| Background:Hepatic fibrosis is the pathological process of excessive deposition of extracellular matrix(ECM)by various pathogenic factors.At the cellular and molecular level,this progressive process is characterized by abnormal activation of HSCs and TGF.Activation of hepatic stellate cells is a key event in hepatic fibrosis as these cells become the primary source of extracellular matrix after injury.After various acute or chronic liver injuries,hepatic stellate cells are activated from a static state and begin to activate,mainly manifested as proliferation,enhanced cell vitality and secretion and synthesis of a large number of extracellular matrix.The pathogenesis of hepatic fibrosis is a very complex and dynamic process involving a variety of cell types,cytokines and growth factors,as well as leading to repeated scar repair of the liver,which eventually leads to cirrhosis.miRNAs are a class of non-coding single-stranded smallRNA molecules with about 17-22 nucleotides,which regulate gene expression as a post-transcriptional regulator.miRNA binds to mRNA through complete or partially complementary bases,mainly including: degradation of target mRNA,inhibition of mRNA translation,recruitment of histone deacetylase and other factors in the nucleus,silencing of DNA expression and amplification of corresponding miRNA,playing a corresponding biological role.miRNA regulates cell proliferation,differentiation,apoptosis,invasion and metastasis in many physiological and pathological processes.More and more literature reports suggest that miRNA is also involved in the process of fibrosis.MicroRNA-338(miR-338)is a newly discovered miRNA,which plays a crucial role in a variety of cancers.The abnormal expression of miR-338 is closely related to the cell proliferation,invasion,early detection and clinicopathological variables of liver cancer,colorectal cancer,gastric cancer and neuroblastoma.CDK4 is a type of cyclin-dependent protein kinase,which plays an important role in the regulation of cell proliferation.As a member of the CDK family,cyclin dependent kinase 4(CDK4)is a type of oncogene located on the long arm of chromosome 12,which regulates the DNA replication and mitosis phases of the cell cycle.Enzymes are the rate-limiting factors for the G1/S phase transition,and cells can only enter the proliferation cycle process in their activated state.CDK4/6 inhibitors combined with endocrine drugs can regulate the expression level of related miRNAs in human breast cancer cells,thereby inhibiting the proliferation of human breast cancer cells.miR-24 inhibits the proliferation of pancreatic β-cells by reducing the levels of CDK4 and Cyclin D3 proteins,and this effect can be reversed by overexpression of CDK4 and Cyclin D3 proteins .In this study,real-time quantitative PCR was used to detect the expression of miR-338-3p in hepatic stellate cell activation process(resting state activated state),liver tissue of DMN liver fibrosis animal model(s0-s4),liver tissue of HBV related liver fibrosis patients and hepatic stellate cells before and after TGF-β1 stimulation.It was confirmed that miR-338-3p was significantly decreased in hepatic stellate cell activation process,DMN induced rat liver fibrosis model,liver tissue of patients with liver fibrosis and hepatic stellate cells stimulated by TGF-β1,and negatively correlated with the severity of liver fibrosis.By constructing miR-338-3p overexpression lentiviral vectors,the effects of miR-338-3p on the proliferation,apoptosis,migration and activation of rat hepatic stellate cells were studied.Through the verification of the miR-338-3p target gene,it was found that miR-338-3p targets the downstream gene CDK4,and the expression of miR-338-3p and CDK4 in liver fibrotic tissues showed a negative linear correlation.Overexpression of CDK4 can reverse the effect of miR-338-3p on hepatic stellate activation.Aims:To observe the effect of miR-338-3p on the activation,proliferation,migration and other functions of hepatic stellate cells,and to explore the relationship between miR-338-3p and the biological behavior of liver fibrosis,so as to provide a new theoretical basis for expanding the treatment strategy of liver fibrosis.Methods:(1)Detection of the relationship between miR-338-3p and activation of hepatic stellate cells1.Isolate,identify,and culture rat primary hepatic stellate cells,and detect the changes in the expression level of miR-338 during natural activation.2.Detect the expression level of miR-338-3p in 5 pairs of normal liver tissues and cirrhotic tissues by quantitative PCR(qRT-PCR)method.3.Construct a DMN liver fibrosis animal model,and detect the expression of miR-338-3p in tissues of various stages of liver fibrosis(S0-S4)by quantitative PCR technology.4.Stimulate hepatic stellate cells with TGF-β1 to detect the expression level of miR-338-3p.(2)Detect the effect of miR-338-3p on the proliferation,migration and activation of hepatic stellate cellsReal-time PCR were used to verify the overexpression and inhibition of miR-338-3p.CCK-8,plate clone experiment,flow cytometry,Transwell migration test and scratch migration test were used to detect the growth,proliferation and migration of hepatic stellate cells.At the same time,real-time-PCR and western blot were used to detect the mRNA and protein expression of hepatic stellate cell activation markers including type Ⅰ collagen,desmin and α-SMA.(3)miR-338-3p regulates the proliferation and activation of hepatic stellate cells by inhibiting the expression of CDK41.Use bioinformatics analysis,luciferase and Western blot to find the target gene of miR-338-3p and verify it.2.Detect the expression of target gene CDK4 in 3 pairs of normal liver tissues and liver fibrotic tissues by fluorescence quantitative PCR(qRT-PCR).The expression of miR-338-3p in the corresponding tissues was used for regression analysis to analyze the correlation between the expression of miR-338-3p and CDK4 in liver fibrotic tissues.3.Construct sh-CDK4 lentivirus to transfect hepatic stellate cells to verify the expression of CDK4 mRNA and protein before and after infection.Observe cell growth and proliferation by CCK-8 and plate cloning experiments,and verify the migration ability by transwell and scratch experiments.At the same time,real-time-PCR and western blot were used to detect the mRNA and protein expression of hepatic stellate cell activation markers including type Ⅰ collagen,desmin and α-SMA.4.Construct miR-338-3p overexpression hepatic stellate cells,and verify the proliferation and migration ability through CCK-8,transwell and wound healing assay.(4)Statistical analysis: the data were expressed as mean standard deviation,and the data were processed by SPSS 22.0 statistics.Double-tailed T test was used for statistical analysis between the two groups.*P<0.05 was considered statistically significant.Results:Through gene microarray and quantitative PCR,the results indicated that the expression of miR-338-3p in primary hepatic stellate cells of rats decreased with the activation of hepatic stellate cells.Luciferase reporter gene and Western blot experiments showed that miR-338-3p regulated the activation and proliferation of hepatic stellate cells by targeting CDK4.In addition,when HSCs were treated with transforming growth factor,the results indicated that the expression of miR-338-3p decreased rapidly,and the level of miR338-3p increased after the TGF-β specific inhibitor SB431542 intervened.Conclusion:(1)Through gene array and quantitative PCR,the results suggest that miR-338-3p is significant reduced in hepatic stellate cell activation process,DMN liver fibrosis animal model,liver fibrotic tissue of patients,and hepatic stellate cells stimulated by TGF-β1.After the intervention of TGF-β1 specific inhibitor SB431542,the level of miR-338-3p increased.(2)miR-338-3p overexpression in hepatic stellate cells significantly inhibits the activation,proliferation of hepatic stellate cells,but has no effect on migration function.(3)Luciferase reporter gene and Western blot found that miR-338-3p regulates the activation and proliferation of hepatic stellate cells by targeting CDK4,and this effect can be reversed by overexpression of CDK4The results of the study found that the miR-338-3p/CKD4 signaling pathway is involved in the occurrence and development of liver fibrosis by regulating the activation and proliferation of hepatic stellate cells. |
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