Study On The Regulatory Mechanism Of Hsa＿circ＿0017639 To Gastric Cancer Proliferation And Metastasis By Targeting MiR-224-5p/USP3 Axis

Part Ⅰ Study on the hsa＿circ＿0017639 expression and its effects on proliferation and metastasis of gastric cancerObjective:To study the hsa＿circ＿0017639 expression and its effects on proliferation and metastasis of gastric cancer.Methods:1.Expression of hsa＿circ＿0017639 expression was detected in human GC cell lines(MGC803,MKN-28,SGC7901,BGC823)and normal human gastric epithelial cells GES-1 using real-time quantitative polymerase chain reaction(RT-qPCR).2.Subcellular localization of hsa＿circ＿0417639 in human GC tissue was verified via fluorescence in situ hybridization(FISH).3.Genomic loci of hsa＿circ＿0017639 was analyzed by bioinformatics analysis.4.Hsa＿circ＿0017639 expression by RT-qPCR,cell proliferation by CCK-8 assay and colony formation,and cell migration by transwell assays were detected and assessed in MGC803 and MKN-28 cells after transfection with a hsa＿circ＿0017639 silencing vector(si-circ0017639)or negative control(NC).Subcutaneous xenografts from indicated MKN-28 cell clones after transfection with si-circ0017639 or NC were excised from nude mice.Results:1.RT-qPCR results showed that the expression of hsa＿circ＿0017639 in four gastric cancer cell lines was significantly higher than that in GES-1,and the difference was statistically significant(P<0.001).The highest expression of hsa＿circ＿0017639 was found in MGC803 and MKN-28 cells.2.FISH assays demonstrated that hsa＿circ＿0017639 was mainly localized in the cytoplasm of gastric cancer tissue cells.3.Bioinformatics analysis suggested that hsa＿circ＿0017639 was formed from exons of the SFMBT2 gene,and the mature hsa＿circ＿0017639 was 536-bp splice length.Hsa＿circ＿0017639 was located at chr10:7290509-7327916.4.Results of RT-qPCR validated that hsa＿circ＿0017639 expression decreased significantly in both MKN-28 and MGC803 cells after hsa＿circ＿0017639 downregulation compared with NC group(P<0.001).CCK8 and clone formation assays showed that downregulation of hsa＿circ＿0017639 suppressed cell proliferation in both MKN-28 and MGC803 cells(P<0.001).Transwell migration assays illustrated that the MKN-28 and MGC803 cell migratory capacity diminished after hsa＿circ＿0017639 being silenced(P<0.001).Hsa＿circ＿0017639 downregulating lentiviral MKN-28 cells were used to form tumors as nude mice xenografts.Hsa＿circ＿0017639 downregulation suppressed tumor growth(P<0.001).Conclusion:The expression of hsa＿circ＿0017639 was significantly increased in gastric cancer cell lines,and the highest expression was found in MGC803 and MKN-28 cells.Then MGC803 and MKN-28 cells were selected for additional study.Hsa＿circ＿0017639 was predominantly localized to the cytoplasm.Bioinformatics analysis suggested that hsa＿circ＿0017639 was assembled with exons from the SFMBT2 gene with a 536-bp splice length.Hsa＿circ＿0017639 was located at chr10:7290509-7327916.Downregulation of hsa＿circ＿0017639 decreased proliferation and migration of both GC cells in vitro and tumor growth in vivo experiments.Part Ⅱ Hsa＿circ＿0017639 targeting miR-224-5p/USP3 in gastric cancerObjective:Explore the downstream targets of hsa＿circ＿0017639,and to clarify the targeting relationship between hsa＿circ＿0017639 and its downstream miR-224-5p and ubiquitin-specific protease 3(USP3).Methods:1.Bioinformatics analysis could predict miRNAs that might bind to hsa＿circ＿0017639.And the downstream target genes of miRNA were predicted.2.The miRNAs binding to hsa＿circ＿0017639 in MKN-28 cells were analyzed by RNA pull down assays.3.The luciferase report assay verified the signal molecules of miRNA-related pathway bound to hsa＿circ＿0017639.Wild-type hsa＿circ＿0017639(hsa＿circ＿0017639-WT)or mutant hsa＿circ＿0017639(hsa＿circ＿0017639-Mut)luciferase reporter vectors were constructed and transfected into HEK293T with miR-224-5p mimic or NC respectively.Luciferase reporter assay was used to detect whether miR-224-5p was the target of hsa＿circ＿0017639.And USP3-3’UTR wild-type(USP3 3’-UTR-WT)or USP3-3’UTR mutant(USP3 3’-UTR-Mut)luciferase reporter vectors were constructed and transfected into HEK293T with miR-224-5p mimic or NC respectively.Luciferase report assay was used to detect whether USP3 was the target of miR-224-5p.Results:1.Bioinformatics analysis predicted that there was correlation between hsa＿circ＿001763 and miR-224-5p,and that miR-224-5p could target USP3.2.RNA pull down analysis showed that miR-224-5p was the only miRNA that was abundantly pulled down by a hsa＿circ＿0017639 probe from MKN-28 cells,and miR-224-5p was absorbed by hsa＿circ＿0017639.3.Luciferase report assay showed that hsa＿circ＿0017639 inhibited luciferase activity in hsa＿circ＿0017639-WT cells,and the difference was statistically significant(P<0.01),but did not affect activity in hsa＿circ＿0017639-Mut cells,which indicated that hsa＿circ＿0017639 could target miR-224-5p.Luciferase report assay also showed that miR-224-5p inhibited luciferase activity in USP3 3’-UTR-WT cells,and the difference was statistically significant(P<0.01),but was not inhibited in USP33’-UTR-Mut cells,which suggested that miR-224-5p could target USP3.Conclusion:Bioinformatics analysis and experimental verification of circRNA and miRNA interaction suggested that hsa＿circ＿0017639 acted as the miRNA sponge of miR-224-5p,and USP3 was the downstream target gene of miR-224-5p.All these results suggested that there was a targeted signaling pathway between hsa＿circ＿0017639 and miR-224-5p/USP3 in gastric cancer.Part Ⅲ The role of hsa＿circ＿0017639 targeting miR-224-5p/USP3 axis to gastric cancer proliferation and metastasisObjective:The function loss or gain experiments were used to analyze the signal pathways related hsa＿circ＿0017639 and miR-224-5p/USP3,and the possible role of hsa＿circ＿0017639 targeting miR-224-5p/USP3 signal axis to gastric cancer proliferation and metastasis was preliminarily studied.Methods:1.MGC803 and MKN-28 cells were transfected with NC,si-circ0017639,si-circ0017639+miR-224-5p inhibitor,or si-circ0017639+USP3 overexpression vector(USP3),respectively.The changes in expression of hsa＿circ＿0017639,miR-224-5p and USP3 were analyzed by RT-qPCR,cell proliferation was detected by plate cell clone formation assay,and cell migration was assessed by transwell assays.2.MGC803 and MKN-28 cells were transfected with NC,miR-224-5p mimic,or miR-224-5p mimic+USP3 overexpression vector(USP3),respectively.RT-qPCR was used to analyze the expression changes of miR-224-5p and USP3,cell proliferation changes were detected by plate cell clone formation assay,and cell migration changes were assessed by transwell assays.Results:1.RT-qPCR results showed that the downregulation of hsa＿circ＿0017639 in MGC803 and MKN-28 cells after si-circ0017639 transfection significantly inhibited the expression of hsa＿circ＿0017639,but promoted the expression of miR-224-5p,while decreased the expression of USP3.And RT-qPCR analysis showed that miR-224-5p inhibitor transfection of MGC803 and MKN-28 cells significantly inhibited miR-224-5p expression,but had no effect on expression of hsa＿circ＿0017639.RT-qPCR analysis showed that the USP3 overexpression vector transfection of MGC803 and MKN-28 cells had no effect on the expression of hsa＿circ＿0017639 and miR-224-5p.RT-qPCR analysis showed that the expression of USP3 was significantly increased after the transfection of MGC803 and MKN-28 cells with miR-224-5p inhibitor or USP3 overexpressing vector,which suggested that hsa＿circ＿0017639 could regulate the expression of USP3 by targeting miR-224-5p.Cell proliferation and migration of MGC803 and MKN-28 cells were significantly inhibited by downregulation of hsa＿circ＿0017639 after si-circ0017639 transfaction,according to clone formation assay and transwell assay.Clone formation assay and cell migration assay showed that the proliferation and migration of MGC803 and MKN-28 cells could be restored through downregulation of miR-224-5p after miR-224-5p inhibitor transfection or by overexpression of USP3 after USP3 overexpression vector transfection of MGC803 and MKN-28 cells.2.RT-qPCR analysis showed that after MGC803 and MKN-28 cells were transfected with miR-224-5p mimic and overexpressed,the expression of miR-224-5p was increased and the expression of USP3 was decreased.And RT-qPCR analysis showed that MGC803 and MKN-28 cells overexpressed USP3 after transfection with USP3 overexpressing vector,miR-224-5p expression did not recover,but USP3 expression significantly increased.Cell proliferation and migration of MGC803 and MKN-28 cells after transfection with miR-224-5p mimic were reduced by clone formation assay and transwell assay.And clone formation assay and transwell assay of cell migration showed that overexpression of USP3 in MGC803 and MKN-28 cells could reverse miR-224-5p induced cell proliferation and migration inhibition,while the overexpression of USP3 in MGC803 and MKN-28 cells promoted the proliferation and migration of gastric cancer cells.Conclusion:All these functional experiments results suggested that miR-224-5p and USP3 were downstream of hsa＿circ＿0017639,and USP3 was downstream target gene of miR-224-5p.In summary,hsa＿circ＿0017639 regulated gastric cancer proliferation and metastasis by targeting the miR-224-5p/USP3 signaling axis.