| Backgrouds:Circular RNAs?CircRNAs?are single stranded covalently enclosed Circular RNAs,without 5'caps and 3'poly A tails,which are resistant to RNA enzymes.CircRNAs were stable and abundant,which were more than 10 times of the corresponding linear mRNAs,with specific expression in tissue and development stage.The specifity of circRNAs in tumor tissues and cells have been confirmed to be closely related to clinicopathological parameters and prognosis,such as TNM,lymph node metastasis and vascular invasion,which may be potential biomarkers and molecular targets.CircRNAs are essential regulators,such as directly targeting protein,influencing the promoters of targeted genes,regulating subcellular localization of protein,being endogenous competitive RNAs?ce RNAs?,involved in the regulation of biological behaviors in cancer.At present,the specific expression in gastric cancer?GC?and the effect on the biological behaviors of GC are not clear.Hsacirc1483 was located on chromosome 5,with ribosomal binding sites and expressed in both normal brain tissue and glioblastoma.Functional prediction indicated that hsacirc1483 may be related to DHX29,and combine to RNA-binding protein EIF4A3.Studies have shown that DHX29 was RNAJ helicase,which co?ld reduce the translation of protein and damage cell proliferation after knocked down.Armen et al.confirmed that silenced DHX29 in HeLa could destroy polymers,inhibite translation and cell proliferation.EIF4A3 encoding the death box protein family,was a key factor in regulating embryonic development,spermatogenesis,cell growth and division.Therefore,hsacirc1483 expression may be involved in cell differentiation and proliferation.Whether hsacirc1483 was involved in proliferation and differentiation of GC needed further explore.Let-7c,a member of the let-7 family,playing an important part in regulating cell differentiation and proliferation.For example,down expression of let-7 induced the transformation of CD8 T cells into cytotoxic T lymphocytes,mediated the TCR signaling pathway,promoted cell proliferation,and stimulated immune response in the body.In acute myelogenous leukemia,let-7c promoted the transformation of leukemia cells from granular to mononuclear by inhibiting PBX2,and induced the mature phenotypic transformation of cells.Let-7b inhibited osteogenic differentiation of apical stem cells,reduced the transcription of osteogenic markers by down-regulating the expression of the matrix metalloproteinase 1?MMP1?.Recently,let-7c hsa been found to be affected by non-coding RNAs.The bioinformatics analysis of our team showed that hsacirc1483 regulated let-7c,which needs to be further confirmed.Pepsinogen C?PGC?is the terminal differentiation factor of gastric mucosa.Our team focused on the regulation of PGC 3'UTR,and found that there were 5 miRNAs screened in luciferase trials targeting PGC 3'UTR.We showed that serum let-7c is negatively related to the expression of PGC in serum,exhibiting that PGC may be targeted by let-7c.It indicated that let-7c may be involved in differentiation of gastric cancer cells,which needs to be further confirmed.Purposes:1 To investigate the differential expression of hsacirc1483 in GC and adjacent tissues,and the correlation between clinicopathological parameters and PGC,clarifying the diagnostic performance of hsacirc1483 in distinguishing GC from normal tissues.2 To explore the effect of hsacirc1483 on the biological behavior of GC cells.3 To explicit whether hsacirc1483 participated in the differentiation of GC through targeting to let-7c.Methods:Part I:Differential expression of hsacirc1483 in GC and paracancer tissues and the correlation with PGC,the differentiation gene of gastric mucosa.1 GC and paracancerous normal tissues of 57 pairs of patients from November 2013to September 2017 in the affiliated hospital of China medical university were enrolled.Real-time quantitative PCR?Real-time PCR?was to evaluate the expression of hsacirc1483,in order to analysis the relationship between hsacirc1483 and PGC mRNA,as well as the correlation with clinical pathological parameters in GC.This study was approved by the ethics committee of the affiliated hospital of China medical university.2 The expression of hsacirc1483 and PGC mRNA was detected by total RNA extraction,cDNA first strand synthesis and real-time PCR?real-time PCR?.The relative expression levels of hsacirc1483 and PGC mRNA were represented by 2-?ct.3 Mann-whitney U nonparametric test was used to analyze the significance of hsacirc1483 expression in GC and adjacent tissues between two groups.The sensitivity and specificity of hsacirc1483 expression to distinguish gastric cancer from normal tissues were analyzed by receiver operating characteristic curve?ROC?.Spearman's correlation coefficient was used to analyze the correlation between hsacirc1483 and PGC.Part II:The effect of hsacirc1483 on the biological behaviors of GC cell1 The construction of hsacirc1483 overexpressed plasmid:GV486 was selected as hsacirc1483 overexpressed plasmid vector and confirmed by enzyme digestion.The sequence of hsacirc1483 was chemically synthesized,then digested with KpnI/BamHI enzyme.And the enzyme products were connected to the linear expression vector,identified by PCR.2 Screening of lower and moderate expression of hsacirc1483 grounded on the basics in GC cell:we detected the expression of hsacirc1483 in MGC803,HGC27,SGC7901,BGC823,AGS and MKN45,as well as the normal gastric mucosa epithelial cell,GES-1,through total RNA extraction,reverse transcription and Real-time PCR,to select three cell lines with hsacirc1483 lower and moderate expression:HGC27,BGC823 and AGS for subsequent experiments.3 The detection of cell transfection efficiency:by cell culture and transient transfection,we transfected hsacirc1483 overexpression plasmid into BGC823?AGS?HGC27,medium was replaced in the time of 24h after transfection.Green fluorescence was to observe using fluorescence microscope,transfection efficiency of plasmid above 50%was chosed in following experiments.4 Detection of differentiation biomarkers in hsacirc1483 overexpressed cells:overexpressed and control plasmid of hsacirc1483 cell precipitation was collected after transfection 24h.Through total RNA extraction,reverse transcription and Real-time PCR,we detected transfection efficiency of hsacirc1483 and the mRNA expressions of PGC,SOX2,CDX2,TFF2.Meanwhile,protein expressions of PGC,SOX2,CDX2,TFF2 were detected by protein extraction,SDS-PAGE electrophoresis and western blot.5 Proliferation detection of hsacirc1483 overexpressed BGC823 cells:overexpressed and control plasmids of hsacirc1483 were digested and centrifuged after transfection 24h,then laid in 96-well plates for cell proliferation toxicity detection?CCK-8?in 24h,48h,and 72h in the time of cultured in 96-well plates.6.Statistical analysis:the relative expressions of hsacirc1483 and PGC,SOX2,CDX2,TFF2 mRNA in the cells were calculated as 2-??ct.Graphpad Prism 6 was used for statistical analysis and graph processing.Paired sample t test was to compare the differential expressions between the two groups.All datas were denoted as mean±standard deviation.Part III:The molecular mechanism of hsacirc1483 targeting to let-7c in the regulation of gastric cancer cell differentiationDual-luciferase assay:hsacirc1483 3'-UTR region was constructed into the downstream of reporter luciferase in the 3'-UTR reporter system,and the luciferase activity was observed after overexpression of let-7c,so as to reflect the inhibitory effect of let-7c on hsacirc1483 quantitatively.Four groups were set up in luciferase assay:hsacirc1483+let-7c,hsacirc1483-nc+let-7c,hsacirc1483-mut+let-7c,hsacirc1483-mut+let-7c-nc.2 The screening of higher and moderate expression of let-7c in GC cell lines:we detected the expression of let-7c in MGC803,HGC27,SGC7901,BGC823,AGS and MKN45,as well as the normal gastric mucosa epithelial cell,GES-1,through total RNA extraction,reverse transcription and Real-time PCR,to select three cell lines with let-7c with higher and moderate expression.3 The detection of cell transfection efficiency:by cell culture and transient transfection,we transfected let-7c mimics into BGC823 and AGS,let-7c inhibitors into BGC823 and HGC27 Medium was replaced in the time of 6h after transfection.Green fluorescence was to observe using fluorescence microscope,transfection efficiency of plasmid above 50%was chosed in following experiments.4 Detection of differentiation biomarkers in let-7c overexpressed/silenced cells:cells precipitation with let-7c mimics/inhibitors were collected in the time of 48h after transfection.Through total RNA extraction,reverse transcription and Real-time PCR,we detected transfection efficiency of let-7c and mRNA expressions of PGC,SOX2,TFF2.Meanwhile,protein expressions of PGC,SOX2,CDX2,TFF2 were detected by protein extraction,SDS-PAGE electrophoresis and western blot.5 Detection of differentiation markers in the gastric cancer cells with overexpression of both hsacirc1483 and let-7c:cells with overexpression of both hsacirc1483and let-7c were collected in the time of 48h after transfection,in order to examine the mRNA and protein expression of PGC,SOX2,CDX2 and TFF2.6 Statistical analysis:relative expressions of hsacirc1483,PGC,SOX2,CDX2 and TFF2mRNA,let-7c in the cells were denoted as 2-??ct.Graphpad Prism 6 was used for statistical analysis and graph processing.Paired sample t test was used to compare the differential expressions between the two groups.All datas were denoted as mean±standard deviation.Results:Part I:Differential expression of hsacirc1483 in GC and paracancer tissues and the correlation with PGC,the differentiation gene of gastric mucosa.1.The results indicated that hsacirc1483 expression was remarkably different between cancer and paracancer tissues in 57 pairs.The expression of hsacirc1483 in cancer(GC 2-?CT6.92×10-6,(3.17×10-6,1.90×10-4))was notably lower than that in paracancer tissues(ANT 2-?CT1.53×10-5,(6.61×10-6,8.31×10-4))?p<0.001?.2.In order to further evaluate the diagnostic efficacy of hsacirc1483,we analyzed the area under ROC curve,sensitivity and specificity.The area under ROC curve of hsacirc1483 was 65.19%?95%CI 0.55,0.75,p<0.001?,sensitivity and specificity was 50.90%and 75.4%respectively.3.Spearman's correlation coefficient was to analyze the relationship between hsacirc1483 and PGC in tissue and paracaner.The results showed that the expression of hsacirc1483 was significantly positively correlated with PGC in both cancer and adjacent normal tissues?cancer tissues:r=0.31,p=0.049,adjacent tissues:r=0.38,p=0.01?.Part II:The effect of hsacirc1483 expression on the biological behaviors of gastric cancer cells1.Screening of cell lines:the expression of hsacirc1483 in three generations of gastric cancer cell lines,including MGC803,HGC27,SGC7901,BGC823,AGS and MKN45 as well as GES-1,the normal gastric mucosal epithelial cell,were detected by Real-time PCR to choose cells with lower and moderate expression of hsacirc1483.Three gastric cancer cell lines were in the following experiment,HGC27,BGC823 and AGS.2.The overexpression efficiency of hsacirc1483:the transfection efficiencies of hsacirc1483 overexpression plasmid were higher than 50%in BGC823,HGC27and AGS.The overexpression efficiencies of hsacirc1483 plasmid in BGC823,HGC27 and AGS were 25,000 times,1200 times,20571 times repectively,higher than that in control?p<0.05?.3.Effect of hsacirc1483 overexpression on BGC823 proliferation:we compared BGC823 transfected with hsacirc1483 overexpression with control plasmid to evaluate the proliferation.It was found that there was no statistical difference of proliferation between the two groups?p>0.05?.4.The effect of hsacirc1483 overexpressed vector on the expression of differentiation markers in gastric cancer cells.?1?The mRNA levels of PGC,SOX2 were 1.52±0.14,1.90±0.23 respectively in hsacirc1483 overexpressed BGC823 cells,strikingly higher than that in control group,?mRNAs levels in control were set to 1??p<0.05?.The mRNA level of TFF2was 0.82±0.03 in hsacirc1483 overexpressed BGC823 cells,strikingly downregulated comparing with the control group?p<0.05?.We did not find the mRNA level of CDX2.The gray value of TFF2 protein was 0.09±0.08 in BGC823with hsacirc1483 overexpression plasmid,significantly lower than that in control?gray value was 0.12±0.09??p<0.05?.There was no protein expressions of PGC,SOX2,and CDX2.?2?The mRNA expression of PGC was 1.36±0.12 in HGC27 with hsacirc1483overexpression,increased remarkably contrasting with the control?mRNAs levels in control were set to 1??p<0.05?.?3?The gray value of PGC protein in AGS with hsacirc1483 overexpression was0.47±0.01,upregulated significantly comparing with the control?0.21±0.01??p<0.001?.While the gray values of CDX2 and TFF2 were 0.13±0.04,0.04±0.01,decreased notably contrasting with the control?0.29±0.08,0.04±0.01??p<0.05?.Part III:The molecular mechanism of hsacirc1483 targeting to let-7c in the regulation of differentiation in gastric cancer cellDouble luciferase assay:by comparing with control?circRNA-nc+let-7c,luciferase activity in control were set to 1?,the luciferase activity of wild plasmid of hsacirc1483 3'UTR together with let-7c co-transfection declined obviously?p<0.05?,the luciferase activity of mutant plasmid of hsacirc1483 3'UTR with let-7c co-transfection had no distinct change,and the same results were observed in other two groups:wild plasmid of hsacirc1483 3'-UTR+let-7c-NC?mutant plasmid of hsacirc1483 3'UTR+let-7c-NC.2.Regulatory relationship of hsacirc1483 and let-7c in gastric cancer cells.?1?Cell screening:we detected the expression of let-7c in MGC803,HGC27,SGC7901,BGC823,AGS and MKN45 gastric cancer cell lines,as well as GES-1,the normal gastric mucosa epithelial cell.Three cell lines were in the following research:HGC27 with let-7c high expression,BGC823 and AGS with let-7c moderate expression.?2?The efficiencies of let-7c overexpression/silence:the expression of let-7c mimics were 130 and 460 times in BGC823 and AGS with let-7c mimics,higher than that in control.The expression of let-7c inhibitors were 0.56±0.18,0.87±0.08 in BGC823and AGS,decreasing by 31%?p<0.05?and 10%?p>0.05?,comparing with the control.?3?The expression of hsacirc1483 were 0.23±0.18,0.54±0.40,decreasing notably in BGC823 and AGS transfected with let-7c mimics?p<0.05?.The expression of hsacirc1483 were 1.78±0.38,1.71±0.36,increasing notably in BGC823 and AGS transfected with let-7c inhibitors?p<0.05?.?4?The expressions of let-7c were 0.88±0.02,0.72±0.08,declining notably in BGC823 and HGC27 with hsacirc1483 overexpression?p<0.05?.3.Influence of let-7c on differentiation of gastric cancer cells?1?The mRNA levels of PGC,SOX2 were 0.49±0.05,0.71±0.03,downregulated strikingly in BGC823 with let-7c overexpression?p<0.05?,while the mRNA level of TFF2 was 1.75±0.13 in BGC823 with let-7c upregulation,increased strikingly comparing with the control group?mRNAs levels in control were set to 1??p<0.05?.We did not find the mRNA level of CDX2.The gray value of TFF2 protein was 0.44±0.06 in BGC823 with let-7c overexpression,significantly higher than that in control?gray value was 0.25±0.15??p<0.05?.There were no protein expressions of PGC,SOX2,and CDX2.?2?The mRNA levels of PGC was 0.87±0.05,declined obviously in AGS with let-7c upregulaion?p<0.05?.While PGC mRNA had upregulated trends in BGC823 and HGC27 with let-7c inhibitors,whose expression were 2.40±1.58,1.73±0.53.The gray value of PGC protein was 0.02±0.00 in AGS with let-7c overexpression,significantly lower than that in control?gray value was 0.07±0.01??p<0.05?.The gray values of CDX2 and TFF2 protein were 0.84±0.04?0.60±0.06 increasing significantly in AGS with let-7c overexpression?p<0.05?.4.The effect of co-overexpression of and let-7c on the differentiation markers of gastric cancer cells.?1?`The mRNA levels of PGC,SOX2 were 0.68±0.05,0.37±0.06 in BGC823 with co-overexpression of hsacirc1483 and let-7c,notably decreased comparing with the control?hsacirc1483 overexpression,mRNAs levels in control were set to 1?.The mRNA level of TFF2 was 1.37±0.01 in BGC823 with co-overexpression of hsacirc1483 and let-7c,significantly increased comparing with the control?hsacirc1483 overexpression,mRNAs levels in control were set to 1?.The gray value of TFF2 protein was 0.44±0.06 in BGC823 with hsacirc1483 and let-7c co-overexpression,significantly higher than that in hsacirc1483 overexpression?gray value was 0.45±0.18??p<0.05?.?2?The gray value of PGC protein was 0.27±0.02 in AGS with hsacirc1483 and let-7c co-overexpression,significantly lower than that in hsacirc1483overexpression?gray value was 0.47±0.01??p<0.05?.And the gray value of CDX2,TFF2 protein were 0.60±0.01,0.34±0.00 in AGS with hsacirc1483 and let-7c co-overexpression,significantly higher than that in hsacirc1483 overexpression?CDX2:0.13±0.04,TFF2:0.04±0.01??p<0.01?.Conclusion:Hsacirc1483 notably decreased in GC tissues,and possessed good diagnostic efficacy in distinguishing GC from normal tissues.The expression of hsacirc1483 was positively correlated with the level of PGC mRNA in tissues.3.Hsacirc1483 upregulation had no effect on the proliferation of GC cells.4.Hsacirc1483 combined with let-7c,and they were negatively regulator relationship between them.5.Hsacirc1483 increased the expression of PGC mRNA and protein,SOX2 mRNA,down-regulated the expression of CDX2 protein,TFF2 mRNA and protein by targeting to let-7c,affecting the differentiation of GC cells. |
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