| Gastric cancer（GC）is the fifth most common malignant tumor in the world and the fourth leading cause of cancer-related death.Due to the atypical early symptoms of GC and the lack of specific molecular markers,most patients were already in the advanced stage at the time of visit,and the 5-year overall survival rate was still less than 30%.Therefore,identifying the molecular mechanism of GC and its specific biomarkers is very important for exploring more accurate diagnosis and treatment of GC.Epigenetic studies have shown that circular RNAs（circ RNAs）play an important role in the occurrence,development,recurrence and metastasis of cancer.Therefore,the exploration of circ RNA related to GC aims to lay a foundation for the early diagnosis,prognosis and precise treatment of GC.Part One The expression,malignant biological behavior and clinicalsignificance of hsa＿circ＿0006646 in gastric cancer tissues and cellsObjective:To investigate the expression and malignant biological behavior of hsa＿circ＿0006646 in GC tissues and cells,and analyze the correlation between its expression level and the survival of GC patients.Methods:1.The Gene Expression Omnibus（GEO）dataset GSE163416 was analyzed to screen up-regulated circ RNAs in GC tissues.Among all candidate circ RNAs,hsa＿circ＿0006646 showed the most significant up-regulation.2.35 pairs of tumor tissues and corresponding normal gastric mucosal epithelial tissue were collected from GC patients who received gastroscopic biopsy in the Fourth Hospital of Hebei Medical University from October 2019to November 2020.Quantitative real-time polymerase chain reaction（q RT-PCR）was used to detect the expression of hsa＿circ＿0006646 in 35 pairs of GC tissues and corresponding normal gastric mucosal epithelial tissue.3.Chi-square method was used to analyze the correlation between the expression level of hsa＿circ＿0006646 and clinical features.4.Kaplan-Meier method was used to analyze the correlation between hsa＿circ＿0006646 expression level and survival of GC patients.5.The expression levels of hsa＿circ＿0006646 in 4 GC cell lines（AGS,HGC27,MKN45 and MKN74）and normal gastric epithelial cells（GES-1）were detected by q RT-PCR.6.The circular structure of hsa＿circ＿0006646 was predicted by circ Base database and verified by Sanger sequencing and agarose gel electrophoresis.7.RNase R digestion assay was used to analyze the stability of hsa＿circ＿0006646.8.Hsa＿circ＿0006646 small interfering RNA（si RNA）has been transfected into AGS and HGC27 cells through Lipofectamine TM2000.GC cell lines with low expression of hsa＿circ＿0006646 were constructed（si-hsa＿circ＿0006646#1,si-hsa＿circ＿0006646#2,si-hsa＿circ＿0006646#3）,and transfected with si RNA-NC as control group.q RT-PCR was used to detect the transfection efficiency,and the two knockdown sequences with the highest transfection efficiency were selected for subsequent experiments.9.CCK-8 assay,colony formation,wound healing assay and Transwell invasion assay were used to detect the effects of hsa＿circ＿0006646knockdown on proliferative activity,colony formation activity,migration ability and invasion ability of GC cells.10.Western blot assay was used to detect the effect of hsa＿circ＿0006646 knockdown on EMT-related proteins in GC cells.Results:1.GEO dataset GSE163416 was used to analyse that among all candi-date circ RNAs with up-regulated expression in GC tissues,hsa＿circ＿0006646showed the most significant expression.2.The results of q RT-PCR showed that hsa＿circ＿0006646 m RNA expression was significantly up-regulated in GC tissues compared with normal gastric mucosal epithelial tissue（P<0.001）.3.Chi-square analysis showed that the expression level of hsa＿circ＿0006646 was positively correlated with TNM stage and lymph node invasion（P<0.05）.4.Kaplan-Meier survival analysis showed that the overall survival（OS）of the hsa＿circ＿0006646 high expression group was significantly shortened compared with the hsa＿circ＿0006646 low expression group（P<0.05）.5.The results of q RT-PCR showed that hsa＿circ＿0006646 m RNA expression was significantly increased in GC cells（AGS,HGC27,MKN45and MKN74）compared with normal gastric epithelial cells（GES-1）（P<0.001）.6.The circular structure of hsa＿circ＿0006646 was confirmed by Sanger sequencing and agars gel electrophoresis,which was consistent with the predicted results of circ Base database.7.The results of RNase R digestion assay showed that hsa＿circ＿0006646was more resistant to RNase R inhibition than its linear transcript（P<0.05）.8.The results of q RT-PCR showed that after knocking down hsa＿circ＿0006646,the expression level of hsa＿circ＿0006646 in si-hsa＿circ＿0006646#1,si-hsa＿circ＿0006646#2 and si-hsa＿circ＿0006646#3groups was significantly lower than that in si-NC group（P<0.01）.It was suggested that hsa＿circ＿0006646 low expression GC cell line was successfully constructed,and among the three groups of knockdown sequences,si-hsa＿circ＿0006646#1 and si-hsa＿circ＿0006646#2 groups had higher knockdown efficiency（P<0.001）.9.The results of CCK-8 assay showed that the proliferation ability of GC cells AGS and HGC-27 in sh-hsa＿circ＿0006646#1 and sh-hsa＿circ＿0006646#2 groups was significantly decreased compared with that in sh-NC group（P<0.001）.10.The results of colony formation assay showed that the colony forma-tion ability of GC cells AGS and HGC-27 in sh-hsa＿circ＿0006646#1 and sh-hsa＿circ＿0006646#2 groups was significantly inhibited compared with that in sh-NC group（P<0.001）.11.The results of the wound healing assay showed that the migration ability of GC cells AGS and HGC-27 in sh-hsa＿circ＿0006646#1 and sh-hsa＿circ＿0006646#2 groups was significantly decreased compared with that in sh-NC group（P<0.001）.12.The results of the transwell invasion assay showed that the invasion ability of GC cells AGS and HGC-27 in sh-hsa＿circ＿0006646#1 and sh-hsa＿circ＿0006646#2 groups was significantly decreased compared with that in sh-NC group（P<0.001）.13.The results of Western blot showed that the expression of EMT-related protein E-cadherin in GC cells AGS and HGC-27 in sh-hsa＿circ＿0006646#1 and sh-hsa＿circ＿0006646#2 groups was increased and the expression of N-cadherin,Vimentin and Snail proteins were decreased compared with that in sh-NC group（P<0.01）.Summary:1.Hsa＿circ＿0006646 is significantly highly expressed in GC tissues and cells,and its high expression is closely related to poor prognosis of GC patients,which may be a biomarker affecting the prognosis of GC patients.2.Hsa＿circ＿0006646 can promote the proliferation,migration,invasion and EMT process of GC cells,and play a role in promoting cancer in GC.Part Two Hsa＿circ＿0006646 promotes progression of gastric cancerby activating Wnt/β-catenin signaling pathway through the mi R-665/HMGB1 axisObjective:To investigate the role of hsa＿circ＿0006646 as a competitive binding of ce RNA to mi R-665 to up-regulate the activation of Wnt/β-catenin signaling pathway by HMGB1,thus inducing EMT and promoting the occurrence and development of GC.Methods:1.Bioinformatics software circ Bank and circ Interactome were used to predict three mi RNAs that could bind to hsa＿circ＿0006646.2.q RT-PCR was used to detect the expression changes of three mi RNAs after hsa＿circ＿0006646 knockdown,and the mi RNA with the most significant expression differences was selected as the research object.3.Fluorescence in situ hybridization（FISH）was used to detect the subcellular localization of hsa＿circ＿0006646 and mi R-665.4.q RT-PCR was used to detect the effect of knockdown and overexpression of mi R-665 on the expression of hsa＿circ＿0006646.5.The binding site of hsa＿circ＿0006646 and mi R-665 was verified by dual luciferase reporter assay.6.RNA immunoprecipitation（RIP）was used to further confirm the existence of endogenous binding between hsa＿circ＿0006646 and mi R-665.7.The four databases of Fun Rich,mi RWalk,Target Scan and mi RDB were used to predict the target genes binding to mi R-665.8.The binding sites of mi R-665 and HMGB1 were verified by dual luciferase reporter assay.9.Western blot was used to detect the influence of mi R-665 on the expression of HMGB1 protein in GC cells AGS and HGC27 after overexpression of mi R-665.10.q RT-PCR and Immunohistochemical staining（IHC）methods were used to analyze mi R-665 and HMGB1 expression levels in GC and corresponding normal gastric mucosal epithelial tissue.The correlation between HMGB1 and the expression of hsa＿circ＿0006646 and mi R-665 was analyzed.11.Lipofectamine TM2000 was transfected into AGS and HGC27 cells by lipofectaminetm2000.The cells were divided into sh-NC,sh-hsa＿circ＿0006646#1,sh-hsa＿circ＿0006646#1+mi RNA inhibitor NC,sh-hsa＿circ＿0006646#1+mi R-665 inhibitor,sh-NC+mi RNA mimic NC and sh-NC+mi R-665 mimic six groups.12.Western blot was used to detect the expression of HMGB1,Wnt/β-catenin signaling pathway related proteins（β-catenin,Wnt3a and c-Myc）and EMT-related proteins（E-cadherin,N-cadherin,Vimentin and Snail）in AGS and HGC-27 cells of six groups.13.Rescue experiments were conducted to confirm the effects of hsa＿circ＿0006646 on proliferation,migration,invasion,EMT and other malignant biological behaviors of AGS and HGC-27 cells through mi R-665/HMGB1 axis.Results:1.The prediction results of bioinformatics software circ Bank and circ Interactome showed that the three mi RNAs binding hsa＿circ＿0006646were mi R-139-3p,mi R-1322 and mi R-665,respectively.2.q RT-PCR results showed that knockdown of hsa＿circ＿0006646 could lead to increased expression levels of three mi RNAs,among which mi R-665showed the most significant increase（P<0.001）,and mi R-665 played an inhibitory role in GC.Therefore,mi R-665 was selected for subsequent experimental studies.3.FISH assay results showed that hsa＿circ＿0006646 and mi R-665 were co-located in the cytoplasm.4.q RT-PCR results showed that knockdown or overexpression of mi R-665 can lead to increased or decreased expression of hsa＿circ＿0006646.5.Double luciferase reporter assay results confirmed the binding site of hsa＿circ＿0006646 and mi R-665.6.RIP results further confirmed the endogenous binding between hsa＿circ＿0006646 and mi R-665.7.The target protein HMGB1 of mi R-665 was predicted using Fun Rich,mi RWalk,Target Scan and mi RDB databases.8.The results of the double luciferase reporter assay showed that mi R-665 could bind to the 3’-UTR region of HMGB1.9.Western blot results showed that overexpression of mi R-665 inhibited the expression of HMGB1 in GC cells AGS and HGC-27（P<0.001）.10.The results of q RT-PCR and IHC showed that compared with the corresponding normal gastric mucosal epithelial tissue,the expression of mi R-665 in GC tissues was decreased,and its expression level was negatively correlated with the expression of hsa＿circ＿0006646（P<0.05）.The expression level of HMGB1 in GC tissues was positively correlated with the expression of hsa＿circ＿0006646 and negatively correlated with the expression of mi R-665（P<0.001）.11.Western blot results showed that compared with the sh-NC group,the expression of HMGB1,related proteins of Wnt/β-catenin signaling pathway（β-catenin,Wnt3a and c-Myc）were decreased in sh-hsa＿circ＿0006646#1 group,and the expression of EMT-related protein E-cadherin was increased.N-cadherin,Vimentin and Snail were decreased（all P<0.01）.HMGB1 expression and the related proteins of Wnt/β-catenin signaling pathway（β-catenin,Wnt3a,and c-Myc）of sh-NC+mi R-665 mimic group were significantly reduced compared with those of sh-NC+mi R-NC mimic group.E-cadherin expression increased,N-cadherin,Vimentin and Snail expression decreased（all P<0.01）.Compared with sh-hsa＿circ＿0006646#1+mi R-NC inhibitor,HMGB1 expression was significantly increased in sh-hsa＿circ＿0006646#1+mi R-665 inhibitor group.The expression of related proteins of Wnt/β-catenin signaling pathway（β-catenin,Wnt3a and c-Myc）increased,the expression of E-cadherin decreased,and the expression of N-cadherin,Vimentin and Snail increased（all P<0.01）.These results suggest that knockdown of mi R-665 expression can save the HMGB1,related proteins of Wnt/β-catenin signaling pathway downregulation and EMT reversal caused by hsa＿circ＿0006646 knockdown.These results indicate that hsa＿circ＿0006646 up-regulates the expression of HMGB1 through mi R-665,activates the Wnt/β-catenin signaling pathway,and thus induces the EMT of GC,and hsa＿circ＿0006646 is involved in the occurrence and development of GC as an oncogene.12.Rescue experiments showed that knockdown of hsa＿circ＿0006646inhibited proliferation,colony formation,migration and invasion of GC cells（P<0.001）.Low expression of mi R-665 reversed the decreased proliferation,colony formation,migration,and invasion of GC cells induced by hsa＿circ＿0006646 knockdown（P<0.001）.Summary:1.Hsa＿circ＿0006646 and mi R-665 were co-located in the cytoplasm of GC cells,and their expression levels were negatively correlated.2.Mi R-665 can target the 3’-UTR region of HMGB1 and inhibit the expression of HMGB1,and the expression level of the two is negatively correlated.3.Hsa＿circ＿0006646 activated Wnt/β-catenin signaling pathway by up-regulating HMGB1 through sponge adsorption of mi R-665 to induce EMT process,thus promoting the occurrence and development of GC.Part Three In vivo experimental study of hsa＿circ＿0006646/mi R-665/HMGB1 axis affecting the occurrence and development of gastric cancerObjective:To explore the carcinogenic effect of hsa＿circ＿0006646/mi R-665/HMGB1 axis in nude mice,and lay a theoretical foundation for the treatment of GC.Methods:1.A stable AGS cell line with low expression of hsa＿circ＿0006646 was constructed,and the cells were divided into hsa＿circ＿0006646 control group（sh-NC）andhsa＿circ＿0006646lowexpressiongroup（sh-hsa＿circ＿0006646#1）.2.The cell suspensions of sh-NC and sh-hsa＿circ＿0006646#1 groups were injected subcutaneously into the groin of 4-week-old male BALB/c nude mice to construct the GC transplantation tumor model in nude mice.3.The nude mice were weighed every 3 days.The tumor volume of transplanted mice was measured in vitro with vernier caliper every week,and the tumor growth curve was drawn.After 4 weeks,all nude mice were sacrificed by cervical dislocation,and the primary tumor was removed and weighed.4.Western blot and IHC were used to detect the expressions of HMGB1,Wnt/β-catenin signaling pathway related proteins and EMT-related proteins in the xenograft tissues of nude mice.Results:1.GC cell line AGS with low expression of hsa＿circ＿0006646 was successfully constructed.2.The tumor growth curve showed that the tumor volume of sh-hsa＿circ＿0006646#1 group was significantly lower than that of sh-NC group（P<0.001）.The tumor size and weight in sh-hsa＿circ＿0006646#1 group were significantly smaller than those in sh-NC group（P<0.001）.3.Western blot results showed that,compared with sh-NC group,the expression of HMGB1,Wnt/β-catenin signaling pathway related proteins（β-catenin,Wnt3a and c-Myc）and EMT-related proteins（N-cadherin,Vimentin and Snail）decreased,E-cadherin expression was elevated in sh-hsa＿circ＿0006646#1.4.IHC results showed that,compared with the sh-NC group,the expression of HMGB1 andβ-catenin in the sh-hsa＿circ＿0006646#1 group decreased.Summary:1.Knockdown hsa＿circ＿0006646 expression significantly inhibits the growth of transplanted tumor in nude mice.2.Hsa＿circ＿0006646 plays a carcinogenic role in nude mice through the mi R-665/HMGB1 axis.Conclusions:1.Hsa＿circ＿0006646 is highly expressed in GC tissues and cells,and its high expression is closely related to the poor prognosis of GC patients,and plays a role in promoting cancer in GC.2.Hsa＿circ＿0006646 induces EMT to promote GC occurrence and development by activating Wnt/β-catenin signaling pathway through mi R-665/HMGB1 axis.Therefore,hsa＿circ＿0006646 can be used as a potential predictive biomarker and therapeutic target for GC. |
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