| ObjectiveHepatocellular carcinoma is a common malignant tumor worldwide,and it is a serious danger to the mankind.Recent studies have shown that hepatitis virus infection,especially hepatitis B virus,is the leading cause of liver cancer.According to clinical survey data,about 10%of viral hepatitis will develop into chronic active hepatitis,while 50%of chronic active hepatitis may develop into liver cirrhosis,and 9.9%-16.6%of liver cirrhosis will develop into cancer.There are many people with hepatitis B virus in China at present.The incidence and the number of patients in China are the highest in the world.The therapeutic drugs for chronic HBV infection are mainly nucleoside analogues(such as adefovir dipivoxil,lamivudine,etc.)and interferon alpha,which can effectively inhibit the replication of the virus,delay the disease progression.But,there are still some drawbacks,such as the disease recrudescence and virus resistance after long-term nucleoside(acid)analogues treatment,adverse reactions of interferon,etc.It is impossible to achieve a complete cure for HBV.Recently,CRISPR/Cas9 system is able to destruct HBV cccDNA,indicating a efficient strategy for the cure of chronic hepatitis B.It has been found that Stat3 as an oncogene is highly expressed and continuously activated in many hematological and solid tumor,and also exhibits high expression in HBV-infected livers.In recent years,small interfering RNA,decoy oligonucleotides and other techniques which can directly inhibit the expression of Stat3 from the mRNA level,have shown a good anti-tumor effect in vitro and in vivo.Currently,HBV infection is known to be an important risk factor for HCC development and the HBV x gene is known to be the smallest open reading frame in the HBV genome and consists of 462 nucleotides.It is most easily integrated into the host genome.The x protein plays an important role in the transcription of HBV DNA.In hepatic carcinoma with HBV infection,HBx and Stat3 can interact with each other,HBx can promote Stat3 phosphorylation,and phosphorylated Stat3 enters into the nucleus and further promotes the replication of HBV and cancer deterioration.The current study found that silencing the expression of both Stat3 and HBx gene showed a promising anti-tumor effect.So whether the combined strategy of silencing two genes has the effect of "One plus one is greater than two"?This research expands further research for this issue first.Liver fibrosis is caused by excessive deposition of extracellular matrix,and if this state persists for long,it can easily transform into liver cancer.Reversing the process liver fibrosis can effectively reduce the occurrence of liver cancer.studies have shown that a class of drugs widely used in the treatment of cardiovascular diseases.Calcium channel antagonists have a protective effect on pulmonary fibrosis which can also inhibit the progression of liver fibrosis,cirrhosis.These drugs can reduce the portal vein pressure and serum hyaluronic acid levels.However,little is known about the exact efficacy and mechanisms of nimodipine against early-stage liver fibrosis.Therefore,this study observed the preventive and therapeutic effects of nimodipine on thioacetamide(TAA)-induced hepatic fibrosis in mice and discussed its possible mechanism.Here,our study was divided into two parts:One is that CRISPR/Cas9 gene editing technology was used to construct a bifunctional vector that can knock out the Stat3 and HBx genes simultaneously,and its therapeutic effect was evaluated.The other is therapeutic effects of nimodipine as one of calcium channel antagonist in TAA-induced liver fibrosis in mice.Part Ⅰ Construction of vectors targeting both Stat3 and HBx using CRISPR/Cas9 gene editing technique and their therapeutic e ffe ct on HBV-related liver diseasesMethods1.PX333 was digested with Bsal,linear fragments were recovered by agarose gel,and three pairs of sgRNAs targeting Stat3 gene and two pairs of sgRNAs targeting HBx gene were annealed to become complementary paired double strands.Then the reco vered fragments and the complementary double-strands were ligated with T4 ligase,the products were transformed into Escherichia coli and single clones were selected for sequencing.2.The protein level of Flag-tag in PX333 was detected by Immunofluorescence,and the protein level of Stat3 was detected by Western Blot.3.The proliferation of HepG2.2.15 cells transfected with vector knocking out Stat3 gene was detected by MTT assay,the confluence of HepG2.2.15 cells was monitored by JuLI Stage Living cell monitoring system every six hours.4.The migration of HepG2.2.15 cells transfected with vector knocking out Stat3 gene was detected by scratch test;the gene levels of VEGF,MMP9,IL-10 and TGF-β were detected by quantitative PCR.5.We amplify the gene sequence targeted by Stat3-sgRNA1 and Stat3-sgRNA2 vector by quantitative PCR.After T7E1 digestion,the mutation of the target site was detected by agarose gel electrophoresis.The amplification sequences of target site were ligated with the M5 Hper pTOPO-Blunt sequencing plasmid,and after trans formation,single clones were sequenced6.The protein levels of HBx and Stat3 in HepG2.2.15 cells transfected with vector knocking out HBx gene were detected by Western Blot;the gene level of HBx gene was detected by quantitative PCR;the level of HBsAg in the supernatant of HepG2.2.15 cells was detected by ELISA.7.After HBx-sgRNA2 vector was digested with BbSI,the linear fragment was recovered by agarose gel.Then,the synthesized sgRNAl sequence that targets the Stat3 gene and the sgRNA sequences that target the HBx gene were annealed to form complementary paired double strands.Finally,the recovered linear fragment and the sgRNA complementary paired double strand were connected by T4 ligase,and the products were transformed into Escherichia coli,and single clones were selected for sequencing to verify the successful construction of tandem vector.8.The silencing effect of vectors such as Stat3-sgRNA1,HBx-sgRNA1,HBx-sgRNA2,Stat3-sgRNA1-HBx-sgRNA2,HBx-sgRNA1-HBx-sgRNA2,HBx-sgRN A2-HBx-sgRNA2 was detected by CCK8 assay,and the confluence on HepG2.2.15 cells was also monitored by JuLI Stage Living cell monitoring system every six hours.9.The apoptosis of HepG2.2.15 cells transfected with different plasmids was detected by Annexin V-PI staining.Results1.Sequencing results showed that three sgRNA sequences targeting Stat3 and two sgRNA sequences targeting HBx were successfully inserted into the PX333 vector.2.The results of immunofluorescence showed that the CRISPR/Cas9 vectors could be normally expressed in HepG2.2.15 cells.Western blot analysis showed that Stat3-sgRNA1 and Stat3-sgRNA2 could reduce the protein level of Stat3.3.Stat3-sgRNA1 can inhibit the proliferation of HepG2.2.15 cells by MTT assay.JuLI Stage Living cell monitoring system showed the same results,and the Stat3-sgRNA2 can also affect the proliferation of HepG2.2.15 cells.4.Stat3-sgRNA1 and-sgRNA2 plasmids could inhibit the migration of HepG2.2.15 cells after treated for 24h and 36h.Both Stat3-sgRNA1 and Stat3-sgRNA2 could reduce the gene levels of VEGF and MMP9,and have the tendency to reduce the gene levels of IL-10 and TGF-β.5.T7E1 digestion results showed that Stat3-sgRNA1 can mutate the gene of the target site.Sequencing results showed that gene editing such as base mutation occurred near the Stat3 target site in HepG2.2.15 cells after transfected with Stat3-sgRNA1 or Stat3-sgRNA2 vectors.6.HBx-sgRNA2 vector can significantly reduce the protein and gene level of HBx.Both HBx-sgRNAl and HBx-sgRNA2 could reduce the level of HBsAg in the supernatant of HepG2.2.15 cells.7.Sequencing results showed that the oligo DNA of Stat3-sgRNA 1,HBx-sgRAN1 or HBx-sgRAN2 was successfully ligated into the HBx-sgRNA2 vector.8.The vectors such as Stat3-sgRNAl,Stat3-sgRNA1-HBx-sgRNA2,HBx-sgRNA1-HBx-sgRNA2,and HBx-sgRNA2-HBx-sgRNA2 all inhibited the proliferation of HepG2.2.15 cells.The results of JuLI Stage Living cell monitoring system showed that Stat3-sgRNAl-HBx-sgRNA2 significantly inhibited the proliferation of Hep G2.2.15 cells.9.Vectors such as Stat3-sgRNA1-HBx-sgRNA2,HBx-sgRAN 1-HBx-sg-RNA2,and HBx-sgRAN2-HBx-sgRNA2 all promoted HepG2.2.15 cell apoptosis significantly,and Stat3-sgRNA1-HBx-sgRNA2 vector was the best to promote apoptosis,and the early and late apoptosis showed the same trend.Conclusions1.This study successfully constructed three vectors targeting Stat3 and two vectors targeting HBx according to CRISPR/Cas9 system,and their silencing effects were evaluated that Stat3-sgRNA1 can inhibit the proliferation and migration,promote thr apoptosis of HepG2.2.15 cells.HBx-sgRNA2 can inhibit the expression of HBx protein and reduce the production of HBsAg.2.This study successfully constructed three different tandem sgRNA vectors:Stat3-sgRNA1-HBx-sgRN A2,HBx-sgRAN1-HB x-s g-RNA2,and HBx-sgRAN2-HBx-sgRNA2.Stat3-sgRNA1-HBx-sgRNA2,a Stat3 and HBx dual-targe ting vector,significantly inhibited HepG2.2.15 cell proliferation and promoted apoptosis.3.This study silenced two kinds of molecules Stat3 and HBx closely related to the occurrence and development of hepatocellular carcinoma using the novel CRISPR/Cas9 gene editing technology,and preliminarily proved that it can inhibit the proliferation,migration,and promote apoptosis of HepG2.2.15 cells.It provides a new strategy and experimental basis to study the effect in the treatment of HBV-related liver disease by silencing of Stat3 and HBx simultaneously.Part Ⅱ Nimodipine reverses the progress of liver fibrosis induced by TAAMethods1.Hepatic fibrosis model was established by intraperitoneal injection of TAA in mice and treated with nimodipine at the same time.The mice were sacrificed after injection of TAA for 12 times.The collagen deposition in the liver was detected by Sirus Red staining.The protein levels of type Ⅰ collagen and α-SMA were detected by Western Blot.2.The gene levels of α-SMA,Collal,Acta2,AFP,GPC-3,TIMP1,MMP2,MMP9,TGF-β,1L-10 and TNF-α were detected by quantitative PCR.3.Hepatic inflammation was detected by HE staining,and the levels of ALT and AST in serum were determined by ALT and AST kits.4.The protein level of MAPK signaling pathway was detected by Western Blot.5.The spleen and liver were weighed and the spleen weight/body weight ratio and liver weight/body weight ratio were calculated.Liver monocytes were counted.The absolute number and ratio ofNK and NKT cells in the liver and spleen were detected by flow cytometry.And the selected mononuclear cells were cultured in vitro,the proportion of NK and NKT cells was measured after treatment with different doses of nimodipine.At the same time,NK active markers such as NKG2D,IFN-y and CD107a were detected by flow cytometry.6.The absolute number,proportion and activation of total T lymphocytes,CD4+T cells,CD8+T cells and CD4+T/CD8+T ratio in the liver and spleen were detected by flow cytometry.7.The proportion of MDSCs in the liver and spleen was detected by flow cytometry.8.The activation and apoptosis of hepatic stellate cells in liver frozen section was detected by immunofluorescence staining.9.The proliferation of LX2 cells treated with different doses of nimodipine was detected by MTT;the confluence of LX2 cells was continuously monitored by JuLI Stage Living cell monitoring system,the cycle of LX2 cells after nimodipine treatment was detected by PI staining.10.The apoptosis rate of LX2 cells treated with different doses of nimodipine was detected by AnnexinV-PI staining at 24h,the protein levels of Bax,Caspase3,Bcl-xl,p-JNK were detected by Western blot,the levels of anti-apoptosis gene such as Bc1-2,Bcl-xl,proapoptotic gene such as Bax,as well as CyclinDl and TGF-β were detected by quantitative PCR.Results1.Sirus Red staining showed that the area of positive staining of liver Sirius red was increased in the TAA model group compared with the control group,and collagen deposition was observed around the blood vessels.After nimodipine treatment,liver collagen deposition was significantly reduced in mice.The protein levels of α-SMA and Col1al were significantly down-regulated in the nimodpine treatment group compared with the TAA group.2.The levels of liver cancer and fibrosis related molecules such as α-SMA,Collal,TIMP1,AFP,GPC-3 and immunosuppressive molecule such as TGF-β were decreased after treatment with nimodipine compared with TAA group.The gene levels of IL-10 and TNF-α were significantly decreased,and Acta2 had a particular tendency to decrease,while the gene levels of matrix metalloproteinases MMP2 and MMP9 were increased.3.HE staining showed that a small amount of inflammatory cells infiltrated around the blood vessels and the cytopathic vacuolar-like structure of the hepatocytes,accompanied with the pathological phenomenon of bile duct hyperplasia between the interlobulars in the TAA model group.Afterwards,the infiltration of inflammatory cells in the liver of mice was reduced after treatment with nimodipine.At the same time,the levels of ALT and AST in the serum increased significantly in the TAA model group,which was reduced to normal levels by nimodipine treatment.4.The protein level of phosphorylated-JNK,-Stat3 was increased in the liver of the TAA model group compared with the control group,which was down-regulated after treatment with nimodipine,while the Erk and p38 signaling pathways were not significantly affected.5.Nimodipine can reverse the liver weight/body weight ratio and spleen weight/body weight ratio caused by TAA.Flow cytometry results showed that the total number of liver mononuclear cells of in TAA group and nimodipine treatment group was significantly increased.The total number of NK and NKT cells in the nimodipine group was significantly higher than that in the TAA group.Further analysis found that the proportion of NK cells increased but the proportion of NKT was lower than that of the control group.However,nimodipine does not activate mouse NK and NKT cells in vitro,which indicated that nimodipine influence NK or NKT cells indirectly.6.Flow cytometry results showed that the treatment with TAA upregulated the proportion of CD8+T cells,which was decreased by the treatment with nimodipine.At the same time,TAA down-regulated the proportion of CD4+T cells,which was increased by nimodipine and was more significant in the spleen.In addition,nimodipine reduced the activation of CD8+T and CD4+T cells,reversed the decrease in CD4+/CD8+T ratio induced by TAA.This similar phenomenon was also observed in the spleen.7.TAA upregulated the proportion of MDSCs,which was significantly inhibited by nimodipine treatment.8.The expression of α-SMA in TAA model group was significantly higher than that in control group,which was significantly decreased after nimodipine treatment.Co-localization of α-SMA and TUNEL revealed that nimodipine treatment can induce hepatic stellate cell apoptosis in the liver.9.Nimodipine inhibited the proliferation of LX2 cells even at a lower concentration of 10 nM,which was concentration-dependent.JuLI Stage Living cell monitoring system showed the same results.The proportion of LX2 cells in the G1 phase increased significantly after treatment with nimodipine,and the proportion of cells in the S phase and G2 phase decreased significantly,indicating the cells arrested in the G1 phase.When LX2 cells were treated with higher concentrations of nimodipine(50 nM),significant apoptotic peaks were observed.10.Nimodipine could promote the apoptosis of human hepatic stellate cell line LX2 cells in a concentration-dependent manner,but did not induce pro-apoptotic effects at a low dose of 10 nM.The levels of pro-apoptotic proteins such as Bax,Caspase3,p-JNK,and JNK increased and the levels of anti-apoptotic protein such as Bcl-xl decreased with the increase dose of nimodipine.The levels of anti-apoptotic genes such as Bcl-2,Bcl-xl and TGF-beta was decreased,the level of CyclinDl was decreased,and the level of pro-apoptotic gene such as Bax was increased.The above results indicated that nimodipine can inhibit the proliferation,cell cycle and promote the apoptosis of LX2 cells by activating the JNK pathway.Conclusions1.This study found that nimodipine can reduce the deposition of collagen,the inflammatory response,and the expression of inflammatory pathway-associated molecules,thereby playing a role in anti-fibrosis in the TAA-induced liver fibrosis.2.This study found that nimodipine may change the immune microenvironment by increasing the number of NK and NKT cells,altering the ratio of CD4+T,CD8+T and CD4+T/CD8+T in liver and decreasing the number of MDSCs to display anti-fibrosis effect.3.In this paper,nimodipine can inhibit the activation of hepatic stellate cells in vivo and promote the apoptosis of hepatic stellate cells by activating the JNK pathway in vitro.4.This study provides experimental basis for the application of calcium channel antagonists for hepatic fibrosis therapy,and provides new ideas for the development of new uses of old drugs. |
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