The Reciprocal Interaction Between LncRNA CCAT1 And MiR-375-3p Contribute To The Downregulation Of IRF5 Gene Expressi On By Solasonine In HepG2 Human Hepatocellular Carcinoma Cells

Objective:The main purpose of this study was to explore the possible molecular mechanism of solasonine,one of the active components in Chinese herbal medicine Solanum nigrum L,in inhibiting the proliferation of hepatocellular carcinoma cells,to explore possible molecular signaling pathways and drug targets.It is hoped to provide a more accurate and sufficient theoretical basis for the clinical application of the anti-tumor effect of Chinese herbal medicine.Methods:In vitro experiments,we first demonstrated that solasonine can inhibit the proliferation of HCC cells by MTT and EdU staining and determined the corresponding effective drug dose.Then,Western blot and Real-time PCR were used to screen out proteins(SP1-transcription factor specific protein 1,IRF5-interferon regulatory factor 5)and RNA(microRNA-375-3p,LncRNA CCAT1-colon cancer related transcription factor 1)related to solasonine inhibiting the proliferation of HCC cells.Finally,we adopted the transient transfection method to detect the expression of related proteins and RNAs by exogenous overexpression of SP1,IRF5,miR-375-3p and CCAT1,and then determined the mutual regulation between related proteins and RNAs.In addition to these experimental methods,we also used the double luciferase reporter method to detect the effect of solasonine on the activity of IRF5 promoter,the interaction between SP1 and IRF5 promoter region,and the interaction between miR-375-3p and CCAT1.RNA-binding protein immunoprecipitation was used to further verify the interaction between miR-375-3p and CCAT1.In vivo experiment:HepG2-Luc cells labeled with luciferase reporter gene were first used to construct a model of tumor-bearing mice in vivo experiment.Mice in each group were treated with different doses of solasonine.Then,we used small animal in vivo imager to detect the changes of tumor-bearing fluorescence value before and after drug administration in each group of mice,and measured the size of tumor on the body surface of mice regularly.After death,the tumor on the body surface of mice was stripped off and weighed,so as to continuously observe the effect of solasosine treatment on tumor growth in mice.Finally,Western Blot and Real-time PCR were used to detect the corresponding protein and RNA expression in mouse surface tumors to verify whether the results of in vivo experiments were consistent with those of in vitro experiments.Results:In vitro experiments:1.MTT results suggested that solasonine could inhibit the proliferation of human HepG2 hepatoma cells and QGY-7703 cells in a time-dose dependent manner.EdU staining also confirmed that solasonine could inhibit the proliferation of HCC.2.Real-time PCR detection found that when the concentration of solasonine was 45μ M,it could promote the expression of miR-375-3p and inhibit the expression of CCAT1.The difference was statistically significant.The expression of CCAT1 can be inhibited by exogenous overexpressed miR-375-3p,while the over expression of CCAT1 can inhibit the expression of miR-375-3p.In other words,the expression of miR-375-3p or CCAT1 will be affected by the expression of another party,and there is a relationship of mutual regulation between them.3.miR-375-3p mimics and CCAT1 3’-UTR double luciferase labeling assay results showed that CCAT1 3’-UTR WT+mimics group was significantly different from CCAT1 3’-UTR MUT+mimics group,CCAT1 3’-UTR MUT+NC group,and CCAT13’-UTR WT+ NC group.Overexpression of miR-375-3p significantly inhibited luciferase activity in CCAT1 3’-UTTR WT plasmid.These results suggest that CCAT1 and miR-375-3p may have mutual binding.In the RIP experiment,compared with IgG negative control group,CCAT1 and miR-375-3p expression levels of Ago2 antibody group were significantly increased,with statistically significant differences.These experimental results further confirmed the possibility of mutual binding between CCATl and miR-375-3p.4.In HepG2 and QGY-7703 cells,exogenous increase of CCAT1 or inhibition of the expression of miR-375-3p would promote cell growth.5.Western Blot showed that solasonine could inhibit the expression of SP1 and the inhibition was enhanced with the increase of solasonine dose.Both the HepG2 cell line and the QGY-7703 cell line suggested that the expression of SP1 was significantly reduced when the concentration of solasonine was in 30μ M,40p M and 50μ M.Exogenous overexpression of CCAT1 can promote the expression of SP1;while overexpression of miR-375-3p can inhibit the expression of SP1.The results indicated that,in HepG2 and QGY-7703 cells,CCAT1 and miR-375-3p can jointly regulate the expression of SP1.Exogenous overexpression of SP1 could reverse the inhibitory effect of solasonine on CCAT1 and reverse the promoting effect of solasonine on the expression of miR-375-3p.These results indicated that SP1 was in the downstream pathway of L CCAT1 and miR-375-3p,but SP1 could regulate the expression of both.6.Western Blot showed that solasonine could inhibit the expression of IRF5 and the inhibition was enhanced with the increase of solasonine dose.The expression of IRF5 in HepG2 cells was significantly decreased when the concentration of solasonine in 40 and 50μM,and the expression of IRF5 in QGY-7703 cells was significantly decreased when the concentration of solasonine in 30,40 and 50μ M.The difference was statistically significant compared with the blank group.Solasonine can not only down-regulate the expression of IRF5 protein,but also reduce the activity of IRF5 promoter.Compared with the control group,IRF5 promoter activity in the SS treatment group decreased significantly,and the difference was statistically significant7.Exogenous overexpression of SP1 can reverse the down-regulation effect of solasonine on IRF5 protein and promoter activity.The binding of SP1 in the promoter region of IRF5 was verified.SP1 regulates the expression of IRF5 by binding to the IRF5 promoter region.8.Exogenous overexpression of IRF5 can reverse the inhibitory effect of solasonine on CCAT1 and promote the expression of miR-375-3p,and promote the proliferation of liver cancer cells.IRF5 has feedback regulation effect on both CCAT1 and miR-375-3p.In vivo animal experiments:In vivo animal experiments also showed that solasonine can inhibit the proliferation of liver cancer cells,reduce the expression of CCAT1,SP1 and IRF5,and promote the expression of miR-375-3p.Conclus ions:Solasonine can inhibit the growth of HCC cells by activating miR-375-3p and inhibiting the expression of CCAT1,SP1 and IRF5.Under the action of solasonine,miR-375-3p and CCAT1 can regulate each other and jointly inhibit the expression of downstream SP1 protein.After the expression of SP1 protein was inhibited,the expression of IRF5 protein was finally inhibited.In vitro experiments also confirmed that solasonine acts through RNA and proteins consistent with in vivo experiments.In part,this study revealed a new mechanism by which solasonine inhibited the growth of hepatocellular carcinoma cells and suggested that IRF5 could be used as a target for the treatment of hepatocellular carcinoma.