| BackgroundHepatocellular carcinoma(HCC)is the one of the most common malignant tumors in the world,has become the third most important factor in the worldwide death of cancer.According to the survey,70-85% of malignant liver cancer and HCC are closely related,while the incidence of liver cancer in China accounted for about half of this value.China’s southern is a high incidence of HCC,which is the hardest area in Guangxi HCC.Guangxi area within the scope of liver cancer mortality showed a sustained increase in the trend.The main reason for the high death of liver cancer is the recurrence of liver cancer metastasis,but the mechanism of liver cancer metastasis recurrence is not fully clear,there is still a lack of effective and reliable treatment and prevention strategies.Therefore,to carry out the study of liver cancer metastasis and molecular regulation mechanism,and to take effective treatment measures and prevention strategies,is to enhance the treatment of liver cancer patients to improve the prognosis of the breakthrough.MicroRNAs(mi RNAs)are a class of about 17-25 nucleotides in length,non-coding single-stranded small molecule RNA,a regulatory factor for the expression of endogenous genes that are present in eukaryotic cell organisms.They are involved in the regulation of gene expression play an important role in a wide range of participation in a variety of pathophysiological processes,and the occurrence and development of tumors are closely related.They interact with the target gene m RNA 3’UTR,the target gene after the level of regulation of transcription,and then mediat complex biological metabolism and proliferation processes,including cellular differentiation,apoptosis,other processes.However,the abnormal expression of mi RNAs in the body,and a variety of diseases,including malignant tumors are closely related to the occurrence and development of the tumor,as a cancer gene or tumor suppressor gene.Recently,more and more closely related to the invasion and metastasis of mi RNAs are closely concerned with the majority of scholars,mi RNAs can participate in cell adhesion,matrix degradation,EMT,angiogenesis and other aspects of play a regulatory role.Such as mi R-130 b,mi R-10 b,mi R-151,mi R-221,mi R-21 and so on,inhibit the metastasis of mi R-34 a,Let-7 family,mi R-126 and so on.A large number of studies have shown that the loss of mi RNA expression is closely related to the occurrence and development of HCC,which can play an important role in the formation of HCC and is directly or indirectly involved in the occurrence,development,invasion and distant metastasis of HCC.The mi R-130 b gene has been shown to play a different regulatory role in different cell background or tumor microenvironment.For example,mi R-130 b can promote tumor invasion and metastasis,so mi R-130 b is called transfer factor.In malignant glioma,mi R-130 b promotes cell proliferation and inhibits apoptosis by inhibiting cell cycle and inhibiting pro-apoptotic genes.In addition,it has been confirmed that the mi R-130 b gene expression is up-regulated during the carcinogenesis of hepatocellular carcinoma stem cells,suggesting that the up-regulated mi R-130 b gene may be involved in the development and progression of hepatocellular carcinoma through the maintenance of biological characteristics of tumor stem cells.Notch-Dll1 gene is a Notch ligand Delta-like 1(Delta Like Canonical Notch Ligand 1,Dll1),located in the chromosome 6q27 protein coding gene,is a classic and biological evolution in a highly conservative Notch signal network(Dll1,Dll3,Dll4,Jagged1 and Jagged2),which play a decisive role in regulating a series of biological events such as cell division,differentiation,proliferation,apoptosis and tumorigenesis,and even almost Involving all aspects of life metabolism process.Previous studies have shown that Notch-Dll1 can be involved in melanoma angiogenesis,cell adhesion and metastasis through signal transduction between cells.Selective overexpression of Notch-Dll1 can save the inhibitory effect of mi R-130b-3p,silencing Notch-Dll1 to promote cell invasion and migration inhibition,play an important role in the invasion of breast cancer cells.However,mi R-130 b involved in liver cancer invasion and metastasis of the downstream target gene is what? They are how to control the target gene? Is interferon targeting mi R-130 b effectively reversing the metastatic phenotype of hepatocellular carcinoma cells? These problems are not yet clear,therefore,to explore the mi R-130 b in the occurrence and development of liver cancer and its molecular regulation mechanism to improve the diagnosis and treatment of liver cancer and find a new target is the need to solve the problem.ObjectiveTo investigate the expression of mi R-130 b in hepatocellular carcinoma(HCC),with explore the relationship between the expression of mi R-130 b and the clinicopathological features and prognosis of patients.To investigate the effect of mi R-130 b on malignant phenotype of hepatocellular carcinoma,with predict and verify the function of target gene Mi R-130 b in the invasion and metastasis of hepatocellular carcinoma and its molecular mechanism,in order to find a new liver cancer treatment target to provide a theoretical basis.Methods1.46 cases of hepatocellular carcinoma were collected(30 cases were diagnosed by pathology and 16 cases were not transferred).The samples of fresh liver cancer and 10 cases of normal liver tissue,The expression of mi R-130 b was detected by real-time quantitative PCR(q RT-PCR).To analyze the relationship between the expression of mi R-130 b gene and clinical and pathological parameters of liver cancer patients with hepatocellular carcinoma.2.To observe the effects of mi R-130 b gene on biological behavior of HCC cells in vitro: Using a synthetic mi R-130 b mimetic,transfection of MHCC97 H +and MHCC97L-subgroups,respectively,and overexpression and inhibition of mi R-130 b gene expression in hepatocarcinoma cells.The mi R-130 b gene was observed for MHCC97H+ and MHCC97L-subgroups with(MTT method),cell cycle(flow cytometry),apoptosis(flowway Annexin V staining),migration(Transwell experiment),invasion(Transwell experiment),cell proliferation(flow cytometry)assay were used to the impact of biological behavior.3.The mi R-130 b overexpression of lentiviral vector was transfected into MHCC97H+ and MHCC97L-cells.To observe the effect of mi R-130 b gene on the proliferation of hepatocarcinoma cells by subcutaneously implanting nude mice into overexpression and inhibition of mi R-130 b gene.And to detect the expression of Notch-Dll1,p53,Bcl-2,SOX2,E2F3,MET,CCND1 and Nanog in the transplanted tumor.4.Prediction and validation of mi R-130 b targets: By using Target Scan Release 7.1 and Pic Tar,then predicted the target gene of mi R-130 b and analyzed comprehensively.The downstream target gene Notch-Dll1,which could regulate mi R-130 b gene,was screened.The negative regulation of mi R-130 b on Notch-Dll1 was verified by luciferase reporter assay.After overexpression or inhibition of mi R-130 b in hepatocellular carcinoma cells,the changes of target Notch-Dll1 m RNA and protein were detected by q RT-PCR and western blotting.Construction of plasmids Notch-Dll1 to inhibit Notch-Dll1 was transfected into MHCC97 H cells.Enhance the invasion and migration of hepatocellular carcinoma cells by invasion(Transwell test)and migration(Transwell test).The expression of Notch-Dll1 in hepatocellular carcinoma was detected by immunohistochemistry and the relationship between them was analyzed.Results1.Expression of mi R-130 b in HCC tissues: Compared with normal liver tissue,the expression level of mi R-130 b in metastatic group and non-metastatic group was significantly higher than that in normal liver tissue.However,the level of mi R-130 b expression in metastatic group was significantly higher than that in normal liver tissue(P< 0.05).2.The expression of mi R-130 b gene in the clinicopathological parameters of hepatocellular carcinoma showed that: mi R-130 b gene overexpression,and liver cancer metastasis,tumor differentiation was significantly correlated(P <0.05).But not with age,sex,tumor size,tumor number and TNM stage.The high expression of mi R-130 b suggests that the prognosis of HCC is poor(P<0.01),and it is an independent risk factor for the overall survival of patients with hepatocellular carcinoma(P <0.01),which is closely related to prognosis.3.Up-regulation of mi R-130 b low expression of MHCC97L-subgroup cells in the cell proliferation capacity was significantly enhanced,the percentage of apoptotic cells in UR+LR was significantly reduced,invasion and migration capacity significantly enhanced,the difference was significant(P<0.05).In contrast,inhibition of mi R-130 b high expression of MHCC97H+ subgroup cells,proliferation capacity was significantly reduced,the percentage of apoptotic cells in UR+LR increased significantly,invasion and migration capacity was significantly reduced,the difference was significant(P<0.05).But up-regulated mi R-130 b low expression of MHCC97L-or inhibited the mi R-130 b overexpression of MHCC97H+ subgroup cells to increase or decrease the percentage of cells in S+G2 phase,but the difference was no significant(P>0.05).4.Inhibition of MHCC97H+ subgroups cells of mi R-130 b expression,the expression of Notch-Dll1 and SOX2,Nanog and E2F3 in the transplanted tumor tissues were significantly higher than those in other groups,the difference was significant(P<0.05).Overexpression of MHCC97L-subgroups cells of mi R-130 b genes,the expression of Notch-Dll1 and Bcl-2,CCND1,Nanog and MET in the transplanted tumor tissues was significantly higher than that in other groups,the difference was significant(P<0.05).All the results showed that Notch-Dll1 and Nanog could be mi R-130 b involved in the in vitro invasion and metastasis of hepatocarcinoma cells with possible target proteins.5.In vivo experiments showed that the overexpression mi R-130 b MHCC97H+ and MHCC97L-subgroups cells had a significant increase in tumor proliferative ability compared with the control group(P <0.05).6.The results of bioinformatics software predictions suggest that :Notch-Dll1 may be a target of mi R-130 b.Further luciferase reporter gene experiments confirmed that the mi R-130 b gene could inhibit the activity of the fluorescent reporter gene containing its complementary binding site.Overexpression of mi R-130 b has a negative regulatory effect on Notch-Dll1 protein levels.Down-regulation of Notch-Dll1 expression by si RNA can also promote the migration and invasion of hepatocarcinoma cells,and the effect of mi R-130 b overexpression is similar.It was confirmed that the mi R-130 b gene was involved in the invasion and metastasis of hepatocellular carcinoma through transcriptional level,negative regulation of the target gene Notch-Dll1 gene.Conclusions1.mi R-130 b gene was significantly up-regulated in hepatocellular carcinoma,especially in patients with metastatic liver cancer,and is closely related to the prognosis of patients,Suggesting that mi R-130 b gene plays an important role in the development of hepatocellular carcinoma and which indicated that mi R-130 b acts as oncogenes in HCC carcinogenesis and progression.2.mi R-130 b gene is expected to be a new prognostic indicator of liver cancer.3.Overexpression of mi R-130 b gene can promote the invasion and metastasis ability and in vivo proliferative ability of hepatocellular carcinoma cells in vitro.Conversely,inhibition of mi R-130 b gene expression,can reduce the invasion and metastasis and proliferation capacity,suggesting that mi R-130 b plays an important role in the metastasis of hepatocellular carcinoma.4.The mi R-130 b gene regulates the protein expression level of its target gene Notch-Dll1 by negative regulation.And thus promote the invasion and metastasis of liver cancer.Both of which are expected to be potential for the treatment of new targets for liver cancer. |
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