Study On The Function And Partial Mechanisms Of CircASPH In Regulating The Proliferation And Apoptosis Of Hepatic Stellate Cells In Liver Fibrosis

Chronic liver disease is a kind of disease that seriously damages health.Many patients with liver disease commonly experience hepatic fibrosis(HF).The typical feature of HF is that the synthesis of extracellular matrix(ECM)is greater than degradation,and a large amount of ECM accumulates in the liver,leading to abnormal proliferative lesions of connective tissue in the liver,mainly collagen fiber hyperplasia.Hepatic fibrosis is usually reversible.If it is not controlled,this fibrous scar will lead to cirrhosis,so cirrhosis is an irreversible lesion.Taking liver fibrosis as a breakthrough,it is of great significance to explore the biomarkers in its pathological process for early clinical diagnosis.Activated hepatic stellate cells(HSCs)have the characteristics of Myofibroblast-like cells,which secrete a lot of collagen to form ECM,so the activation of HSCs is called the core event of HF.Non-coding RNAs(nc RNAs)are a class of RNAs that do not have the ability to translate into proteins.Circular RNA(circ RNA)is an important member of the nc RNAs family.With the application of high throughput screening technology,the expression and molecular functions of circ RNA in various diseases have gradually been revealed.The research team found through high throughput screening that multiple circ RNAs are closely related to the occurrence and development of liver fibrosis,among which circ FBXW4 inhibits the progress of liver fibrosis through mi R-18b-3p/FBXW7 axis.However,the function and molecular mechanism of circASPH in liver fibrosis are still unclear.According to literature reports,circASPH is involved in the occurrence of various diseases and plays an important regulatory role in cell proliferation and apoptosis.Since hepatic stellate cells are important effector cells in liver fibrosis,we investigated the effects of circASPH on activation,proliferation,and apoptosis of hepatic stellate cells and related regulatory mechanisms.First,we investigated the expression of circASPH in primary hepatic stellate cells of mice with liver fibrosis.Extracted primary HSCs from mice,q-RT-PCR results showed that the expression of circASPH increased in primary HSCs from liver fibrosis mice.The expression of circASPH in tissues and serum of patients with clinical liver fibrosis was further studied.Fluorescence in situ hybridization experiments showed that the expression of circASPH increased in liver tissue of patients with liver fibrosis.RNA was extracted from the serum of patients with liver fibrosis.The results of q-RT-PCR experiments showed that the expression of circASPH in the serum of patients with liver fibrosis increased.We also tested the expression of circASPH in TGF-β1 stimulated LX-2 cells was changed,and the q-RT-PCR results showed that the expression of circASPH increased in activated LX-2 cells in vitro.To explore the function of circASPH in vivo,we constructed a recombinant adeno-associated virus(r AAV)that silences circASPH.After modeling liver fibrosis,pathological sections of the liver were taken for HE staining,Sirius red staining,Masson staining α-SMA immunohistochemical staining and detection of serum ALT,AST,TC,TG levels.Detection of col1α1 and α-SMA expression in primary HSC by q-RT-PCR.The results showed that compared with the virus control group,the liver fibrosis damage in mice silencing circASPH was significantly reduced,and the liver damage indicators in serum were decreased.After silencing circASPH,the expression of col1α1 and α-SMA decreases and inhibits the activation of primary HSCs.Further in vitro,si RNA transfection was used to silence circASPH and lentivirus overexpression of circASPH to explore the function of circASPH in LX-2 cells.Q-RT-PCR,western blot,immunofluorescence staining,TUNEL and Ed U staining were used to detect the effects of circASPH on the activation,proliferation,and apoptosis of LX-2 cells.The experimental results showed that silencing circASPH inhibited the activation and proliferation of LX-2 and promoted the occurrence of apoptosis in LX-2.On the contrary,overexpression of circASPH promotes the activation and proliferation of LX-2 and inhibits its apoptosis.Next,we continued to explore the mechanism of circASPH regulating hepatic stellate cells.Circ RNA in the cytoplasm can act as a mi RNA sponge and bind to mi RNA.Nuclear cytoplasmic separation experiments have shown that circASPH is mainly expressed in the cytoplasm.Therefore,circASPH can bind to mi RNA.The website database predicts the potential binding mi RNA sites of circASPH,and the dual luciferase reporting experiment and RNA pull down experiment explore the binding relationship between circASPH and mi RNA.The results showed that circASPH targeted regulation of mi R-139-5p.In LX-2 cells,mi R-139-5p was overexpressed or silenced.The effects of mi R-139-5p on activation,proliferation,and apoptosis of hepatic stellate cells were investigated using q-RT-PCR,Western blot,TUNEL,and Ed U staining.The results showed that mi R-139-5p inhibited the expression of col1α1 and α-SMA,inhibited the proliferation and promoted the apoptosis of LX-2 cells.It has been reported in the literature that Notch1 participates in the activation of hepatic stellate cells and has the effect of promoting fibrosis.Notch1 plays an important role in the proliferation and apoptosis of many cells.Bioinformatics analysis revealed that mi R-139-5p and Notch1 protein have base complementary pairing sites.Western blot was used to detect the expression of Notch1 in mouse primary HSC and LX-2 cells in vitro.The experimental results showed that the expression of Notch1 in primary HSCs of mice with liver fibrosis increased.Silencing circASPH,Notch1 expression decreased.After simultaneously silencing circASPH and mi R-139-5p,the inhibitory effect of si-cir ASPH on Notch1 expression was cancelled out.Conclusion: The expression of circASPH is elevated in hepatic stellate cells of liver fibrosis,which promotes the activation and proliferation of hepatic stellate cells by targeting the mi R-139-5p/Notch1 axis,thereby promoting the progression of liver fibrosis.CircASPH may be an important biomarker for the clinical diagnosis and treatment of liver fibrosis.