| Objectives: Hepatocellular carcinoma(HCC)is one of the most common malignancies in the world.At present,surgical resection is the preferred treatment for hepatocellular carcinoma,but postoperative recurrence is easy and the 5-year survival rate is low.Although a large number of new drugs and therapies have been used in recent decades to improve the survival rate of patients,the insensitivity of chemoradiotherapy and the resistance of targeted drugs are still difficult problems in clinical HCC treatment.CHD1L(Chrodomain Helicase/Adenosine Triphosphatase DNA Binding Protein 1-Like,CHD1L)gene is located in region 1q21 of chromosome,belonging to SWI2/ Sn F2-related ATPase superfamily.It is a chromatin remodeling enzyme,which plays an important role in maintaining chromatin stability and repairing broken DNA.Abnormal amplification and high expression of CHD1 L have been found in a variety of tumors,and it plays an important role in malignant proliferation of tumor cells,epithelial-mesenchymal transformation,chemotherapy resistance,and invasive metastasis.Tumors with high expression of CHD1 L are prone to relapse and metastasis,and with short survival time and poor prognosis in patients.Therefore,CHD1 L can be used as a marker and therapeutic target for malignant progression of tumor.Lanatoside C belongs to the Cardiac glycosides(CGS)drugs,which are widely used in the treatment of cardiovascular diseases.However,it has been reported that lanatoside C can inhibit the growth of tumor cells in a small concentration.In our experiment,we found that the semiinhibitory concentration(IC50)of different hepatocellular carcinoma cells treated with lanatoside C was different.Is this difference related to the expression level of CHD1 L in hepatocellular carcinoma cells? And does lanatoside C inhibit HCC growth by inhibiting CHD1 L expression? It has been reported that in the process of mediating apoptosis of tumor cells,the level of autophagy is increased,so whether the effect on autophagy is related to the expression of CHD1L? Therefore,we want to clarify the regulatory relationship between lanatoside C and CHD1 L by lanatoside C,and preliminarily explore the possible mechanism of action,this paper intends to study this.Methods: 1.IC50 values of lanatoside C treated QCY-7703,PLC-8024,Hep G2 and HUH-7 were detected by CCK8.Colony formation experiments was used to detect the effect of lanatoside C on cell proliferation after treatment with IC50 a concentrations.2.q RT-PCR was used to detect the effect of Hep G2 and HUH-7 treated with IC50 concentrations of lanatoside C on CHD1 L expression in hepatoma cells.The basic CHD1 L expression levels in LO2,Hep G2,HUH-7,PLC-8024 and QCY-7703 cells were detected by Western Blot.GFPVector/CHD1 L plasmid was used to construct overexpressed CHD1 L cells,q RT-PCR and Western Blot were used to detect the overexpression efficiency,and CCK8 was used to detect the effect of overexpressed CHD1 L on the IC50 of lanatoside C.3.Western Blot results showed that the expression of CHD1 L was inhibited by lanatoside C,LC3-II/I was increased,p62 level was decreased,LC3-II/I was decreased after overexpression of CHD1 L,and p62 level was increased.Fluorescence detection of immune cells showed that flosside promoted the aggregation of LC3 particles to form autophagosomes.After overexpression of CHD1 L,LC3 particles were uniformly distributed and no autophagosomes were formed.4.Mi RNAs target gene prediction analysis,dual luciferase reporting system and cell biology were used to identify mi RNAs and their target genes based on CHD1 L expression differences,and to study their mutual regulatory mechanisms.Results: 1.The results of CCK8 showed that the IC50 of floside C in QCY-7703,PLC-8024,Hep G2 and HUH-7 were 114.3 n M,147.3 n M,188.9 n M and198.5 n M,respectively.The results of plate cloning experiment showed that the proliferation capacity of Hep G2 and HUH-7 was significantly decreased after the treatment with lanatoside C.2.q RT-PCR results showed that the expression level of CHD1 L decreased after treatment with IC50 concentrations of lanatoside C.Western Blot results showed that CHD1 L protein expression in Hep G2 and HUH-7 cells was relatively high,while that in PLC-8024 and QCY-7703 cells was relatively low.q RT-PCR and Western Blot showed that CHD1 L overexpressed cells were successfully constructed.The results of CCK8 showed that the IC50 in the CHD1 L overexpression group was higher at 597.4 n M than that in the control group at 84.3 n M.3.Western Blot analysis was performed to detect the effects of lanatoside C on the expression of CHD1 L and autophagy related proteins LC3 and p62 in hepatocellular carcinoma cells Hep G2 and hepatocellular carcinoma cells PLC-8024 overexpressing CHD1 L with IC50 concentration.4.Bioinformatics analysis of mirnas targeting CHD1 L showed a total of 16 mirnas,and comprehensive analysis selected mi R-32-5p,mi R-363-3p,and mi R-330-5p for follow-up detection.q RT-PCR and detection showed that the expression level of mi R-32-5p increased and the expression level of mi R-363-3p decreased after drug treatment,and the basic expression levels of mi R-32-5p and mi R-363-3p were lower in Hep G2 and HUH-7 cells with high expression of CHD1 L.The basic expression levels of mi R-32-5p and mi R-363-3p were higher in PLC-8024 and QCY-7703 cells with low expression of CHD1 L.The expression of CHD1 L after transfection with mi R-32-5p,mi R-363-3p inhibitor and mimic was detected by q RT-PCR and Western Blot.The expression of mi R-32-5p was negatively correlated with CHD1 L,while that of mi R-363-3p was not negatively correlated with CHD1 L.Dual luciferase reporting system detection proved that mi R-32-5p could target binding to the 3’UTR end of CHD1 L,and the results of CCK8 and Colony formation experiments showed that the cell proliferation ability was enhanced when mi R-32-5p was down-regulated,while the cell proliferation ability was reduced when mi R-32-5p was up-regulated.Conclusions: 1.Lanatoside C can inhibit the proliferation of hepatocellular carcinoma cells.2.Lanatoside C plays an anti-proliferation role by inhibiting CHD1L;3.Lanatoside C promoted autophagy of hepatocellular carcinoma cells,while CHD1 L reversed lanatoside C induced autophagy;4.Lanatoside C inhibits the expression of CHD1 L through mi R-32-5p;5.mi R-32-5p inhibited the proliferation of hepatoma cells. |
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