miR-375-NAT10 Axis Dysfunction Promotes Oral Cancer Development and Mediates Cdk7 Stabilizing and Cell Cycle Progression

Background: Oral cancer is the most common cancer with poor prognosis and outcome for the patients due to the challenging diagnosis and limited treatment possibilities. However, the molecular underpinnings behind the malignant progression of oral cancer remain incompletely understood. Methods: The expression proling of NAT10 and CDK7 in oral cancer patients were assessed by IHC, qPCR and western blots. ShRNA was used to silence gene expression. The biological function of NAT10 and CDK7 in cholangiocarcinoma was investigated using in vitro and in vivo studies including, transwell cell migration, plate cloning, CCK8, shRNA interference, western blots, ow cytometry and xenograft mouse model. The underlying molecular mechanism was determined by western blots and immunoprecipitation. Results: In this study, we demonstrated that deregulation of miR-375-NAT10 axis is among the most causes in inducing the acquisition of a tumorigenesis phenotype in oral cancer cells. NAT10 is abundant in oral cancer tissue. and its protein level is positively correlated with poor overall survival. Increased the level of NAT10 promotes oral cancer cell proliferation in vitro as well as xenograft tumorigenicity in vivo. Most importantly, NAT10 regulates cancer cell proliferation through stabilizing CDK7 thus regulating the cell cycle. NAT10 as an acetyltransferase is responsible for CDK7 acetylation at lysine 328 (K328Ac). Moreover, it was found that the expression of miR-375 is abnormally alleviated in oral cancer tissues. Bioinformatics analysis revealed a targeted complementary binding site between miR-375 and NAT10. Decreased expression of miR-375 promotes expression of NAT10. Conclusion: Our study showed that NAT10 plays a strong carcinogenic role in oral cancer tumorigenesis by acetylating CDK7 at K382 thus promotion stability. Moreover, NAT10 may serve as a target for miR-375. Therefore, targeting may provide a new and effective strategy to inhibit the tumorigenicity of oral cancer.

N-acetyltransferase 10 (NAT10) is a member of the GNAT family of KATs and has been documented to acetylate RNA [14][15][16] , NAT10 or a homologous enzyme in other species increased the formation of ac4C on tRNA, rRNA, and mRNA, thereby maintaining the accuracy of protein translation and stabilizing the mRNA 15,17 . Transcriptional cofactor Che-1, hTERT and T-tubulin were also documented as substrates of NAT10 14,18−20 . Moreover, deregulation of NAT10 has been implicated in Hutchinson Gilford progeria syndrome 15,21,22 . NAT10 was associated with cancer by demonstrating that it could signi cantly promote cell growth in epithelial ovarian cancer and breast cancer 23,24 . NAT10 also has a potential role in increasing melanogenesis and melanoma growth 25,26 . However, NAT10 inhibits cell proliferation and expression of NAT10 decreases in human colorectal carcinomas 14 . These ndings suggest that NAT10 has multifaceted functional roles in cancers. However, the underlying functions and mechanisms of NAT10 in oral cancer progression remain unknown.
Among the CDKs regulating DNA transcription, CDK7 is the most critical one. It activates mRNA synthesis by phosphorylating the carboxyl terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) 27,28 . CDK7 and its partners, cyclin H and MAT1, were originally isolated as a CDK-activating kinase (CAK), which is necessary for the complete activation of CDKs in cell cycle 29,30 . CDK7 de cient mouse model de ciency showed early embryonic lethality 31 , which indicates its importance in development. Recently, CDK7 has been increasingly recognized for its role in breast cancer, T cell acute lymphoblastic leukemia, gastric cancer, small cell lung cancer, neuroblastoma and ovarian cancer [32][33][34][35][36] .
As an anticancer target, the inhibition of CDK7 with small molecules THZ1 has been considered to be a promising cancer therapy 37,38 . There are several ongoing preclinical studies and clinical trials to examine CDK7 inhibitors in advanced solid malignancies.

Patients
This study was approved by the Ethical Committee of Foshan Stomatological Hospital. All patients signed an informed-consent document for diagnosis and research on tissue specimens before being enrolled in the project.

Animals
All the animal experiments were approved by Animal Care and Use Committee of Foshan University. Then, extensive efforts were made to minimize the pain of the animals used in the study.
In vivo tumor growth assay A total of 12 male athymic Balb/c nude mice (aged 6 weeks old and weighing 20 ~ 23 g) were injected with about 1 × 107 OECM1 cells resuspended with 200 µL PBS. Mice were treated with a remodelin dose of 100 mg per kg orally after injection, until the end-point. Tumor volumes was measured every 3 to 4 days (2 times a week). When the tumor grew into a certain size, the tumor size was calculated with the following formula:(π × length × width2)/6. After the mice were euthanized, the tumor size and weight were measured.

Cell lines and cell culture
The HOK, HSC3, OECM1, KG005, SCC4 and SCC25 cell lines were purchased from the Cell Resource Center of the Shanghai Institute of Life Sciences, Chinese Academy of Sciences and authenticated by short-tandem repeat testing. Each cell line was mycoplasma-free and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scienti c, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scienti c, Inc.) and 1% penicillin-streptomycin (Gibco; Thermo Fisher Scienti c, Inc.) in an incubator at 37 ˚C with 5% CO 2 .

Construction of lentivirus and stable transfection into cell lines
The lentiviral vector encoding the human NAT10 gene was constructed by Genechem Co., Ltd. Lentiviral vector coding shRNA targeting NAT10 and shRNA negative control were designated as shNAT10 and shNC, respectively. The target sequences were as follows: shNAT10 #1, 5'-AUGGAACACUGAACAUAAATT-3'; and shNAT10 #2, 5'-GGCCAAAGCUGUCUUGAAATT − 3'. The cells were transfected with recombinant lentivirus-transducing units in the presence of polybrene (6 µg/ml) according to the manufacturer's instructions and stable NAT10 knockdown clones were selected by 2.5 µg/ml puromycin (A1113803; Gibco; Thermo Fisher Scienti c, Inc.). The selected pools of knockdown cells were then collected and cultured within 10 passages for use for subsequent experiments. Tables S2 and S3 provide detailed information on the expression structure and primers used for molecular cloning.

Co-Immunoprecipitation Assay and Immunoblotting
For immunoblotting analysis, modi ed RIPA lysis buffer (50 mM Tris-HCl, pH = 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) with supplements of protease inhibitors as well as phosphatase inhibitors (Bimake, Houston, USA) were used to lyse cells. The BCA protein assay reagent (Yeasen, Shanghai, China) was used to examinate protein concentrations. Cell extracts were rst decomposed by SDS-PAGE and then applied to PVDF membrane (Billerica millipore, USA). Then, they were incubated by the appropriate primary antibodies. Later, enhanced chemiluminescent substrate kit (Yeasen) was utilized to analyze the speci c signals of indicated antibody. For immunoprecipitaton of endogenous proteins, primary antibodies or control IgG were used to incubate cell extracts in a rotating incubator overnight at temperature of 4°C, then the products were incubated for another 3 h with protein A/G magnetic beads (Sigma-Aldrich, St. Louis, MO, USA). The beads were washed three times using lysis buffer before immunoblotting analysis.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted by TRIzol reagent (Invitrogen; Thermo Fisher Scienti c, Inc.) and reverse transcribed using the ThermoScript RT-PCR system (cat. no. 11731015; Invitrogen; Thermo Fisher Scienti c, Inc.) according to the manufacturer's protocol. qPCR with SYBR-Green (cat. no. 1708886; Bio-Rad Laboratories, Inc.) were performed on an iQ5 Multicolor Real-Time PCR Detection system (Bio-Rad Laboratories, Inc.). The PCR conditions were as follows: 95 ˚C for 5 min followed by 40 cycles of 95 ˚C for 30 s and 57 ˚C for 30 s. GAPDH was used as an internal control. All reactions were run in triplicate and quanti ed with 2-ΔΔCq 62 .

Cell proliferation assay
Cell proliferation was measured by cell count methods. For CCK-8 assay (Keygen Biotech), the cells were inoculated in 96-well plates in triplicate and observed every day, 10 µl CCK-8 and 100 µl fresh medium were added to each well and at speci c time-points and then incubated for 3 h at 37 ˚C, the absorbance was measured at 450 nm using a microplate reader (ST-360; KHB). For colony-formation assay, stable cells were inoculated in six-well plates in triplicate and cultured for 14-21 days. The cells were then xed with 4% paraformaldehyde and stained with crystal violet staining solution. Visualize colonies were counted.

Flow cytometric evaluation
The cells were then collected and washed with cold PBS. Annexin V-FITC reagent kit (cat. no. A211-01; Vazyme Biotech Co., Ltd.) was used to perform analysis. The cells were re-suspended in annexin V binding buffer with the nal concentration of 1x10 6 cells/ml and incubated with AnnexinV-FITC for 15 min at 4 ˚C in the presence of propidium iodide. Samples were analyzed using a BD FACSCalibur ow cytometer (BD Biosciences) and then analyzed were performed with FlowJo software (FlowJo 10.5, LLC).

Dual-luciferase reporter gene assay
Page 6/23 The genomic DNA was extracted from OECMs. Then the NAT10 3′UTR sequence containing the putative binding site for miR-375 was ampli ed. GeneArt™ site-directed Mutagenesis PLUS System (ThermoFisher, Waltham, MA, USA) was used to mutate the sequences. The target sequences were collected after gel electrophoresis of the ampli ed products, and then inserted into the pmirGLO dualLuciferase miRNA Target expression vector (Promega, Madison, WI, USA) to construct the reporter vectors: NAT10 wild type (WT) and NAT10 mutant (MUT). After the sequence of the reporter vectors were sequenced by Sangon Biotech (Shanghai, China) with sequencing, the reporter vectors were transfected into the cells respectively, together with miR-375 mimics or NC mimics. The medium was discarded after 48 h. The cells were washed with PBS and then lysed with cell lysis buffer. After centrifugation at 3000×g for 5 min, the supernatant was collected and the luciferase activity was detected by dual luciferase reporter assay system (Promega, Madison, WI, USA).

Statistical analysis
All experiments were performed at least 3 independent experiments independently, and all values were expressed as the means ± standard deviation. The SPSS 20.0 software (IBM Corp.) was used for statistical analysis. A two-tailed unpaired Student's t-test with Welch correction or one-way ANOVA followed by Dunnett's adjustment (for dose-response effects) or the Bonferroni test (for multiple comparisons) was used to determine the statistical signi cance of the differences in the measured variables. A value of p < 0.05 was considered to indicate a statistically signi cant difference.

Results
Increased mRNA and protein level of NAT10 in oral cancer tissues In order to identify the regulatory molecules that play a major role in the progression of oral cancer, we searched the public database (http://tumorsurvival.org) and found that patients with high expression of NAT10 had signi cantly poor prognosis in HNSCC patients ( Fig. 1A and Supplementary Figure S1A). Subsequently, immunohistochemical staining was performed in 80 samples from oral cancer patients who underwent surgical resection in the Foshan Stomatological Hospital. The results also con rmed the negative regulatory effect of NAT10 on the prognosis of oral cancer patients ( Fig. 1B-1C). The expression of NAT10 genes was investigated in oral tumor mRNA expression pro les. Analysis of publicly available microarray data revealed a highly signi cant upregulation of NAT10 mRNA in oral tumors compared to normal oral tissues (Fig. 1D). In order to con rm the expression of NAT10 in oral cancer, RT-PCR was performed in oral tumor containing 21 normal and 54 tumor samples. Results con rmed the elevated expression of NAT10 with signi cant fold change in all oral tumor tissue (Fig. 1E). We also observed that the expression of NAT10 increased with the progression of tumor stage in the public database (Fig. 1F). We con rmed the elevated expression of NAT10 with the progression of tumor staging using RT-PCR in 54 oral tumor samples (Fig. 1G).
NAT10 promotes the proliferation of oral cancer cells We investigated the expression level of NAT10 in oral cancer cell lines. We observed that the expression of NAT10 in OECM1 and KG005 was in the middle level among those cell lines ( Supplementary Fig. S1B), we selected OECM1and KG005 for the next proliferation experiments. we overexpressed NAT10 in OECM1 and KG005 cells ( Fig. 2A-2B), and considerable promotions of growth in CCK8 and plate cloning assays were observed in both OECM1 and KG005 cells with the overexpression of NAT10 (Fig. 2C-2G). We treated OECM1 and KG005 cells with NAT10 enzymatic inhibitor, remodelin. We observed that the addition of remodelin signi cantly inhibited proliferation of OECM1 and KG005 cells in CCK8 and plate cloning assays (Fig. 2H-2L). Furthermore, we designed two NAT10 shRNAs to down-regulated NAT10 in OECM1and KG005 cells. As shown in Fig. 2M-2N, the protein level of NAT10 signi cantly reduced in OECM1and KG005 cells after NAT10 knockdown (Fig. 2M-2N). And the NAT10 knockdown obviously inhibited the proliferation of OECM1 and KG005 cells in CCK8 and plate cloning assays (Fig. 2O-2S).
Thus, these results suggested that NAT10 promotes the proliferation of oral cancer cells in vitro.
NAT10 advances the tumorigenesis of oral cancer by upregulating CDK7 expression to promote the cell cycle To further characterize the regulatory effect of NAT10 on cell proliferation. We next investigated the effect of NAT10 on cell cycle. As shown in Fig. 3A-3B, the population of cells arrested at G2/M phase was signi cantly increased in NAT10-knockdown OECM1 cells (Fig. 3A-3B). In addition, the number of cells arrested at G2/M phase was high in remodelin-treated OECM1 cells (Fig. 3C-3D). To elucidate how NAT10 suppression delays cell cycle progression, an integrated bioinformatics platform (BioGRID) for investigating the protein interaction network (https://thebiogrid.org) was utilized to predict the partners of NAT10. We performed GO analysis to the molecules predicted by the website that interact with NAT10, we observed that these molecules are enriched in rRNA processing, DNA repair, cell cycle and so on (Fig. 3E). Considering the effect of NAT10 on cell cycle, we noticed that CDK7 may serve as a binding partner for NAT10 (Fig. 3E). CDK7 has essential role in both the cell-division cycle and transcription. To achieve this, we next examined whether CDK7 interacts with NAT10. The interaction between CDK7 and NAT10 at the endogenous protein levels was validated in OECM1 and KG005 cells by co-immunoprecipitation with an anti-CDK7 antibody and anti-NAT10 (Fig. 3F). Then we explored the interaction between CDK7 and NAT10 at the exogenous protein levels in HEK293T cells. We also obtained a similar result at the exogenous protein levels (Fig. 3G). By silencing NAT10, we discovered that the protein level of CDK7 was signi cantly decreased in OECM1 and KG005 cells (Fig. 3H-3I). Overexpression of NAT10 increase the level of CDK7 in OECM1 and KG005 cells (Fig. 3J-3K). The treatment of remodelin have a negative regulation on expression of CDK7 in OECM1 and KG005 cells (Fig. 3L-3M). These ndings might indicate NAT10 promotes the protein level of CDK7. Meanwhile, the positive correlation between NAT10 and CDK7 was observed in 6 clinical sample (Fig. 3N). Therefore, these results provided the important clues that NAT10 may promote the tumorigenesis of oral cancer via upregulating CDK7 level.

NAT10 upregulates CDK7 expression on protein level
We next further study whether the regulation of NAT10 on CDK7 is at the RNA level or protein level. The results demonstrated that NAT10 depletion by two respective shRNAs did not affect mRNA levels of CDK7 but signi cantly reduced its protein levels in OECM1 and KG005 cells, and the increased NAT10 also have no in uence in mRNA levels of CDK7 (Fig. 4A-4D). Furthermore, addition of MG132 with OECM1 and KG005 cells rescued decreased CDK7 induced by knockdown of NAT10 (Fig. 4E-4F). Those results suggested NAT10 play a regulatory role for CDK7 on protein level. As shown in Fig. 4G, H, the halflife of CDK7 in OECM1 cells expressing shNAT10 #1 and shNAT10 #2 was remarkable shorter than that in cells expressing shNC, indicating that NAT10 enhances the stability of CDK7 protein (Fig. 4G-4H). The treatment of remodelin also decrease half-life of CDK7 in OECM1 cells (Supplementary Figure S2A-S2B). In agreement with these observations, OECM1 cells were transfected with pCDH and NAT10. After 48 hours of transfection, cells were treated with 10 µM MG-132 for 6 hours and then total cellular lysates were subjected to IP assays with anti-CDK7 antibody. Immunoblotting analysis showed that NAT10 knockdown signi cantly decreased the ubiquitination of CDK7 protein (Fig. 4I).
CDK7 is acetylated at lysine 328 by NAT10 As NAT10 is a novel KAT with intrinsic acetyltransferase activities (41-46), we next investigated whether NAT10 acetylates CDK7. Results showed that ectopic expression of wildtype NAT10 but not its catalytically inactive mutant (G641E) enhanced acetylation of CDK7 in OECM1 and KG005 cells (Fig. 5A-5B). Conversely, knockdown of NAT10 using two independent shRNAs reduced acetylation of CDK7 (Fig. 5C-5D). In support of these ndings, inhibition of NAT10 activity using the small molecule inhibitor remodelin also decreased acetylation of CDK7 (Fig. 5E). For further veri cation, we predicted the acetylation site lysine 382 of CDK7 through the PLMD website (http://plmd.biocuckoo.org) and mutated lysine 382 to arginine. The results showed that the mutation of lysine 382 signi cantly weaken the acetylation of CDK7 (Fig. 5F), which indicated that lysine 382 is the major acetylated site of CDK7. NAT10 play a regulatory role on acetylation and stability of CDK7. Glutamine (Q) is a mimic of acetylation mutation. To achieve the effect of K382 mutation on stability of CDK7, we treated OECM1 cells with CHX. the half-life of CDK7 in OECM1 cells expressing K328Q was remarkable longer than that in cells expressing wildtype (Fig. 5G), indicating that acetylation enhances the stability of CDK7 protein.

NAT10 promotes tumorigenesis of oral cancer through regulating CDK7
We also elaborate relative expression of NAT10 and CDK7 in clinical sample. The positive correlation between NAT10 and CDK7 was con rmed in those clinical sample (Fig. 6A-6B). The results demonstrated that engraft tumors in mice treated with remodelin grew lower than those treated DMSO, but CDK7 reoverexpression partly restored growth rate of engraft tumors (Fig. 6C-6E). CDK7 re-overexpression restored growth rate of remodelin treated OECM1 cells in vitro (Fig. 6F-6H). CDK7 re-overexpression also restored proliferation of OECM1 cells expressed shNAT10 (and supplementary FigureS3A-S3C). The treatment of remodelin arrested OECM1 cells in G2/M phase, but CDK7 re-overexpression impaired the arrest (Fig. 6I-6J). CDK7 is necessary for the complete activation of CDKs in cell cycle. We examined phosphorylation of CDK1 and CDK2, results suggested addition of remodelin and NAT10 knockdown signi cant reduced phosphorylation of CDK1 and CDK2 but re-overexpression of CDK7 in NAT10 knockdown cells restore the phosphorylation (Fig. 6K-6L). Those results demonstrated that NAT10 promotes cell cycle and tumorigenesis of oral cancer through regulating CDK7. miR-375 targeted and regulated NAT10 in oral cancer microRNAs (miRNAs) have been demonstrated to participate in the pathological process of oral cancer. We tended to investigate the upstream microRNAs of NAT10, which also play a major role in oral cancer. we determine upstream microRNA of NAT10 analysis based on the public miRNA predicted website (http://www.targetscan.org). We observed that NAT10 was one of the potential targets of miR-375 (Fig. 7A). Thus, dual-luciferase reporter gene assay was performed, and it was observed that miR-375 mimics remarkably reduced the luciferase activity of NAT10 WT reporter but not NAT10 MUT (Fig. 7B). Consistently, miR-375 inhibitor increased the luciferase activity of NAT10 WT reporter but not NAT10 MUT (Supplementary Figure S4A). We further determined the expressions of NAT10 using qRT-PCR and Western blot. It was found that the over-expression of miR-375 inhibited but inhibition of miR-375 increase the expression levels of NAT10 at both mRNA and protein levels ( Fig. 7C-7E and Supplementary Figure S4B-S4D). We also veri ed the negative correlation between NAT10 and miR-375 in clinical sample (Fig. 7F). Collectively, it was con rmed that NAT10 serve as a target of mi-375 in oral cancer. Subsequently, we observed the expression of miR-37 in oral cancer is remarkably upregulated than normal tissue in predicted website also in our clinical sample (Fig. 7G-7I). Those results con rmed that miR-375 serve as upstream of NAT10 and CDK7 in oral cancer.

Discussion
Oral squamous cell carcinoma arises from the oral cavity, which includes hard and soft plate, tongue, oor of mouth, buccal mucosa and alveolar rim, oropharynx, hypopharynx and larynx of head and neck 39,40 . It is one of the six most common cancers in the world. Annually, approximately 45,780 new cases of OSCC in the United States, 61,400 in Europe, and 274,000 in Asia [41][42][43] . The OSCC is the leading cause of cancer deaths in the East Asian countries 44 .
Important risk factors in the development of the disease are tobacco, betel quid, alcohol, age, gender and sunlight, although a role for candida and the human papilloma virus has also been documented 45,46 . The increase in alcohol consumption is associated with an increased incidence rate of OSCCs as tobacco consumption decreases 43,47 . When tobacco and alcohol are taken together, the carcinogenic action of tobacco and alcohol is synergistic 43,47 . The risk of oral cancer of alcoholics and smokers is 38 times higher than that of abstainers 48 . In addition, considering the amount of alcohol consumed by binge drinking, it is considered to be of particular importance in the malignant development of young people 49,50 . Historically, the risk of oral cancer increased with age 51,52 . Many patients however do not have history of any those habits and/or evidence of previous viral infection 53 . Therefore, additional factors are likely to cause OSCC in this subgroup. Genetic aberrations and predispositions are likely to be important and previous studies have identi ed a number of oncogenes and tumor suppressor genes that are involved in the pathogenesis of OSCC [54][55][56] . Better understanding of these genetic factors and molecular mechanisms underlying the oral carcinogenesis and metastasis is important to improve the diagnosis, therapeutic options, prognosis and prevention of OSCCs.
Recently, microRNAs (miRNAs) have been documented potentially culminate in the development and progression of oral cancer. Different from deregulation of mRNA expression levels, miRNA expression alterations are mostly due to promoter-related changes 1,57,58 . The role of miRNA deregulation in oral cancer have been explored in several studies, therein, presenting compelling evidence for the involvement of miRNAs in angiogenesis, tumor progression, invasion, etc 58-60 . However, the regulatory mechanisms behind miRNA expression deregulation at a genome scale remained largely unknown.
NAT10 has been reported to promote formation of ac4C on tRNA, rRNA, and mRNA and protein acetylation 14,17 . Limited literature reported that its dysfunction plays an important role in some tumors 61 , and its regulatory role in oral cancer remains unknow. Here we demonstrated a new acetylated substrate CDK7 for NAT10, and con rmed the regulatory role of NAT10 in cell cycle. NAT10 enzymatic inhibitor, remodelin, signi cantly inhibit the proliferation of oral cancer cells in vitro and in vivo, which provides a research foundation for the targeted therapy of NAT10 in oral cancer.
In this article, we also investigated the role of small RNA mediated regulation of oncogene in oral cancer. We have extensively described the role of miR-375 in regulating carcinogenesis in OSCC. We also discussed the role of miRNA mediated transcriptional repression of NAT10 in oral cancer. A comprehensive understanding of the miRNA regulation of oncogene in oral cancer may help to develop better strategies for the diagnosis, molecular targeted therapy and overall prognosis of oral cancer.

Conclusion
Our results unveil NAT10 as a potential diagnostic, prognostic and therapeutic tool for oral cancer, which provides a novel approach for use in noninvasive screening of oral cancer. Findings presented here show that NAT10 plays a progressive role in oral cancer progression (Fig. 8). NAT10 exerts its tumorigenic progressive functions through acetylated-dependent of CDK7. NAT10 functions as a tumor promoter to participate in cell cycle of oral cancer. Deregulated miR-375 increased the transcriptional level of NAT10. Consequently, evaluating the therapeutic potential of NAT10 in oral cancer deserve large-scale studies. Availability of data and material The datasets generated and/or analyzed in this study are available from the respective author upon reasonable request.

Competing interests
The authors declare that there is no con ict of interest regarding the publication of this paper.     (G-H) 100 μg/mL of CHX was used to treat OECM1 Cells which stably expressed both shNC, shNAT10 #1 and shNAT10 #2 for the indicated times, which were analyzed by immunoblotting. (I) OECM1 cells stably expresseing pCDH and NAT10 were performed immunoprecipitation, then subjected to immunoblotting analysis with the indicated antibodies. OECM1 cells were treated with 20 μM MG-132 for 6 h before harvest. #2 were subjected to immunoprecipitation, then analyzed by immunoblotting using indicated antibody.
(E) OECM1 cells treated with DMSO or remodelin were subjected to immunoprecipitation, then analyzed by immunoblotting using indicated antibody. (F) OECM1 cells stably expressing pCDH, CDK7 and NAT10 K328R were subjected to immunoprecipitation, then analyzed by immunoblotting using indicated antibody. (G-H) 100 μg/mL of CHX was used to treat OECM1 Cells which stably expressed both CDK7 K328Q and CDK7 for the indicated times, which were analyzed by immunoblotting.

Supplementary Files
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