Mir-454-3p induced WTX deficiency promotes hepatocellular carcinoma progressions through regulating TGF-β signaling pathway

Background: Wilms tumor gene on X chromosome (WTX) is an X-linked tumor suppressor gene in Wilms tumor; however, however, the molecular mechanism of WTX in the occurrence and development of HCC has not been reported. Methods: The expression of miR-454-3p and WTX wre analyzed in 32 matched human HCC and normal tissue samples. The molecular mechanisms of miR-454-3p/WTX/TGFβ signaling in cell proliferation, migration, invasion and autophagy were investigated in vitro and in vivo. Results: WTX expression was downregulated in HCC tissues; lower WTX levels were associated with poor HCC patient outcomes. WTX loss triggers the activation of TGF-β signaling, which promotes HCC cells proliferation, migration, invasion and autophagy. Further mechanistic study showed that the aberrant upregulation of miR-454-3p was identified as the reason of WTX loss in HCC. Conclusions: WTX is a tumor suppressor gene in HCC, miR-454-3p/WTX/TGFβ signaling will provide a new direction for the diagnosis and treatment of HCC.


Introduction
Hepatocellular carcinoma (HCC) is a common malignant tumor, ranking fourth in incidence and third in mortality among tumors [1][2][3][4]. Although early-stage hepatocellular carcinoma patients can be treated with surgery, 70% of patients will recurrence and metastasize within five years [5,6]. Due to the lack of therapeutic biomarkers for effective diagnosis, the overall survival rate of HCC patients is poor [7,8]. Therefore, to search for efficient therapeutic targets involving in the progression of HCC is becoming urgent.
WTX (Wilms Tumor gene on X chromosome) is an X-linked tumor suppressor discovered in the Wilms tumor [9][10][11]. WTX forms a complex with β-TrCP2, β-catenin, APC, and AXIN1 to promote the ubiquitination of β-catenin protein, which inhibits the activation of WNT signaling pathway in Wilms tumors [12][13][14]. Recent studies have shown that WTX can regulate the occurrence and development of gastric cancer by controlling AKT1 expression [15]. However, the biological function of WTX in liver cancer is still unknown.
MicroRNAs (miRNAs) are non-coding RNA composed of [19][20][21][22][23][24][25] nucleotides that can complementally bind to the mRNA of target genes and inhibit post-transcriptional processes [16,17]. More and more studies show that miRNAs play important biological functions in the pathogenesis of Ivyspring International Publisher tumors, indicating that they can be used as new targets for tumor diagnosis and treatment [18][19][20]. Mir-454-3p has been confirmed to be abnormally expressed in a variety of cancers, down-regulated in non-small cell lung cancer and breast cancer, and up-regulated in glioma and cervical cancer [21][22][23][24]. However, the biological mechanisms of miR-454-3p in HCC needs further explored.
In our study, we identified to identify the regulatory mechanism of WTX in the occurrence and development of HCC. We found that downregulation of WTX in HCC was due to the abnormal increase of miR-454-3p, which is related to the poor prognosis of patients. WTX loss promoted HCC cells proliferation, migration, invasion and autophagy through the activation of the TGF-β signaling. And we determined that miR-454-3p/WTX/TGF-β signaling axis regulates the occurrence and development of HCC.

Tissue specimens
In total, 32 matched human HCC and normal liver samples were collected from patients undergoing liver cancer resection at Liaocheng People's Hospital (Liaocheng, P. R. China). The collection and use of specimens were carried out with the informed consent of all patients and the approval of the Medical Ethics Committee of Liaocheng People's Hospital.

Cell culture
HepG2 and Huh7 cell lines were purchased from American Type Culture Collection (Manassas, VA). All cell lines were cultured in DMEM (DMEM, BI, Israel) supplemented with 10% FBS at 37 °C in a 5% CO2 humidified incubator.
To generate stable WTX overexpressing cells, we infect HCC cells with a lentivirus expressing WTX or a negative control (Genechem, Shanghai, China), and use 500 ng/mL puromycin for resistance selection. After 6 days, the cells were collected and verified by real-time RT-PCR.

Transwell invasion and migration assays
Chamber invision ability were performed with the transwell 24-well Boyden chamber (Corning, USA). 5×10 4 cells were seeded into chambers and incubated for 24 h. The cells were fixed with formaldehyde for 20 minutes, stained with 0.1% crystal violet and photographed under a microscope for statistics.

Cell proliferation and scratch-wound assays
Cell proliferation was measured using MTS Assay Kit (Sigma). For cell scratch-wound assays, cells were inoculated in 6-well plates and transfected 12 hours later. A horizontal line was drawn on the monolayers with a 200 µL tip. At 0, 24 and 48 h, they were photographed and analyzed under a microscope.

EdU assay
HCC cells were inoculated in 24-well plates, transfected after 12 hours, and then transfected with 50 μM edu was incubated and finally fixed with 4% paraformaldehyde. The images were taken and analyzed under confocal laser microscope.

Autophagy assay
The HCC cells were inoculated in 24-well plates, and 48 hours after transfection, the cells were stained with CYTO-ID autophagy detection kit. The images were taken and analyzed under confocal laser microscope.

Immunohistochemistry
Firstly, paraffin sections were dewaxed and gradient dehydrated, then antigen repaired, and finally stained.

Animal studies
All animal experiments were conducted in accordance with the guidelines of the Animal Care Committee of Wuhan University of science and technology (P. R. China). The mice used in this experiment were all male BALB/c nude mice aged 4 weeks. All mice were purchased from Charles River Laboratories (P. R. China) and raised in a sterile animal room. HepG2 or Huh7 stable cell lines with a total amount of 1×10 7 cells were subcutaneously injected into the lower right flank of Nude mice. All mice were euthanized 28 days later and tumors were excised carefully and final tumor weight measured.

Statistical analysis
All experiments were independently repeated more than three times, and the experimental results were expressed as means ± SEM. Statistical analysis was performed using the SPSS20 software (SPSS, USA). P < 0.05 was defined as statistically significant.

WTX is downregulated in HCC tissues
To explore the biological function of WTX in Hepatocellular carcinoma (HCC), immunohistochemical (IHC) staining was performed on the collected 32 HCC and paired normal tissues. The results of immunohistochemistry showed that WTX expression was downregulated in HCC tissues ( Figure 1A-B). Western blotting was further implemented and verified WTX loss in HCC ( Figure  1C-D). Kaplan-Meier survival curves showed that lower WTX expression levels in HCC tissues were significantly associated with poor prognosis of HCC patients ( Figure 1E). It suggests that WTX can be a new target for diagnosis and treatment of HCC.

WTX inhibits the malignant potential of HCC cells
To explore the biological mechanism of WTX in HCC, HepG2 and Huh7 cell lines were transfected with WTX expression plasmid (pWTX) or control plasmid (pcDNA3.1) (Figure 2A). MTS assay showed that WTX inhibited the proliferation of HCC cells ( Figure S1A). IF staining also revealed that WTX negatively regulated Ki67 (a cell proliferation marker) protein expression in HCC cell lines ( Figure 2C). Furthermore, EdU assays showed that DNA replication in WTX-overexpressing HCC cells was significantly reduced ( Figure 2D). In an in vitro wound-healing assay, compared with control cells, the overexpression of WTX inhibited the migration ability of cells ( Figure 2E). As shown in Figure 2F, cell migratory and invasive ability were significantly inhibited compared with control cells. Interestingly, we also found that WTX inhibited rapamycin-induced autophagy ( Figure 2B and G). In addition, WTX knockdown promoted proliferation, migration, invasion and autophagy in HCC cells ( Figure S2).
To investigate whether WTX inhibits tumorigenicity in vivo, we constructed stable WTX-overexpressing cells (Lv-WTX-HepG2 and Lv-WTX-Huh7) and control cells (Lv-Con-HepG2 and Lv-Con-Huh7). We injected Lv-WTX cells or Lv-Con cells subcutaneously into the right abdomen of nude mice. All mice were euthanized 28 days later. As expected, the average volume and weight of the tumors injected with Lv-WTX cells were significantly reduced ( Figure 3A-B). In addition, Ki67 and LC3B in the experimental group tumors were significantly reduced ( Figure 3C).

Aberrant miR-454-3p upregulation relates to WTX loss in HCC
To understand the regulatory mechanism of WTX, we used bioinformatics algorithms to predict 5 potential binding miRNAs in WTX 3'-UTR ( Figure  4A). Interestingly, only miRNA-454-3p was significantly increased ( Figure 4B). The analysis of starBase showed that the expression of miRNA-454-3p in the tissues of HCC patients was higher than that of normal tissues ( Figure 4C). The survival analysis from the TCGA database also showed that the survival of HCC patients with high expression of miR-454-3p was poor ( Figure 4D). Similarly, by detecting the 32 cases of HCC and matched normal liver tissue samples, we found that miR-454-3p in HCC was significantly higher than that in neighboring tissues, which was negatively correlated with the expression of WTX ( Figure 4E-F). To verify whether miR-454-3p can directly bind to the WTX3′ UTR, luciferase assays were performed ( Figure 4G). The results conrmed that miR-454-3p could directly regulate WTX ( Figure 4H). In addition, after miR-454-3p mimics transfection, WTX was also significantly reduced in HCC cells ( Figure 4I).

WTX inhibits HCC progression and miR-454-3p
could directly regulate WTX, we investigated whether miR-454-3p have a similar effect on HCC tumorigenesis? To test this hypothesis, HepG2 and Huh7 cells were transduced with miR-454-3p mimics or miR-454-3p mimic NC, WTX protein level was detected by western blot ( Figure 5A). As expected, miR-454-3p significantly promoted the proliferation ability of HCC cells (Figure 5B-D). As shown in Figure  5E, 5F, cell migratory and invasive ability were significantly enhanced in the miR-454-3p mimics groups. Interestingly, autophagy also increased significantly in the miR-454-3p mimics groups ( Figure  5G), the opposite experimental results appeared in the miR-454-3p inhibitor groups ( Figure S3 and Figure   S4).

miR-454-3p -WTX axis regulates TGF-β signaling in HCC cells
Genes related to tumorigenesis were verified by RT-qPCR. Obviously, only TGF-β2 is regulated by WTX ( Figure 6A and Figure S5A). In addition, a positive correlation between TGFβ2 and miR-454-3p was observed in HCC tissues, and a negative correlation with WTX ( Figure 6B-C). In addition, after WTX overexpression or miR-454-3p inhibition, TGFβ2 mRNA decays faster, indicating that they both interfere with the conversion of TGFβ2 mRNA ( Figure  6D and Figure S5B). miR-454-3p mimics caused an increase in TGFβ2 mRNA levels ( Figure 6E and Figure  S5C), which was restored by overexpression of WTX ( Figure 6F and Figure S5D). In addition, after transfection of miR-454-3p mimics, the levels of TGFβ2, N-cadherin and phosphorylated SMAD3 increased, and E-cadherin decreased ( Figure 6G-H). In overexpressing WTX cells, the levels of TGFβ2, N-cadherin and phosphorylated SMAD3 decreased, and E-cadherin increased ( Figure 6I-J). Collectively, miR-454-3p activates TGFβ2 signaling by targeting WTX in HCC cells.

Discussion
In this study, we demonstrated that high expression of WTX inhibited the malignant potential of HCC cells. In addition, through the analysis of clinical samples, we found that WTX expression is reduced in HCC tissues, and lower levels of WTX are associated with poorer prognosis of HCC patients. Our data shows that WTX acts as a tumor suppressor gene in HCC, which is consistent with previous reports on the biological functions of WTX in several other cancers, including gastric cancer [15], colon cancer [11], and cervical cancer [26]. These results indicate that WTX can be used as a new target for tumor prevention, diagnosis and treatment. This study found the biological function of WTX in HCC for the first time. Our data showed that decreased WTX expression could activate TGF-β2 signal transduction in HCC cells. It is well known that abnormal activation of TGF-β2 signal can lead to tumor development and metastasis [27][28][29][30]. Therefore, regulating the activity of TGF-β2 signaling may be one of the biological functions of WTX in HCC. RT-qPCR and Western blot farther conrmed that WTX negatively regulated TGF-β2 signaling activity and inhibited HCC proliferation. Our research showed that the absence of WTX could cause abnormal activation of TGF-β2 signal in HCC, thereby promoting the occurrence and development of HCC.
Abnormal expression of miRNA often leads to the silencing of target genes [17,31,32]. To explore the molecular mechanism leading to the loss of WTX, we screened miR-454-3p as a candidate for regulating WTX expression through bioinformatics algorithms. It was reported that miR-454-3p is up-regulated in breast cancer [22], nasopharyngeal carcinoma [33] and cervical cancer [24], and plays a role as an oncogene. We haved verified that miR-454-3p could directly regulate WTX through luciferase activity experiments. Our research confirms that the miR-454-3p/WTX/ TGF-β2 signaling axis regulates the occurrence and development of HCC, providing new strategies and targets for the diagnosis and treatment of HCC.
In summary, our research revealed the biological function of WTX in HCC and determined the molecular mechanism of WTX loss. Mir-454-3p inhibits WTX expression, activates the downstream TGF-β2 signaling pathway, increases cell inhibits HCC proliferation, migration, invasion and autophagy. The study also showed that miR-454-3p/ WTX/TGF-β2 signaling may provide a new target for the diagnosis and treatment of HCC.