LncTUG1 identification and its regulation of HCC progression in vitro
To sieving the differentially expressed lncRNAs in HCC, we used the NCBI, TCGA and LncRNA disease public databases (http://www.cuilab.cn/LncRNAdisease). Venn diagram analysis identified two differentially expressed lncRNAs in the three databases: LncTUG1 and ZnRD1-ASP (Figure 1a). The expression of LncTUG1 and ZnRD1-ASP were measured in HCC tissues by way of StarBase database (http://starbase.sysu.edu.cn), and the results displayed that compared with normal tissues, LncTUG1 and ZnRD1-ASP expressions were both rised in HCC tissues (Figure 1b). In addition, the higher LncTUG1 expression, the worse prognosis of sufferers (P<0.05) , while ZNRD1 was not (P>0.05) (Figure 1c). We verified high expression of LncTUG1 in HCC cells utilized qRT-PCR (Figure 1d).
In order to find out the act of LncTUG1 on HCC, LncTUG1 was knocked down in Huh7 cells by shRNA, and the capacities of proliferation, invasion and apoptosis were measured adopting CCK-8, Transwell and flow cytometry assays severally. Results displayed that in Huh7 cells with TUG1 knockdown, the invasion and proliferation capacity reduced evidently and the level of apoptosis rased (Figure 1e–h). These findings revealed that LncRNA TUG1 is overexpressed in HCC and was relevent to the evolvement of hepatocellular carcinoma.
LncTUG1 activated the mTOR/S6K pathway and regulated HCC cells malignant behavior
To examine the mechanism by which LncTUG1 may promote HCC progression, we first selected genes related with LncTUG1 and make GO enrichment analysis in StarBase (http://starbase.sysu.edu.cn) and Metascape (http://metascape.org/gp/index). On account of it remarkably affects the evolement of HCC[18-20], the mTOR/S6K pathway was selected for study (Figure 2a). In order to confirm the activation of mTOR/S6K pathway in HCC cells, we examined the p-mTOR and p-S6K levels by western blot. The p-p70S6K and p-mTOR expressions were both obviously increased in HCC cells (Figure 2b). Next, we used mTOR inhibitor or agonist to treat Huh7 cells and measured the levels of apoptosis, proliferation and invasion by Transwell, CCK-8 and flow cytometry, respectively. When cells treated with the inhibitor, the apoptosis level increased significantly, and the proliferation and invasion decreased dramatically. In the meantime, the agonist displayed opposite results (Figure 2c–e).
Next, we further to confirm the regulation of LncTUG1 to mTOR/S6K. In cells with LncTUG1 knockdown, addition of the mTOR agonist can lead to reduced cell proliferation and invasiveness while increased the level of cell apoptosis (Figure 2f–h). The above results indicated that LncTUG1 regulates cell proliferation, apoptosis and invasion via the mTOR/S6K pathway in HCC.
LncTUG1 regulates the progression of HCC through activated RRAGD-mTOR/S6K pathway
To more closely examine the mechanism by which LncTUG1 regulates the mTOR/S6K pathway, we examined genes related to mTOR pathway and genes regulated by LncTUG1 in HCC and obtained three genes. We analyzed the association of the three genes on HCC patient prognosis by Kaplan–Meier Plotter (http://kmplot.com/analysis/). Results displayed that high RRAGD expression is relevent to the poor prognosis (P<0.05) while not in patients of high RPS6 or TSC2 expression; furthermore, high RRAGD expressions were observed in HCC tissues and cells (Figure 3a–c).
RRAGD, a GTP binding protein, functions as a molecular switch through mTOR pathway[21]. To study the role of RRAGD in HCC, siRNA was used to knockdown RRAGD. Knockdown of RRAGD repressed cell proliferation and invasion (Figure 3d–f), while apoptosis level increased (Figure 3g).
Results of Western blot displayed that RRAGD activates the mTOR/S6K pathway (Figure 4a). We activated the mTOR pathway by mTOR agonist in cells transfected with si-RRAGD. The results showed that addition of mTOR agonists reversed the effects of cell proliferation, apoptosis, and invasion produced by the downregulation of RRAGD (Figure 4b–d).
We examined RRAGD after knockdown of TUG1 and found that the level of RRAGD decreased significantly after TUG1 knockdown (Figure 4e). After knocking down LncTUG1 in Huh7 cells, the proliferation and invasion were inhibited and apoptosis increased which can be reversed by the overexpressed RRAGD (Figure 4f–h). These results showed that LncTUG1 activates the mTOR/S6K pathway in HCC cells by upregulating RRAGD, and promotes the progression of HCC.
LncTUG1 is involved in HCC progression by directly targeting miR-144-3p in Huh7 cells
Our results above suggest that LncTUG1 can regulate HCC progression through RRAGD, but the specific mechanism is unclear. To more closely explore the mechanism of LncTUG1 in HCC, we examined potential target miRNAs of LncTUG1 in the LncACTdb2.0 (http://www.bio-bigdata.net/LncACTdb/index.html) database. We identified four miRNAs associated with TUG1 in HCC: miR-29a-3p, miR-29b-3p, miR-29c-3p and miR-144-3p. In addition, prediction by TargetScan (http://www.targetscan.org/vert_71/) revealed 1810 miRNAs may bind to the 3′UTR of RRAGD mRNA. In conclusion, miR-144-3p is the only common miRNA in these results (Figure 5a).
The analysis of miR-144-3p utilized StarBase (http://starbase.sysu.edu.cn) revealed that compared with normal samples, miR-144-3p expression was dramatically repressed in HCC (Figure 5b). In addition, qRT-PCR results displayed that the miR-144-3p mRNA significantly reduced in cells (Figure 5c).
Next, we utilized luciferase reporter assays to elucidate the regulatory relationship between LncTUG1 and miR-144-3p. Results anounced that miR-144-3p was the target molecular of LncTUG1 (Figure 5d). miR-144-3p expression rised in LncTUG1-knockdown cells (Figure 5e). These results suggested a negative regulatory relationship between TUG1 and miR-144-3p.
In order to elucidate how miR-144-3p effects on HCC, we overexpressed miR-144-3p in Huh7 cells. Overexpressed-miR-144-3p depressed the proliferation and invasion while apoptosis level increased (Figure 6a–d) . Overexpression of miR-144-3p also remarkably reduced activation of the mTOR/S6K pathway and RRAGD expression (Figure 6e–f).
So as to expound how LncTUG1 affects miR-144-3p on cell proliferation, invasion and apoptosis, rescue experiments were performed. LncTUG1 in HCC cells attenuated the positive effect of the miR-144-3p inhibitor on cell proliferation and invasion. Cell apoptosis was inhibited by miR-144-3p inhibitor; however, these effects were eliminated by knockdown of LncTUG1 in Huh7 cells (Figure 6g–i). Moreover, knockdown of LncTUG1 reversed the RRAGD expression induced via miR-144-3p inhibitors (Figure 6j). Those ablve results announced that TUG1 accelerates cell proliferation, invasion while inhibits apoptosis by means of targeting miR-144-3p in HCC.
miR-144-3p regulated HCC progression through targeting RRAGD mRNA 3′-UTR
Whether RRAGD is the target gene of miR-144-3p was proved utilizing the luciferase reporter assay (Figure 7a). Next we examined whether the neoplasm inhibitory effect of miR-144-3p involves RRAGD. Results announced that miR-144-3p could reverse the effects of RRAGD on cell proliferation, invasion and apoptosis (Figure 7b–d). Furthermore, Overexpressed-miR-144-3p reversed the RRAGD-induced activation of mTOR/S6K pathway (Figure 7e).
LncTUG1 promotes the development of HCC in vivo through miR-144-3p/RRAGD-mTOR/S6K pathway
In order to elucidate the effect of LncTUG1 on HCC progression in vivo, we evaluated tumor formation in nude mice. Mices were injected into sh-TUG1-expressing Huh7 cells to construct tumor model in vitro. Results proved that tumor volume was evidently reduced (Figure 8a–c). In addition, IHC staining certificated that the RRAGD expression decreased in neoplasms from mice injected with sh-TUG1-expressing Huh7 cells (Figure 8d). Western blot and qRT-PCR results announced that RRAGD expression and the activation of mTOR/S6K were significantly reduced and miR-144-3p was increased in subcutaneous xenograft nude mouse models derived from sh-LncTUG1-expressing Huh7 cells (Figure 8e–f). These results suggest that LncTUG1 facilitates the HCC tumor growth in vivo, and these effects may occur via its regulation of the miR-144-3p/RRAGD-mTOR/S6K pathway.