TET3 Promotes HCC Proliferation And Metastasis Via lncRNA ARAP1-AS1

Qiuyu Zhuang Mengchao Hepatobiliary Hospital of Fujian Medical University Xuechun Xu Mengchao Med-X Center, Fuzhou University Zhiguo Dai Mengchao Med-X Center, Fuzhou University Xiaoyuan Zheng Mengchao Hepatobiliary Hospital of Fujian Medical University Wuhua Guo Mengchao Hepatobiliary Hospital of Fujian Medical University Xiaolong Liu Mengchao Hepatobiliary Hospital of Fujian Medical University Yingchao Wang Mengchao Hepatobiliary Hospital of Fujian Medical University Bixing Zhao (  bixingzhao@gmail.com ) Mengchao Hepatobiliary Hospital of Fujian Medical University


Introduction
Hepatocellular carcinoma (HCC) ranks the most common primary liver cancer and is one of the leading causes of cancer related death worldwide (1). Although partial hepatectomy or liver transplantation is available for patients with early-stage HCC, most of HCC patients are diagnosed at an advanced stage and miss the best treatment period owing to the lack of early symptoms and detection (2). Therefore, further understanding the molecular mechanism underneath HCC development is fundamental for the designs and inventions of more effective diagnostic and therapeutic approach in the future.
Epigenetic characteristics have been emerging as the main features of cancer (3). Epigenetic regulation, such as DNA/RNA methylation and histone modi cation, refers to functionally relevant changes to the genome that do not involve a change in the nucleotide sequence. As an important epigenetic modi cation, DNA methylation has been validated to be critical for the regulation of gene expression, histone modi cation and other biological processes (4,5). Accordingly, aberrations of DNA methylation and proteins involved in DNA methylation process are signi cantly correlated with tumor malignancy and prognosis of patients (6).
Ten-eleven translocation enzyme (TET) family catalyzes the oxidation of 5-methylcytosine (5mC) to 5hydroxymethylcytosine (5hmC) and causes DNA demethylation (7). The TET family comprises three members: TET1, TET2, and TET3 with different cellular locations and biological functions (8). Here we focused on TET3, which was identi ed to be upregulated in HCC tissues and associated with the poor prognosis of HCC patients. Increasing evidence indicates that TET3 participates in the tumorigenesis of glioblastoma, ovarian cancer, breast cancer and several other types of cancers (9,10), but the functions of TET3 in HCC tumorigenesis remains to be elusive.
In the present study, we identi ed that TET3 accelerated the proliferation and metastasis of HCC cells.
Mechanical assay revealed that lncRNA ARAP1-AS1 as a downstream target of TET3 played critical roles in regulating the biological function of TET3. In conclusion, our ndings suggest that TET3 may exert as a potential tumor regulator to involve in HCC progression, which provided a new insight into HCC tumorigenesis.

Cell culture
The human hepatoma cell line HepG2 and human embryonic kidney cell HEK293T were purchased from the American Type Culture Collection (ATCC). The SMMC-7721 and Huh7 cell lines were obtained from the Chinese Academy of Science (Shanghai, China). All the cell lines were cultured in Dulbecco's modi ed eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (ExCell) at 37°C and 5% CO 2 . Viral production, transduction, and siRNA transfection Lentivirus was produced as previous description (11) with sight modi cation. Brie y, lentiviral constructs were packaged into lentivirus with lipofectamine 3000 (Invitrogen) and viral supernatants were harvested at 48 h post transfection. To generate stable-expressing cells, cells were infected with the viral supernatant and selected by puromycin (1 μg / mL). siRNAs (Sangon Biotech) speci cally targeting TET3 and lncRNA ARAP1-AS1 were transfected with lipofectamine 3000 (Invitrogen). Sequences of siRNAs are listed in Table 1.

Statistical analysis
Statistical analysis of data was performed with GraphPad Prism 9.0. *p<0.05 was regarded as statistically signi cant. **p<0.01, ***p<0.001, ****p<0.0001. All the data were shown as means ± SD through at least three independent experiments.

TET3 is upregulated in HCC tumor tissues and indicates the poor prognosis of HCC patients
According to the RNA sequencing data of an HCC sample cohort with 65 pairs of tumor and their corresponding peri-tumor tissues, which was described in our previous work (11), we identi ed that TET3 was signi cantly upregulated in HCC tumor tissues ( Fig 1A). To further con rm this result, we detected TET3 expression in an expanded HCC sample cohort containing 103 pairs of HCC and the corresponding peri-tumor liver tissues by qRT-PCR. A consistent result was observed that TET3 was upregulated in HCC tumor tissues (Fig 1B). Similarly, TET3 was also identi ed to be upregulated in HCC according to The Cancer Genome Atlas (TCGA) RNA-Seq dataset (Fig 1C). Further Kaplan-Meier analysis of TCGA data revealed that high TET3 expression was signi cantly associated with poor overall survival (OS) and poor relapse-free survival (RFS) of patients with HCC (Fig 1D and E). Moreover, TET3 expression was gradually elevated from grade 1 to grade 4 in HCC according to TCGA dataset (Fig 1F). Taken together, our data indicated that TET3 is upregulated in HCC tumor tissues and its high expression is correlated with poor prognosis of HCC patients, suggesting the possible contribution of TET3 to HCC tumorigenesis.
TET3 is required for the proliferation and metastasis of HCC cells In order to investigate the biological function of TET3 in HCC, we rst knocked down TET3 in Huh7 cells with two independent siRNA sequences (siTET3 #1 and #2). qRT-PCR was performed to con rm the e ciency of these two sequences (Fig 2A). Huh7 with TET3 knockdown showed reduced proliferative capacity compared with the control cells in CCK8 assay (Fig 2B). Consistent results were observed in colony formation assay, in which TET3 knockdown signi cantly impaired the colony formation of Huh7 cells (Fig. 2C). To con rm the role of TET3 in HCC cell proliferation, we overexpressed TET3 in HepG2 cells and proved that TET3 was successfully overexpressed by Western blot (Fig. 3A). Overexpression of TET3 in HepG2 signi cantly stimulated cell proliferation (Fig. 3B) and colony formation (Fig. 3C). Similar results were obtained in SMMC-7721 cells that TET3 overexpression increased the proliferating ability and colony-formation ability (Fig. 3D-E). Taken together, the results from both our knockdown and overexpression experiments indicated that TET3 may serve as an oncoprotein to promote HCC cell proliferation.
Additionally, the effects of TET3 on cellular migration and invasion were also assessed in vitro. The transwell assays were performed and the results showed that TET3 overexpression markedly induced the migratory and invasive ability of both HepG2 and SMMC7721 cells compared with the control cells ( Fig.   4A and B). Thus, our results suggested that beside proliferation, TET3 also plays an important role in promoting HCC metastasis.

LncRNA ARAP1-AS1 is the key downstream mediator of TET3 in HCC progression
To identify the downstream mediator(s) of TET3, we performed RNA-seq analysis in TET3 stableexpressing HepG2 and its corresponding control cells. TET3 is an epigenetic eraser which oxidizes 5mC to 5hmC. It has been reported that 5hmC is positively associated with gene expression (12). Therefore, the upregulated genes in the cells with TET3 overexpression were the reasonable downstream candidates (Fig. 5A). Accordingly, we screened the top 50 upregulated genes for their contribution to HCC proliferation and their transcriptional dependence on TET3. Our attention was captured by lncRNA ARAP1-AS1, which was upregulated to two folds in TET3-overexpressing HepG2 cells. It has been reported that ARAP1-AS1 plays important roles in promoting tumorigenesis and metastasis in several cancer types but not in HCC (13)(14)(15)(16)(17)(18)(19). qRT-PCR analysis indicated that the expression of ARAP1-AS1 was regulated by TET3, as we observed an upregulation of ARAP1-AS1 upon TET3 overexpression in both HepG2 and SMMC-7721 cells, which is ideally consistent with the RNA-seq results (Fig. 5B).
Next, we investigated whether ARAP1-AS1 was involved in TET3-dependent HCC proliferation. We transfected siRNA against ARAP1-AS1 or the control siRNA in TET3-overexpressing HepG2 and the cells were subjected to proliferation assay. The knockdown e ciency of ARAP1-AS1-targeting siRNA was veri ed with qRT-PCR (Fig. 5C). Rescue experiments indicated that knockdown of ARAP1-AS1 abolished the induction of proliferation by TET3 overexpression in CCK8 assay (Fig. 5D). Consistent results were observed in colony formation assay, in which ARAP1-AS knockdown reduced the colony formation capacity induced by TET3 overexpression (Fig. 5E). Taken together, these results indicated that TET3 transcriptionally regulated lncRNA ARAP1-AS1, which is required for TET3-mediated proliferation of HCC cells.

Discussion
Recently, the function of DNA methylation and enzymes involved in DNA methylation processes in HCC have attracted much attention and widen the understanding of HCC pathogenesis (20)(21)(22). Our study indicated that TET3, was upregulated in HCC tissues and was positively correlated with poor prognosis of HCC patients. Our study also veri ed that TET3 was required for the proliferation, migration and invasion of HCC cells. In terms of mechanism, our study identi ed that TET3 in uenced HCC progression by regulating lncRNA ARAP1-AS1.
In human genome, 5hmC is a common epigenetic modi cation in promoters, gene bodies and gene regulatory elements (23). Recent studies have suggested that 5hmC modi cation is associated with cancer pathobiology, with the global reduction of 5hmC in both hematological and solid tumors, including colon, liver, lung, skin, prostate, and breast tumors (23)(24)(25)(26). As one of the enzymes that catalyze 5hmC formation, the upregulation of TET3 we observed in HCC tissues is contrary to the previously described global reduction of 5hmC (22). This discrepancy might be due to the redundant function of TET family member to catalyze 5hmC formation. In two cohorts in which genomic 5hmC contents of HCC tissues were signi cantly decreased, the expression level of TET2 in tumor tissues was signi cantly decreased (22). This work indicated that among three members of TET family, downregulation of TET2 is responsible for the global loss of 5hmC in HCC. This result was consistent with the biological function of TET2 which has been previously demonstrated to repress HCC tumorigenesis (27). Meanwhile, our data indicated that TET3 was upregulated in HCC tissues and promoted the proliferation and metastasis of HCC cells.
LncRNAs have been reported to regulate tumor cell proliferation, metabolism, and metastasis, and thus have been attracted much attention as a novel and important regulator of cancer development. To address the underlying mechanism of TET3 in HCC, we performed RNA-seq in control and TET3overexpressing HCC cells. lncRNA ARAP1-AS1 together with other upregulated genes were selected as potential downstream targets. Previous work has proved that the upregulation of lncRNA ARAP1-AS1 promotes the proliferation and metastasis in several cancer types but unknown in HCC (13)(14)(15)(16)(17)(18). Our ndings indicated that ARAP1-AS1 upregulated by TET3 exerts similar roles in HCC as in other cancer types. However, further studies are required to clarify the speci c mechanisms underlying TET3 increase the expression of ARAP1-AS1. Moreover, increasing evidence indicates that several signaling pathways has been demonstrated to mediate biological functions of ARAP1-AS1 (14,16,17), but the speci c effector of TET3/ARAP1-AS1 axis in regulating HCC tumorigenesis remains to be explored.

Conclusion
In this study, we demonstrated that TET3, a member of TET family which catalyzes the conversion from 5mC to 5hmC, accelerates proliferation and metastasis of HCC cells via lncRNA ARAP1-AS1. Our ndings exempli ed the critical role of DNA methylation proteins in HCC tumorigenesis and implied that TET3 may have attractive translational potential of HCC therapy.