miR-488-5p mitigates hepatic stellate cell activation and hepatic fibrosis via suppressing TET3 expression

Downregulation of miR-488-5p is detected in activated HSCs

First, the LX-2 cells were cultured with medium containing TGF-β1 (10 ng/mL) for 0, 3, 6, 12 and 24 h. As shown in the Fig. 1a, b, the expression of α-SMA was upregulated by degrees based on the results of WB and IF staining. Afterwards, we performed the qRT-PCR assay, the data revealed that as the relative level of α-SMA increased in the activated HSCs, the relative level of miR-488-5p was gradually downregulated (Fig. 1c, d). To sum up, our study presented that miR-488-5p is downregulated in the activated HSCs.

Fig. 1figure 1

miR-488-5p is downregulated during HSCs activation. LX-2 cells were activated by TGF-β1 (10 ng/mL) for respective time. a, b Western blot (WB) and Immunofluorescence (IF) analysis of the expression level of α-SMA in LX-2 cells. c, d qRT-PCR analysis of the level of α-SMA and miR-488-5p in activated LX-2 cells. Data represent means ± SEM of at least three independent experiments. *p value < 0.05

Downregulation of miR-488-5p is detected in multiple hepatic fibrosis models

For purpose of detecting the effect of miR-488-5p in hepatic fibrotic tissues, we established three mouse liver fibrosis models. CCl4, BDL and HFD were administrated to the mice to, respectively, develop liver fibrosis. As shown in the Fig. 2a–c, HE, Sirius Red and Masson staining results presented the severity of fibrotic level caused by CCl4, BDL and HFD. Compared with the respective control (CTL) group, the histological structure was seriously damaged in CCl4 and BDL group, meanwhile the lipid vacuoles were abundant in the HFD group. At the same time the massive deposition of collagen was presented in CCl4, HFD and BDL group. Compared to their respective CTL group, the mRNA level of fibrosis-related markers containing α-SMA, tissue inhibitor of metalloproteinases 1 (TIMP-1) and Collagen-I were significantly upregulated (Fig. 2d–f). In the meantime, the level of miR-488-5p was decreased in the CCl4, BDL and HFD group, compared with their respective CTL group (Fig. 2g). To sum up, our study presented that miR-488-5p is downregulated in multiple liver fibrosis models, indicating that miR-488-5p participates in the development of hepatic fibrosis.

Fig. 2figure 2

miR-488-5p is downregulated in multiple hepatic fibrosis models. Hepatic fibrosis models including CCl4, HFD and BDL were established for further research. a HE, Masson and Sirius Red staining of hepatic specimens from CCl4 (8 weeks), HFD (24 weeks) and BDL (2 weeks). b, c The Masson-positive and Sirius Red-positive areas were quantified in each group. df qRT-PCR analysis of the mRNA level of α-SMA, TIMP-1 and collagen-I in each group. g qRT-PCR analysis of the mRNA level of miR-488-5p in each group. Data represent means ± SEM of at least three independent experiments. *p value < 0.05

miR-488-5p regulates cellular proliferation, apoptosis and activation level of HSCs

For further determining the precise effect of miR-488-5p in the activated HSCs during fibrosis, we transfected the LX-2 cells with miR-SCR as control or miR-488-5p mimics. As presented in the Fig. 3a, the relative level of miR-488-5p was remarkably increased in LX-2 cells after miR-488-5p mimics transfection. As presented in the Fig. 3b, LX-2 cells with miR-488-5p overexpression were characterized with the lower protein level of fibrosis-related markers which contained a-SMA, TIMP-1, and collagen-I. Moreover, the cellular proliferative capability was inhibited in the LX-2 cells with miR-488-5p overexpression, based on the cell viability and clone formation results (Fig. 3c, d). At the same time, the IF staining revealed that the lower intensity of a-SMA was found in the LX-2 cells with miR-488-5p overexpression (Fig. 3e). Meanwhile, transfection with miR-488-5p mimics could result in the higher apoptosis index of LX-2 cells (Fig. 3f). As mentioned above, miR-488-5p overexpression was able to contribute to the repressed expression of fibrotic markers, cellular viability and the promoted apoptotic level of HSCs.

Fig. 3figure 3

miR-488-5p regulates cellular proliferation, apoptosis and activation level of HSCs. a qRT-PCR analysis of the mRNA level of miR-488-5p in LX-2 cells. b WB analysis of the protein level of α-SMA, collagen-I and TIMP-1 in LX-2 cells. c Detection of the cell viability was dependent on CCK8 assay. d Clone formation of LX-2 cells and quantified. e IF tests the expression of α-SMA in LX-2 cells. f Flow cytometry evaluates the apoptosis index of LX-2 cells and the quantification. Data represent means ± SEM of at least three independent experiments. *p value < 0.05

TET3 is a direct target of miR-488-5p and upregulated in activated HSCs and fibrotic liver tissues

For the purpose of elucidating the underlying mechanism of miR-488-5p regulating HSCs activation, the online bioinformatics analyses for seeking the target gene of miRNA were performed. The intersection of two databases, containing miRWalk and TargetScan, showed that tet methylcytosine dioxygenase 3 (TET3) was a probable target gene of miR-488-5p. After consulting plentiful literatures, we found that tet methylcytosine dioxygenase 3 (TET3) was closely related with fibrosis [14, 15]. For the purpose of determining whether the effect of miR-488-5p was dependent on TET3, first, we performed the WB and qRT-PCR assays and our data revealed that the relative level of TET3 were increased in HSCs activated by TGF-β1 for 24 h (Fig. 4a, b). After performing the dual‐luciferase reporter assay, we observed that the co‐transfection with miR‐488-5p mimic and pGL3‐TET3‐WT 3′UTR contributed to the reduced luciferase activity (Fig. 4c, d). Meanwhile, the expression of TET3 was remarkably repressed in the miR‐488-5p-overexpressed LX-2 cells (Fig. 4e). Subsequently, the higher protein level of TET3 was detected in three hepatic fibrosis models (Fig. 4f). Moreover, the expression of TET3 was remarkably higher in patients with hepatic fibrosis, based on the WB, Masson and IHC staining (Fig. 4g, h). Meanwhile, the qRT-PCR data revealed that miR‐488-5p was downregulated in human fibrotic liver tissues which indicated miR-488-5p could serve as a potential target for hepatic fibrosis (Fig. 4i). Collectively, the data identified that TET3, as a direct target of miR‐488-5p, were remarkably upregulated in activated HSCs, liver fibrosis models and fibrotic liver specimens from the patients.

Fig. 4figure 4

miR-488-5p targets TET3 via binding onto the 3′ UTR of TET3 mRNA directly. a, b WB and qRT-PCR analysis of the expression level of TET3 in LX-2 cells activated by TGF-β1 (10 ng/mL) for 0 and 24 h. c Predicted miR-488-5p targeting sequence in TET3 3′UTR (WT TET3 3′UTR). Target sequences of TET3 3′UTR were mutated (MUT TET3 3′UTR). d Dual-luciferase reporter assay of LX-2 cells transfected with WT TET 3′UTR or MUT TET3 3′UTR reporter together with miR-488-5p mimics or miR-SCR. e WB analysis of the expression of TET3 in LX-2 cells transfected with miR-488-5p mimics or miR-SCR. f WB analysis of the expression of TET3 in CCl4, HFD and BDL group as well as their respective control (CTL) group. g WB analysis of the expression of TET3 in normal and fibrotic liver tissues from patients. h Immunohistochemical staining of Masson and TET3 in hepatic samples from patients. i qRT-PCR analysis of the expression level of miR-488-5p in normal and fibrotic liver tissues from patients. Data represent means ± SEM of at least three independent experiments. *p value < 0.05

Effect of miR-488-5p in HSCs activation depends on TET3

Few studies have reported that TET3 played an essential role in activating TGF-β pathway, which is closely involved in the liver fibrosis [14, 16]. For the purpose of further judging that the effect of miR-488-5p in HSCs activation depended on TET3, LV-TET3 or LV-NC was used to transfect the miR‐488-5p-overexpressed LX-2 cells. As shown in the Fig. 5a, as the expression of TET3 decreased in the miR‐488-5p-overexpressed LX-2 cells, so did the expression of TGF-β, p-SMAD2 and p-SMAD3. Meanwhile, overexpression of TET3 in LX-2 could contribute to reversing the inhibitory effect of miR-488-5p in TGF-β/SMAD2/3 pathway. In addition, the expression of fibrosis-related makers containing TIMP-1, collagen-I and a-SMA was upregulated in the LX-2 cells transfected with miR-488-5p mimics and LV-TET3 (Fig. 5b). Then, we performed the CCK8 assay and clone formation assay; the data showed that the cellular proliferative capabilities were promoted in the LX-2 cells transfected with miR-488-5p mimics and LV-TET3 (Fig. 5c–e). At the same time, the IF staining data revealed that the higher intensity of a-SMA was detected in the LX-2 cells transfected with miR-488-5p mimics and LV-TET3 (Fig. 5f). Meanwhile, the promoted apoptosis index of the miR‐488-5p-overexpressed LX-2 cells was counteracted by TET3 overexpression (Fig. 5g, h). Collectively, our data demonstrated that the effect of miR-488-5p in HSCs activation depended on TET3.

Fig. 5figure 5

The effect of miR-488-5p in HSCs activation depends on TET3. LX-2 cells transfected with miR-SCR or miR-488-5p mimics or miR-488-5p mimics + LV-TET3 or miR-488-5p mimics + LV-NC were activated by TGF-β1 (10 ng/mL) for 24 h for further research. a WB analysis of the relative expression of TET3, TGF-β, p-SMAD2, SMAD2, p-SMAD3 SMAD3 in LX-2 cells from each group. b WB analysis of the relative protein level of TIMP-1, collagen-I and α-SMA in LX-2 cells from each group. c The cell viability of LX-2 cells from each group was detected by CCK8 assay. d, e Clone formation of LX-2 cells was quantified in each group. f IF analysis of the expression of α-SMA in LX-2 cells from each group. g, h Flow cytometry evaluates the apoptosis index of LX-2 cells and the quantification. Data represent means ± SEM of at least three independent experiments. *p value < 0.05

miR-488-5p contributes to the reduced degree of hepatic fibrosis

Based on our results described above, we discovered that miR-488-5p participated in the HSCs proliferation, apoptosis and activation in vitro. For the purpose of sequentially exploring the effect of miR-488-5p in liver fibrosis in vivo, the mice from Control, CCl4, HFD and BDL group were, respectively, subjected to the transfection with ago-miR control (CTL) and ago-miR-488-5p. As presented in the Fig. 6a, the qRT-PCR results revealed that the level of miR-488-5p was upregulated in the group treated with ago-miR-488-5p. Meanwhile, our data revealed that miR-488-5p overexpression was helpful to mitigate liver fibrosis, as the histological structural damage was significantly alleviative in CCl4 and BDL group; meanwhile, the number of lipid vacuoles was reduced in the HFD group (Fig. 6b). Moreover, the reduced amount of collagen deposition was demonstrated in the liver tissues from CCl4, HFD and BDL group which were transfected with ago-miR-488-5p, according to the Masson and Sirius Red staining results (Fig. 6b–d). Therefore, the above data suggested that miR-488-5p alleviated liver fibrosis from multiple liver fibrosis models.

Fig. 6figure 6

miR-488-5p contributes to the reduced degree of liver fibrosis. The mice in the control, CCl4, BDL and HFD groups were treated with ago-miR control (CTL) or ago-miR-488-5p. a The mRNA level of miR-488-5p in each group was detected in by qRT-PCR. b HE, Masson and Sirius Red staining of liver sections from the mice in each group. c, d The quantification of Masson-positive and Sirius Red-positive areas in each group. Data represent means ± SEM of at least three independent experiments. *p value < 0.05

miR-488-5p inhibits TET3 expression in fibrotic liver tissues

To identify that miR-488-5p inhibited liver fibrosis by targeting TET3 which was related with TGF-β/SMAD2/3 pathway, first, we evaluated the α-SMA expression of liver tissues through IHC staining; our data showed that α-SMA expression was remarkably inhibited in the liver tissues from CCl4, HFD and BDL group which were transfected with ago-miR-488-5p (Fig. 7a, b). Next, the protein expression of Timp1, collagen I and α-SMA was detected by WB assay which presented that miR-488-5p overexpression could significantly inhibit the expression of the above fibrosis-related makers in liver fibrosis tissues (Fig. 7c). Sequentially, the qRT-PCR analysis revealed that relative mRNA level of TET3 in liver tissues was significantly lower in the CCl4, HFD and BDL group which were transfected with ago-miR-488-5p compared to those transfected with ago-miR control (CTL) (Fig. 7d). Eventually, the WB results showed transfection with ago-miR-488-5p could reduce the expression of TET3, TGF-β, p-SMAD2 and p-SMAD3 in the fibrotic liver samples from CCl4, HFD and BDL group. To sum up, our study determined that miR-488-5p mitigates liver fibrosis by inhibiting TET3/TGF-β/SMAD2/3 pathway.

Fig. 7figure 7

miR-488-5p inhibits the expression of TET3 in liver fibrosis tissues. a, b The expression of α-SMA in liver sections from the mice in each group was detected by immunohistochemical staining and quantified. c WB analysis of the relative expression of TIMP-1, collagen-I and α-SMA in liver tissues of mice in each group. d qRT-PCR analysis of the level of TET3 in liver tissues of mice in each group. e WB analysis of the expression of TET3, TGF-β, p-SMAD2, SMAD2, p-SMAD3 SMAD3 in liver tissues of mice in each group. Data represent means ± SEM of at least three independent experiments. *p value < 0.05

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