CASE Suppresses Migration Of HepG2 Cells Transfected With Smad3 WT,Smad3 3S-A And Smad3 EPSM Via The MAPK Pathway

Background Hepatocellular carcinoma(HCC),It is known as "the king of cancer",because of its insidious onset,long incubation period,high degree of malignancy,short survival time,poor quality of life,and the intractable treatment.So it is important to develop new drugs to prevent and cure HCC.Compound Astrag alus and Salvia miltiorrhiza extract(CASE)is mainly of Astragalus and salvia extract Astragalosides and astragalus polysaccharides,salvianolic acid constituents in the best proportion.TGF-?1 signaling pathway has been proved to be one of the most closely signaling pathways related to HCC.It is play a different role in different stages of tumor growth.On the one hand,the inhibitory effect of TGF-?1 on tumorigenesis and develo pment is the phosphorylation of Smad3 Cregion(p Smad3C),by activat ing the TGF-?1 receptor(TGF-?1 type I,TR?I);on the other hand,the effect of TGF-?1 transfer cancer-promoting signals promote tumorigenesis signals is through the activation of MAPK signal pathway to promote the linker phosphorylated Smad3(p Smad3L).T? RI/p Smad3 C transfer tumor suppressor signal,MAPK/p Smad3 L signaling promotes tumorigenesis and development.Our previous studies showed that CASE against hepatocellular carcinoma may be related to the amount of p Smad3C/3L,then we construct three plasmids Smad3 WT,Smad3 EPSM and Smad3 3S-A stably transfected HepG2 cell lines and validated.In the early studies have shown that CASE play a role in the inhibition of TGF-?1 induced proliferation and promote apoptosis of the transfected cells with different extents.However,the effect of CASE on the selective p Smad3 C/3L HepG2 cell scratch was not reported.Objectives1.The effect of CASE on the apoptosis and proliferation of HepG2 cell with selective upregulation of p Smad3C/3L has been identified,On this basis,we continued to observe the effect of CASE on the migration of HepG2 cells with selective up regulation of p Smad3C/3L.2.In order to clarify the molecular mechanism of CASE effect on scratch in He p G2 cells with the selective upregulation of p Smad3C/3L,we observe the effect of CASE on the activation of MAPK signaling pathway in HepG2 cells with hig h expression of p Smad3C/p Smad3 Land transplanted tumor of nude mice model.3.To make clear the effect of MAPK pathway activation on cell migration,prol iferation,apoptosis and cell cycle of HepG2 cells with selective up regulation of p Smad3C/3L.Methods1.The cell scratch assay was used to observe the migration ability of HepG2 cells transfected with Smad3 WT,Smad3 EPSM and Smad3 3S-A three plasmids under CASE intervention in vitro The three plasmids of Smad3 WT,Smad3 EPSM and Smad3 3S-A transfect ed HepG2 cell have been successively constructed and verified.cell culture,scratc h injury,with or without treated with 9 pmol/L TGF-?1 in the absence or presence of CASE(20mg/L).Pictures were taken at 0,12,24 h time point under the mic roscope.2.The expression of MAPKs protein in transplanted tumor model of nude mice was detected by Western blot method Nude mice were inoculated with Smad3 WT,Smad3 EPSM and Smad3 3S-A three plasmid transfected HepG2 cells.The nude mice were concomitantly admini stered CASE at the doses of 310 mg/kg/d.we detect the expression of p-ERK1/2,p-JNK1/2,p-p38 by Western-blot,the expression of non-phosphorylated ERK1/2,JNK1/2 or p38 was measured as the reference,respectively.3.Western blot method was used to detect the expression of MAPKs protein in HepG2 cells transfected with Smad3 WT,Smad3 EPSM and Smad3 3S-A under CASE interference After incubation,with or without CASE(20mg/L)treatment,treated with 9 pmol/L TGF-?1 before 1 hours the end of cell culture,Western-blot method was used to detect the expression of p-ERK1/2,p-JNK1/2,p-p38 JNK1/2,we measure the expression of non-phosphorylated ERK1/2,pp38 or JNK1/2 as the reference,respectively.4.The cell scratch assay was used to observe the migration ability of three kind s of MAPK inhibitor on HepG2 cells transfected with Smad3 WT,Smad3 EPSM and Smad3 3S-A three plasmids Cell cultures were subjected to scratch injury,in the absence or presence of three MAPK inhibitors,while treated with 9 pmol/L TGF-?1 co-culture,Pictures were taken at 0,12,24 h time point after wounded.5.The cell proliferation of three kinds of MAPK inhibitor on HepG2 cells transfected with Smad3 WT,Smad3 EPSM and Smad3 3S-A three plasmids be detect ed by MTT assays.Cell culture,Before the end of the experiment 5h,with or without three kinds of MAPK inhibitor treatment,with or without 9 pmol/L TGF-?1were cultured.we use MTT to detected cell proliferation.6.Flow cytometry was used to detect the effects of three MAPK inhibitors on the apoptosis of HepG2 cells transfected with the three plasmids Cell culture,Before the end of the experiment 5h,with or without three kind s of MAPK inhibitor treatment,Before the end of the experiment 1h,with or wi thout 9 pmol/L TGF-?1.Cell apoptosis were measured by flow cytometry.Results1.The effect of CASE on the migration of HepG2 cells transfected with Smad3 WT,Smad3 EPSM and Smad3 3S-A three plasmids Cell scratch test results showed that CASE could inhibit the migration of HepG2 cells transfected with three plasmids to scratch wounds in different degrees,compared with Smad3 WT or Smad3 3S-A plasmid group,CASE had the strongest inhibitory effect on the migration ability of HepG2 cells transfected with Smad3 EPSM plasmids induced by TGF-?1.2 Effects of CASE on MAPK signaling pathway2.1 Effects of CASE on MAPK signaling pathway of in transplanted tumor model of nude mice The results showed that CASE could inhibit the expression of p-ERK in transfected with Smad3 EPSM,Smad3 3S-A plasmid groups.The effect of CASE on p-ERK expression in transfected Smad3 EPSM plasmid group was more obvious.CASE coulddown regulate the expression of p-JNK in three kinds of plasmids in different degrees.Compared with Smad3 EPSM and Smad3 WT plasmid group,CASE decreased the most obvious p-JNK in transfected Smad3 3S-A plasmid group.CASE up regulate the expression of p-p38 in different degrees.Compared with the transfected Smad3 WT plasmid group,the ability of CASE to regulate pp38 in the transfected Smad3 EPSM cells is more obvious.2.2 Effects of CASE on MAPK signaling pathway of HepG2 cells transfected with Smad3 WT,Smad3 EPSM and Smad3 3S-A three plasmids induced by TGF-?1.The results showed that CASE could inhibit up-regulation of p-ERK in HepG2 cells transfected with Smad3 EPSM and Smad3 by TGF-?1,and the effect of CASE on the expression of p-ERK down regulated by transfection of Smad3 EPSM plasmid group was more obvious than that of 3S-A.CASE could down regulate the expression of p-JNK in three kinds of plasmids in different degrees,and the expression of p-JNK in Smad3 EPSM-HepG2 cells was significantly lower than that of Smad3 3S-A-HepG2 cells.CASE can up regulate the expression of p-p38 in different degrees,and compared with the transfection of Smad3 WT plasmid group,the ability of CASE to enhance the ability of p-p38 in transfected Smad3 EPSM and Smad3 3S-A plasmid group is more obvious.3 Effect of three kinds of MAPK inhibitor on the cellular function of HepG2 cells transfected with Smad3 WT,Smad3 EPSM and Smad3 3S-A three plasmids induced by TGF-?1.3.1 Effects of three kinds of MAPK inhibitors on the migration of HepG2 cells transfected with the three plasmid induced by TGF-?1.Three kinds of MAPK inhibitors could inhibit the migration of HepG2 cells transfected with the three plasmid induced by TGF-?1.Compared with transfected Smad3 WT,Smad3 3S-A plasmid groups,three kinds of MAPK inhibitors had thestrongest inhibitory effect on the migration ability of HepG2 cells transfected with Smad3 EPSM plasmids induced by TGF-?1.3.2 Effects of three kinds of MAPK inhibitors on the proliferation of HepG2 cells transfected with the three plasmid induced by TGF-?1.The results of MTT showed that the three inhibitors can inhibit the proliferation of TGF-?1 in different degrees.Compared with the HepG2 cells transfected with Smad3 WT or Smad3 3S-A,transfected with Smad3 EPSM plasmid had the strongest inhibitory effect on proliferation.3.3 Effects of three kinds of MAPK inhibitors on the cell apoptosis of HepG2 c ells transfected with the three plasmid induced by TGF-?1.According to the results of apoptosis showed that the three kinds of MAPK inhibitors had the most obvious effect on the apoptosis of Smad3 EPSM plasmid group,but Smad3 3S-A plasmid group was the weakest.Conclusions CASE may reduce the cell migration ability of Smad3 WT,Smad3 EPSM,Smad33S-A plasmid transfected HepG2 cells by inhibiting the activation of ERK,JNK pathway and stimulating the activation of p38 pathway,and migration gap may be related to p Smad3C/3L.