UCA1 group was significantly lower than that of LV-NC(P<0.05),and the expression of UCA1-shRNAgroup and si-F group was significantly higher than that of the corresponding control group(P<0.05).In addition,Western blot found that the expression of key protein molecules PI3K,AKT,and P-AKT in the LV-UCA1 group was significantly lower than that of LV-NC(P<0.05),and the expression was significantly higher in the UCA1-shRNAgroup and si-F group than that in the corresponding control group(P<0.05).The expression of PTEN in the LV-UCA1 group was significantly higher than that of the LV-NC group(P<0.05),while that in the UCA1-shRNAgroup and si-F group was significantly lower than that in the corresponding control group(P<0.05).These results suggest that LncRNA-UCA1/hnRNP F/FGFR2 Ⅲc signal axis may be involved in regulating the EMT process of esophageal squamous cell carcinoma via the PI3K-AKT signal pathway4.Mechanism of the occurrence and development of esophageal cancer by LncRNA-UCA1/miR-18a-5p/SORBS2 axis4.1.lncRNA-miRNA-mRNA co-expression network was constructed based on TCGA databaseUsing the RNA sequencing data of 173 esophageal cancer patients in the TCGA database,a total of 82 miRNA with statistical differences between the esophageal cancer tissues and the adjacent normal tissues were screened out,among which 35 were up-regulated and 47 were down-regulated There were 1398 miRNAs,of which 725 were up-regulated and 673 were down-regulated.The up regulated miRNA and down-regulated miRNAs with statistical differences were selected for analysis using the online bioinformatics prediction software of Targetscan and miRanda,Cytoscape software was used to construct ceRNA co-expression network with LncRNA-UCA1 as the core.Results showed that 7 miRNA,including miR-18a-5p,miR-196b-5p,and miR-452-5p,were important nodes in ceRNA with LncRNA-UCA1 as the core.The binding capacity of these 7 miRNA and LncRNA-UCA1 was preliminarily verified through DLR experiment,and the results showed that only miR-18a-5p and miR-196b-5p were statistically different from the control(P<0.05).However,only miR-18a-5p was significantly reduced compared with the control group(P<0.05).This suggests that LncRNA-UCA1/miR-18a-5p/SORBS2 acting axis may be closely related to esophageal cancer4.2.LncRNA-UCA1 can be used as a molecular sponge to adsorb miR-18a-5pResults of ms2-rip combined with qRT-PCR showed that miR-18a-5p was significantly enriched in the experimental group co-transfected with GV127-MS2-UCA1 and pMS2-GFP(P<0.05),while miR-18a-5p was not significantly enriched in the control group co-transfected with GV127-MS2 and pMS2-GFP(P>0.05).DLR test results showed that miR-18-5p significantly decreased the fluorescence signal value of the reported plasmid containing wild-type LncRNA-UCA1(P<0.05)without decreasing the fluorescence signal value of the reported plasmid containing mutant(P>0.05).This indicates that miR-18a-5p can be used as a "molecular sponge" LncRNA-UCA1 competitive adsorption,which may affect the expression level of its target gene SORBS24.3.LncRNA-UCA1 participates in the regulation of tumor growth by competing with SORBS2 and binding miR-18a-5pDLR experimental results showed that miR-18a-5p significantly reduced the fluorescence signal value of wild-type pmirGLO-SORBS2(WT)(P<0.05),but did not affect the fluorescence signal value of mutant pmirGLO-SORBS2(WT),which indicated that SORBS2 was a miR-18a-5p target gene,and the two could co-valently bind.qRT-PCR and Western blot showed that overexpression of LncRNA-UCA1 could significantly up-regulate SORBS2 level(P<0.05),and interference LncRNA-UCA1 could significantly down-regulate SORBS2 level(P<0.05).QRT-PCR showed that the expression of SORBS2 in EC patients was significantly lower than that in normal adjacent tissues(P<0.05).In the TCGA database,the expression of SORBS2 in EC tissues was significantly lower than that in paracancerous tissues(P<0.05).The results of CCK8 rescue experiment showed that co-transfection of LncRNA-UCA1 and miR-18a-5p in EC109 cells could partially rescue the growth promoting capacity of EC109 cells after the single transfection of mi-18a-5p.Co-transfection of miR-18a-5p and SORBS2 and EC109 cells partially reversed the inhibition of growth of EC109 cells after single SORBS2 transfection.The results indicated that LncRNA-UCA1/miR-18a-5p/SORBS2 acting axis played an important regulatory role in the proliferation capacity of esophageal cancer cells in vitro5.Preliminary study on the mechanism of LncRNA-UCA1 in NMBA and MCLR induced esophageal cancer5.1.the expression pattern of LncRNA-UCA1 in EC109 cells and Het-IA cells of esophageal squamous cell carcinoma treated with NMBA and MCLR in an urgent and chronic manner,respectivelyThe results of qRT-PCR showed that LncRNA-UCA1 expression was significantly down-regulated at 12h,24h,48h,and 72h after NMBA,MCLR alone and combined with toxic esophageal squamous cell carcinoma(EC109)in comparison with Blank group(P<0.05).The results of qRT-PCR showed that in the Het-IA cells separately infected with NMBA and MCLR,with the combination of 10 and 20 generations,both of which had malignant transformation,the NMBA group and Blank group were significantly reduced by 2.42 times(10 generation and 20 generation)(P<0.05),while the combination group was significantly lowered by 3.84 times(10 generations)and 14.29 times(20 generations)(P<0.05).These results indicated that NMBA and MCLR could induce the down-regulation of LncRNA-UCA1 expression in the cell model of malignant Het-IA,suggesting that LncRNA-UCA1 was involved in the occurrence and development of esophageal cancer induced by NMBA and MCLR.5.2.FGFR2 Ⅲb and Ⅲc participate in the EMT induced by NMBA and MCLR through the PI3K-AKT signaling pathwayQRT-PCR results showed that FGFR2 Ⅲb in the NMBA group was 2.35 times higher than that in the Blank group(P<0.05).Ⅲc was increased 5.85 times and 5.32 times in the NMBA group and MCLR+NMBA group respectively(P<0.05).Western blot results showed that compared with Blank group,the level of Ⅲb in NMBA group was significantly increased(P<0.05),and the level of Ⅲc in NMBA and NMBA+MCLR group was also significantly increased(P<0.05).Western blot results showed that key molecules in the PI3K-AKT signaling pathway,such as PI3K,AKT and P-AKT proteins,were significantly up-regulated in the NMBA and NMBA+MCLR groups(P<0.05),and PTEN protein expression was significantly down-regulated in the NMBA and NMBA+MCLR groups(P<0.05),suggesting that NMBA and MCLR can induce the activation of PI3K-AKT signaling pathway.Western blot results showed that compared with Blank group,the level of interstitial cell marker n-cadherin was significantly increased in the NMBA group and the combination group(P<0.05),and the level of epithelial marker e-cadherin was significantly decreased in the NMBA group and the combination group(P<0.05),suggesting that NMBA and MCLR can induce EMT These results indicated that variable splicing of FGFR2 was involved in the activation of PI3K-AKT signaling pathway and promoted the EMT process of NMBA and MCLR induced esophageal cancer5.3.LncRNA-UCA1/miR-18a-5p/SORBS2 axis was involved in the occurrence and development of esophagus induced by nitrite and algal toxinThe expression of miR-18a-5p and SORBS2 were detected separately in 20 generation cells of NMBA and MCLR separately and combined with toxicity induced Het-IA induced by qRT-PCR The results showed that miR-18a-5p was significantly up-regulated in NMBA,MCLR,NMBA+MCLR groups compared with Blank group(P<0.05),and SORBS2 was significantly down-regulated in NMBA and NMBA+MCLR groups(P<0.05).Western blot results showed that the protein expression level of SORBS2 was significantly down-regulated in the MCLR+NMBA group(P<0.05).The results indicated that LncRNA-UCA1/miR-18a-5p/SORBS2 axis was involved in the occurrence and development of esophagus induced by nitrosamine and algal toxinsConclusion1.This study through microarrays select a is closely related to esophageal cancer development lncRNA-UCA1,and found that lncRNA-UCA1 in esophageal cancer cells and tissues were lower expression,its low expression level is negatively correlated with the risk of esophageal cancer,and closely related with lymph node metastasis2.This study through the body function experiment confirmed lncRNA-UCA1 expression can significantly inhibit the EC cells in vitro proliferation,invasion and migration ability.Animal studies have found that the overall overexpression of lncRNA-UCA1 can inhibit the growth of EC cells in nude mice,and inhibit the distant liver metastasis of EC cells.It is suggested that lncRNA-UCA1 play a role of tumor suppressor in esophageal cancer development3.This study was the first to discover that lncRNA-UCA1 and hnRNP F bind to each other in esophageal cancer cells,further regulating the production of two alternative splicing variants Ⅲb andⅢc of FGFR2,affected the ratio of two variable splicing bodies,and ultimately affected PI3K-AKT signaling pathway mediated EMT progression4.In this study,we found lncRNA-UCA1 could negative regulate the target gene SORBS2 expression by blocking the miR-18a-5p,and promoted the protein exression of SORBS2,and play a role of inhibited the proliferation of EC cells5.In this study,we found that NMBA and MCLR could inhbited the expression of lncRNA-UCA1 in the malignant Het-1A cell,on the one hand,its could act as a molecular sponge to combind miR-18-5p,and downregulated the expressiong of SORBS2 which is a tumor suppressor gene,and promoted the proliferation ability of tumor cells;on the other hand,its could activated PI3K-AKT signals to promote the EMT through the signal axis of lncRNA-UCA1/hnRNP F/FGFR2 Ⅲc.