METTL3-mediated m6A modification of lncRNA TSPAN12 promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma through SENP1-dependent deSUMOylation of EIF3I

doi:10.21203/rs.3.rs-2466161/v1

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Research Article

METTL3-mediated m6A modification of lncRNA TSPAN12 promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma through SENP1-dependent deSUMOylation of EIF3I

https://doi.org/10.21203/rs.3.rs-2466161/v1

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Background

Our previous study revealed that lnc-TSPAN12 was remarkably upregulated in HCC and correlated with poor survival. However, the role of lnc-TSPAN12 in regulating epithelial-mesenchymal transition (EMT) and metastasis in hepatocellular carcinoma (HCC) remains uncharacterized.

Methods

The biological function of lnc-TSPAN12 in HCC metastasis was investigated by loss- and gain-of-function experiments both in vitro and in vivo. RNA pull-down, RNA immunoprecipitation and fluorescence in situ hybridisation were performed to examine the interaction between lnc-TSPAN12 and Eukaryotic Translation Initiation Factor 3 subunit I (EIF3I)/Sentrin/SUMO-Specific Protease 1 (SENP1) (SUMO: small ubiquitin-like modifier). EIF3I ubiquitination and SUMOylation modifications were analyzed using co-immunoprecipitation and western blot.

Results

Functional assays showed that lnc-TSPAN12 positively regulated the migration, invasion and EMT of HCC cells in vitro and accelerated liver metastasis in patient-derived tumour xenograft metastasis models in vivo. Importantly, methyltransferase like 3 (METTL3) mediated N6-methyladenosine modification is responsible for the upregulation of lnc-TSPAN12, contributing to lnc-TSPAN12 stabilization. Mechanistically, lnc-TSPAN12 physically interacts with EIF3I/SENP1 and functions as a scaffold to facilitate the binding of SENP1 to EIF3I, which suppresses EIF3I SUMOylation and ubiquitin degradation, ultimately activating Wnt/β-catenin signalling to promote EMT and metastasis in HCC.

Conclusions

Our findings elucidate the regulatory mechanism of lnc-TSPAN12 involved in metastasis of HCC and highlight the lnc-TSPAN12-EIF3I/SENP1 axis as a promising therapeutic target for the disease.

Lnc-TSPAN12

Hepatocellular carcinoma

Long noncoding RNAs

EIF3I

SENP1

N6-methyladenosine

EMT

Primary liver cancer is a highly aggressive malignancy, serving as the sixth most frequently diagnosed cancer and the third leading cause of cancer-related death globally [1]. Hepatocellular carcinoma (HCC) is the most common subtype of primary liver cancer, accounting for approximately 75–85% of all cases [1]. Despite the advancements made in targeted therapies and immunotherapies, the 5-year survival rate of HCC patients remains poor, which is attributed to frequent postoperative recurrence and metastasis [2]. Thus, elucidating the molecular mechanisms underlying HCC metastasis and identifying novel molecular targets will contribute to the development of effective treatment modalities for this lethal disease.

Long non-coding RNAs (lncRNAs) are a class of RNA molecules that are over 200 nucleotides in length and without protein-coding capacity. In recent years, lncRNAs have been identified as important regulators in various biological processes, such as chromatin modification, transcriptional activation or inhibition, post-transcriptional regulation and protein function regulation [3–7]. Increasing evidence has demonstrated that lncRNAs are frequently dysregulated and play a vital role in the occurrence and progression of various cancers, including HCC [8–10]. Therefore, further identification of HCC-related lncRNAs and exploration of their potential functions in HCC are necessary.

Tumour metastasis is a complex process involving multiple steps and factors. The epithelial-mesenchymal transition (EMT), a process in which epithelial cells acquire a more migratory and invasive mesenchymal phenotype, is regarded as the initiation and key step in tumour metastasis [11]. During EMT, epithelial tumour cells lose their cell polarity and adhesion and increase their migration and invasion ability, making it easier to infiltrate into adjacent tissues or enter blood vessels [12]. Accumulating studies have indicated that EMT is closely relevant to tumour metastasis, stemness and drug resistance [13–15]. Recently, lncRNAs have been reported to exert significant effects on EMT and thereby promote tumour metastasis by regulating key EMT transcription factors [16–17]. However, the underlying molecular mechanisms of lncRNAs in EMT regulation in HCC remain poorly understood.

The N6-methyladenosine (m6A) RNA modification, as the most abundant endogenous modification in eukaryotic mRNAs and lncRNAs, is a dynamic and reversible process catalyzed by methyltransferases (also called writers: methyltransferase-like 3 (METTL3), METTL14 and WT1-associated protein (WTAP), removed by demethylases (also called erasers: fat-mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5) and recognized via m6A-specific binding proteins (also called readers: YTH domain-containing family protein (YTHDF1/2/3), insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1/2/3) [18, 19]. This modification is extensively involved in regulating various aspects of RNA processing and metabolism, including alternative splicing, stability, nucleation, translation and RNA-protein interactions [20]. Furthermore, the crucial role and mechanism of m6A modification in cancer occurrence and progression have drawn increasing attention [21]. The highly expressed METTL3 in gastric cancer promotes m6A modification of hepatoma-derived growth factor (HDGF) mRNA, whereas the m6A reader IGF2BP3 directly recognizes and binds to the m6A site of HDGF mRNA to enhance its stability, thereby promoting tumour angiogenesis and glycolysis [22]. METTL3-mediated m6A modification has been identified as a critical factor in the resistance of HCC to sorafenib therapy, which significantly promotes HCC resistance to sorafenib by suppressing the stabilization of its key downstream target Forkhead box protein O3 (FOXO3) mRNA through a YTHDF1-dependent manner [23]. However, whether m6A modification is involved in lncRNA regulation and the role of m6A in lncRNA transcripts in HCC metastasis remain largely unknown.

Our previous study revealed that lnc-TSPAN12 was remarkably upregulated in HCC and correlated with poor prognosis, and it can serve as a potential microvascular invasion-related biomarker in HCC [24]. In this study, we further confirmed that lnc-TSPAN12 upregulation is attributable to m6A modification mediated by METTL3, leading to lnc-TSPAN12 stabilization. In addition, lnc-TSPAN12 promoted invasion, metastasis and EMT of HCC cells in vitro and in vivo. Mechanistically, lnc-TSPAN12 can bind to Eukaryotic Translation Initiation Factor 3 subunit I (EIF3I) and enhance Sentrin/SUMO-Specific Protease 1 (SENP1)-mediated EIF3I deSUMOylation (SUMO: small ubiquitin-like modifier). The increased crosstalk between SENP1 and EIF3I competitively inhibited EIF3I ubiquitination and degradation, leading to the enhancement of EIF3I stability. Collectively, our results suggest that lnc-TSPAN12 plays a pivotal role in HCC metastasis and may act as a promising therapeutic target of HCC.

Cell culture

Human liver cancer cell lines (Huh7 and Hep3B) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Huh7 and Hep3B cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco). All media were supplemented with 10% foetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Sigma). All cell lines were cultured in a humidified atmosphere containing 5% CO₂ at 37°C.

Wound-healing assay

Different groups of transfected Huh7 and Hep3B cells were seeded in a six-well plate and grew until ~ 80% confluence. The cell monolayer was wounded by scratching with a 200 µl pipette tip. Cell movement was captured by an Olympus microscope (Olympus, Tokyo, Japan) at 0 and 72 h.

Transwell assay

Transwell chambers (Corning, New York, USA) were used to perform migration and invasion assays. A total of 2.5 × 10⁵ cells were seeded into the upper chamber of each insert with 100 µL serum-free medium and 600 µL medium with 10% FBS added to the lower chambers. After incubation at 37°C for 24 h, the cells were fixed with methanol and stained with 1% crystal violet. The cells on the top side of the membrane were removed using cotton swabs, and those that had invaded the underside of the membrane were counted and imaged under a microscope (Olympus, Tokyo, Japan).

RNA isolation, reverse transcription and quantitative real-time PCR (qRT-PCR)

The total RNA from frozen tissue samples or cultured cells was isolated using TRIzol reagent (Takara, Japan) following the manufacturer’s instructions. The first-strand complementary DNA was synthesised using iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). qRT-PCR was performed using SYBR green (Bio-Rad) under a cycling protocol (40 cycles) with 10 s denaturation (95°C), 20 s annealing (60°C) and 15 s extension (72°C). All primers and shRNA sequences used in this study are listed in Additional file 2: Table S1, S2. The relative fold changes in the expression were analyzed using the 2^−△△CT method.

Western blot

Cells were lysed with RIPA buffer with protease inhibitor cocktail (Roche), and protein samples (20 µg) were separated by SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked in 5% non-fat milk and incubated with primary antibodies at 4 ℃ overnight. After incubation with secondary antibodies at room temperature for 1 h, the signal intensities were visualized by a chemiluminescence instrument and analyzed with Image J software. Information of antibodies used in this study can be found in Additional file 2: Table S3.

RNA immunoprecipitation (RIP)

The RIP experiment was performed using a Magna RIP Kit (Millipore, Schwalbach, Germany) in accordance with the manufacturer’s instructions. Briefly, lysates were prepared in RIP lysis buffer containing protease inhibitor cocktail for per immunoprecipitation. A total of 5 µg interest antibody was incubated with Protein A/G Magnetic Beads for 30 min at room temperature. The RIP lysate was added to each bead–antibody complex in the RIP immunoprecipitation buffer and incubated overnight at 4°C. Each immunoprecipitant was resuspended in proteinase K buffer and incubated at 55°C for 30 min. Purified RNA was obtained and analyzed using qRT-PCR assay.

Methylated RIP (MeRIP)-qPCR

MeRIP was performed following the protocols of the Magna MeRIP m6A Kit (Millipore, Schwalbach, Germany). Briefly, 18 µg mRNA was chemically fragmented into approximately 100 nt-long fragments using a fragmentation buffer. Then, the Magna ChIP Protein A/G Magnetic Beads were incubated with m6A-specific antibody in immunoprecipitation buffer for 1 h at room temperature. The mixture was incubated with the MeRIP reaction mixture at 4°C overnight. The m6A enrichment was analyzed via RT-qPCR analysis.

RNA binding protein pull-down assay

RNA binding protein pull-down assay was performed using the Thermo Fisher Scientific™ RNA pulldown Kit (Thermo Fisher Scientific, Pittsburgh, PA, USA) following the manufacturer’s instruction. Sense and antisense strands of lncRNA cDNA were synthesised by Ribo Biotech (RiboBio, Guangzhou, China). Biotin-labelled lncRNAs were obtained using in vitro transcription with T7 RNA polymerase and Biotin RNA Labeling Mix (Roche). Cell lysates extracted from HCC cells were incubated with 50 pmol purified biotinylated transcripts for 1 h at 4°C with rotation. Then, streptavidin magnetic beads were added to the cell protein lysate to precipitate the RNA–protein complex. The beads were washed thrice and boiled in SDS buffer to retrieve proteins for western blot analysis.

Fluorescence in situ hybridization (FISH)

The lnc-TSPAN12 FISH probe was synthesised by GenePharma Co., Ltd (Shanghai, China). A FISH kit was used to detect the subcellular localization of lnc-TSPAN12 using a confocal microscope (Zeiss, Germany) in accordance with the manufacturer’s protocol (RiboBio). The protein expressions of SENP1 and EF3I in HCC cells were determined by immunofluorescence with anti-SENP1 and anti-EF3I antibodies (1:100; Abcam, Cambridge, UK), respectively, as previously described.

Immunoprecipitation (IP)

HCC cells were lysed with lysis buffer on ice for 1 h. The samples were clarified by centrifugation at 14000 g for 30 min, and the supernatants were incubated with the specified antibodies at 4°C overnight. Protein–antibody complexes were captured with Protein A/G agarose beads (Life Technologies, 20421) at 4°C for 2 h mixing by rotation. Then, the beads were washed, boiled and subjected to immunoblotting analysis.

Co-immunoprecipitation (Co-IP) assay

Co-IP was performed as follows. Briefly, the treated HCC cells were collected and lysed in ice-cold IP lysis buffer. The cell lysates were used for Co-IP and incubated with anti-Flag antibodies at 4 ℃ overnight and human anti-IgG as a negative IP control, respectively. The immune complexes were incubated with protein A/G agarose beads for 2–3 h. Then, beads were washed at least 5 times, treated and boiled for 5 min with 1× SDS sample buffer. Western blot assays were performed as described previously.

Immunohistochemistry (IHC) Staining

The xenograft tissues were fixed in 10% neutral formalin and embedded in paraffin. IHC staining was performed on paraffin-embedded xenograft tissues. Briefly, paraffin-embedded slices were dried at 65°C for 4 h. They were deparaffinised by xylene for 10 min twice and soaked in ethyl alcohol (100%, 100%, 95%, 90%, 80%, and 70%) for 2 min and soaked in purified water. After antigen retrieval by microwave in sodium citrate buffer for 16 min, the evaporated water was replaced every 8 min. The slices were cooled at room temperature. After washing with phosphate buffered saline (PBS) thrice, incubation with 3% H₂O₂ for 15 min and washing with PBS three times again, the slices were blocked in 10% sheep serum albumin for 40 min and incubated overnight at 4°C with primary antibodies. On the second day, the slices were rewarmed for 30 min and washed thrice with PBS. Then, the secondary antibody was added to the slices for 40 min incubation at room temperature. Colour development was performed using 3ʹ3-diaminobenzidine tetrahydrochloride as a chromogen, and the slices were counterstained using haematoxylin for 2 min, washed with water, soaked in 100% ethyl alcohol for 1 min and air dried. Finally, the slices were sealed with Neutral Balsam.

In vivo experiment

All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the West China Hospital, Sichuan University, China. For the construction of the liver metastasis model, five male nude mice were inoculated per group. A total of 2 ×10⁶ Huh7^NC, Huh7^sh-TSPAN12#1 and Huh7^sh-TSPAN12#2 cells were injected into the spleens of the mice. Then, the cells were transported to the liver for the spontaneous formation of metastatic lesions.

Statistical analysis

Statistical analysis was performed using SPSS 24.0 (IBM Corporation, Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). Data are presented as mean ± standard error of the mean. Student’s t-test or one way analysis of variance was performed to evaluate the differences between two or more than two groups, respectively. The cumulative survival curves were analyzed using the Kaplan–Meier method, with the log-rank test being used for comparison. P < 0.05 was considered statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Lnc-TSPAN12 promotes HCC cells metastasis in vitro and in vivo

Our previous study demonstrated the high expression of lnc-TSPAN12 in HCC and its association with unfavourable clinicopathological features and poor prognosis. To further determine the potential role of lnc-TSPAN12 in HCC, we knocked down or overexpressed lnc-TSPAN12 in Huh7 and Hep3B HCC cell lines by transfecting shRNA (pEX-2) or overexpressing a plasmid (pcDNA3.1) and confirmed the transfection efficiency by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis (Additional file 1: Fig. S1A-B). Wound healing and transwell assays showed that knockdown of lnc-TSPAN12 evidently inhibited the migration and invasion of Huh7 and Hep3B cells in vitro when compared with the scramble control group (Fig. 1A-D, Additional file 1: Fig. S1C, D). By contrast, the ectopic expression of lnc-TSPAN12 enhanced migration and invasion of HCC cells (Fig. 1A-D, Additional file 1: Fig. S1C, D).

EMT has been recognized as a critical process in HCC metastasis [25, 26]. Thus, we aimed to determine whether lnc-TSPAN12 is involved in inducing EMT in HCC cells. As shown in Fig. 1E, F, knockdown of lnc-TSPAN12 significantly increased the expression of epithelial cell marker E-cadherin but decreased those of mesenchymal cell markers Vimentin and Fibronectin. Nevertheless, overexpression of lnc-TSPAN12 presented the opposite result. To better demonstrate this phenomenon, we used confocal microscopy after immunofluorescence staining. The expressions of E-cadherin and Vimentin in Huh7 cells were consistent with those observed in western blot analysis (Fig. 1G, H). Collectively, our data suggest that lnc-TSPAN12 may promote HCC metastasis by regulating EMT.

To further explore the promoting effects of lnc-TSPAN12 on HCC cell metastasis, we firstly constructed stable lnc-TSPAN12-knockdown Huh7 HCC cells (sh-lnc-TSPAN12#1, 2) and established patient-derived xenograft (PDX) models for liver metastasis by injection into the inferior hemispleen of nude mice. As shown in Fig. 2A-D, after knockdown of lnc-TSPAN12 in Huh7 cells, the numbers and luciferase activity of metastatic nodules in the liver significantly decreased compared with those of the control cells. Haematoxylin and eosin staining showed that the metastatic foci in the sh-lnc-TSPAN12#1,2 cells dramatically shrunk in the liver sections (Fig. 2E). Immunohistochemical staining analysis revealed that knockdown of lnc-TSPAN12 led to a reduced level of Vimentin and an increased level of E-cadherin in xenograft tumour tissues (Fig. 2F). Taken together, these findings suggest that lnc-TSPAN12 may function as an oncogenic driver in the metastasis of HCC.

Lnc-TSPAN12 specifically interacts with EIF3I

Given that the function of lncRNA is related to its subcellular localization, we firstly detected the distribution of lnc-TSPAN12 by RNA fluorescence in situ hybridization (FISH) in Huh7 cell. The result suggested that lnc-TSPAN12 was predominantly present in the cytoplasm (Additional file 1: Fig. S2A). To elucidate the potential molecular mechanisms of lnc-TSPAN12 in HCC metastasis, we performed RNA-seq analysis to investigate the difference in the gene expression profiles of Huh7 cell before and after lnc-TSPAN12 knockdown. Hierarchical clustering showed that a total of 4,505 genes (FDR＜0.05 and｜log2FC｜> 1.0) were significantly altered in lnc-TSPAN12 knockdown Huh7 cell when compared with the control cell (Additional file 1: Fig. S2B, Table S4). KEGG enrichment analysis revealed that the enriched differential genes belonged to several key biological processes and signal pathways, including cell adhesion, gap junctions, extracellular matrix receptor interactions and TGF-β, which are closely associated with EMT and cancer metastasis (Additional file 2: Fig. S2C). These data suggest that lnc-TSPAN12 may be a critical modulator in HCC metastasis.

To further investigate the regulatory role of lnc-TSPAN12 in HCC metastasis, we performed RNA pull-down assay combined with mass spectrometry (MS) to screen lnc-TSPAN12-interacting proteins. In brief, the extracted proteins were subjected to silver staining and SDS-PAGE analysis, and several distinct bands were selected for mass spectrum analysis (Fig. 3A and Additional file 2: Table S5). We noted that EIF3I, the top ranked lnc-TSPAN12-interacting protein in band 1, has been shown to play critical roles in cancer progression and β-catenin pathway [27, 28]. Importantly, Wnt/β-catenin signalling is deemed as a powerful driver of EMT [26]. Therefore, we selected this protein for subsequent validation. We further identified EIF3I as specific lnc-TSPAN12-binding protein by western blot (Fig. 3B). In addition, RNA immunoprecipitation (RIP) assays were adopted with antibodies against EIF3I to demonstrate the association between EIF3I and lnc-TSPAN12 (Fig. 3C). Furthermore, lnc-TSPAN12 FISH followed by immunofluorescence of EIF3I verified the colocalization of lnc-TSPAN12 and EIF3I in the cytoplasm of Huh7 cell, further supporting their interaction (Fig. 3D). Taken together, these data suggest that lnc-TSPAN12 physically interacts with EIF3I.

To identify the binding region of lnc-TSPAN12 that interacts with EIF3I, we constructed a series of lnc-TSPAN12 truncated fragments based on the predicted secondary structure of lnc-TSPAN12 in the catRAPID database (http://service.tartaglialab.com/page/catrapid_group, Fig. 3E) [29] and then performed RNA pull-down assays with these deletion mutant constructs. It was shown that the 430–493 nt fragment of lnc-TSPAN12 was responsible for its interaction with EIF3I (Fig. 3F). EIF3I is a protein containing the WD40 domain, with seven WD40 repeats covering almost the entire sequence of EIF3I [30]. Next, to further determine the domain of EIF3I that accounted for its interaction with lnc-TSPAN12, we performed RIP assays with FLAG-tagged full-length and five truncated EIF3I. As shown in Fig. 3G, the deletion of RNA-binding domains (128–325 and 218–325 aa) of EIF3I dramatically abolished the association between this protein and lnc-TSPAN12, indicating that these domains may be essential for the interaction with lnc-TSPAN12.

Lnc-TSPAN12 increases the stability of EIF3I protein

To investigate whether a cross-talk exists between lnc-TSPAN12 and EIF3I, we firstly measured the EIF3I levels in lnc-TSPAN12-knockdown HCC cells. The mRNA levels of EIF3I remained unaltered after knockdown of lnc-TSPAN12 (Additional file 1: Fig. S3A). However, knockdown of lnc-TSPAN12 led to a remarkable decrease in EIF3I protein level, suggesting that lnc-TSPAN12 can increase the levels of EIF3I protein at the post-transcriptional level (Fig. 4A). Similarly, immunohistochemical staining analysis against previous xenograft tumour tissues displayed that lnc-TSPAN12 knockdown was also correlated with the decreased level of EIF3I (Additional file 1: Fig. S3B). Through treatment with the protein synthesis inhibitor cycloheximide (CHX), we observed that knockdown of lnc-TSPAN12 in Huh7 and Hep3B cells accelerated the degradation of EIF3I and shortened its half-life (Fig. 4B, C). Moreover, following treatment with the proteasome inhibitor MG132, the down-regulation of endogenous EIF3I in HCC cells induced by knockdown of lnc-TSPAN12 was prevented, and this finding was accompanied by increased EIF3I ubiquitination levels, which indicates that EIF3I degradation through the ubiquitin-proteasome pathway may be inhibited by lnc-TSPAN12 (Fig. 4D, Additional file 1: Fig. S3C). We further discovered that several E3 ligases can mediate the ubiquitination and degradation of EIF3I in the Ubibrowser database (Additional file 1: Fig. S3D) (http://ubibrowser.ncpsb.org/) [31].

Previous study has shown that the lysine at 298 served as the major ubiquitination site of EIF3I [32, 33]. We then mutated this lysine to alanine (K298A mutant) and evaluated whether this mutant can block the EIF3I ubiquitination degradation. We measured the ubiquitination and expression level of EIF3I in lnc-TSPAN12 knockdown HCC cells co-transfected with a lentiviral vector expressing a FLAG-tagged EIF3I K298A mutant (FLAG-EIF3I-K298A) and a lentiviral vector expressing a FLAG-tagged wild-type EIF3I (FLAG-EIF3I-WT). Indeed, knockdown of lnc-TSPAN12 can reduce the stability of EIF3I and facilitate its degradation via enhancing its ubiquitin modification, whereas K298A mutant significantly abolished the promoting effect of lnc-TSPAN12 knockdown on EIF3I ubiquitination degradation, thus increasing the expression level of EIF3I (Fig. 4E, F). These data indicated that lnc-TSPAN12 has a negative impact on the process of EIF3I ubiquitination. Therefore, we further determined the effect of lnc-TSPAN12 on the binding of EIF3I to its predicted ubiquitin E3 ligases, including SMURF1 and STUB1. Co-immunoprecipitation (co-IP) experiments showed that knockdown of lnc-TSPAN12 enhanced the interaction between EIF3I and SMURF1, but with no evident changes on the binding between EIF3I and STUB1 (Fig. 4G). Thus, lnc-TSPAN12 can block the ubiquitination and degradation of EIF3I by attenuating its binding to the ubiquitin E3 ligase SMURF1.

Lnc-TSPAN12 enhancement of SENP1-mediated EIF3I deSUMOylation inhibits its ubiquitination

We further investigated the mechanism of lnc-TSPAN12-mediated ubiquitination and degradation of EIF3I. SUMOylation is now recognized as a crucial ubiquitin-like, post-translational modification (PTM) of proteins by SUMO proteins, which can regulate the stability, subcellular localization and interaction of the targeted substrate proteins [34–36]. Intriguingly, previous studies have shown that SUMOylation and ubiquitination often occur at the same lysine residues of a substrate protein, and SUMOylation can sometimes promote the ubiquitination and degradation of modified proteins [37, 38]. Of note, apart from EIF3I, a SUMO1-specific protease SENP1 was also found in band 4. RNA pull-down followed by western blot and RIP assay were performed, and the results showed that SENP1 was also directly bind to lnc-TSPAN12 (Fig. 5A, B). FISH followed by immunofluorescence of SENP1 further verified the colocalization of lnc-TSPAN12 and SENP1 in the cytoplasm of Huh7 cell (Additional file 1: Fig. S4A). In addition, the 430–493 nt fragment of lnc-TSPAN12 was also confirmed to be responsible for its interaction with SENP1(Additional file 1: Fig. S4B). Given that SENP1 is responsible for the removal of SUMO family [39], we wondered whether SENP1 acts as a lncRNA-binding protein and gives lnc-TSPAN12 the capacity for deSUMOylation- and/or deubiquitination-related PTM on EIF3I. As expected, lnc-TSPAN12 knockdown had minimal effect on the expression of SENP1 protein (Additional file 1: Fig. S4C). However, co-IP assay results showed that knockdown of lnc-TSPAN12 decreased the combination of SENP1 and EIF3I, proposing that lnc-TSPAN12 may serve as a scaffold to promote the binding of SENP1 to EIF3I (Fig. 5C). Moreover, after knockdown of lnc-TSPAN12, the binding of EIF3I with SUMO1, SUMO2 and SUMO3 was enhanced to varying degrees, indicating the facilitation of lnc-TSPAN12 to EIF3I deSUMOylation (Fig. 5D).

We next investigated whether SUMOylation of EIF3I exerts influences on the combination of EIF3I with ubiquitination enzyme and ubiquitination of EIF3I. We found that silencing of SENP1 increased the combination of EIF3I with SMURF1 and SUMO1, as well as enhancement of EIF3I ubiquitination (Fig. 5E, F). On the other hand, the binding of EIF3I with SUMO1, SUMO2, SUMO3 and SENP1 remained unchanged after silencing of SMURF1 (Fig. 5G), implying that SUMOylation of EIF3I can mediate its ubiquitination, whereas the ubiquitination of EIF3I had no effect on its SUMOylation level. Coincidentally, K298 is also the underlying SUMOylation site according to the bioinformatics website prediction(http://ubibrowser.ncpsb.org/ubibrowser). To verify this prediction, we mutated K298 and detected the changes in EIF3I SUMOylation level after knockdown of lnc-TSPAN12 in Huh-7 cell. Accordingly, this mutant remarkably reduced the EIF3I SUMOylation (Fig. 5H). Similarly, the intracellular level of EIF3I was obviously decreased in sh-lnc-TSPAN12 + Flag-EIF3I-K298A group when compared with that of the FLAG-EIF3I-WT (Fig. 5H). Consequently, these findings show that lnc-TSPAN12 enhances SENP1-mediated deSUMOylation of EIF3I K298, thereby restraining the ubiquitination of EIF3I.

Lnc-TSPAN12 exerts EMT-promoting functions in HCC by regulating EIF3I/SENP1

Considering the effect of lnc-TSPAN12 on EIF3I stability, we hypothesized that lnc-TSPAN12 may exert its biological effects through interacting with EIF3I/SENP1 in HCC. We observed that specific shRNA for EIF3I/SENP1 remarkably reduced the migration and invasion abilities of Huh7 cell (Fig. 6A, B). In addition, the increased cell migration and invasion in lnc-TSPAN12-overexpressing HCC cell was reversed by EIF3I/SENP1 knockdown (Fig. 6A, B). These data suggest that knockdown of EIF3I/SENP1 can reverse the lnc-TSPAN12-induced metastasis in HCC.

To further elucidate the role of lnc-TSPAN12 in Wnt/β-catenin signalling, we firstly examined the effects of lnc-TSPAN12 on the expression of downstream target genes of the Wnt/β-catenin signalling. As shown in Fig. 6C, D, the mRNA and protein expression levels of cyclin D1, c-myc and Axin2 were significantly down-regulated in Huh-7 cells with lnc-TSPAN12 knockdown. We next investigated whether lnc-TSPAN12 can activate Wnt/β-catenin signalling through regulating the EIF3I/SENP1 activity to induce EMT in HCC. Consistent with these results, western blot findings further showed that ectopic lnc-TSPAN12 expression increased the expression levels of β-catenin along with its downstream target genes and induced EMT in HCC, whereas knockdown of EIF3I (Fig. 6E) or SENP1 (Fig. 6F) alleviated lnc-TSPAN12-mediated Wnt/β-catenin signalling activation. Collectively, our results demonstrate that EIF3I and SENP1 are required for lnc-TSPAN12 to activate Wnt/β-catenin signalling, which consequently induces EMT and promote metastasis in HCC.

m6A modification is responsible for lnc-TSPAN12 upregulation in HCC

Preliminary studies have reported that m6A modifications are widespread in mRNA and lncRNA and functionally regulate the transcriptome to affect RNA translation, export, localization and stability [40, 41]. We then determined whether m6A modification is associated with lnc-TSPAN12 upregulation in HCC. Methylated RIP qPCR analysis of HCC cells was performed. The results showed that the m6A methylation level of lnc-TSPAN12 was highly enriched in Huh7 and Hep3B cells (Fig. 7A). Our bioinformatics analysis suggested that METTL3, the crucial m6A methyltransferase, was evidently upregulated in HCC (Fig. 7B). Moreover, the overall survival (Fig. 7C) and disease-free survival (Fig. 7D) of HCC patients with high METTL3 expression were also significantly lower than those with low METTL3 expression, indicating its prognostic value in HCC.

To investigate the effects of METTL3 on lnc-TSPAN12 upregulation in HCC, we thus knocked down the expression of METTL3 using shRNA in Huh7 and Hep3B cells. As shown in Fig. S5A, the knockdown efficiency was verified by both western blot. We found that METTL3 knockdown was associated with remarkably decreased lnc-TSPAN12 expression level and m6A level of lnc-TSPAN12 when compared with the control group (Fig. 7E, F). Afterward, we treated two HCC cells with methylation inhibitor 3-DAA and observed that the expression level of lnc-TSPAN12 was significantly reduced (Fig. 7G). Finally, RNA FISH assay displayed that METTL3 and lnc-TSPAN12 were colocalized in the cytosol (Fig. 7H).

Furthermore, METTL3 knockdown suppressed the invasion and migration abilities of Hep3B cell (Additional file 1: Fig. S5B, C). Consistently, after knockdown of METTL3 in Hep3B and Huh7 cells, the expression of E-cadherin was significantly upregulated, whereas those of Vimentin and Fibronectin were significantly downregulated (Additional file 1: Fig. S5D). The above findings raised the possibility that the transcriptional stability of lnc-TSPAN12 was enhanced by METTL3-mediated m6A modification, which may partially be responsible for the upregulation of lnc-TSPAN12 in HCC.

Emerging evidence indicates that lncRNAs play key roles in the progression of various types of human cancers. Our previous research revealed that lnc-TSPAN12 was remarkably upregulated in HCC, especially in HCC with microvascular invasion, and correlated with a poor survival [24]. Here, we demonstrated that lnc-TSPAN12 presented a strong oncogenic activity by promoting the migration, invasion and EMT of HCC cells in vitro and in vivo. Importantly, lnc-TSPAN12 bound to EIF3I and then reinforced SENP1-mediated EIF3I deSUMOylation by facilitating the interaction between SENP1 and EIF3I, thereby restraining the ubiquitination and proteasomal degradation of EIF3I. These findings suggest that lnc-TSPAN12 has an important influence on HCC metastasis and may act as a promising therapeutic target of HCC.

Over the past two decades, the significance of EMT process in cancer progression and metastasis has been well-established. However, the underlying molecular mechanisms of lncRNAs in EMT regulation still need to be further elucidated. By means of gain- and loss-of-function experiments, we determined that lnc-TSPAN12 can trigger the migration, invasion and EMT process of HCC cells in vitro and in vivo. Consistent with our study, lncRNA TTN-AS1 can function as a competing endogenous RNA to regulate the expression of CDK5 by sponging miR-142-5p, thereby inducing EMT in lung adenocarcinoma progression [42]. In addition, lncRNA RP11 prompts the dissemination and EMT of colorectal cancer cells via upregulation of the key EMT transcription factor Zeb1[43]. Significantly, our study confirmed the regulatory role of lnc-TSPAN12 in EMT and HCC metastasis, which is realized by the interaction of lnc-TSPAN12 with EIF3I to activate Wnt/β-catenin signalling.

Cytoplasmic lncRNAs generally exert their biological functions through acting as miRNA sponge or physically interacting with regulatory proteins. Our RNA pull-down assay and MS data identified that lnc-TSPAN12 directly binds to EIF3I and subsequently acts as a scaffold to promote the binding of SENP1 to EIF3I, thus enhancing the stability of EIF3I. EIF3I is a subunit of the eIF3 complex, which influences mRNA translation initiation, cell cycle regulation, proliferation and tumorigenesis [30]. Accumulating evidence has revealed the high expression of EIF3I in various human cancers, such as colon adenocarcinoma [27], HCC [28] and melanoma [44], which indicates its potential role in tumorigenesis and progression. Although EIF3I overexpression contributes to tumorigenesis, no study has yet to be investigated the lncRNA-mediated PTM of EIF3I thus far. Here, we presented evidence that SENP1-mediated EIF3I deSUMOylation blocks the ubiquitination and degradation of EIF3I and ultimately improves its stability. These findings confirm that lnc-TSPAN12 is involved in the regulation of PTM of EIF3I and provide new insights into the functional relationship between lncRNA and protein SUMOylation modification.

As one of the most prevalent internal RNA modifications, m6A can modulate all stages of the RNA life cycle [20]. In the past several years, m6A has been substantiated to affect the targeted mRNA or lncRNA and participate in the development of various cancers [21,45]. However, studies on m6A modification in lncRNAs are still limited, especially in HCC. For instance, METTL3-mediated m6A modification results in LINC00958 elevation through stabilizing its RNA transcript [46]. Another recent study reported that LNCAROD upregulation in HCC was maintained by increased m6A methylation-mediated improvement of its RNA stability [47]. In this study, we revealed that m6A was enriched within lnc-TSPAN12 in HCC cells. Furthermore, METTL3-mediated m6A modification in lnc-TSPAN12 improved its RNA stability, which indicates that m6A modification may be partially responsible for the elevation of lnc-TSPAN12 in HCC.

In summary, the novel m6A regulated lnc-TSPAN12 exerts a pro-metastatic role in HCC progression through the lnc-TSPAN12/EIF3I axis. The upregulation of lnc-TSPAN12 in HCC is partly attributed to the METTL3-mediated m6A modification. High expression of lnc-TSPAN12 promotes EMT and metastasis of HCC by acting as a scaffold to facilitate the binding of SENP1 to EIF3I, leading to EIF3I stabilization and activation of Wnt/β-catenin signalling (Fig. 8). Importantly, our findings provide new insights into HCC metastasis and may offer a potential therapeutic target to broaden the anti-metastatic approach options for HCC.

LncRNAs

Long non-coding RNAs

EMT

Epithelial-mesenchymal transition

HCC

Hepatocellular carcinoma

EIF3I

Eukaryotic Translation Initiation Factor 3 subunit I

SENP1

Sentrin/SUMO-Specific Protease 1

TSPAN12

Tetraspanin 12

SUMO

small ubiquitin-like modifier

PDX

Patient-derived tumour xenograft

METTL3

Methyltransferase like 3

m6A

N6-methyladenosine

WTAP

WT1-associated protein

FTO

Fat-mass and obesity-associated protein

ALKBH5

AlkB homolog 5

YTHDF

YTH domain-containing family protein

IGF2BP

Insulin-like growth factor 2 mRNA-binding protein

HDGF

Hepatoma-derived growth factor

FOXO3

Forkhead box protein O3

FISH

Fluorescence in situ hybridization

KEGG

Kyoto Encyclopedia of Genes and Genomes

Mass spectrometry

RIP

RNA immunoprecipitation

CHX

Cycloheximide

SMURF1

Smad ubiquitylation regulatory factor-1

STUB1

STIP1 homology and U-box-containing protein 1

PTM

Post-translational modification

ATCC

American Type Culture Collection

DMEM

Dulbecco’s Modified Eagle Medium

FBS

foetal bovine serum

qRT-PCR

Quantitative real-time PCR

Immunoprecipitation

IHC

Immunohistochemistry

PBS

Phosphate buffered saline

PAGE

Polyacrylamide gel electrophoresis.

Ethics approval and consent to participate

This study was reviewed and approved by the Ethnics Committees of West China Hospital of Sichuan University (Chengdu, China). The study was conducted in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects.

Consent for publication

Not applicable.

Competing interests

No potential conflicts of interest were disclosed.

Availability of data and materials

All data generated during this study are available in this article and its supplementary files and from the corresponding author upon reasonable request.

Funding

This work as supported by the National Natural Science Foundation of China (No. 82002578), 1·3·5 project for disciplines of excellence–Clinical Research Incubation Project, West China Hospital, Sichuan University (No. 20HXFH021), 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (No. ZYJC21049).

Authors’ contributions

J. Lu and X. Xiong considered and designed the study. B. Li, J. Xu, D. Peng, and G. Nie performed experiments. B. Li, X. Xiong, N. Wen and Y. Wang did data analyses. J. Lu and B. Li wrote the manuscript. J. Lu, B. Li and X. Xiong contributed to the revision of manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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No competing interests reported.

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METTL3-mediated m6A modification of lncRNA TSPAN12 promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma through SENP1-dependent deSUMOylation of EIF3I

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Abstract

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Introduction

Methods

Cell culture

Wound-healing assay

Transwell assay

Western blot

RNA immunoprecipitation (RIP)

Methylated RIP (MeRIP)-qPCR

RNA binding protein pull-down assay

Fluorescence in situ hybridization (FISH)

Immunoprecipitation (IP)

Co-immunoprecipitation (Co-IP) assay

Immunohistochemistry (IHC) Staining

In vivo experiment

Statistical analysis

Results

Discussion

Conclusions

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