Research On The Mechanism Of MiR-514a-3p Influencing The Biological Behaviors By Targeting EGFR In Clear Cell Renal Cell Carcinoma

Objective: Explore the differential expression profiling of mi RNAs between clear cell renal cell carcinoma(cc RCC)tissues and paired normal tissues in online mi RNA microarray database and analyze the correlation between the expression difference of mi R-514a-3p in the cc RCC tissues and clinicopathological parameters of patients.Methods: According to the chip data in the online mi RNA chip database ym500v2,the differences of mi RNA expression profiles between 496 cases of primary cc RCC tissues and 71 cases of normal renal tissues were analyzed,and fold change>3 and p<0.05 were set as significant expression differences.The expression of mir-514a-3p in 20 pairs of clinical specimens of cc RCC and four types of renal cell carcinoma(RCC)cell lines were detected by real-time q RT-PCR.Independent sample t test was used to reveal the relationship between the expression of mir-514a-3p and the clinicopathological parameters of cc RCC.Results: A total of 54 mi RNAs were found to be differentially expressed in cc RCC tissues and adjacent normal tissues by mi RNA profiling analysis,27 of which were significantly up-regulated in cc RCC tissues,and 27 of which were significantly down-regulated.The q RT-PCR results showed that the expression of mi R-514a-3p was significantly down-regulated in cc RCC tissues or RCC cell lines compared to normal tissue or normal renal tubular epithelial cell(all p<0.01).The t test analysis showed that there was no significant correlation between mi R-514a-3p expression and clinicopathological parameters in cc RCC tissue(all p>0.05).Conclusion: The mi RNA expression profiles of cc RCC tissues and normal adjacent tissues were significantly different,among of which mi R-514a-3p was most significantly down-regulated,and the result was verified in RCC cell lines.However,no significant correlation was found between expression changes of mi R-514a-3p and clinicopathological parameters in cc RCC tissue.Therefore,mi R-514a-3p may play the role of tumor suppressor gene in cc RCC.Objective: To confirm the targeted regulatory action and mechanism of mi R-514a-3p on target genes in cc RCC.Methods: The potential target genes of mi R-514a-3p were selected by using the online mi RNA target gene prediction software.The expression level of target gene m RNA was detected by real-time q RT-PCR and the correlation analysis was used to reveal the correlation between mi R-514a-3p and target gene expression.The chemically synthesized mi R-514a-3p mimic and inhibitor were transfected into RCC cell lines respectively.Real-time q RT-PCR and Western blot were used to detect the expression of m RNA and protein in target gene.A dual luciferase reporter plasmid vector containing the wild or mutant type(Wt or Mut)target gene 3?-UTR was constructed and the regulatory mode of mi R-514a-3p on target gene was explored by dual-luciferase assay.Results: EGFR was screened as a potential target gene for mir-514a-3p in online mi RNA target gene prediction software.Real-time q RT-PCR results showed that the expression of EGFR m RNA was significantly higher in cc RCC than in normal adjacent tissues(p<0.0001),and correlation analysis revealed that EGFR expression was negatively correlated with mi R-514a-3p(p<0.05).Compared with transfected mimic NC or inhibitor NC,there was no significant change in EGFR m RNA in renal cell carcinoma cell lines after transfection with mi R-514a-3p mimic or inhibitor(all p>0.05),but the EGFR protein was significantly down-regulated after transfected with mi R-514a-3p mimic(all p<0.05).Conversely,the expression of EGFR protein was significantly up-regulated after transfection with mi R-514a-3p inhibitor in RCC cell lines(all p<0.05).Dual luciferase reporter assays showed that after transfection with mi R-514a-3p mimic,the luciferase activity of Wt EGFR 3?-UTR in RCC cells was significantly lower than that of the control group(all p<0.01),while the luciferase activity of the Mut EGFR 3?-UTR had no significant change(all p>0.05).Conversely,the luciferase activity of Wt EGFR 3?-UTR in RCC cells after transfection with mi R-514a-3p inhibitor was significantly higher than that of the control group(all p<0.05),but there was no significant change in the Mut EGFR 3?-UTR(all p>0.05).Conclusion: The expression of EGFR in cc RCC was significantly higher than that of the normal adjacent tissues,and it was negatively correlated with the expression of mi R-514a-3p.Moreover,mi R-514a-3p directly inhibits the translation of EGFR at the post-transcriptional level,rather than causes it to cleave.Objective: To investigate the effects of mi R-514a-3p and EGFR on the biological behavior of RCC cells and its mechanism.Methods: The chemically synthesized small molecule inhibitor si-EGFR was transfected into RCC cells,and the effects of EGFR on the ability of proliferation,invasion and migration of RCC cells were detected by the experiments of colony formation,invasion and migration respectively.Similarly,the chemically synthesized mi R-514a-3p mimic and mi R-514a-3p inhibitor were transfected into RCC cells,and the change of viability,proliferation,invasion,and migration ability of RCC cells were observed using MTS assay,colony formation assay,and invasion and migration assay.To establish a cc RCC model of nude mouse and periodically inject chemically synthesized mi R-514a-3p agomir or control into the tumor to observe changes in tumor growth.Western blot was used to detect the changes in the molecular expression of key signaling pathways in the EGFR downstream.Results: Compared with the control group,the ability of proliferation,invasion,and migration of RCC cells were significantly reduced after transfection with si-EGFR(all p<0.05).After transfection with mi R-514a-3p inhibitor,the viability,proliferative capacity,invasion and migration ability of RCC cells were significantly higher than those of the control group(all p<0.05).Conversely,the viability,proliferation,invasion and migration ability were significantly decreased after transfection with mi R-514a-3p mimic in RCC cells(all p<0.05).In vivo experiments showed that the volume and weight of subcutaneous xenografts in nude mice injected with mi R-514a-3p agomir were significantly lower than those in the control group(p<0.05).Western blot results revealed that the expression of AKT,p-AKT,and ERK 1/2 in the mimic or inhibitor group was not significantly different from that in the control group after transfection with mi R-514a-3p mimic or inhibitor in RCC cells,but there was a significant difference in the expression of p-ERK1/2 between the groups.The expression of p-ERK 1/2 in RCC cells after transfection with mi R-514a-3p mimic was significantly down-regulated compared to the control group.Conversely,the expression of p-ERK 1/2 in RCC cells after transfection with inhibitor was significantly higher than that in the control group.Conclusion: EGFR can promote the proliferation,invasion and migration of RCC cells,while mi R-514a-3p can inhibit the viability,proliferation,invasion and migration through EGFR.Moreover,mi R-514a-3p inhibits the growth of subcutaneous xenografts in nude mice.From the perspective of molecular mechanism,mi R-514a-3p functions as a tumor suppressor through the EGFR/MAPK/ERK pathway but not the EGFR/PI3K/AKT pathway.