MicroRNA-100 Targeting SNAI1 Induced Epithelial Mesenchymal Transition Inhibits Invasion And Metastasis Of Hepatocellular Carcinoma

BackgroundAccording to pathological types,HCC can be divided into hepatocellular carcinoma,cholangiocarcinoma and mixed hepatocellular carcinoma,90% of which belong to hepatocellular carcinoma.Therefore,this study focuses on hepatocellular carcinoma.Because of poor prognosis and high mortality,HCC is called "king of cancer"The occurrence and development of HCC are characterized by insidious onset,clinical symptoms,atypical signs and rapid progression of the disease.The morbidity and mortality of HCC are increasing year by year.Because the survival rate of HCC is low and the incidence is occult,the majority of liver cancer has been diagnosed in the middle and late stages and has lost the opportunity of effective treatment.Therefore,the early diagnosis of liver cancer is very important.The prognosis of HCC is poor.The invasion and metastasis of HCC is the key factor affecting the prognosis and long-term survival of HCC patients.At present,the mechanism of the invasion and metastasis of HCC remains unclear,with in-depth study of liver cancer,especially the progress of molecular biology,gene therapy of hepatocellular carcinoma has become targeted therapy in clinical application is very promising,seek the effective treatment method has become the new research hotspot at home and abroad.The invasion and metastasis of hepatocellular carcinoma is a complicated process of multiple steps,including the degradation of extracellular extracellular.barrier is the initial step of tumor invasion and metastasis.Tiny RNA(MicroRNAs,miRNAS)is a short,highly conserved,endogenous,non coding RNA found in eukaryotes,capable of regulatory function,with a length of about 21-25 nucleotides(NT).MicroRNAs(miRNAS)plays an important role in the occurrence and development of malignant tumors.A large number of MicroRNAs(miRNAS)target genes are tumor suppressor genes or oncogenes,and multiple MicroRNAs(miRNAS)molecules have been shown to be involved in many aspects of HCC invasion and metastasis.Small RNA can recognize specific target mRNA or inhibit protein translation,thus transcription level plays a negative regulatory role.More than half of the human MicroRNAs(miRNAS)gene is located in fragile sites or contain tumor tumor related genes,and MicroRNAs(mi RNAS)has a wide range of biological functions,including cell proliferation,apoptosis,metabolism,differentiation and tumor growth control and forming process.The major molecular biological mechanism of MicroRNAs(mi RNAS)silencing target genes is achieved by specifically combining the target gene 3-UTR with MicroRNAs(miRNAS)conserved seed region sequences(about 2-7 nucleotides(NT)).RNA technology is a gene silencing technology developed in recent years.It uses double stranded RNA to degrade mRNA with homologous sequence,thus silencing the expression of corresponding gene.Epithelial mesenchymal transition(epithelial-mesenchymal,transition,EMT)is the process of epithelial cell transition from epithelial state to mesenchymal state,and it is also an important feature of cell metastasis.Epithelial mesenchymal transition plays an important role in the deterioration and development of tumors and can significantly enhance the invasive ability of tumor cells.In the course of EMT,the loss of epithelial function and the acquisition of mesenchymal function significantly enhanced the migration and invasion ability of cells.E-cadherin is a specific molecular marker of epithelial cells,Vimentin is a specific marker of interstitial cells,so Vimentin protein obtained and the loss of E-cadherin protein showed that the cells had epithelial mesenchymal transition.SNAI1(snail)is a transcription factor closely related to EMT.It can inhibit the expression of E-cadherin and promote the migration and invasion of tumor.Epithelial mesenchymal transition plays a key role in tumor invasion and metastasis,and is closely related to the prognosis of patients with[17-19].Abnormal expression of SNAI1 has been observed in epithelial mesenchymal transition of various tumors.In recent years,more and more evidences show that SNAI1 plays an important role in the regulation of EMT.In addition,SNAI1 induced downregulation of E-cadherin has been shown to play a key role in triggering epithelial mesenchymal transition in tumor cells.The invasion and metastasis of tumor cells is a multi-stage complex process,including circulating in the blood transport of the surrounding tissue invasion and distant metastasis foci formation and other aspects in the process of HCC cells,epithelial mesenchymal transition is considered to be a feature of events.MiRNA-100 is located on chromosome 11q24.1,the size of about 22 nucleotides and is located on human chromosome 11q24.1,the seed sequence region containing two GC,the other end is TA,the structure characteristics of the target gene less species,seed sequences with strong.Previous studies have shown that miRNA-100 exhibits different expression patterns in different lesions.Previous studies have found that the expression of miRNA-100 is down regulated in bladder cancer cells and tissues.Overexpression of miRNA-100 in vitro can inhibit the invasion and metastasis of bladder cancer cells.It has been proved that in many tumor cells,miRNA-99a/100 regulates and alters the value-added migration function of mTOR through transcription of [31-32].In our previous studies,we found that miRNA-100 is highly expressed in normal human hepatocytes,but is low expressed in HCC cells.Sui Chengguang's study found that overexpression of mi RNA-100 inhibited the migration and invasion of HCC by inhibiting the expression of mTOR,and by blocking up the occurrence of EMT by up regulating the expression of E-cadherin.In recent years,the role of mi RNA-100 in the occurrence and development of hepatocellular carcinoma in concern,but the specific role of miRNA-100 in hepatocellular carcinoma and how to play caused by specific mechanisms of hepatocellular carcinoma invasion and metastasis is not very clear,this study to further explore the possible mechanism that miRNA-100 induce hepatocellular carcinoma cell invasion and migration,thus providing new the clues for clinical diagnosis and treatment of hepatocellular carcinoma,establish the molecular biology basis for finding new target in the treatment of hepatocellular carcinoma.ObjectiveThrough this experiment,to explore the effect of expression of microRNA-100 in hepatocellular carcinoma and its invasion and migration of hepatocellular carcinoma cells,and to study the occurrence and development of microRNA-100 in hepatocellular carcinoma,to further explore the possible mechanism of microRNA-100 induced hepatocellular carcinoma cell invasion and migration,lay the molecular basis for the search for new treatment targets hepatocellular carcinoma.Method1.using qRT-PCR method to study the expression of microRNA-100 in hepatocellular carcinoma tissues and corresponding adjacent tissues,and the relationship between the microRNA-100 expression and prognosis of patients with hepatocellular carcinoma and the clinical pathological characteristics of the increase.2.using qRT-PCR method to detect HepG2,Huh7,Hep3 B,MHCC97L,microRNA-100 and MHCC97 H SMMC7721 expression in hepatocellular carcinoma cell lines and human immortalized liver cell line LO2,to further explore the expression level of microRNA-100 on the invasion of hepatocellular carcinoma metastasis and extent of influence.3.immunohistochemistry method was used to detect the expression levels of SNAI1 protein,Vementin protein and E-cadherin protein in HCC tissues,and the correlation between the expression of them and microRNA-100 was analyzed.4.microRNA-100 inhibitor and microRNA-100 mimics were transfected with MHCC97 H and SMMC7721 cells,then Transwell invasion test and scratch test were used to detect the changes of cell invasion and migration ability.5.after transfection of MHCC97 H and SMMC7721 cells by microRNA-100 mimics and microRNA-100 inhibitor,the expression of Vementin and E-cadherin protein in cells was detected by Western-blot assay.6.microRNA-100 mimic and microRNA-100 inhibitor were transfected into MHCC97 H cells with SNAI1 overexpression vector,and then the cell migration and invasion ability were detected by scratch test and Transwell invasion test.7.microRNA-100 mimics and microRNA-100 inhibitor were transfected into MHCC97 H and SMMC7721 cells,then the expression of SNAI1,mRNA and protein in cells were detected by qRT-PCR and Western-blot methods,respectively.8.using luciferase reporter genes to verify whether microRNA-100 was inhibited by binding to-UTR 3 and SNAI1 regions.9.microRNA-100 mimics and microRNA-100 inhibitor were transfected into MHCC97 H cells with SNAI1 overexpression vector,and the expression of E-cadherin and Vementin protein in cells was detected by Western-blot assay.Result1.The expression of 1.microRNA-100 in hepatocellular carcinoma tissues than in paracancerous tissues was significantly reduced,and the down-regulation of microRNA-100 expression and intrahepatic metastasis,late TNM stage was significantly correlated with high Edmonson grade(P<0.05);further by COX regression analysis and Kaplan-Meier survival curves showed that the decrease of microRNA-100 indicates that the hepatocellular carcinoma patients than short disease-free survival(HR = 2.7;P = 0.003).2.HCC cell lines were all detected to detect the expression of microRNA-100 and microRNA-100 in MHCC-97 H cells was the lowest,while in normal immortalized LO2 cells express the highest.In HCC tissues,the expression level of microRNA-100 in metastasis group was significantly lower than that in non metastasis group(P<0.001).3.overexpression of microRNA-100 inhibited migration and invasion of MHCC97 H and SMMC7721 cells;knockdown of miR-100 promoted migration and invasion of MHCC97 H and SMMC7721 cells.4.In MHCC97 H and SMMC7721 cells,knockdown of microRNA-100 promoted Vimentin protein expression,inhibited the expression of E-cadherin protein;overexpression of microRNA-100 promotes the expression of E-cadherin protein,inhibited the expression of Vimentin protein.5.In 70 hepatocellular carcinoma tissues,the level of microRNA-100 was positively correlated with the expression of E-cadherin protein(r=0.639,P<,0.01),and negatively correlated with the expression of Vimentin protein(r=-0.456,P<,0.01).6.SNAI1 is the direct target of microRNA-100.7.In MHCC97 H and SMMC7721 cells,overexpression of microRNA-100 inhibited the expression of SNAI1,mRNA and protein;knockdown of microRNA-100 promoted the expression of SNAI1,mRNA and protein.8.In 70 cases of HCC,the level of microRNA-100 was negatively correlated with the expression of SNAI1 protein(r=-0.615,P<,0.001).microRNA-100 affects cell migration,invasion and epithelial mesenchymal transition by acting on SNAI1.Conclusion1.the expression of microRNA-100 reduced inhibition or deletion of hepatoma cells,down-regulation of microRNA-100 by promoting the expression of SNAIL and promote cell epithelial mesenchymal transition,which caused the cell invasion and metastasis.2.in hepatocellular carcinoma,downregulation of microRNA-100 promotes cell invasion,migration,and epithelial mesenchymal transition.The down-regulation of microRNA-100 expression in.3.The expression of miRNA-100 in hepatocellular carcinoma is lower than in noncancerous tissue,which is closely related to multiple malignant clinical pathological indexes and the poor prognosis of patients with hepatocellular carcinoma.4.microRNA-100 is expected to be a potential prognostic indicator for HCC patients.