ZC3H13 Inhibits the Progression of Hepatocellular Carcinoma through m6A-PKM2-Mediated Glycolysis and Enhances Chemosensitivity

Objective N6-Methyladenosine (m6A) is the most prevalent RNA epigenetic modulation in eukaryotic cells, which serves a critical role in diverse physiological processes. Emerging evidences indicate the prognostic significance of m6A regulator ZC3H13 in hepatocellular carcinoma (HCC). Herein, this study was conducted for revealing biological functions and mechanisms of ZC3H13 in HCC. Methods Expression of ZC3H13 was examined in collected HCC and normal tissues, and its prognostic significance was investigated in a public database. Gain/loss of functional assays were presented for defining the roles of ZC3H13 in HCC progression. The specific interactions of ZC3H13 with PKM2 were validated in HCC cells via mRNA stability, RNA immunoprecipitation, and luciferase reporter and MeRIP‐qPCR assays. Moreover, rescue experiments were carried out for uncovering the mechanisms. Results ZC3H13 expression was downregulated in HCC, and its loss was in relation to dismal survival outcomes. Functionally, overexpressed ZC3H13 suppressed proliferation, migration, and invasion and elevated apoptotic levels of HCC cells. Moreover, ZC3H13 overexpression sensitized to cisplatin and weakened metabolism reprogramming of HCC cells. Mechanically, ZC3H13-induced m6A modified patterns substantially abolished PKM2 mRNA stability. ZC3H13 facilitated malignant behaviors of HCC cells through PKM2-dependent glycolytic signaling. Conclusion Collectively, ZC3H13 suppressed the progression of HCC through m6A-PKM2-mediated glycolysis and sensitized HCC cells to cisplatin, which offered a fresh insight into HCC therapy.


Introduction
Liver carcinoma represents the most frequent fatal malignant disease across the globe [1]. Among all liver carcinoma patients, hepatocellular carcinoma (HCC) occupies over 90% [2]. Patients' survival outcomes are dismal. Merely 5%-15% of patients benefit from radical resection, only for those in the earlier stages [3]. erapeutic strategies for advancedstage patients contain transarterial chemoembolization (TACE) as well as oral sorafenib [4]. Nevertheless, <33% of patients do not respond to this therapy as well as develop marked chemotherapy resistance within 6 months from starting therapeutic intervention [5]. Moreover, long-term usage of chemotherapeutic agents causes toxic response as well as chemotherapeutic inefficiency [6]. erefore, neither TACE nor chemotherapeutic agents can remarkedly improve the outcome of liver cancer. In-depth exploration is required for finding a better way to treat liver cancer. N 6 -methyladenosine (m 6 A) is the most prevalent form of internal mRNA modification [7]. m 6 A modification has been proposed as the most frequent chemical modified form in eukaryotic mRNAs [8], which is of importance for controlling diverse cellular and biological events like RNA stability, translation, and splicing [6]. As estimated, about 0.1%-0.4% of adenosine in mRNAs may be modified via m 6 A, with a mean of 2-3 m 6 A modified sites per transcript [9]. m 6 A modification patterns are dominated through methyltransferase complex ("writer"), demethylase ("eraser"), and RNA-binding protein ("reader") [10]. Emerging evidences highlight the significance of deregulation of m 6 A modification in liver carcinogenesis [9].
rough comprehensive analyses of m 6 A regulators in TCGA-HCC project, Liu et al. proposed that METTL3, YTHDF2, and ZC3H13 acted as independent prognostic indicators of HCC outcomes [11]. METTL3 expression exhibited a frequent upregulation in HCC and promoted HCC development via YTHDF2-dependent posttranscriptional silence of SOCS2 [12]. Another study proposed the mechanisms of SUMOylated METTL3-mediated Snail mRNA homeostasis during HCC progression [13]. HBXIP triggered metabolism reprogramming of HCC cells through METTL3-dependent m 6 A modified HIF-1α [14]. e hepatic microenvironment facilitated HCC proliferation and metastases through METTL3-mediated m 6 A modification of YAP1 [15]. YTHDF2 triggered HCC stem cell phenotype as well as metastases through modulating OCT4 expression via an m 6 A modification manner [16]. YTHDF2 deletion fueled inflammation as well as vascular abnormalization in HCC [17]. YTHDF2 weakened cellular proliferation and growth through destabilization of EGFR mRNA in HCC [18]. Nevertheless, to date, no experimental evidences have confirmed the biological significance of ZC3H13 in HCC pathogenesis.
Herein, we observed the biological roles of m 6 A regulator ZC3H13 in HCC as well as addressed the underlying mechanisms. Our data suggested that ZC3H13 suppressed the progression of HCC with m 6 A-PKM2-mediated glycolysis and sensitized HCC cells to cisplatin. us, our findings highlighted the critical functions of ZC3H13-mediated m 6 A modification in HCC and provided a promising therapeutic regimen against HCC.

Patients and Specimens.
Primary HCC as well as adjacent control tissue specimens from 30 patients in the People's Hospital of Changshou Chongqing were harvested for this study. e inclusion criteria were as follows: (i) patients with pathologic diagnosis of HCC and (ii) patients who received curative removal. Meanwhile, patients with distant metastases at diagnosis were excluded. Informed consent was acquired from each patient. is research was carried out in line with the guidelines of the Ethics Committee of the People's Hospital of Changshou Chongqing and approved following the ethical standards of World Medical Association Declaration of Helsinki.

Cell
Culture. Human normal liver cells L-O2 as well as human liver cancer cell lines HUH-7, Hep3B, HepG2, and SMMC-7721 were retrieved from ATCC (USA). All cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) as well as 1% streptomycin/penicillin. Cells were grown in a humidified environment of 5% CO 2 at 37°C.
2.6. Transfection. Short interfering RNA (siRNA) against ZC3H13 as well as PKM2 was synthesized for specifically silencing ZC3H13 as well as PKM2 expressions in Hep3B and HUH-7 cells. HCC cells transfected with scrambled siRNAs acted as si-NC. e full-length ZC3H13 cDNA was synthesized and then subcloned into the pcDNA3.1 vector to establish pcDNA-ZC3H13 overexpression (OE-ZC3H13) plasmid. All plasmids were retrieved from GenePharma (Shanghai, China). Transient transfection was carried out lasting two days via Lipofectamine 3000.

Measurement of Glucose Uptake and Lactate Production.
Glucose uptake as well as lactate production was separately tested through Glucose Uptake Colorimetric Assay Kits (BioVision, USA) and Lactate Colorimetric Assay Kits (BioVision, USA) in Hep3B as well as HUH-7 cells following the manufacturer's protocols.

mRNA Stability Assay. Stability of mRNA assays in
Hep3B as well as HUH-7 cells was evaluated through incubating cells with 5 μg/mL actinomycin D (Act-D, Sigma, USA). Afterwards, cells were harvested at the indicated time points, and mRNAs were drawn for RT-qPCR with GAPDH as the reference control.
2.14. RNA Immunoprecipitation (RIP) Assay. RIPA assay was conducted with Magna RIP RNA-Binding Protein Immunoprecipitation kits (Millipore, USA) in line with the manufacturer's instructions. Hep3B as well as HUH-7 cells were lysed utilizing RIPA lysis buffer. Cell lysate was immunoprecipitated through anti-ZC3H13 antibodies or nonimmunized IgG at 4°C overnight. Afterwards, RNA was purified and RT-qPCR was utilized for measuring the level of PKM2 transcript in ZC3H13 or IgG immunocomplex.

Luciferase Reporter Assay.
Promoter sequence of PKM2 was cloned into pEZX-PL01 control vector containing firefly luciferase as well as Renilla luciferase. Luciferase assay was carried out utilizing Luc-Pair ™ Duo-Luciferase HS Assay kits. In brief, pretreated Hep3B as well as HUH-7 cells were cotransfected by ZC3H13-wild-type (ZC3H13-WT) or ZC3H13-mutation-type (ZC3H13-MUT) as well as 250 ng pEZX-PL01 reporter plasmid (Promega, Shanghai, China) in 12-well plates. Following transfection lasting 6 h, HCC cells were seeded into 96-well plates. Following 36 h, cells were collected and analyzed utilizing Dual-Glo Luciferase Assay system. Activity of firefly luciferase was normalized to that of Renilla luciferase for evaluating the luciferase and transcriptional activity.

Methylated RNA Immunoprecipitation qPCR
(MeRIP-qPCR). 1 μg·m 6 A and IgG antibodies were treated by Protein G Magnetic Beads in 1x reaction buffer at 4°C lasting 3 h as well as treated by 200 μg isolated RNA at 4°C lasting 3 h. Bound RNAs were eluted via incubating by RNAantibodies-conjugated bead plus 100 μL Elution Buffer lasting 30 min at room temperature. Eluted RNAs were extracted through phenol: chloroform method in line with ethanol precipitation. Extracted m 6 A-RIP RNAs were reverse-transcribed as well as quantified via RT-qPCR. IPs enriched rates of transcripts were determined as the ratios of their amounts in IPs to those in the input generated from the same number of cells.

Statistical Analyses.
Statistical analyses were conducted with GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA, USA). Student's t-test and one-or two-way analysis of variance were utilized for comparisons between groups as appropriate. Kaplan-Meier method was conducted for measuring the survival curves, and differences were assessed with log-rank test. P values less than 0.05 were indicative of statistical significance.

ZC3H13 Displays Low Expression in HCC and
Correlates with Survival Outcomes. For investigating the underlying function of ZC3H13 in liver carcinogenesis, this study firstly tested the mRNA expressions of m 6 A methyltransferase ZC3H13 in 369 HCC tissues and 160 normal tissues from TCGA and GTEx projects. In Figure 1(a), ZC3H13 expressions were markedly downregulated in HCC relative to control tissues. Furthermore, similar mRNA expression patterns of ZC3H13 were verified in our cohort comprising of 30 paired cancerous and normal specimens (Figure 1(b)). Analysis of western blotting showed the decreased expression of ZC3H13 protein in HCC relative to normal tissues (Figures 1(c) and 1(d)). Moreover, our in vitro experiments confirmed the decreased mRNA expression of ZC3H13 in human liver cancer cell lines HUH-7, Hep3B, HepG2, and SMMC-7721 relative to human normal liver cells L-O2 ( Figure 1(e)). Kaplan-Meier analysis uncovered that HCC patients with high ZC3H13 expression displayed a remarked survival advantage utilizing the online bioinformatics tool Kaplan-Meier plotter (Figure 1(f )). With the above evidences, ZC3H13 expression was remarkedly downregulated in HCC, which could be implicated in the pathogenesis and progression of HCC.

ZC3H13 Inhibits Cell Proliferation of HCC Cells.
For addressing the effects of ZC3H13 on HCC progression, this study silenced ZC3H13 expression in Hep3B and HUH-7 cells (Figures 2(a)-2(c)), and its expression was upregulated in the two HCC cells (Figures 2(d)-2(f )) due to their relatively lower expression among all HCC cells, as determined with RT-qPCR and western blotting. CCK-8 results demonstrated that ZC3H13 deficiency enhanced cell growth in Hep3B as well as HUH-7 cells (Figures 2(g) and 2(h)). In contrast, cell growth of HCC cells was alleviated through overexpressed ZC3H13 (Figures 2(i) and 2(j)). As depicted in clonogenicity assay, clone formation of Hep3B and HUH-7 cells was remarkedly enhanced through ZC3H13 deficiency (Figures 2(k) and 2(l)). e opposite results were investigated when ZC3H13 was overexpressed (Figures 2(k) and 2(m)). Collectively, ZC3H13 might inhibit cell proliferation of HCC cells.

ZC3H13 Promotes Apoptosis and Suppresses Migration
and Invasion in HCC Cells. TUNEL assays were utilized for evaluating the effects of ZC3H13 on apoptosis of HCC cells. Our data showed that ZC3H13 deficiency reduced cell apoptosis, whereas ZC3H13 overexpression enhanced cellular apoptotic levels of Hep3B as well as HUH-7 cells (Figures 3(a)-3(c)). Transwell assays revealed that migrative capacities of Hep3B as well as HUH-7 cells were enhanced through ZC3H13 deficiency; meanwhile, overexpressed ZC3H13 alleviated the migrative capacities of HCC cells (Figures 3(d)-3(f )). We also noticed the increase in the invasive abilities of Hep3B and HUH-7 cells induced by ZC3H13 knockdown (Figures 3(g) and 3(h)). However, invasive abilities were reduced by ZC3H13 overexpression (Figures 3(g) and 3(i)). Taken together, ZC3H13 promoted apoptosis as well as suppressed migrative and invasive capacities of HCC cells.

ZC3H13 Increases Sensitivity to Cisplatin in HCC Cells.
We assessed the effects of ZC3H13 on sensitivity to cisplatin in HCC cells. Our CCK-8 data suggested that viable Hep3B as well as HUH-7 cells were suppressed as cisplatin was gradually increased (Figures 4(a) and 4(b)). ZC3H13 knockdown prominently reduced the inhibition rates of cisplatin in HCC cells relative to controls. Quantification analysis of IC50 values of cisplatin showed that ZC3H13 knockdown contributed to increased IC50 of cisplatin in Hep3B and HUH-7 cells, indicative of the reduced sensitivity to cisplatin (Figure 4(c)). Meanwhile, overexpressed ZC3H13 elicited the opposite effects (Figures 4(d)-4(f )). Moreover, our results uncovered that apoptotic levels of Hep3B as well as HUH-7 cells were markedly enhanced following treatment with cisplatin lasting 48 h (Figures 4(g)-4(j)). However, ZC3H13 knockdown weakened the inhibitory effects of cisplatin on apoptosis of HCC cells; meanwhile, overexpressed ZC3H13 enhanced the cisplatininduced apoptotic levels. Above data demonstrated that ZC3H13 was capable of enhancing the cisplatin chemosensitivity of HCC cells.

ZC3H13 Reduces Metabolism Reprogramming of HCC Cells.
e Warburg effect represents a sign of metabolism reprogramming of cancer, in which most cancer cells exhibit enhanced glucose uptake as well as lactic acid production when there is sufficient oxygen supply [20]. Herein, we measured the effects of ZC3H13 on bioenergy metabolism levels of HCC cells. Our data demonstrated that ZC3H13 deficiency increased glucose uptake of Hep3B and HUH-7 cells, whereas ZC3H13 overexpression reduced glucose uptake (Figures 5(a) and 5(b)). Moreover, we noticed that lactate production was enhanced by ZC3H13 knockdown, and the opposite results were investigated when ZC3H13 was overexpressed (Figures 5(c) and 5(d)). rough western blotting, the expressions of glycolysis-related proteins GLUT, LDHA, LDHB, and PKM2 were measured in Hep3B and HUH-7 cells (Figure 5(e)). As a result, ZC3H13 overexpression remarkedly decreased the expressions of GLUT, LDHA, LDHB, and PKM2 proteins in HCC cells (Figures 5(f )-5(i)). In conclusion, ZC3H13 modulated metabolism reprogramming of HCC cells.

ZC3H13-Mediated m 6 A Modification Reduces PKM2
mRNA Stability. ZC3H13-silencing and overexpressing HCC cells were treated by Act D. Our results showed that ZC3H13 knockdown remarkedly increased the remaining PKM2 transcripts for Hep3B as well as HUH-7 cells (Figures 6(a) and  6(b)). Rather, ZC3H13 overexpression reduced the remaining PKM2 transcripts in two HCC cells (Figures 6(c) and 6(d)). e data indicated that ZC3H13 could decrease the stability of PKM2 mRNA. Moreover, RIP results showed that anti-ZC3H13 antibody prominently enriched the levels of PKM2 mRNA relative to anti-IgG antibody in Hep3B and HUH-7 cells (Figure 6(e)). However, GAPDH transcript was not detected in ZC3H13 or IgG immunocomplex. us, ZC3H13 possessed the capacity of binding to PKM2 transcript physically. Moreover, we further investigated whether PKM2 3′untranslated region (3′-UTR) was required for ZC3H13 for reducing PKM2 expression. erefore, dual-luciferase assay was carried out. Our data demonstrated that ZC3H13 overexpression remarkedly lowered the luciferase activities of PKM2 3′-UTR reporter vector for Hep3B as well as HUH-7 cells (Figures 6(f) and 6(g)). However, no effect was investigated for the empty vector. us, above data were indicated that ZC3H13 bound to PKM2 3′-UTR. In line with MeRIP-qPCR results, ZC3H13 overexpression reduced the m 6 A levels of PKM2 mRNA for Hep3B as well as HUH-7 cells (Figure 6(h)).
us, the findings indicated that ZC3H13  Journal of Oncology decreased the stability of PKM2 mRNA with an m 6 A-dependent manner.

PKM2 Knockdown Weakens Cell Proliferation and
Metabolic Reprogramming Mediated by ZC3H13 in HCC Cells. We further investigated the effects of interactions of ZC3H13 with PKM2 on HCC progression. We firstly confirmed the successful knockdown of PKM2 for Hep3B as well as HUH-7 cells with si-PKM2 transfections (Figure 7(a)). Afterwards, we assessed the cellular proliferation of PKM2 interacted with ZC3H13 in HCC through CCK-8. Our data demonstrated that PKM2 knockdown induced a prominent reduction in cell viability. Nevertheless, PKM2 knockdown reversed the cell growth mediated by ZC3H13 deficiency in Hep3B and HUH-7 cells (Figures 7(b) and 7(c)). Clone formation of HCC cells was weakened by PKM2 knockdown (Figures 7(d) and 7(e)). However, silencing ZC3H13 remarkedly ameliorated the clone formation induced by ZC3H13 knockdown in HCC cells. By quantitative analyses of glycolysis, we noticed that PKM2 deficiency prominently reduced glucose uptake and lactic acid production for Hep3B as well as HUH-7 cells (Figures 7(f ) and 7(g)). But silencing PKM2 alleviated glucose uptake and lactic acid production induced by ZC3H13 deficiency. Taken together, ZC3H13 alleviated HCC cell proliferation by PKM2-dependent glycolytic signaling.

PKM2 Deficiency Alleviates Migration and Invasion Induced by ZC3H13 in HCC Cells.
e effects of interactions of ZC3H13 with PKM2 on HCC metastasis were investigated through quantification of migration and invasion via transwell assays. Our results demonstrated that PKM2 deficiency remarkedly alleviated the migrative capacities of Hep3B and HUH-7 cells (Figures 8(a) and 8(b)). Additionally, its deficiency reversed the migrative abilities induced by ZC3H13 knockdown in HCC cells. As depicted in

Discussion
m 6 A modification of RNAs acts as a novel layer of epigenetic modulation [8]. e biochemical event exerts critical roles in modulating growth, differentiation, resistance, and metabolic reprogramming of cancer cells via modulation of RNA splicing, translation, and stability [14,21,22]. Several evidences have proposed m 6 A as a major modified type of mRNAs [23][24][25]. In our study, our evidences confirmed the important roles of m 6 A regulator ZC3H13 in HCC progression and uncovered the underlying mechanisms. Our results demonstrated that overexpressed ZC3H13 weakened malignant behaviors of HCC cells through m 6 A-PKM2mediated glycolysis and enhanced chemosensitivity. Consistent with bioinformatics analysis, ZC3H13 expression was downregulated in HCC as well as its loss correlated to dismal survival outcomes [26][27][28]. ZC3H13 weakens proliferative and invasive capacities of colorectal carcinoma cells through inactivating Ras-ERK pathway [29]. ZC3H13 is predictive of immune phenotype and therapeutic responses in renal carcinoma [30]. Our data demonstrated that overexpressed ZC3H13 alleviated proliferation, migration, and invasion as well as aggravated apoptosis in HCC cells, confirming that ZC3H13 exacerbated malignant behaviors of HCC cells. Tumor metastases and chemoresistance act as the major causes of therapeutic failure and increased mortality for HCC [31]. In line with the perspective of precision medicine, it is an urgency for finding novel molecular targets upon developing more effective therapeutic regimen. Herein, ZC3H13 overexpression could sensitize HCC cells to cisplatin, providing novel evidences for HCC chemotherapy. HCC represents a heterogeneous malignancy, characterized by diverse etiological factors, that is implicated in (h) MeRIP-qPCR detecting m 6 A modification levels of PKM2 through immunoprecipitation of m 6 A-modified mRNA for Hep3B as well as HUH-7 cells with empty vector or ZC3H13 overexpression. Ns: not significant; * P < 0.05; * * P < 0.01; * * * P < 0.001; and * * * * P < 0.0001. metabolic alterations [32]. Previous evidences have demonstrated the significance of metabolic normalization to HCC inhibition [33][34][35]. e Warburg effect is fundamental to metabolic reprogramming in HCC progression [36]. Enhanced glucose uptake and lactate production maintain longterm growth of cancer cells. Hopefully, reprogramming of HCC cells may manifest itself as a new insight into developing therapeutic regimen against HCC. Our data demonstrated that ZC3H13 had much potential of inhibiting glycolysis in HCC through modulating metabolism reprogramming. Our further analyses uncovered that ZC3H13-mediated m 6 A modification substantially alleviated PKM2 mRNA stability as well as overexpressed ZC3H13 facilitated malignant behaviors of HCC cells through PKM2-dependent glycolytic signaling. Even so, more selective and efficacious agents activating ZC3H13 will be developed upon HCC therapy in our future studies. ere are several limitations in our study. Firstly, we collected 30 pairs of HCC specimens and matched nontumor specimens, and our results revealed that ZC3H13 expression was downregulated in HCC specimens. However, the sample size is small. e expression of ZC3H13 will be verified in larger HCC cohorts. Secondly, the biological function of ZC3H13 in HCC progression will be investigated through in vivo experiments.

Conclusion
In all, our evidences demonstrated that overexpressed ZC3H13 alleviated malignant behaviors and metabolism reprogramming of HCC cells through mediating the m 6 Amodified PKM2 mRNA. erefore, ZC3H13 possessed the potential as a therapeutic target against HCC. Effective treatments for HCC might be conducted on the basis of the new molecular mechanisms proposed in these observations.