| Part 1 Correlation analysis between mi R-122-5p and CLIC1 and gastric cancerObjective: To investigate the expression and clinical significance of mi R-122-5p and CLIC1 in gastric cancer cells and gastric cancer tissues.Methods: Real-time fluorescence quantitative PCR(q RT-PCR)and western blot were used to detect gastric cancer cell lines(AGS,MGC-803,BGC-823,MKN-45,MKN-28,SGC-7901 and HGC-27)and the expression levels of mi R-122-5p and CLIC1 in gastric cancer tissues.Immunohistochemistry(IHC)experiment was used to detect the expression level of CLIC1 in gastric cancer tissues.To analyze the correlation between mi R-122-5p,CLIC1 and clinicopathological parameters of gastric cancer.Results: 1.Compared with normal gastric mucosal cells GES-1,q RT-PCR results showed that mi R-122-5p expression level in gastric cancer cell lines were significantly reduced(P<0.05),q RT-PCR and western blot results It showed that the expression level of CLIC1 was significantly increased(P<0.05);compared with adjacent normal tissues,q RT-PCR results showed that the expression level of mi R-122-5p in gastric cancer patients was significantly reduced(P <0.05);The results of PCR and IHC showed that the expression level of CLIC1 in gastric cancer patients was significantly increased(P <0.05).2.The expression level of mi R-122-5p was not correlation with age,sex,tumor size,and lymph node metastasis(P> 0.05).It was significantly related to histological typing and TNM pathological staging(P<0.05);the expression level of CLIC1 was not correlation with age,sex,tumor size and histological typing(P>0.05).However,the expression level of CLIC1 was significantly related to lymph node metastasis and TNM pathological stage of gastric cancer(P<0.05).Conclusions: The expression level of mi R-122-5p was significantly down-regulated in gastric cancer cell lines and tissues,which was significantly related to the histological classification and TNM stage of gastric cancer patients;CLIC1 was significantly up-regulated in gastric cancer cell lines and tissues,and was associated with lymph node metastasis and TNM pathology stages.Part 2 mi R-122-5p regulates the expression of CLIC1 in gastric cancer cellsObjective: Explore the mechanism of mi R-122-5p regulating CLIC1 in gastric cancer cells.Methods: Luciferase reporter plasmids containing CLIC1 3’-UTR wild-type(Wt)plasmid and mutant(Mut)plasmid were constructed,and co-transfected with mi R-122-5p mimics or negative control in MGC-803 and AGS gastric cancer cells.Then,the fluorescence values in each group were determined.q RT-PCR and western blot were used to detect the expression level of CLIC1 after transfection with mi R-122-5p mimics and inhibitor.Results: 1.The results of the dual luciferase reporter gene assay showed that the fluorescence values of mi R-122-5p mimics + CLIC1-Wt group were significantly reduced compared to the co-transfected mi R-NC + CLIC1-Wt group(P<0.05);Fluorescence values of mi R-122-5p mimics + CLIC1-Mut group was not change significantly compared with co-transfected mi R-NC +CLIC1-Mut group(P>0.05).2.The results of q RT-PCR and western blot showed that after transfection of mi R-122-5p mimics,the expression level of CLIC1 was significantly decreased(P<0.05);after transfection of mi R-122-5p inhibitor,the expression level of CLIC1 was significantly increased(P<0.05).Conclusions: mi R-122-5p can target and regulate CLIC1.CLIC1 may be one of the target genes of mi R-122-5p.Part 3 mi R-122-5p mediates the effect of CLIC1 on the migration and invasion of gastric cancer cellsObjective: To investigate the effect of mi R-122-5p targeting CLIC1 on the migration and invasion of gastric cancer cells in vitro.Methods: mi R-122-5p mimics,inhibitor,si-CLIC1,OE-CLIC1 were transfected into gastric cancer cells MGC-803 and AGS;mimics + OE-CLIC1,inhibitor + si-CLIC1 groups were co-transfected.The wound healing assay and Transwell assay were used to detect the migration and invasion ability of mi R-122-5p and CLIC1 in gastric cancer cells.Results: 1.The results of the wound healing showed that mi R-122-5p mimics could significantly inhibit the migration ability of gastric cancer cells compared with the control group,and mi R-122-5p inhibitor could promote the migration ability of gastric cancer cells(P<0.05);si-CLIC1 could significantly inhibit the migration ability of gastric cancer cells,OE-CLIC1 could promote the migration ability of gastric cancer cells(P<0.05).2.Transwell results show that compared with the control group,mi R-122-5p mimics could significantly reduce the number of gastric cancer cells through the membrane,inhibitor could increase the number of gastric cancer cells through the membrane(P<0.05);si-CLIC1 could significantly reduce the number of gastric cancer cells;OE-CLIC1 could increase the number of gastric cancer cells(P<0.05).3.The results of the wound healing showed that compared with the mimics group,co-transfection of mimics + OE-CLIC1 could promote the migration ability of gastric cancer cells(P<0.05).Compared with the inhibitor group,co-transfected inhibitor + si-CLIC1 could inhibit the migration ability of gastric cancer cells(P<0.05).4.Transwell results show that,compared with the mimics group,co-transfection of mimics + OE-CLIC1 could significantly increase the number of gastric cancer cell(P<0.05).Compared with the inhibitor group,co-transfection of inhibitor + si-CLIC1 could significantly reduce the number of gastric cancer(P<0.05).Conclusions: mi R-122-5p can target CLIC1 to regulate the migration and invasion ability of gastric cancer cells.Part 4 Mechanism of mi R-122-5p mediating CLIC1’s effect on gastric cancer cell migration and invasionObjective: To explore the mechanism of mi R-122-5p mediated CLIC1 affecting gastric cancer cell migration and invasion.Methods: Western blot was used to detect the expression levels of CLIC1,E-cadherin,Snail and β-catenin,MMP9,ERK,p-ERK,p38,p-p38 protein in each group.Results: 1.Compared with the control group,after transfection with mi R-122-5p mimics,the expression of CLIC1,Snail,β-catenin,MMP9,p-ERK and p-p38 were significantly reduced(P<0.05),E-cadherin expression was significantly up-regulated;ERK and p38 had no significant changes(P> 0.05);CLIC1,Snail,β-catenin,MMP9,p-ERK and p-p38 expression were significantly up-regulated and E-cadherin expression was significantly down-regulated after transfection with mi R-122-5p inhibitor(P<0.05);ERK and p38 had no significant changes(P>0.05).2.Compared with the control group,the expressions of CLIC1,Snail,β-catenin,MMP9,p-ERK and p-p38 protein were significantly reduced,and the expression of E-cadherin was significantly up-regulated after transfection with si-CLIC1(P<0.05);There were no significant changes in ERK and p38(P>0.05);Compared with the control group,the expressions of CLIC1,Snail,β-catenin,MMP9,p-ERK and p-p38 protein were significantly up-regulated,and E-cadherin protein expression was significantly down-regulated after transfection with OE-CLIC1(P<0.05).There were no significant changes in ERK and p38(P>0.05).3.Compared with mimics group,the expressions of CLIC1,Snail,β-catenin,MMP9,p-ERK and p-p38 were significantly increased,and the expression of E-cadherin protein was significantly reduced after co-transfection of mimics +OE-CLIC1(P<0.05).But p-p38 was not change significantly in MGC-803 cells(P>0.05)and β-catenin was not change significantly in AGS cells(P>0.05).ERK and p38 were not change significantly(P>0.05);4.Compared with the inhibitor group,the expression of CLIC1,Snail,β-catenin,MMP9,p-ERK and p-p38 protein was significantly down-regulated,and the expression of E-cadherin protein was significantly up-regulated after co-transfection of inhibitor + si-CLIC1(P<0.05),but MMP9 had no significant change in MGC-803 cells(P>0.05);ERK and p38 had no significant change(P>0.05).Conclusions: mi R-122-5p could mediate CLIC1 to regulate ERK/MAPK pathway,and induce EMT change in gastric cancer cells.Part 5 Based on high-throughput sequencing to study the potential biological role of CLIC1 downstream genes in gastric cancerObjective: To explore the downstream target genes of CLIC1 and their potential biological effects.Methods: High-throughput sequencing technology was used to detect m RNA in CLIC1 silence group,CLIC1 overexpression group and control group.Differentially expressed genes were filtered for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.Results: In CLIC1 silence group,583 differentially expressed genes(340down-regulated and 243 up-regulated genes)were selected;in CLIC1 overexpression group,320 differentially expressed genes(154 down-regulated and 166 up-regulated genes)were screened.The Molecular Function of GO was mainly enriched in extracellular matrix structural constituent and integral binding,the Cellular Component is mainly enriched in extracellular matrix and extracellular region,and the Biological Process was mainly enriched in cell migration and regulation of cell migration.KEGG analysis was mainly enriched in MAPK,PI3K-Akt signaling pathway and Focal adhesion signaling pathways.Conclusions: CLIC1 may affect the migration and invasion of gastric cancer cells through MAPK and PI3K-Akt signaling pathway. |
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