| Part I Bioinformatic research on TGF-beta 1/miRNA/target gene axisTGF-beta 1 promotes the development and progression of squamous cell carcinoma of the skin.Studies have shown that TGF-beta 1 can increase the levels of miR-27a,miR-29b-1,miR-143/145,miR-149,miR-181b,miR-194,miR-196a,and miR-497 in a variety of cancer cells.In order to reveal whether TGF-beta 1 promotes the occurrence and deterioration of cutaneous squamous cell carcinoma through activation of miR-27a,miR-29b-1,miR-143/145,miR-149,miR-181b,miR-194,miR-196a,miR-497 expression,and in turn the regulation of their target genes.Bioinformatics research was first performed to determine the target TGF-beta 1/miRNA/target gene axis.All names of candidate miRNAs were first collected from different databases to eliminate the differences in the nomenclature in different databases.Target genes of the candidate miRNAs miR-27a,miR-29b-1,miR-143/145,miR-149,miR-181b,miR-194,miR-196a and miR-497 were then collected from TargetScan,DIANA MicroT and miRDB database.The top 100 target genes from each database for each individual candidate miRNA were collected and the common genes to three databases were identified and pooled as the candidate genes.A total of 99 candidate genes were identified.Except hsa-miR-181b-5p which did not get the candidate gene,18 candidate genes were identfied for miR-143,8 genes for miR-145,11 genes for miR-149,13 genes for miR-194,22 genes for miR-196a,13 genes for miR-27a,13 genes for miR-29b-1,1 gene for miR-497.DAVID database,Gene Ontology Cellular Component,Molecular Function,Biological Process were used to analyze the function of the candidate genes,and then GO,Uniprot,TSGene database were used to functionally classified candidate genes that was associated with cancer.Targetscan was used to search the base pair match between the candidate miRNA and the candidate genes.The miR-27a-3p was selected for further study.It has been reported that FBXW7 predicted to be targeted by miR-27a-3p plays a role in a variety of squamous cell carcinoma and epithelial cell carcinoma.Therefore,the TGF-beta 1/miR-27a-3p/FBXW7 axis was selected to study its biological effects on cutaneous squamous cell carcinoma.Part II Identification and verification of TGF-beta 1/miR-27a-3p/FBXW7 axis in human cutaneous squamous cell carcinoma A431 cellsIn order to verify and identify the presence of TGF-beta 1/miR-27a-3p/FBXW7 axis in human cutaneous squamous cell carcinoma A431 cells,the effect of TGF-beta 1 on the proliferation of A431 cells and siRNA expression changes were first determined.TGF-beta 1 siRNA and its negative control control-siRNA were transfected into A431 cells and the cell growth was determined by observation using microscopy.The results showed that TGF-beta 1 siRNA inhibited the growth of A431 cells,compared with the control group,which was consistent with that reported in the literature.The levels of candidate miRNAs in TGF-beta 1 siRNA transfected A431 cells were determined using real time quantitative PCR(QPCR).First of all,the detection system of PCR instrument is verified by the standard curve.The dissociation curves in QPCR showed only one peak in each reaction,indicating that the primers were specific,and the PCR amplification reaction was also specific.The results showed that the copy number of hsa-miR-27a,hsa-miR-29b-1,hsa-miR-143 miRNA,hsa-miR-145,hsa-miR-149,hsa-miR-181b,hsa-miR-194,hsa-miR-196a,hsa-miR-497 in TGF-beta 1 siRNA transfected A431 cells was lower than that in the control group,indicating that TGF-beta 1 have the same direction of changes as that of hsa-miR-27a,hsa-miR-29b-1,hsa-miR-143,hsa-miR-145,hsa-miR-149,hsa-miR-181b,hsa-miR-194,hsa-miR-196a,hsa-miR-497 in human cutaneous squamous cell carcinoma A431 cells.The pMIR-Glo-WP-normal,pMIR-Glo-WP-mutation plasmids containing 3 'UTR sequence of FBXW7 gene were constructed and verified by sequencing.They were used to confirm the relationship between miR-27a-3p and FBXW7 gene using the dual luciferase reporter assay in A431 cells.The normal 3 'UTR of FBXW7 gene,the internal control vector plasmid,miR-NC/27a-3p plasmid,the mutation group and and its internal control vector were transfected into A431 cells,the multi-microplate assay of firefly luciferin and Renilla luciferase expression was performed.The results showed that miR-27a-3p could regulate the expression of FBXW7 3 'UTR' luciferase(p<0.01).After the binding site was mutated,the regulatory relationship disappeared.The suggested that miR-27a-3p can regulate the expression of luciferase through the the specific site.MiR-27a-3p mimic was able to specifically bind to the 3 'UTR region of the FBXW7 gene,which inhibited the expression of luciferase.This was consistent with the expected results based on the bioinformatic research based on miRBase and Targetscan databases.Therefore,the presence of TGF-beta 1/miR-27a-3p/FBXW7 axis in human cutaneous squamous cell carcinoma A431 cells was identified and verified.Part III The role of TGF-beta 1/miR-27a-3p/FBXW7 axis in cell proliferation,cell cycle and invasion of the human cutaneous squamous cell carcinoma A431 cellsIn order to study the role of TGF-beta 1/miR-27a-3p/FBXW7 axis in the proliferation,cell cycle and invasion of the human cutaneous squamous cell carcinoma A431 cells,we constructed a recombinant vector containing pcDNA3.1-FBXW7 overexpression and SD1211-si-FBXW7 interference plasmids.The effects of miR-27a-3p and FBXW7 expression on the proliferation of human cutaneous squamous cell carcinoma A431 cells were further studied using these plasmids.MiR-27a-3p mimic,miR-27a-3p inhibitor,pcDNA3.1-FBXW7,SD1211-si-FBXW7 plasmids were transfected into human cutaneous squamous cell carcinoma A431 cells and determined using microscopy and CCK-8 assay.The results consistently showed that miR-27a-3p mimic enhanced proliferation of A431 cells,miR-27a-3p inhibitor suppressed proliferation of A431 cells,pcDNA3.1-FBXW7 suppressed proliferation of A431 cells,and SD1211-si-FBXW7 enhanced proliferation of A431 cells.The effects of these plasmids were enhanced with prolonged time.The results suggested that miR-27a-3p promoted the proliferation of A431 cells,and FBXW7 suppressed proliferation of A431 cells.In order to study the miR-27a-3p and FBXW7 expression on human cutaneous squamous cell carcinoma A431 cell cycle,miR-27a-3pmimic,miR-27a-3p inhibitor,pcDNA3.1-FBXW7,SD1211-si-FBXW7 plasmids were transfected into human cutaneous squamous cell carcinoma A431 cells and the cell cycles were determined by flow cytometry.The results showed that the proportion of cells in G0/G1 phase in the miR-27a-3p-mimic transfection group was decreased,that in M/G2 phase did not change,that in S phase was increased.The proportion of cells in G0/G1 phase in the miR-27a-3p_inhibitor transfection group was decreased,that in M/G2 phase was increased,that in S phase was decreased.The proportion of cells in G0/G1 phase in the pcDNA3.1-FBXW7 transfection group did not change,that in M/G2 phase was dramatically increased,that in S phase was decreased.The proportion of cells in G0/G1 phase in the SD1211-si-FBXW7 transfection group was decreased,that in M/G2 phase did not change,that in S phase was increased.All transfection groups were compared with untransfected groups.The results suggested that miR-27a-3p promoted transition of A431 cells from G0/G1 phase into S phase cells and arrest in M/G2 phase.FBXW7 promoted transition of A431 cells from S phase to M/G2 phase and arrest in M/G2 phase.In order to study the effects of miR-27a-3p and FBXW7 expression on invasion of human cutaneous squamous cell carcinoma A431 cells,miR-27a-3pmimic,miR-27a-3p inhibitor,pcDNA3.1-FBXW7,SD1211-si-FBXW7 plasmids were transfected into human cutaneous squamous cell carcinoma A431 cells and the cell invasion was determined by Transwell invasion assay.The results showed that the invasion ability of the miR-27a-3p-mimic group was increased,that of the the miR-27a-3p-inhibitor transfection group was decreased,that of the pcDNA3.1-FBXW7 transfection group was decreased,and that of the SD1211-si-FBXW7 transfection group was increased.The results suggested that miR-27a-3p promoted the invasion ability of A431 cells,while FBXW7 inhibited the invasion ability of A431 cells. |
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