Inhibition Of MiR-155 Signaling In ImDC Was Activated Effects On Proliferation And Apoptosis Of Hepatic Stellate Cells

Liver cirrhosis is a major public health problem that perplexes researchers at home and abroad.About 2 million people die from chronic liver disease worldwide every year.The most common pathological process of end-stage liver disease is liver fibrosis,which can progress sequentially to cirrhosis,liver dysfunction and failure,and even to liver malignant tumors.Liver fibrosis is a key factor affecting the development and prognosis of liver diseases,which makes the study of improving liver fibrosis conducive to improving the long-term quality of life of patients with liver disease and reducing the medical burden of society.Therefore,people’s research direction is as much as possible on the intervention of early disease occurrence and development.Hepatic stellate cells（HSCs）are the central key cells of liver fibrosis,which are in a quiescent state when they are not stimulated by internal or external sources.When inflammatory cells are chemotactic and inflammatory molecules are secreted in large quantities,HSCs are activated and participate in the process of liver fibrosis.Immature dendritic cells（imDCs）can not only induce immune tolerance,but also secrete anti-inflammatory factors.Studies have shown that imDCs can improve the process of fibrosis through anti-inflammatory related mechanisms.miR-155 has been found to be widely involved in the maturation and function of DC.Related experimental studies have found that knocking down or low expression of miR-155can maintain the immature state of DC,and play the role of anti-inflammatory and immune tolerance induction.In this study,we investigated the effect of lentiviral transfection of rat bone mark-derived immature dendritic cells with antisense miR-155 on the proliferation and apoptosis of activated rat hepatic stellate cells（HSCS）in vitro.Objective（s）:To construct rat microRNA-155（microRNA-155）lentiviral vector,identify,induce and culture rat bone marrow-derived dendritic cells,and transfuse recombinant anti-miR-155 lentiviral vector into rat immature dendritic cells.To investigate the effect of rat bone mark-derived imDC with low expression of miR-155 on the proliferation and apoptosis of activated rat hepatic stellate cells.Methods:1.Induction,culture and identification of rat bone marrowderived imDCs:bone marrow cells of tibia and femur were obtained from rats and induced with rr GM-CSF and rr IL-4（10ng/m L）for 7 days to obtain rat imDCs,and then LPS was added to obtain rat m DC.Immunophenotyping and ultrastructure of DC were observed by flow cytometry and transmission electron microscope.2.Construction and identification of antisense miR-155 lentiviral vector:the lentiviral vector was digested and linked with the annealed double-stranded DNA of the target gene antisense miR-155,and the obtained product was cultured in competent cells.The cultured vectors were sequenced.The lentivirus vector was packaged,concentrated and purified,and the virus titer was determined by limiting dilution method.3.Identification of miR-155 expression in imDC:The recombinant lentiviral vector was transfected into imDC of rats,and the expression of miR-155 in imDC was detected by RT-q PCR.4.Activation of HSC:HSCS were stimulated with 5ng/ml TGF-βfor 48h in a cell incubator.5.Experimental grouping:（1）HSC blank control group;（2）TGF-βinduced activated HSC group;（3）TGF-βinduced HSC and imDC co-culture group;（4）TGF-βinduced activated HSC and imDC^anti-miR-155co-culture group;（5）TGF-βinduced HSC and m DC co-culture group;6.Study on the inhibitory effect of imDC with low expression of miR-155 on activated hepatic stellate cells:HSC were co-cultured with imDC/imDC^anti-miR-155/m DC at a ratio of 1:2 through Transwell chamber（pore size0.4μm）to establish a two-cell co-culture system.imDC/imDC^anti-miR-155/m DC was seeded in the upper chamber,and activated HSC was seeded in the lower chamber.After co-culture for 48h,the apoptosis rate of HSCs was detected by flow cytometry,and the proliferation of HSCs was detected by EdU.Results:1.Mononuclear cells were isolated from rat bone marrow cells and induced by rr GM-CSF and rr IL-4 for 7 days to obtain imDCs,and LPS was added to obtain m DC.The cell surface markers identified by flow cytometry were consistent with the typical phenotypes of imDC and m DC.The morphologic features of imDC and m DC were observed under light microscope and transmission electron microscope.It met the requirements of subsequent co-culture experiments.2.The antisense miR-155 lentiviral vector was constructed,and the cultured vector was sequenced.The vector sequence contained the target gene.3.The rat anti-miR-155 lentiviral vector was successfully constructed by linking anti-miR-155 with lentiviral vector.The recombinant lentiviral vector was used to infect rat imDC.RT-q PCR showed that miR-155 was significantly down-regulated in imDC,and the target gene was successfully expressed in target cells.The transfected imDC^anti-miR-155did not transform into m DC under the stimulation of LPS,and still maintained the expression state of imDC.4.HSCs were successfully activated after adding TGF-βunder microscope,and the cells were in the morphology of fibrocytes.5.Flow cytometry showed that both imDC and imDC ^anti-miR-155could promote the apoptosis of activated HSCs,and the apoptosis effect of imDC ^anti-miR-155group was more obvious（P<0.0001）,while m DC had no effect on the apoptosis of activated HSCs(apoptosis rate:A:HSC group:9.54±1.60%;B:HSC+TGF-βgroup 9.12± 1.38%;C:HSC+TGF-β+imDC group:22.07±0.75%;D:HSC+TGF-β+imDC^anti-miR-155group:38.37±0.93%;E:HSC+TGF-β+m DC group:8.56±2.02%)6.EdU results showed that both imDC and imDC^anti-miR-155could inhibit the proliferation of activated HSCs compared with HSCs activation group(imDC group:P<0.01;imDC^anti-miR-155group:P<0.0001),and imDC^anti-miR-155had a stronger ability to inhibit the proliferation of activated HSCs（P<0.0001）.m DC group failed to inhibit the proliferation of activated HSCS.（m DC group:P=0.3149）Conclusion（s）:1.imDC^anti-miR-155,which inhibits the expression of miR-155,can maintain an immature state under an inflammatory background.2.imDC^anti-miR-155could inhibit the proliferation of activated HSCs,and the inhibitory effect was stronger than that of untransfected imDC,while m DC had no effect on the proliferation of activated HSCs.3.imDC^anti-miR-155could promote the apoptosis of activated HSCs,and its promoting effect was stronger than that of untransfected imDC,while m DC had no effect on the apoptosis of activated HSCs.