Roles Of Transforming Growth Factor-beta1-Smad Signaling Pathway In The Development Of Hepatocellular Carcinoma

It is now generally accepted that hepatocellular carcinoma developsby genetic alterations. Analysis of the molecular mechanism makes itpossible to develop a more targeted approach to prevention and treatmentof this cancer.The transforming growth factor-β1 (TGF-β1)-Smad signalingpathway has an important role in carcinogenesis. TGF-β1 acts as a ligandin tumor genesis suppressing cell growth in early stage and promotingcell growth in advanced stage. Smads are essential signal transducers ofTGF-β1 signaling pathway. Smad4 is an action substrate of TGF-β1receptor and conducts a central role in TGF-β1 signaling pathway.Mutation or lower expression of Smad4 has been observed in many kindsof tumors. In recent years, many investigations have revealed that themajor factors in TGF-β1-Smad signaling pathway such as receptors,Smad proteins, up-stream and down-stream regulators and cross-talkamong many signaling pathways function in gastrointestinal cancers, lungcancer and so on. However, in hepatocellular carcinoma, the precise rolesof TGF-β1-Smad are still not well understood. And there is also muchdifference in the existed data, some of which are even conflictive. In thepresent study, based on the clinical pathologic analysis, we investigated the roles of TGF-β1-Smad signaling pathway in the development ofhepatocellular carcinoma.ObjectivesTo explore the roles of TGF-β1-Smad signaling pathway in thepathogenesis and development of hepatocellular carcinoma.Methods(1) The expressions of TGF-β1, typeⅡtransforming growth factor-betareceptor (TβR-Ⅱ), nuclear factor-kappaB (NF-κB), Smad4 and Smad7proteins in 36 cases of hepatocellular carcinoma (HCC) and surroundingHCC tissue were separately detected by immunohistochemistry. ThemRNA and protein expression of Smad4 were further measured byRT-PCR and Western blot assays in human hepatocelluar line L02 andhepatocelluar carcinoma cell line SMMC-7721.(2) Three small hairpin RNA (shRNA) sequences targeting Smad4were designed by software and cloned into the expression vectorpGCsi-H1/Neo/GFP. DNA sequencing was used to confirm that theplasmids were constructed correctly. The constructed plasmids withdifferent shRNA and the control plasmid were transiently transferred intoAD293 cell line for 72 h. Fluorescene quantitative PCR was used todetect the efficiency of RNA interference (RNAi) against Smad4.(3) The effective sequence of small interfering RNA (siRNA) targetingSmad4 gene was confirmed in the previous study. The complementaryDNA containing both sense and antisense oligo-DNA of the targeting sequence was designed, synthesized and cloned into the pLVTHM vector,which contained H1 promoter and GFP. The resulting lentiviral vectorcontaining Smad4 shRNA was named LT32, and it was confirmed byPCR and sequencing. 293T cells were cotransfected with lentiviral vectorLT32, pCMV-dR8.74 and pMD2G. All virus stocks were produced bycalcium phosphate-mediated transfection. The titer of virus was testedaccording to the expression level of GFP.(4) Human hepatocelluar carcinoma cell line SMMC-7721 wastransfected with Smad4 RNAi negative control lentivirus (LTN) andSmad4 RNAi lentivirus (LT32), respectively. The cells were treated withTGF-β1 at the concentration of 100 ng/ml. To determine cell growth, cellviability was assayed by exclusion of 0.2% trypan blue and then the cellnumbers were counted under the light microscopy. Cell proliferation wasalso detected by MTT reduction assay. Cell apoptosis was evaluated byPI staining and TUNEL staining. Transwell chamber assay wasperformed to determine the effect of TGF-β1 on the invasion ofSMMC-7721 cells. The protein expressions of genes related to cellgrowth, apoptosis and invasion including p16, p21, p53, caspase 3, andVEGF, were determined by Western blot assay, respectively. The mRNAexpressions of matrix metalloproteinase (MMP) -2 and MMP-9 weredetected by reverse transcription polymerase chain reaction (RT-PCR),respectively.Results(1) The expressions of TGF-β1 and Smad7 in HCC tissue were higherthan that in surrounding HCC tissue. But TβR-Ⅱand Smad4 proteins were on the opposite way. There was tightly consistency between theprotein expressions of TGF-β1 and NF-κB in HCC tissue. This studysuggests that the protein expressions of TGF-β1, TβR-Ⅱ, NF-κB, Smad4and Smad7 in HCC tissue, which are major factors of TGF-β1-Smadsignaling pathway, are abnormal. They may play important roles in thedevelopment of hepatocellular carcinoma. The action of TGF-β1 isweakened in hepatic carcinoma cells because of abnormality of TGF-β1receptor (such as TβR-Ⅱ) and post-receptor (such as Smad4 and Smad7)factors. NF-κB may cause TGF-β1 activation and production. In addition,the mRNA and protein expression of Smad4 in SMMC-7721 cells werelower than that in L02 cells, further demonstrated the substantial role ofSmad4 in HCC.(2) Construction and identification of the shRNA expression vector forSmad4 gene: Double enzyme (HindⅢand BamHⅠ) digestion analysisand DNA sequencing confirmed that samd4 specific shRNA expressionvectors were constructed successfully. The constructed plasmids withdifferent shRNA and the control plasmid were transiently transferred intoAD293 cell line for 72h. Fluorescene quantitative PCR analysis showedthat the inhibition rate of Smad4 mRNA expression in AD293 cellstransferred with shRNA expression vector psiSmad4-1, psiSmad4-2 andpsiSmad4-3 was 39.0%, 8.8% and 73.8%, respectively, which indicatedSmad4 mRNA expression was specifically inhibited and the effectivesequence of siRNA targeting Smad4 gene was the third one.(3) Construction and identification of the lentiviral vector for RNAinterference against Smad4 gene: According to the effective siRNAsequence of Smad4 confirmed in the previous study, lentivirus vector producing shRNA against Smad4, which was constructed and identifiedby PCR and DNA sequencing, was named LT32. PCR analysis showedthat the inhibition rate of Smad4 expression in cells transfected withlentivirus LT32 was 60%, indicating that Smad4 expression waseffectively inhibited.(4) To explore the effects of inhibition of Smad4 expression by RNAion TGF-β1-induced antiproliferation in SMMC-7721 cells: Weperformed RNA interference to further investigate the role of Smad4 inTGF-β1-induced growth inhibition of SMMC-7721 cells. As comparedwith the control, mere transfection with Smad4 RNAi negative controllentivirus LTN had no effect on SMMC-7721 cell growth. Transfectionwith LTN prior to TGF-β1 treatment also did not affect TGF-β1-inducedgrowth inhibition of SMMC-7721 cells. Transfection with Smad4 RNAilentivirus LT32, however, markedly weakened the antiproliferation effectof TGF-β1. Furthermore, although mere transfection with lentivirus LTNhad no effect on SMMC-7721 cell growth, the inhibition of Smad4expression by RNAi caused the cellular growth inhibition inSMMC-7721 cells.(5) Roles of p16 and p21 in TGF-β1-induced growth inhibition ofSMMC-7721 cells: After 36h exposure of SMMC-7721 cells to TGF-β1,the protein expression of p16 was up-regulated .It was 1.55-fold of thecontrol. Transfection with Smad4 RNAi negative control lentivirus LTNand Smad4 RNAi lentivirus LT32 both had no effect on p16 expression inSMMC-7721 cells. Transfection with LTN prior to TGF-β1 treatmentalso did not affect TGF-β1-induced up-regulation of p 16 expression.However, up-regulation of p16 expression induced by TGF-β1 could be weakened by silencing of Smad4, indicating that TGF-β1-induced growthinhibition of SMMC-7721 cells was associated with the up-regulation ofp16. And p16, may be as a down-stream target gene of Smad4, involvedin the signal transduction of TGF-β1. In addition, to SMMC-7721 cells,no matter under the induction of TGF-β1, or the silence of Smad4, p21expression was not obviously changed, suggesting that p21 might notplay an important role as a down-stream gene of Smad4 in the aboveprocess.(6) To explore the effects of inhibition of Smad4 expression by RNAion TGF-β1-induced apoptosis of SMMC-7721 cells: As compared withthe control, mere transfection with Smad4 RNAi negative controllentivirus LTN and Smad4 RNAi lentivirus LT32 both did not induceSMMC-7721 cell apoptosis. Transfection with LTN prior to TGF-β1treatment also did not affect TGF-β1-induced apoptosis of SMMC-7721cells. However, transfection with lentivirus LT32, markedly weakenedthe apoptosis induced by TGF-β1, which indicated that TGF-β1-inducedSMMC-7721 cell apoptosis was Smad4-dependent.(7) Roles of p53 and caspase 3 in TGF-β1-induced apoptosis ofSMMC-7721 cells: In SMMC-7721 cells, no matter under the inductionof TGF-β1, or the silence of Smad4, the expression of p53 was notobviously affected, suggesting that p53 might be neither associated withTGF-β1-Smad signaling pathway nor involved in the course ofTGF-β1-induced apoptosis. But companied with TGF-β1-inducedSMMC-7721 cell apoptosis, caspase 3 was activated. And the activationof caspase 3 by TGF-β1 could be inhibited by silencing of Smad4,indicating that TGF-β1-induced apoptosis of SMMC-7721 cells may be associated with the activation of caspase 3 which was Smad4-dependent.(8) Role of TGF-β1-Smad signaling pathway in the invasion ofSMMC-7721 cells: Transwell chamber assay was performed to determinethe invasion of SMMC-7721 cells. After 36 h exposure of SMMC-7721cells to TGF-β1, the number of cells cross matrigel increased 20%,indicating that TGF-β1 could promote the cellular invasion. As comparedwith the control, transfection with Smad4 RNAi lentivirus LT32 alsoincreased the invasion ability of SMMC-7721 cells. But transfection withLT32 prior to TGF-β1 treatment, neither inhibited TGF-β1-inducedSMMC-7721 cell invasion, and nor further cooperated to promote thecellular invasion.(9) Effects of TGF-β1-Smad signaling pathway on the expressions ofMMP-2, MMP-9 and VEGF in the invasion of SMMC-7721 cells:Companied with the increase of SMMC-7721 cell invasion induced byTGF-β1 and the silencing against Smad4, the expression of MMP-2 wasup-regulated significantly, while MMP-9 was not affected. These data notonly suggested that the invasion of SMMC-7721 cells was closelyassociated with MMP-2, not MMP-9, but indicated that the up-regulationof MMP-2 induced by TGF-β1 was not Smad4-dependent. In addition,we found that the expression of VEGF was up-regulated by TGF-β1.Transfection with Smad4 RNAi negative control lentivirus LTN prior toTGF-β1 treatment did not affect TGF-β1-induced up-regulation of VEGFexpression. However, up-regulation of VEGF expression induced byTGF-β1 could be weakened by silencing of Smad4, indicating that VEGFmay be as a down-stream target gene of Smad4, involved in the signaltransduction of TGF-β1. (10) Roles of MAPK pathway in the signal transduction of TGF-β1:Associated with TGF-β1-induced biological effects, JNK and p38 werephosphorylated and activated. Although the expression of Erk1/2 was notaffected, its activation still kept in a high level. As compared with thecontrol, mere transfection with Smad4 RNAi negative control lentivirusLTN and Smad4 RNAi lentivirus LT32 both did not phosphorylate andactivate JNK and p38. Transfection with LTN prior to TGF-β1 treatmentalso did not affect TGF-β1-induced activtion of JNK and p38. However,transfection with lentivirus LT32, markedly inhibited the activation ofJNK and p38 induced by TGF-β1. These data indicated that activation ofJNK and p38 participated in the signal transduction of TGF-β1 and theywere regulated by Smad4.Conclusion(1) The expressions of TGF-β1, TβR-Ⅱ, NF-κB, Smad4 and Smad7proteins in HCC tissue, which are major factors of TGF-β1-Smad signaltransduction pathway, are abnormal, suggesting that they may playimportant roles in the pathogenesis and development of hepatocellularcarcinoma. The inhibitory effect of TGF-β1 weakened in hepaticcarcinoma cells may be because of the abnormality of TGF-β1 receptor(such as TβR-Ⅱ) and post-receptor factors (such as Smad4 and Smad7).And NF-κB may paly a role in the production and activation of TGF-β1.(2) Considering that Smad4 is an action substrate of TGF-β1 receptorand plays a central role in TGF-β1 signaling pathway, we constructedSmad4 RNAi plasmid pGCsi-H1/Neo/GFP/shRNA and Smad4 RNAilentivirus LT32 successfully, which may provide a novel applicable strategy for exploring the role of TGF-β1-Smad pathway incarcinogenesis and gene therapy.(3) In human hepatocelluar carcinoma cell line SMMC-7721, TGF-β1might induce cell growth inhibition by up-regulating the expression ofp16 and cellular apoptosis by activating caspase 3 in a Smad4-dependentmanner. And TGF-β1 could increase the expressions of MMP-2 andVEGF to create a suitable microenvionment in favour of SMMC-7721cell invasion. MAPK pathways such as JNK and p38 could interact withSmad4, involved in the signal transduction progress of TGF-β1 inSMMC-7721 cells.Taken together, TGF-β1-Smad signaling pathway, associated with thegenes such as p16, caspase 3, MMP-2, VEGF, JNK and p38, inducedbroadly biological effects including cell growth inhibition, apoptosis andinvasion in human hepatocelluar carcinoma cell line SMMC-7721. Adeeper and full understanding of the function of TGF-β1-Smad signalingpathway may provide significant insights into the pathogenesis,development and prognostic of hepatocellular carcinoma and a novelapplicable strategy for gene therapy.