| Objective:1.To explore the expression of hsa?circ?0000478 in human colon cancer and its function in proliferation,invasion,migration and cells cycle;2.To analyze the downstream genes and proteins that may have binding sites with hsa?circ?0000478,and to verify their relationship with experiments.3.To explore the expression of mir-624-3p and ZEB1 in human colon cancer.Methods:1.To explore the expression of circ RNA in colon cancer cells and clarify the research object,we selected 4 pairs of colon cancer and adjacent tissues for high-throughput sequencing.The circ RNAs with different expression level were analyzed,and the six groups of circ RNAs with the most obvious expression differences were selected.The chromosomal locations and regulated mi RNAs of these 6 circ RNAs were analyzed by bioinformatics software Targetscan.64 cases of colon cancer tissues and their adjacent tissues were collected,and q RT-PCR was used to verify the expression of 6 circrnas in colon cancer tissues and adjacent tissues.2.The expression levels of hsa?circ?0000478,mi R-624-3p,and ZEB1 in colon cancer cells and normal colon epithelial cells were verified by q RT-PCR.3.To confirm that hsa?circ?0000478 binds to mi R-624-3p and mi R-624-3p binds to ZEB1,we cloned the full-length sequence of hsa?circ?0000478,the ZEB1-3'UTR plasmid and mi R-624-3p mimics and co-transfect to LOVO cells.Then we tested luciferase activity.The results proved that hsa?circ?0000478 had a binding site to mi R-624-3p and mi R-624-3p had a binding site to ZEB1.4.We constructed si RNA sequence to inhibit the expression of hsa?circ?0000478 and set the cell blank control group(control)and negative control group(NC,containing empty virus,empty plasmid,negative control sequence).MTT and transwell test were used to detect the effect of inhibiting the expression of hsa?circ?0000478 on the proliferation,invasion and migration of colon cancer cell LOVO.5.The hsa?circ?0000478 overexpression vector was constructed and transfected into LOVO cells.The effects of target gene overexpression on colon cancer cell proliferation,invasion,and migration were detected by MTT and Transwell methods.6.Annexin V-APC / PI dual staining flow cytometry was used to detect the effects of inhibition and overexpression of hsa?circ?0000478 on colon cancer cell apoptosis,and PI single staining was used to detect the effects of inhibition and overexpression of hsa?circ?0000478 on cell cycle.Results:1.High-throughput sequencing of 4 samples revealed differences in 1817 circ RNAs,of which1236 were up-regulated and 581 were down-regulated.High-throughput sequencing of 4 samples revealed differences in 1817 circ RNAs,of which 1236 were up-regulated and 581 were down-regulated.Hsa?circ?000047,hsa?circ?0000780,hsa?circ?0014879,hsa?circ?0001615,hsa?circ?0010575,and hsa?circ?401801 have the most significant differences in expression.Targetscan software analyzed the chromosomal positions of these circ RNAs,and found that most of the circular RNAs were located in the gene exons.The results of q RT-PCR on 64 cases colon cancer tissues and adjacent tissues showed that the expression levels of hsa?circ?0000478,hsa?circ?0014879,hsa?circ?0001615,and hsa?circ?0007840 in the experimental group were higher than those in the control group.The results of q RT-PCR experiments on 100 samples showed that the average expression levels of hsa?circ?00070478,hsa?circ?0014879,hsa?circ?0001615,and hsa?circ?0007840 in cancer tissues were much higher than those in adjacent tissues,and the difference was statistically significant(P <0.05).The expression of hsa?circ?0010575 and hsa?circ?401801 in the two tissues was not significantly different(P> 0.05).The average expression of hsa?circ?0000478 in cancer tissues was the most significant compared with the control group.The software further analyzed the downstream mi RNA corresponding to circ RNA,and found that hsa?circ?0000478has a binding effect with mi R-624-3p,and mi R-624-3p also has a target binding site for ZEB1.ZEB1 is an important regulator of EMT.Therefore,this study selected the hsa?circ?0000478 /mi R-624-3p / ZEB1 pathway as the next research object.2.q RT-PCR results showed that the expression of hsa?circ?0000478 and ZEB1 in colon cancer cells were higher than normal colonic epithelial cells FHC,while the expression of mi R-624-3p decreased.3.The luciferase experiment results showed that after co-transfection of hsa?circ?0000478full-length sequence plasmid and mi R-624-3p into LOVO cells,the intracellular luciferase activity was decreased than the control group,and the difference was statistically significant,P<0.05.4.The results of MTT and Transwell experiments showed that inhibiting the expression of hsa?circ?0000478 could inhibit the proliferation,invasion and migration of colon cancer cell LOVO.5.The hsa?circ?0000478 over-expression vector was constructed.The results of the vector verification showed that the expression efficiency of hsa?circ?0000478 in the transfection group was 138 times that of the control group(P <0.05).6.After the vector of over expressing hsa?circ?0000478 was introduced into LOVO cells,the proliferation,invasion and migration of colon cancer cell were promoted7.Flow cytometry experiments showed that interferencing the expression of hsa?circ?0000478can promote the apoptosis of colon cancer cells and cause G1 phase arrest of the cell cycle.And hsa?circ?0000478 over-expression goroup had no obvious effect on cell cycle.Conclusion:1.Hsa?circ?0000478 is highly expressed in colon cancer cells.Inhibiting the expression of this gene can inhibit the proliferation,invasion and migration of colon cancer cells.In addition,the cell cycle of colon cancer cells were blocked in the G1 phase and apoptosis was promoted.2.Overexpression of hsa?circ?0000478 can promote proliferation,invasion,and migration of colon cancer cells.3.The binding site of hsa?circ?0000478 to mi R-624-3p,mi R-624-3p to ZEB1 were confirmed.This result indicates that hsa?circ?0000478 may have an effect on colon cancer cell proliferation,invasion,and migration through the hsa?circ?0000478 / mi R-624-3p / ZEB1 pathway. |
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