Study On The Mechanism Of Hsa?circ?001988/miR-197-3p/FBXW7 Axis In Regulating Gastric Cancer Cell Biological Behavior

Objective: With the development of RNA sequencing and bioinformatics technology,circ RNAs have been proved to have multiple functions in human cells.Many studies have reported the expression profile of circular RNAs in GC.which have found that circ RNAs have potential value in regulating cell function and play an important role in the development and progression of gastric cancer.Hsa?circ?001988 is one of the circ RNAs,which has been reported taking part in the regulation of colon cancer,breast cancer,non-small cell cancer and clear cell carcinoma of kidney.However,the role of hsa?circ?001988 in GC has not been reported.In addition,more evidences demonstrated circ RNAs participated in tumorigenesis and progression via sponging miRNAs and regulating targeted genes.In present study,we aimed to find the expression patterns of hsa?circ?001988 in GC tissues and cell lines,analyze the relationship between the expression and clinicopathological factors.Further study the effect of hsa?circ?001988 on GC cell proliferation,migration and invasion both in vivo and vitro.In addition,based on the available bioinformatic prediction,we found hsa?circ?001988-related RNA binding protein U2AF65 and EIF4A3,miR-197-3p and its downstream FBXW7.Therefore,a series of experiments were conducted to explore and elucidate the potential underlying molecular mechanisms of U2AF65/EIF4A3-mediated hsa?circ?001988 in inhibiting GC cell proliferation,migration and invasion by sponging miR-197-3p.Methods:1.Total three hundred and forty-one GC tissues ernd matched normal mucosa samples were collected after surgical resection in China Medical University Affiliated Hospital.Quantative real-time quantitative polymerize chain reaction(q RT-PCR)was applied to detect the expression of hsa?circ?001988 in GC tissues,matched normal epithelium samples and GC cell lines.The relationship between the expression of hsa?circ?001988 and clinicopathological features was analyzed.2.The overexpressing hsa?circ?001988 and vector plasmids were transfected into BGC823 cells via liposome tranfection.The stable overexpressing hsa?circ?001988BGC823 cell line was constructed through G418 pressure screening.Theexpression of hsa?circ?001988 in AGS cells were kocked down by si RNA interference.The silencing effiency was confirmed by q RT-PCR assay.CCK-8 assay,flow cytometry,cell cycle distribution,cell wound healing assay,Transwell migration and invasion assays were performed to access the effect of different hsa?circ?001988 expression on cell proliferation,clone-formation,cell cycle distribution,cell migration and invasion abilities.After overpressing hsa?circ?001988 in BGC823 cells,the experiments above were repeated.In addition,xenografted tumor formation assay was used to detect the effect of overexpressing hsa?circ?001988 BGC823 on tumor growth in vivo.3.The expression of miR-197-3p and FBXW7 was detected in 33 GC tissues and matched normal mucosal tissues by q RT-PCR assay.The correlation between hsa?circ?001988,miR-197-3p and FBXW7 was analyzed in GC tissues.The expression of CCDC6 was detected in 69 GC tissues and matched normal mucosal tissues by q RT-PCR assay.KMPLOT website was used to analyze the effect of FBXW7 and CCDC6 on the prognosis of 631 patients with GC from GEO.The effect of hsa?circ?001988 on the mRNA expression of miR-197-3p ? FBXW7 ? CCDC6 was assessed by q RT-PCR assay.The effect of hsa?circ?001988 on the protein expression of Cyclin D1,YAP1,E-cadherin?Vimentin?N-cadherin was detected by western blot assay.4.The mRNA and protein expression of U2AF65 was detected in 33 GC tissues and matched normal mucosal tissues by q RT-PCR assay and western blot assay.Human Protein Atlas database was used to analyze the effect of U2AF65 on the prognosis of 354 patients with GC from TCGA.RIP experiment was used to verify the interaction between U2AF65,EIF4A3 and hsa?circ?001988.After knocking down the expression of U2AF65 and EIF4A3 in AGS and BGC823 with si RNA interference,q RT-PCR assay was used to detect the variation of hsa?circ?001988 expressions.Results:1.The expression of hsa?circ?001988 in GC tissues was lower than that in paracancerous normal epithelium samples(P<0.001).The low expression of hsa?circ?001988 was closely related to WHO histological types,Lauren types and depth of invasion(P<0.05).ROC curve was established accordin to the expression of hsa?circ?001988.The results showed that the area under the ROC curve was 0.708,the sensitivity and specificity was 0.716 and 0.566,respectively.Compared with human immortalized gastric mucosa cell line,the expression of hsa?circ?001988 in GC cell line was significantly decreased.The expression was highest in AGS cell lines and lowest in BGC823 cell line.2.The results of q RT-PCR assay verified the successful establishment of overexpressing hsa?circ?001988 BGC823 cell line,while the expression of hsa?circ?001988 in AGS cells was obviously declined via si RNA intterference.CCK8 study demonstrated that silencing hsa?circ?001988 in AGS cells promotedcells proliferation ability.The results of clone-formation assay showed that knocking down hsa?circ?001988 in AGS cells facilitated the cells colony formation ability.The flow cytometry assay displayed that knocking down hsa?circ?001988 in AGS cells induced the decrease of G0/G1 phase ratio.Wound-healing assay demonstrated that knocking down hsa?circ?001988 in AGS cells induced the increased migration ability.Transwell migration and invasion assay displayed that knocking down hsa?circ?001988 in AGS promoted the migrative and invasive ability.While overexpressing hsa?circ?001988 in BGC823 cells induced the opposite effect.Furthermore,xenografted tumor formation was dramatically inhibited by overexpressing hsa?circ?001988 in BGC823 cells.3.Compared with non-cancerous normal musosal tissues,the expression ofmiR-197-3pwere obviously increased in 33 GC tissues,while FBXW7 expression was significantly decreased in GC.Moreover,the expression of miR-197-3p was respectively negatively related to hsa?circ?001988 and FBXW7 expressions in GC.However,the expression of FBXW7 in GC was positively related to hsa?circ?001988 expression.4.The expression of CCDC6 was obviously decreased in 69 GC tissues compared to matched normal musosal tissues.KMPLOT database demonstrated that the median overall survival time in 386 patients with GC in FBXW7 high-expressed group was 65 months,significantly longer than that in 245 patients with low-expressed FBXW7(34.1months,P=0.0041).The first progression time in 303 patients with GC in FBXW7high-expressed group was 49.47 months,significantly longer than that in 219 patients with low-expressed FBXW7(23.2 months,P=2.7e-06).The median overall survival time in 265 patients with GC in CCDC6 high-expressed group was 100.8 months,significantly longer than that in 366 patients with low-expressed CCDC6(31.2 months,P=2.7e-06).The first progression time in 239 patients with GC in CCDC6high-expressed group was 12.31 months,significantly longer than that in 283 patients with low-expressed CCDC6(8.4 months,P=5.4e-06).5.Silencing hsa?circ?001988 expression in AGS cells,the expression of miR-197-3p was elevated,while the downstream gene FBXW7 and CCDC6 mRNA expression was decreased.Overexpressing hsa?circ?001988 expression in BGC823 cells inhibited the expression of miR-197-3p.The downstream gene FBXW7 and CCDC6 mRNA expression was increased.6.Silencing hsa?circ?001988 expression in AGS cells,the protein expression of Cyclin D1,YAP1,Vimentin and N-cadherin was elevated,while the protein expression of E-cadherin was decreased.Overexpressing hsa?circ?001988 expression in BGC823 cells inhibited the protein expression of Cyclin D1,YAP1,Vimentin and N-cadherin,while the protein expression of E-cadherin was increased.7.Compared with non-cancerous normal musosal samples,the expression of U2AF65 was obviously decreased in GC tissues,the difference was statistically significant(P<0.01).The results of Human Protein Atlas showed that in total three hundred fifty-four patients with GC,5-years survival in 91 patients with U2AF65 low expressed was obviously shorter than in 263 U2AF65 high expressed patients(Log rank survival analysis,P=4.80e-2).The results of RIP experiments verified the interaction between U2AF65,EIF4A3 and hsa?circ?001988,the enrichment of U2AF65 and EIF4A3 targeted hsa?circ?001988 was respectively 8.691±0.796,12.74±1.795.Silencing U2AF65 and EIF4A3 in AGS and BGC823 cells significantly down-regulated the expression of hsa?circ?001988.Conclusion: 1.The expression of hsa?circ?001988 in GC tissues and cell lines was significantly lower than in normal gastric mucosa tissues and human immortalized gastric mucosa cell line.Its expression was closely related to WHO histological types,Lauren types and depth of invasion.Hsa?circ?001988 might have certain potential value for the diagnosis of GC.2.Weakened the expression ofhsa?circ?001988 could significantly promoted GC cell proliferation,clone-formation,migration and invasion,and decreased the ration of G0/G1 phase;while up-regulating hsa?circ?001988 might inhibit GC cell proliferation,clone-formation,migration and invasion,induce G0/G1 phase arrested and suppress xenografted tumor formation.All above suggest that hsa?circ?001988 might play an onco-suppressor role in GC progression.3.Hsa?circ?001988 could inhibit GC cell proliferation,migration and invasion possibly through sponging miR-197-3p and promoting FBXW7 expression.Moreover,hsa?circ?001988 might regulate the downstream gene CCDC6,Cyclin D1,YAP1 and EMT-related proteins E-cadherin,Vimentin and N-cadherin.4.U2AF65 was low-expressed and played as an onco-suppressor gene in GC.The low expression of U2AF65 was intimately related to the poor prognosis of patients with GC.U2AF65 and EIF4A3,acts as RNA binding proteins,could enrich and promote the cyclizing and expression of hsa?circ?001988,thus they play vital roles in gastric cancer progression.