The Mechanism Of MiR-217 Negative Regulation Of MTDH On Proliferation And Invasion Of Hepatocellular Carcinoma Cells

BackgroundHepetocellular carcinoma(HCC)is the fifth most common cancer worldwide and the third leading cause of cancer mortality.Because of the early nonspecific manifestation and easy to relapse after treatment,the overall prognosis of HCC patients is poor,which is a major medical problem threatening human health.Therefore,it is important to search for biomarkers and effective therapeutic targets for early diagnosis.In recent years,it has been proved that miR-217 has been expressed in gastric cancer,prostate cancer and other malignant tumors.However,the mechanism of miR-217 in HCC is still unclear.MTDH is a single transmembrane protein containing 582 amino acids,which is highly expressed in a variety of tumors,and its expression level is positively correlated with tumor progression.The expression of MTDH was correlated with the stage,stage and prognosis of cancer progression.MTDH is a potential therapeutic target in the process of cancer by regulating multiple signaling pathways involved in the process of cancer.MTDH.At present,there are few studies on the regulation mechanism of MTDH expression in liver cancer.MTDH can indirectly regulate the expression of MTDH in colorectal cancer,and miRNA is an indirect regulator of miRNA.In breast cancer,head and neck squamous cell carcinoma,miRNA is an example of direct regulation of MTDH.However,the relationship between miR-217 and MTDH is not clear.Based on the study of HCC tissues and HepG2 cells,we investigated the role and mechanism of MTDH in the development and progression of hepatocellular carcinoma(HCC)by miRNA-217.Objective1.Test the change of miR-217 and MTDH Mrna/protein in HCC tissues and the normal adjacent liver tissues and find the relationship.2.Make sure the relationship of miR-217 and MTDH,test the effect of miR-217 to MTDH 3'-UTR and the expression of MTDH.3.Test the proliferation,apoptosis,migration,and invasion of HepG2 cells transfected with miR-217 mimics.Method1.HCC tissues and the normal adjacent liver tissues were collected from 20 patients in Nanfang Hospital,Southern Medical University,China.Quantitative real-time RT-PCR were used to measure the expression of miR-217 and MTDH mRNA in tissues,and Western Blot were used to test the expression of MTDH protein.2.Targetscan was used to shown that miR-217 bound to the 3'-UTR of MTDH gene.The expression plasmid for MTDH 3'-UTR wild type or mutation were constructed and transfected with miR-217 mimics into HepG2 cells.The firefly and renilla luciferase activities were examined to shown the function of miR-217 to MTDH 3'-UTR.qPCR and Western Blot were used to examine the expression of MTDH mRNA and protein in HepG2 cells when transfected with miR-217 mimics.3.HepG2 cells were transfected with miR-217 mimics,the proliferation of HCC cells was measured using MTT and flow cytometry.Flow cytometry can also used to test the apoptosis of HepG2 cells.The migration and invasion of HepG2 cells transfected with miR-217 mimics were tested by Transwell.Result1.Compared with the matched normal tissues which acquired from pathology department,Southern Medical University,miR-217 expression level in HCC tissues significantly decreased(40%).Compared with the normal tissues,the mRNA expression of MTDH significantly upregulated in HCC tissues,as well as the expression of MTDH protein.2.We transfected HepG2 cell lines with miR-217 mimics,the erpression of MTDH protein was significantly increased.The dual luciferase report assay was performed to confirm the relationship of miR-217 and MTDH,after cotransfection with the 3'-UTR of wild-type MTDH and miR-217,the relative luciferase activity was significantly decreased in HepG2 cells.While the luciferase activity of the mutant construct shown little change,indicating that miR-217 inhibited MTDH transcription via directly targeting the 3'-UTR of MTDH.3.Overexpression of miR-217 in HepG2 cells by CCK-8 assay and flow cytometry cycle found in blank and negative compared with the control group,over expression of miR-217 can significantly inhibit the proliferation of HepG2 cells,and increased in G1 phase and decreased S phase HepG2 cells;at the same time,the over expression of miR-217,promote the apoptosis of HepG2 cells;Transwell the experimental results show that the expression of miR-217 after the transfer to the lower chamber in cells significantly reduced,that migration and invasion of HepG2 cells was significantly inhibited by miR-217.Conclusion1 the expression of miR-217 was down regulated and the expression of MTDH was up-regulated in HCC tissues.2 miR-217 targeted to MTDH and inhibited its expression.3 miR-217 could significantly inhibit the proliferation,invasion and migration of HepG2 cells and promote the apoptosis of HepG2 cells.