Interaction Of MicroRNA-21/145 And Smad3-Domain-Specific Phosphorylation In Hepatocellular Carcinoma

Primary liver cancer is the third cause of human cancer death in today’s society,and one of the most common malignancies in China.The mortality of primary liver cancer is preceded only by gastric cancer and esophageal cancer.Among them,hepatocellular carcinoma(HCC)accounts for about 90% of primary liver cancer.HCC is one of the hotspots in the field of research because of its hidden disease,short survival period,poor prognosis and high mortality rate.Although molecular targeting drugs increase the survival cycle of patients with HCC,but HCC treatment is still facing a huge challenge.Existing studies suggest that HCC development is associated with multiple signaling pathways,including TGF-b/Smad,which interacts directly or indirectly with micro RNAs and plays an important role in cancer progression.The expression of micro RNA-21 was up-regulated in HCC,while micro RNA-145 was down-regulated,but how these two micro RNAs interact with Smad3 phospho-isoform(p Smad3 L and p Smad3C)switch downstream of TGF-β/MAPK signaling remains unresolved.This study investigated the interaction between micro RNA-21/145 and p Smad3C/3L in HCC.Purpose:1.It has been reported that miR-21 is up-regulated and mi R-145 is down-regulated in HCC.In this experiment,we established transplanted tumor model,and injected subcutaneously into nude mice with agomir/antagomir to up-regulate micro RNA-145 or down-regulate micro RNA-21.And how these influenced HCC growth and tumor cell apoptosis.2.Smad3 is an important molecular downstream of the TGF-b/Smad pathway,and the phosphorylation of Smad3 at C-terminal and Link-region(p Smad3 C and p Smad3L)mediates tumor suppressor or activation respectively.In this experiment,we used the specific high expression p Smad3 C or p Smad3 L stable transfection cell lines(Smad3WT,Smad3 EPSM,Smad3 3S-A)to inject subcutaneously into nude mice,and to establish transplanted tumor model.Then we investigate that how do increased p Smad3C/3L influence HCC tumor growth and tumor cell apoptosis.3.In the transplanted tumor model,down-regulated micro RNA-21 and up-regulated micro RNA-145 effected on the expression of p Smad3 C and p Smad3 L.And specific-overexpressin of p Smad3 C or p Smad3 L effected on the expression of micro RNA-21 and micro NRA-145.We investigated the interaction between micro RNA-21/145 and p Smad3 C / 3L in HCC and their interaction effected on HCC progression.Methods:1.Animal models: Specific pathogen free(SPF)grade male BALB/c nude mice(14-18 g),aged 5 weeks were purchased from Vital River Laboratory Animal Technology Company(Beijing,China).The mice were kept in super clean laminar flow cabinet under the SPF conditions at the Experimental Animal Center of Anhui Medical University(Anhui,China).The mice were maintained under these conditions for 1 week for acclimatization before the commencement of experiments.2.Grouping and treatment of animals: BALB/c nude mice were divided into four groups of six mice each as follows: mi R-21 antagomir NC,mi R-21 antagomir,mi R-145 agomir NC and mi R-145 agomir groups.After adaptation under SPF conditions for 1 week,animals were injected subcutaneously with Hep G2 cell suspension(9×106)to establish xenograft tumors.After tumor formation,mi R-21antagomir(20 μl),mi R-21 antagomir NC,mi R-145 agomir and mi R-145 agomir NC were respectively injected into xenograft every four days,for a total of 28 days.In a separate experiment,BALB/c nude mice were divided into four groups of six mice each: Hep G2,Smad3-WT,Smad3-EPSM and Smad3-3S-A groups.After successful transfection of Hep G2 cells respectively with Smad3-WT,Smad3-EPSM,and Smad3-3S-A plasmid,they were respectively injected into nude mice in each group.Animals were kept under strict observation throughout the experiment.Tumor tissues of each mice was harvested and stored at a temperature of-80 °C until use.H&E staining,immunohistochemistry,and transmission electron microscopy were used to monitor tumor morphology,Smad protein expression and apoptosis respectively.Immunofluorescence was used to monitor cyto-nuclear expression of Smad proteins.Western blot and q RT-PCR were used to determine Smad protein and m RNA outputs respectively.3.Cell culture: The human HCC Hep G2 cell line was purchased from the Chinese Academy of Sciences Cell Bank(Shanghai,China).Hep G2 cell lines were cultured in Dulbecco’s modified Eagle medium(DMEM)supplemented with 10 % fetal bovine serum(FBS).Cell cultures were maintained and incubated at 37 °C in humidified air with 5 % CO2.The Hep G2 cells with conventional culture were seeded at a density of5×105 on six well plates.Hep G2 cells were starved overnight.And transfected with Smad3 WT,Smad3 EPSM,and Smad3 3S-A.The cells were selected with G418(600μg/ml)(Sigma,Shanghai,China)24h after transfection.Amplification of survived cells was used for experiments.And in another experiment,Hep G2 Cells were transfected with micro RNA-145 agomir and micro RNA-21 antagomir.Results:1.Down-regulated microRNA-21 and up-regulated microRNA-145 expressions produced opposing effects on Tumor burden and promoting effects on apoptosis in vivo: Tumor burden and tumor cell survival are two important phenotypic hallmarks of cancer,and they are related to dysregulated TGF-β signaling [43].Tumor transplantation model of cancer involving xenogeneic tumor cells can be achieved in immuno-compromised animals such as nude mice [44].Such cancer models allow for the study and investigation of cancer phenotypic hallmarks and the underlying dysregulated cell signaling and molecular pathways.To study the involvement of micro RNA-21 and 145 in tumor burden and survival in HCC,transplanted Hep G2 cell tumors in nude mice were treated with micro RNA-21 antagomir and micro RNA-145 agomir respectively as against their respective negative controls.Notably,tumor volume was decreased in xenograft tumors treated with micro RNA-21 antagomir and micro RNA-145 agomir compared to those tumors that received micro RNA-21 antagomir NC and micro RNA-145 agomir NC treatments.Pathologically,there was a obvious nucleus shrivel in the micro RNA-21 antagomir and micro RNA-145 agomir group compared to antagomir NC and agomir NC group.And this patho-morphological change correlated with promoting effects on apoptosis.These results preliminarily suggest that down-regulated micro R-21 or up-regulated micro R-145 plays an important role in inhibiting HCC growth and accelerating the apoptosis.2.Down-regulated micro RNA-21 expression suppressed MAPK pathway and up-regulated micro RNA-145 expression switched Smad3 phosphorylation at Linker and C-terminal in HCC: We investigated whether the regulation of MAPK and TβRI by micro RNA-21 and 145 are directly related to Smad3 phosphorylation.However there were not obvious changes in the expression of p Smad3 C and p Smad3 L in the Hep G2 cells transfected with mi R-21 antagomir compared with antagomir NC-group.We further explore the effects of decreased micro RNA-21 on MAPK pathway,the expression of p ERK1/2,p JNK1/2 and pp38 were decreased in micro RNA-21antagomir-group compared to antagomir NC-group.The results indicate that down-regulated mi R-21 expression can suppress the activation of MAPK signaling pathway in Hep G2 cells.Of note,the expression level of p Smad3 C was up-regulated in mi R-145 agomir-group and p Smad3 L expression level was obviously decreased compared with agomir NC-group in vitro and in vivo.These data indicate that micro RNA-145 switches p Smad3 L to p Smad3 C to suppress tumor progression in HCC.3.Smad3 phosphorylation at Linker and C-terminal effects on microRNA-21 and 145 expressions in HCC: Successful transfection of Hep G2 cells with Smad3-WT,Smad3 EPSM,and Smad3 3S-A respectively was confirmed.Then,subcutaneous injection of nude mice with Hep G2 cells successfully transfected with Smad3 WT,Smad3 EPSM,and Smad3 3S-A respectively,p Smad3 C was up-regulated in EPSM-tumors and p Smad3 L was up-regulated in 3S-A-tumors compared to WT-tumors.Tumor volume of EPSM-group was significantly reduced(P<0.01)compared with WT-group.On the contrary,the tumor volume in 3S-A-group were indeed increased(P<0.01)compared with WT-group.Nuclear condensation and shrinkage were observed in EPSM-group compared to WT-group.And this correlated with increased tumor cell apoptosis in EPSM-tumors compared to WT-tumors.In contrast to these observations,tumor cells grew well in 3S-A-group compared to WT-group.And these observations correspondingly led to elevated expression of micro RNA-145 and 21 respectively in3S-A-group and EPSM-group compared to their respective expressions in WT-group in vivo and in vitro.These results suggest that increased p Smad3 C or decreased p Smad3 L expression reduce tumor-burden and promotes apoptosis.And Smad3domain-specific phosphorylation interacts with the expressions of micro RNA-145 and21 in HCC.Conclusions:1.Down-regulated mi R-21 or up-regulated mi R-145 expression suppressed tumor growth and promoted tumor cell apoptosis in HCC.2.Up-regulated mi R-145 significantly increased p Smad3 C expression and decreased p Smad3 L expression.And mi R-145 could promote the shift from p Smad3 L to p Smad3 C in HCC.3.Increased expression of pSmad3 C suppressed tumor growth and promoted tumor cell apoptosis in HCC.And increased p Smad3 L expression promoted tumor growth and suppressed tumor cell apoptosis.4.Increased pmad3 C in HCC resulted in down-regulated mi R-21 expression and up-regulated mi R-145 expression.But increased p Smad3 L led to up-regulated mi R-21 expression and down-regulated mi R-145 expression.5.Micro RNA-145/p Smad3 C exhibited tumor suppressor-like activity to attenuate HCC progression,while micro RNA-21 promotes tumor progression via unknown mechanism.Modulation of Smad3 phosphorylation switch by micro RNAs or naturally derived small molecule drugs may be relevant for therapy against HCC.