| Background Hepatocellular carcinoma(HCC)is one of the most common cancer worldwide.It is a typically aggressive cancer and the third leading cause of cancer-related death in the world.Myriads of causes and risk factors have been identified to function in the development of HCC,while the main treatment is surgical resection with limited other treatment modalities available.Therefore,it is crucial to investigate the molecular mechanism involved in pathogenesis and progression in HCC,and accordingly develop therapeutic strategies targeting specific molecules implicated.Transforming growth factor-beta(TGF-?)signaling pathway plays an important role in orchestrating a microenvironment for tumor cell growth and promotes HCC progression.Its roles evolve in different stages in HCC progression.In the early stage,TGF-?1 inhibits cellular proliferation,while it promotes tumor progression and metastasis in the later stage.Based on current understanding,its paradoxical dual effects relate to phosphorylation of C-terminal or L region of Smad3.p Smad3 C signals for growth inhibition and p Smad3 L signals for oncogenesis,which makes the transition from p Smad3 C to p Smad3 L,a pivotal link in the oncogenesis of HCC.Micro RNAs(mi RNAs)are small endogenous noncoding RNAs that are involved in oncogenesis.In HCC,mi R-21 and mi R-145 have been shown to be expressed and contribute to the maintenance of oncogenic phenotype through the regulation of target gene expression,with mi R-21 as an oncomi R and mi R-145 as an anti-oncomi R. Previous studies demonstrated that mi R-145/mi R-21 interact with p Smad3C/p Smad3 L,which subsequently influences the progression of HCC.Consequently,the intervention of mi R-145/mi R-21 and p Smad3C/3L provides a promising therapeutic approach for HCC.Serials of studies in our group found that Compound Astragalus and Salvia miltiorrhiza extract(CASE)inhibits tumor growth through promoting p Smad3 C and inhibiting p Smad3 L both in vitro and in vivo.Recent study also demonstrates that CASE upregulates mi R-145 and downregulates mi R-21,both in DEN-induced rat HCC model and TGF-?1 stimulated Hep G2 cell model.Hence,we speculate that the therapeutic effect of CASE depends on the crosstalk between mi R-145/mi R-21 and p Smad3C/p Smad3 L in hepatocellular carcinoma.Using two distinct experimental systems: established HCC cell lines transfected with Smad3C/3L mutation plasmids or mi R-21 agomir/mi R-145 antagomir and nude mice xenograft model to further investigated the therapeutic effect of CASE and the molecular mechanisms involved.Objectives 1.To observe the effect of CASE on the biological behavior of tumor cells in vitro and tumor growth in vivo,using mi R-145-knockdown or mi R-21-overexpressed Hep G2 cell line and nude mice xenograft model respectively,in order to illustrate the correlation between tumor suppression of CASE and mi R-145/mi R-21;2.To observe the effect of CASE on TGF-? signaling pathway in mi R-145-knockdo-own or mi R-21-overexpressed Hep G2 cell line and nude mice xenograft model respectively,in order to illustrate the correlation between tumor suppression of CASE and mi R-145/mi R-21 regulating TGF-? signaling pathway;3.To illustrate the involvement of TGF-? signaling pathway in the regulation of mi R-145/mi R-21 expression by CASE,using Hep G2 cell lines stably transfected with Smad3 WT,Smad3 EPSM,or Smad3 3S-A plasmids and nude mice xenograft models.Methods 1.Establish mi R-145-knockdown or mi R-21-overexpressed Hep G2 cell line to observe the effect of CASE on the biological behavior of tumor cells in vitro and possible molecular mechanisms involved.Hep G2 cells were transfected with mi R-145 antagomir,mi R-21 agomir,related negative controls antagomir NC or agomir NC respectively,and then treated with or without CASE(80 ?g/ml)for 24 h.Simultaneously,the cells were stimulated with TGF-?1 for the determined concentrations and time depending on the different purposes.MTT assay was used to evaluate proliferation.Cell wound scratch assay was used to analyze the effects on migration in vitro.Flow cytometry was used to evaluate cell apoptosis.q RT-PCR was used to analyze the mi R-145/mi R-21 expression.Western blot was used to evaluate the expression of T?RII,Smad3,p Smad3 C,p Smad3 L,p ERK1/2,ERK1/2,p JNK1/2,JNK1/2,pp38 and p38.2.Establish mi R-145-knockdown or mi R-21-overexpressed nude mice xenograft models to investigate the effect of CASE on the oncogenesis in vivo and possible molecular mechanisms involved.Hep G2 cell suspensions(9×106)were injected subcutaneously into right flank area to establish nude mice models.36 male BALB/c nude mice were grouped as follows,with 6 mice in each group: antagomir NC group,mi R-145 antagomir group,mi R-145 antagomir+CASE group,agomir NC group,mi R-21 agomir group,and mi R-21 agomir +CASE group.After tumor formation,mi R-145 antagomir(20 ?l),antagomir NC,mi R-21 agomir and agomir NC were respectively injected into xenograft every four days,for a total of 28 days.The mice in CASE groups were intragastrically administrate CASE(310 mg/kg)per day for 36 days,while 0.5% CMC-Na were administrated to the model groups.Mice were sacrificed at the end of experiment,and tumor tissues were harvested.Tumor weight and volume were measured and recorded.Pathological characteristics were evaluated via H&E staining.q RT-PCR was used to analyze the mi R-145/mi R-21 expression.Western blot was used to evaluate the protein level of T?RII,Smad3,p Smad3 C,p Smad3 L,p ERK1/2,ERK1/2,p JNK1/2,JNK1/2,pp38 and p38.3.Further study the effect of CASE on mi R-145/mi R-21 expression using three Hep G2 cell lines stably transfected with Smad3 WT,Smad3 EPSM,or Smad3 3S-A plasmids.The Hep G2 cell lines stably transfected with Smad3 WT,Smad3 EPSM,or Smad3 3S-A plasmids respectively,were established by our group screened using G418.Then all three cell lines were intervened by CASE(80 ?g/ml)and stimulated by TGF-?1(9 pmol/L).q RT-PCR was subsequently used to analyze the mi R-145/mi R-21 expression.4.Establish nude mice xenograft models with Hep G2 cell lines stably transfected with Smad3 WT,Smad3 EPSM,or Smad3 3S-A,to investigate the effect of CASE on the oncogenesis in vivo and possible molecular mechanisms involved.Hep G2 cells stably transfected with Smad3 WT,Smad3 EPSM,or Smad3 3S-A were injected into right flank area to establish nude mice models.48 male BALB/c nude mice were grouped as follows,with 6 mice in each group: Hep G2 group,Hep G2+ CASE group,Smad3 WT-Hep G2 group,Smad3 WT-Hep G2+CASE group,Smad3 EPSM-Hep G2 group,Smad3 EPSM-Hep G2+CASE group,Smad3 3S-A-Hep G2 group,and Smad3 3S-A-Hep G2+CASE group.The mice in CASE groups were intragastrically administrated CASE(310 mg/kg)per day for 36 days,while 0.5% CMC-Na were administrated to the model groups.Mice were sacrificed at the end of experiment,and tumor tissues were harvested.Tumor weight and volume were measured and recorded.Pathological characteristics were evaluated via H&E staining.Transmission electron microscope was used assess the cellular ultrastructures.q RT-PCR was used to analyze the mi R-145/mi R-21 expression.Western blot was used to evaluate the protein level of p Smad3C/p Smad3 L.Results 1.The effect of CASE on the biological behavior of tumor cells in vitro and the regulation of TGF-? pathway 1.1 The effect of CASE on the biological behavior in mi R-145-knockdown or mi R-21-overexpressed Hep G2 cell line Compared with NC group,mi R-145 antagomir remarkably promote cell proliferation(P<0.01)and cell migration(P<0.01),while remarkably inhibit cell apoptosis(P<0.01).CASE treatment could significantly inhibit cell proliferation(P<0.01)and migration(P<0.01),while significantly promote cell apoptosis(P<0.01).Compared with NC group,mi R-21 agomir remarkably promote cell proliferation(P<0.01)and cell migration(P<0.01),while remarkably inhibit cell apoptosis(P<0.01).CASE treatment could significantly inhibit cell proliferation(P<0.01)and migration(P<0.01),while significantly promote cell apoptosis(P<0.01).1.2 The effect of CASE on mi R-145/mi R-21 expression in mi R-145-knockdown or mi R-21-overexpressed Hep G2 cell line Compared with NC group,mi R-145 antagomir decreases mi R-145 expression(P<0.01),while CASE treatment significantly reverses this effect(P<0.01);likewise,mi R-21 agomir increases mi R-21 expression(P<0.01),while CASE treatment significantly reverses this effect(P<0.01). 1.3 The effect of CASE on regulation of TGF-? signaling pathway in mi R-145-knockdown or mi R-21-overexpressed Hep G2 cell line After TGF-?1 stimulation,compared with NC group,the T?RII,p Smad3 L,Smad3,p JNK1/2,and pp38 expression are increased in mi R-145-knockdown Hep G2 cell line,with no remarkable change in p ERK1/2.Treatment with CASE reverses the observed protein changes by mi R-145,at the meantime,inhibits p ERK1/2.Similarly,compared with agomir NC group,the T?RII,p Smad3 L,p ERK1/2,p JNK1/2,and pp38 expression are increased in mi R-21-overexpressed Hep G2 cell line after TGF-?1 stimulation;CASE treatment significantly decreases T?RII,p Smad3 L,p ERK1/2,p JNK1/2,and pp38 expression and increases p Smad3 C,with no change in Smad3.2.The effect of mi R-145-kncokdown or mi R-21-overexpressed on tumor growth in vivo and the therapeutic effect of CASE 2.1 CASE intervention inhibits ontogenetic effect in mi R-145-kncokdown or mi R-21-overexpressed nude mice xenograft model Hep G2 cells were injected into right flank area to establish nude mice models.Compared with antagomir NC group,tumor weight and volume are significantly increased in mi R-145 antagomir injection groups(P<0.01).Furthermore,CASE gavage significantly inhibits tumor weight and volume compared with untreated groups(P<0.01);likewise,compared with agomir NC group,tumor weight and volume are significantly increased in mi R-21 agomir injection groups(P<0.01).Furthermore,CASE gavage significantly inhibits tumor weight and volume compared with untreated groups(P<0.01).2.2 The effect of CASE on pathologic changes in mi R-145-kncokdown or mi R-21-overexpressed nude mice xenograft model Compared with antagomir NC group,tumor cells arrayed as nest,the nuclear was big,deep-stained,pathologic karyokinesis can be seen in mi R-145 antagomir group.In mi R-145 antagomir+CASE group,there were cells with nuclear pyknosis,karyorrhexia,and karyolysis.In mi R-21 agomir group,cells with greater nucleus-to-cytoplasm(N/C)ratio were densely arranged,which suggested mi R-21 promoted the growth of the tumor cells.In the mi R-21agomir+CASE group,cells were showed more nuclear pyknosis and nuclear fragmentation.It was a clue of apoptosis.2.3 The effect of CASE on expression of mi R-145/mi R-21 in mi R-145-kncokdown or mi R-21-overexpressed nude mice xenograft model Compared with antagomir NC group,mi R-145 expression decreases significantly in mi R-145 antagomir group(P<0.01),while CASE gavage significantly reverses the mi R-145 reduction(P<0.01).Comparably,mi R-21 expression increases significantly in mi R-21 agomir group compared with agomir NC group(P<0.01),while CASE treatment also significantly reverses the mi R-21 elevation(P<0.01).2.4 The effect of CASE on regulation of TGF-? pathway in mi R-145-kncokdown or mi R-21-overexpressed nude mice xenograft model Compared with antagomir NC group,the expression of T?RII,p Smad3 L,Smad3,p ERK1/2,p JNK1/2,and pp38 expression are remarkably increased demonstrated by Western blot,after injection of mi R-145 antagomir,with dramatic decrease in p Smad3 C and no change in total ERK1/2,JNK1/2,and p38.After CASE administration,these effects are reversed,demonstrated by remarkable decrease in T?RII,p Smad3 L,Smad3,p ERK1/2,p JNK1/2,and pp38,and increase in p Smad3 C.Similarly,under the influence of mi R-21 agomir,the expression of T?RII,p Smad3 L,p ERK1/2,p JNK1/2,and pp38 expression are increased and no noticeable chan ges in Smad3,p Smad3 C,ERK1/2,JNK1/2,and p38.CASE gavage dramatically intervened these elevations,causing decrease in T?RII,p Smad3 L,p ERK1/2,p JNK1/2,and pp38,and increase in p Smad3 C.3.The effect of CASE on expression of mi R-145/mi R-21 in Hep G2 cell lines stably transfected with Smad3 WT/Smad3 EPSM/Smad3 3S-A CASE treatment significantly upregulates mi R-145 and downregulates mi R-21 in all three cell lines after TGF-?1 stimulation(P<0.01),of which the effect is most prominently seen in Smad3 EPSM-Hep G2 cell lines.4.The therapeutic effect of CASE in nude mice xenograft models established with Hep G2 cell lines stably transfected with Smad3 WT/Smad3 EPSM/Smad3 3S-A 4.1 The effect of CASE on tumor growth and pathologic changes Hep G2 cells stably transfected with Smad3 WT,Smad3 EPSM,or Smad3 3S-A were injected respectively into right flank area to establish nude mice models.CASE gavage inhibits subcutaneous tumor growth to a different extent within different treatment groups.Compared with Smad3 WT-Hep G2+CASE group,the average tumor weight and volume are smaller in Smad3 EPSM-Hep G2+CASE group,while the average tumor weight and volume are larger in Smad3 3S-A-Hep G2+CASE group.When compared to their own untreated group,CASE administration induces cell apoptosis or tissue necrosis in H&E staining.This phenomenon is most prominent in tumors harvested from Smad3 EPSM-Hep G2+CASE group which is demonstrated as nuclear pyknosis,karyorrhexia,and karyolysis;while the tumors harvested from Smad3 3S-AHep G2+CASE group illustrate the opposite side of the spectrum,with hyperchromatic nuclei and conspicuous mitotic figures.Ultrastructural findings using transmission electron microscope are consistent with the above histological findings,with the most apoptotic bodies found in Smad3 EPSM-Hep G2+CASE group,indicating vacuolation;while tissue samples from Smad3 3S-A-Hep G2+CASE group demonstrate early apoptotic changes with slight cellular shrinkage,scarce vacuoles and intact nuclear.4.2 The effect of CASE on mi R-145/mi R-21 expression CASE treatment increases mi R-145 and decreases mi R-21 in all four groups,with the most pronounced finding seen in Smad3 EPSM-Hep G2+CASE group.4.3 The effect of CASE on p Smad3C/p Smad3 L expression Western blot using tissue proteins harvested from each group shows that CASE administration upregulates p Smad3 C and downregulate p Smad3 L.This observation is also most prominent in Smad3 EPSM-Hep G2+CASE group compared with Smad3 WT;while both p Smad3 C and p Smad3 L are downregulated in Smad3 3S-A-Hep G2+CASE group.Conclusions 1.Following upregulation of mi R-145,CASE promotes the transition of p Smad3 L to p Smad3 C via TGF-?/Smad and MAPK pathways,which may serve as a possible mechanism in its tumor suppression effect.2.By downregulation of mi R-21,CASE primarily reverses its regulation on MAPK pathway to inhibit p Smad3L;meanwhile,CASE indirectly promotes p Smad3 C protein expression via TGF-?/Smad pathway.3.Upregulation of p Smad3 C enhances mi R-145 expression and inhibits mi R-21,which synergistically improves the therapeutic effect of CASE in HCC;while upregulation of p Smad3 L impairs the effect of CASE.In summary,CASE exerts its significant anti-hepatocellular carcinoma effects in Hep G2 cells and nude mice xenograft models.On the one hand,CASE regulates mi R-145/mi R-21 to promotes the transition of p Smad3 L to p Smad3 C.On the other hand,p Smad3C/p Smad3 L regulates the expression of mi R-145/mi R-21 to improve or reduce the therapeutic effect of CASE in HCC.In sum,the therapeutic effect of CASE in HCC relies on the interaction between mi R-145/mi R-21 and p Smad3C/p Smad3 L. |
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