| Background:Bladder cancer(BC)is one of the most common malignant tumors of the urinary system,and its incidence has gradually increased in recent years.Although about 75%of the newly diagnosed patients are non-muscle invasive urothelial carcinoma,due to its high recurrence rate,and with the recurrence of bladder cancer,the degree of malignancy gradually progresses.At present,the comprehensive treatment of bladder cancer has achieved a certain effect,but the prognosis of patients with invasive,high grade and late stage bladder cancer is still poor,and the cancer-specific mortality is still high.At present,it is considered that the study of tumor-related genes is very important for the diagnosis and treatment of tumors.Most tumors have been clinically targeted drugs,but there is still no very suitable targeted drugs for bladder cancer.Therefore,the research on gene targets of bladder cancer needs to be strengthened urgently.In the current research on malignant tumors,the occurrence of tumors is considered to be closely related to the abnormal expression of a variety of genes.Tumor suppressor genes and tumor promoting genes play an important role in the development of tumors,and the research on tumor-related genes has gradually become the frontier of the field of tumor treatment.Micro RNA is a kind of short non-coding RNA molecules with a length of about 22 nucleotides,which can complement and bind to the 3’ untranslated region of messenger RNA,regulate gene expression by targeting the translation and degradation of m RNA,and play the role of tumor suppressor or oncogene.Studies have shown that miR-485-5p is involved in the proliferation,apoptosis,migration,and invasion of a variety of tumor cells,and is closely related to the occurrence and development of tumors.It has been confirmed that Mir-485-5p is low expressed in tumors and plays a role of tumor suppressor gene.However,the expression of miR-485-5p is increased in the serum of Kaposi’s sarcoma patients and Kaposi’s sarcoma-associated herpesvirus-infected cells,which promotes cell proliferation and migration.It can be seen that the expression of miR-485-5p is different in different tumors and plays different roles.At present,the role and molecular mechanism of miR-485-5p in bladder cancer are not clear.Studies have shown that MFGE8 can enhance phagocytosis and clearance of apoptotic cells by macrophages.Whether MFGE8 is the target gene of miR-485-5p and whether it affects the occurrence and development of bladder cancer is still unclear.This study investigated the effect of miR-485-5p on the proliferation,migration,and other biological behaviors of bladder cancer,confirmed that MFGE8 as one of the target genes of miR-485-5p was involved in the occurrence and development of bladder cancer,and described its molecular mechanism.Methods:1.qRT-PCR was used to detect the expression level of miR-485-5p in 16 pairs of bladder cancer and adjacent tissues and its relationship with the prognosis of bladder cancer.2.The expression levels of miR-485-5p in bladder cancer cells(T24,EJ,J82,5637,UMUC-3)and immortalized bladder epithelial cells(SV-HUC-1)were detected by qRT-PCR.Two bladder cancer cell lines with the lowest expression of miR-485-5p(T24,EJ)were selected for overexpression,and two bladder cancer cell lines with relatively low expression(5637,UMUC-3)were selected for inhibitorion of miR-485-5p expression for subsequent experiments.3.Overexpression and knockdown of miR-485-5p in bladder cancer cells,and detection of miR-485-5p expression.The growth curve of the cells was observed by CCK-8 assay,the colony formation ability of the cells was detected by plate cloning assay,the migration ability of the cells was detected by Transwell migration assay,and the expression of cell death-related proteins was detected by Western blot.4.The potential target gene of miR-485-5p: MFGE8 was screened by miRBase,Target Scan and miRanda databases.Dual luciferase assay was used to verify the targeting relationship between miR-485-5p and MFGE8.After overexpression or knockdown of miR-485-5p in bladder cancer cells,the change of MFGE8 protein level was detected by Western Blot.5.T24 and EJ were transfected with over express MFGE8,5637 and UMUC-3were transfected with siMFGE8,and the expression of MFGE8 protein was detected by Western Blot.6.MiR-485-5p and MFGE8 co-transfection experiment: CCK-8 experiment was used to observe the cell growth curve,plate cloning experiment was used to detect the cell colony formation ability,Transwell cell migration experiment was used to observe the cell migration ability,and Western Blot experiment was used to detect the expression of cell death-related proteins.Results:1.RT-q PCR detection of miR-485-5p in 16 pairs of bladder cancer and adjacent tissues showed that the expression of miR-485-5p in bladder cancer was significantly lower than that in the corresponding adjacent tissues.2.The expression of miR-485-5p in bladder cancer cell lines(5637,UMUC-3,J82,EJ,T24)was significantly lower than that in normal bladder epithelial cell line(SV-HUC-1).3.After T24 and EJ bladder cancer cells were transfected with miR-485-5p mimic,the expression of miR-485-5p in bladder cancer cells was significantly increased,and functional experiments showed that the proliferation and migration of bladder cancer cells were weakened.5637 and UMUC-3 bladder cancer cells transfected with miR-485-5p inhibitor showed significantly decreased expression of miR-485-5p,and functional experiments showed that bladder cancer cell proliferation and migration were enhanced.Western blot showed that Bcl2 protein expression was decreased and Bax protein expression was increased in cells transfected with miR-485-5p mimic.The expression of Bcl2 protein was increased and the expression of Bax protein was decreased in cells transfected with miR-485-5p inhibitor.4.Dual luciferase assay verified the targeting relationship between miR-485-5p and MFGE8.After T24 and EJ bladder cancer cells were transfected with miR-485-5p mimic,Western blot showed that MFGE8 protein level was decreased.After5637 and UMUC-3 bladder cancer cells were transfected with miR-485-5p inhibitor,the protein level of MFGE8 was increased as detected by Western Blot.5.T24 and EJ cells were transfected with MFGE8 overexpression plasmid,and the MFGE8 protein level was increased by Western Blot.5637 and UMUC-3 cells were transfected with MFGE8 si#3,and the MFGE8 protein level was decreased by Western Blot.6.MiR-485-5p and MFGE8 co-transfection experiment: T24 and EJ bladder cancer cells transfected with miR-485-5p mimic showed decreased proliferation and migration ability,but could be partially reversed by transfection over express MFGE8;The proliferation and migration of 5637 and UMUC-3 bladder cancer cells transfected with miR-485-5p inhibitor were enhanced,but could be partially reversed by transfection of MFGE8 si#3,which verified that MFGE8 was a downstream target gene of miR-485-5p.In addition,Bcl2 expression was increased and Bax expression was decreased in cells co-transfected with miR-485-5pmimic and over express MFGE8 compared with cells transfected with miR-485-5p mimic alone.Conclusion:In this study,qRT-PCR was used to detect the expression level of miR-485-5p in bladder cancer and adjacent tissues,bladder cancer cell lines and bladder immortalized epithelial cells,suggesting that miR-485-5p is lowly expressed in bladder cancer and can increase the expression of MFGE8,which affects the proliferation,migration and apoptosis of bladder cancer.miR-485-5p plays a tumor suppressor role in bladder cancer,and MFGE8 is one of the downstream target genes of miR-485-5p.MiR-485-5p can inhibit the occurrence and development of bladder cancer,which provides an experimental basis for exploring the diagnosis and treatment targets and mechanism of bladder cancer. |
PDF Full Text Request