The Role And Mechanism Of MicroRNA-335-5p In The Cell Proliferation And Metastasis Of Pancreatic Cancer AsPC-1

Part Ⅰ Expression of MicroRNA-335-5p in pancreatic carcinoma tissue and its effect on the biological behavior of pancreatic carcinoma cells.Objective: To analyze the expression of microRNA-335-5p in pancreatic carcinoma and its relationship with clinical prognosis.To investigate the effect of microRNA-335-5p on the proliferation,migration and invasion of pancreatic carcinoma cell line As PC-1.Methods:(1)The differential expression of microRNA-335-5p in pancreatic carcinoma tissue and normal tissues and the correlation between microRNA-335-5p expression,jointly with prognosis of pancreatic patients were analyzed by public database GEO and TCGA.(2)The effects of overexpression or inhibition of microRNA-335-5p on pancreatic carcinoma cells As PC-1 were observed by CCK8 assay,Ed U cell proliferation assay,cell scratch assay,transwell invasion assay and other functional experiments.Results:(1)Analysis the data set GSE71533 in GEO and public data TCGA showed that the expression of microRNA-335-5p in pancreatic cancer tissue was markedly down-regulated compared with normal tissue(p<0.001),and the overall survival time of pancreatic cancer patients with high expression of microRNA-335-5p was significantly longer than that of patients with low expression(p<0.001).(2)Functional experiments show that microRNA-335-5p mimics can significantly inhibit the ability of pancreatic carcinoma cell As PC-1 to proliferate,migrate and invade(p<0.01);In contrast,microRNA-335-5p inhibitors can promote the malignant biological behavior of carcinoma cells(p<0.01).Conclusion:(1)Analysis of the database GEO and TCGA showed that microRNA-335-5p was significantly underexpressed in pancreatic carcinoma tissue,and the low expression of microRNA-335-5p was associated with poor prognosis of pancreatic cancer patients.(2)MicroRNA-335-5p plays an important inhibitory role in the proliferation,migration and invasion of pancreatic carcinoma cells.Part Ⅱ The mechanism of microRNA-335-5p regulating pancreatic carcinoma cell proliferation,migration and invasion through HOXD8.Objective: Find the target gene of microRNA-335-5p and verify it,as well as to reveal the potential molecular mechanism of microRNA-335-5p regulating the proliferation,migration and invasion of pancreatic carcinoma cell line As PC-1.Methods:(1)The target genes of microRNA-335-5p were predicted using bioinformatics analysis websites(Target Scan,miRDB,DIANA).(2)The targeting relationship between microRNA-335-5p and the target gene HOXD8 was verified by dual-luciferase reporter assay.(3)Western blot and q RT-PCR experiments were used to detect the effect of microRNA-335-5p expression on the protein and m RNA levels of the target gene HOXD8.(4)The effects of inhibiting HOXD8 expression on the proliferation,migration and invasion of As PC-1 were observed by CCK8 assay,Ed U cell proliferation assay,cell scratch assay,and Transwell invasion assay.(5)According to the rescue experimental strategy,the function experiments related to the proliferation,migration and invasion of As PC-1 were verified to reveal the potential molecular mechanism of microRNA-335-5p.Results:(1)According to Target Scan,miRDB,and DIANA information,40 candidate target genes were screened,among which HOXD8 had the highest comprehensive score among the three bioinformatics analysis libraries,so HOXD8 was selected for further experiments.(2)Dual-luciferase reporter assay revealed that microRNA-335-5p directly targets HOXD8(p<0.001).(3)By western blot and q RT-PCR experiments,it was found that inhibiting microRNA-335-5p could significantly increase the expression of HOXD8 protein and m RNA.(4)Functional experiments showed that knockdown of HOXD8 could significantly inhibit the proliferation,migration and invasion of As PC-1(p<0.01).(5)Rescue experiments showed that knockdown of HOXD8 attenuated the tumor-promoting effect of the microRNA-335-5p inhibitor on pancreatic carcinoma cell As PC-1(p<0.01).Conclusion:(1)HOXD8 is one of the target genes of microRNA-335-5p,and microRNA-335-5p negatively targets the expression of the regulatory gene HOXD8.(2)Inhibition of HOXD8 expression can significantly reduce the growth,proliferation,migration and invasion of pancreatic carcinoma cells As PC-1.(3)MicroRNA-335-5p inhibits the malignant biological behavior of pancreatic carcinoma cells As PC-1 by targeting HOXD8 gene expression.