M6A epitranscriptomic regulation of KRAS by METTL3 promotes EMT and stromal remodeling through TGF-β/SMAD signaling in cervical cancer.

Epigenetic alteration is a common trait of human cancers. Chemical modifications on DNA and core histone proteins are the major mechanisms for epigenetic regulation. Recently, emerging evidence suggested that reversible chemical modifications on RNA also play a critical role in epigenetic control of gene expression. N6-methyladenosine (m6A) is the most abundant modification found in mammalian mRNA. m6A is involved in regulating mRNA stability, splicing, and translation. However, the implications of m6A modification in human carcinogenesis remain poorly understood. In this study, we investigated the expression of m6A methyltransferases and demethylases in human hepatocellular carcinoma (HCC). We found the major m6A methyltransferase METTL3 was remarkably up-regulated in HCC. High METTL3 expression was associated with poor overall and disease-free survival in HCC patients. Consistent with the METTL3 overexpression, we found that mRNA m6A level was significantly elevated in human HCC. To investigate the roles of METTL3 in human HCC, we employed lentiviral-based shRNA and CRISPR/Cas9 systems to inactivation METTL3 in HCC cell lines. We showed that knockdown and knockout of METTL3 drastically suppressed HCC proliferation and migration in vitro and abolished HCC tumorigenicity and lung metastasis in vivo. In contrast, overexpression of METTL3 by CRISPR/dCas9 SAM system promoted HCC growth. Using RNA-seq and m6A-Seq, we identified tumor suppressor gene SOCS2 as a novel target of METTL3. We detected m6A modification in SOCS2 mRNA and the m6A modification was diminished upon METTL3 knockdown. m6A modification promoted SOCS2 mRNA degradation. We demonstrated that knockdown of METTL3, mutation of m6A modification sites, and treatment of demethylating agent augmented SOCS2 mRNA expression in HCC cells. In addition, knockdown of YTHDF2, an m6A reader protein, also rescued SOCS2 mRNA expression in HCC cells. The above findings together demonstrated that up-regulation of METTL3 lead to m6A modification of SOCS2, which in turn promotes SOCS2 mRNA degradation through YTHDF2 dependent mechanism. Our findings provided a proof-of-concept model to demonstrate the importance of aberrant m6A modification in epigenetic silencing of tumor suppressor genes. Citation Format: Mengnuo Chen, Lai Wei, Cheuk-Ting Law, Felice H. Tsang, Jialing Shen, Carol L. Cheng, Long-Hin Tsang, Daniel W. Ho, David K. Chiu, Joyce M. Lee, Carmen C. Wong, Irene O. Ng, Chun-Ming Wong. Up-regulation of METTL3 promoted m6A modification and epigenetic silencing of SOCS2 in human liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4321.

MicroRNA-31 (miR-31) functions as tumor suppressors or oncogenes that are involved in tumor behavior. However, the function of miR-31 in cervical carcinogenesis remains unclear. The aim of this study was to validate the potential role of miR-31 and BRCA1-associated protein-1 (BAP1) on regulating epithelial-mesenchymal transition (EMT) in cervical cancer. In the present study, qRT-PCR assay revealed that the expression of miR-31 was upregulated in human cervical cancer cells and clinical tissues. Results of wound healing and cell migration assay revealed that knockdown of miR-31 inhibited cell metastasis and migration. Bioinformatic and dual-luciferase reporter gene assay showed that BAP1 was the direct target of miR-31. Furthermore, the results revealed that miR-31 promoted proliferation and EMT in cervical cancer cells and accelerated the development of tumor growth in vivo xenograft experiment by inhibiting BAP1 expression. Overall, these results highlight an important role of miR-31 functioning as an oncomir which could promote EMT in cervical cancer via downregulating BAP1 expression. Thus, downregulation of miR-31 could be a novel approach for the molecular treatment of cervical cancers and other malignancies.

BackgroundDysregulated epithelial–mesenchymal transition (EMT) is involved in cervical cancer metastasis and associated with histone acetylation. However, the underlying molecular mechanisms of histone acetylation in cervical cancer EMT and metastasis are still elusive.MethodsWe systematically investigated the expression patterns of histone acetylation genes and their correlations with the EMT pathway in cervical cancer. The expression of CSRP2BP among cervical cancer tissues and cell lines was detected using Western blotting and immunohistochemistry analyses. The effects of CSRP2BP on cervical cancer cell proliferation and tumorigenicity were examined by cell growth curve, EdU assay, flow cytometry and xenotransplantation assays. Wound healing assays, transwell migration assays and pulmonary metastasis model were used to evaluate the effects of CSRP2BP on cell invasion and metastasis of cervical cancer cells in vivo and in vitro. RNA-seq, chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP) and luciferase reporter assays were used to uncover the molecular mechanisms of CSRP2BP in promoting cervical cancer EMT and metastasis.ResultsWe prioritized a top candidate histone acetyltransferase, CSRP2BP, as a key player in cervical cancer EMT and metastasis. The expression of CSRP2BP was significantly increased in cervical cancer tissues and high CSRP2BP expression was associated with poor prognosis. Overexpression of CSRP2BP promoted cervical cancer cell proliferation and metastasis both in vitro and in vivo, while knockdown of CSRP2BP obtained the opposite effects. In addition, CSRP2BP promoted resistance to cisplatin chemotherapy. Mechanistically, CSRP2BP mediated histone 4 acetylation at lysine sites 5 and 12, cooperated with the transcription factor SMAD4 to bind to the SEB2 sequence in the N-cadherin gene promotor and upregulated N-cadherin transcription. Consequently, CSRP2BP promoted cervical cancer cell EMT and metastasis through activating N-cadherin.ConclusionsThis study demonstrates that the histone acetyltransferase CSRP2BP promotes cervical cancer metastasis partially through increasing the EMT and suggests that CSRP2BP could be a prognostic marker and a potential therapeutic target for combating cervical cancer metastasis.

The modification of methyltransferase-like (METTL) enzymes plays important roles in various cellular responses by regulating microRNA expression. However, how m6A modification is involved in stress granule (SG) formation in the early stage of acute ischemic stroke by affecting the biogenesis processing of microRNAs remains unclear. Here, we established a middle cerebral artery occlusion (MCAO) model in rats and an oxygen-glucose deprivation/reperfusion (OGD/R) model in primary cortical neurons and PC12 cells to explore the potential mechanism between m6A modification and SG formation. The in vivo results showed that the level of infarction and apoptosis increased while SG formation decreased significantly within the ischemic cortex with improved reperfusion time after 2 h of ischemia. Consistent with the in vivo data, an inverse association between the apoptosis level and SG formation was observed in PC12 cells during the reperfusion period after 6 h of OGD stimulation. Both in vivo and in vitro results showed that the expression of METTL3 protein, m6A and miR-335 was significantly decreased with the reperfusion period. Overexpression of the METTL3 and METTL3 gene-knockdown in PC12 cells were achieved via plasmid transfection and CRISPR-Cas9 technology, respectively. Overexpression or knockdown of METTL3 in oxygen-glucose deprivation of PC12 cells resulted in functional maturation of miR-335, SG formation and apoptosis levels. In addition, we found that miR-335 enhanced SG formation through degradation of the mRNA of the eukaryotic translation termination factor (Erf1). In conclusion, we found that METTL3-mediated m6A methylation increases the maturation of miR-335, which promotes SG formation and reduces the apoptosis level of injury neurons and cells, and provides a potential therapeutic strategy for AIS.

Acquisition of epithelial-mesenchymal transition (EMT) by primary carcinoma cells is associated with disrupted epithelial integrity, local invasion, and ultimately metastasis. Little is known about the existence and function of EMT in cervical cancer. This study aims to investigate the regulation of EMT in cervical squamous cell carcinoma. We investigated the molecular events of EMT in surgical specimens, which present the progression of cervical carcinoma. Two cervical cancer cell lines and the primary culture of normal cervical epithelia were used to study the regulatory mechanisms of EMT. The chronic epidermal growth factor (EGF) treatment induces the elongation of cell shape, increases cell scattering, and enhances cell invasion. EGF treatment down-regulates E-cadherin and up-regulates vimentin in cervical cancer cells. These characteristics are consistent with the morphologic changes, molecular events, and functional significance of EMT. EGF receptor (EGFR) signaling inactivates glycogen synthase kinase-3beta, which results in the nuclear accumulation of up-regulated Snail and then leads to EMT program. alpha(5)beta(1) integrin signaling and extracellular matrix fibronectin can modulate EGF-induced EMT. Importantly, the immunofluorescent stainings of surgical specimens indicate that cervical carcinoma progression is accompanied by EGFR overexpression, which is in parallel with decreased E-cadherin and increased vimentin. Up-regulation and nuclear accumulation of Snail correlate with EMT program in tumor tissues. EGF cooperates with alpha(5)beta(1) integrin signaling to induce EMT in cervical cancer cells via up-regulated Snail. Blockade of EGFR activity or expression may provide a potential target for the treatment of cervical cancer progression.

<div>Abstract<p><b>Purpose:</b> Acquisition of epithelial-mesenchymal transition (EMT) by primary carcinoma cells is associated with disrupted epithelial integrity, local invasion, and ultimately metastasis. Little is known about the existence and function of EMT in cervical cancer. This study aims to investigate the regulation of EMT in cervical squamous cell carcinoma.</p><p><b>Experimental Design:</b> We investigated the molecular events of EMT in surgical specimens, which present the progression of cervical carcinoma. Two cervical cancer cell lines and the primary culture of normal cervical epithelia were used to study the regulatory mechanisms of EMT.</p><p><b>Results:</b> The chronic epidermal growth factor (EGF) treatment induces the elongation of cell shape, increases cell scattering, and enhances cell invasion. EGF treatment down-regulates E-cadherin and up-regulates vimentin in cervical cancer cells. These characteristics are consistent with the morphologic changes, molecular events, and functional significance of EMT. EGF receptor (EGFR) signaling inactivates glycogen synthase kinase-3β, which results in the nuclear accumulation of up-regulated Snail and then leads to EMT program. α<sub>5</sub>β<sub>1</sub> integrin signaling and extracellular matrix fibronectin can modulate EGF-induced EMT. Importantly, the immunofluorescent stainings of surgical specimens indicate that cervical carcinoma progression is accompanied by EGFR overexpression, which is in parallel with decreased E-cadherin and increased vimentin. Up-regulation and nuclear accumulation of Snail correlate with EMT program in tumor tissues.</p><p><b>Conclusion:</b> EGF cooperates with α<sub>5</sub>β<sub>1</sub> integrin signaling to induce EMT in cervical cancer cells via up-regulated Snail. Blockade of EGFR activity or expression may provide a potential target for the treatment of cervical cancer progression.</p></div>

<div>Abstract<p><b>Purpose:</b> Acquisition of epithelial-mesenchymal transition (EMT) by primary carcinoma cells is associated with disrupted epithelial integrity, local invasion, and ultimately metastasis. Little is known about the existence and function of EMT in cervical cancer. This study aims to investigate the regulation of EMT in cervical squamous cell carcinoma.</p><p><b>Experimental Design:</b> We investigated the molecular events of EMT in surgical specimens, which present the progression of cervical carcinoma. Two cervical cancer cell lines and the primary culture of normal cervical epithelia were used to study the regulatory mechanisms of EMT.</p><p><b>Results:</b> The chronic epidermal growth factor (EGF) treatment induces the elongation of cell shape, increases cell scattering, and enhances cell invasion. EGF treatment down-regulates E-cadherin and up-regulates vimentin in cervical cancer cells. These characteristics are consistent with the morphologic changes, molecular events, and functional significance of EMT. EGF receptor (EGFR) signaling inactivates glycogen synthase kinase-3β, which results in the nuclear accumulation of up-regulated Snail and then leads to EMT program. α<sub>5</sub>β<sub>1</sub> integrin signaling and extracellular matrix fibronectin can modulate EGF-induced EMT. Importantly, the immunofluorescent stainings of surgical specimens indicate that cervical carcinoma progression is accompanied by EGFR overexpression, which is in parallel with decreased E-cadherin and increased vimentin. Up-regulation and nuclear accumulation of Snail correlate with EMT program in tumor tissues.</p><p><b>Conclusion:</b> EGF cooperates with α<sub>5</sub>β<sub>1</sub> integrin signaling to induce EMT in cervical cancer cells via up-regulated Snail. Blockade of EGFR activity or expression may provide a potential target for the treatment of cervical cancer progression.</p></div>

BackgroundPyruvate kinase isozyme type M2 (PKM2) is a key glycolytic enzyme and is upregulated in multiple human malignancies. However, the role of PKM2 in human cervical cancer (CC) remains elusive. Thus, this study explored the role of PKM2 in CC by detecting its expression patterns in human CC tissues and cell lines and investigated its effects on cell proliferation and invasion.Materials and methodsQuantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry and western blotting assays were used to detect the expression of PKM2 in CC tissues and CC cells. In vitro, we overexpressed and knocked down PKM2 expression in CC cell lines and investigated the biological function and underlying mechanism of PKM2 in cervical carcinogenesis.ResultsThe results showed that PKM2 mRNA and protein were highly expressed in CC tissues and cell lines. Furthermore, increasing PKM2 expression was closely correlated with the clinical stage (P=0.001) and lymph node metastasis (P=0.023). The functional roles of PKM2 were determined using Cell Counting Kit-8, colony formation, and transwell assays. The results showed that PKM2 knockdown inhibited cell proliferation and the migratory and invasive capacities of CC cells, suppressed epithelial–mesenchymal transition (EMT), and inhibited Wnt/β-catenin signaling in vitro. However, overexpression of PKM2 led to increased proliferation and invasion activity as well as the EMT in CC cells.ConclusionTaken together, our study results revealed that PKM2 may act as a molecular target for CC treatment.

Cervical cancer is a common women malignancy worldwide. Recent reports emphasize inactivation of tumor suppressor genes are due to promoter hypermethylation in cervical cancer. Our previous study from methylation array has shown NK6 homeobox 1 (NKX6.1) is hypermethylated and downregulated in cervical cancer cell lines and clinical samples. Nkx6.1 contains a homeobox domain and functions as a transcription factor that regulates insulin-secreting β cell differentiation and neuronal fate determination. However, the biological function of NKX6.1 in cervical cancer remains elusive. Overexpression of NKX6.1 suppressed the transformation and invasive ability of HeLa and CaSki cells in vitro as well as decreased tumor formation and metastatic property in vivo. Conversely, knockdown of NKX6.1 strongly enhanced the transformation and invasiveness of SiHa cells. Epithelial-mesenchymal transition (EMT) has been shown to play a key role in the progression of uterine cervix carcinoma in situ (CIS) to invasive squamous cell carcinoma (SCC). NKX6.1 suppresses cancer invasiveness through coordinately increasing E-cadherin and decreasing Vimentin. Moreover, we demonstrated E-cadherin and vimentin were direct targets of NKX6.1 by luciferase reporter assay, electrophoretic mobility shift assay and chromatin immunoprecipitation quantitative polymerase chain reaction (Chip-qPCR) assay. Collectively, our data demonstrate NKX6.1 acts a tumor suppressor, and inhibits tumor invasion and metastasis by suppressing Epithelial-mesenchymal transition (EMT) in cervical cancer. These data imply that modulation of NKX6.1 expression may be a novel therapeutic option for treatment of cervical cancer. Citation Format: Hsin-Jung Li, Pei-Ning Yu, Yu-Lueng Shih, Ya-Wen Lin. NKX6.1 suppresses cancer invasion and epithelial-mesenchymal transition in cervical cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4108. doi:10.1158/1538-7445.AM2015-4108

N6-methyladenosine (m6A) was the most abundant modification of mRNA and lncRNA in mammalian cells and played an important role in many biological processes. However, whether m6A modification was associated with recurrent spontaneous abortion (RSA) and its roles were still unclear. Methods: Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was used to study the global m6A modification pattern in RSAs and controls. RNA sequencing (RNA-Seq) was used to study the level of global mRNA in two groups. Real-time quantitative PCR (RT-qPCR) was used to verify the level of mRNA of METTL3 and ZBTB4. MeRIP–qPCR was conducted to test the level of ZBTB4 m6A modification in two groups. In order to further explore whether ZBTB4 was the substrate of METTL3, the HTR-8/SVneo (HTR-8) cell line was selected for the knockdown and overexpression of METTL3. To study whether METTL3 regulated the ZBTB4 expression by recognizing ZBTB4 mRNA m6A motifs in coding sequences (CDS), dual-luciferase reporter assay was conducted. RNA stability assays using actinomycin D were conducted to study the RNA stability of the HTR-8 cell line with METTL3 overexpression and knockdown. To illustrate the role of METTL3 in the invasion of trophoblast, matrigel invasion assays and transwell migration assays were conducted using the HTR-8 cell line with METTL3 overexpression and knockdown. Results: A total of 65 genes were found with significant differences both in m6A modification and mRNA expression. We found m6A methyltransferase METTL3 was significantly down-regulated in the RSA group. Through gene function analysis, RT-qPCR, MeRIP–qPCR validation experiment, knockdown, and overexpression of METTL3 in the HTR-8 cell line, ZBTB4 was selected as one target of METTL3. Furthermore, we clarified that METTL3 regulated the expression of ZBTB4 by recognizing ZBTB4 mRNA m6A motifs in the CDS using the dual-luciferase reporter assay and METTL3 regulated the invasion of trophoblast by altering the stability and expression of ZBTB4 by RNA stability, matrigel invasion, and transwell migration assays. Conclusion: Our study revealed the mechanism by which METTL3 regulated the stability and expression of ZBTB4 and the trophoblast migration ability of RSA. A new perspective was provided for exploring the mechanism of embryonic development in RSA patients.

This study aimed to explore the regulatory mechanism of methyltransferase3 (METTL3) -mediated long non-coding RNA (lncRNA) N6-methyladenosine (m6A) modification in the osteogenic differentiation of human adipose-derived stem cells (hASCs) induced by NEL-like 1 protein (NELL-1). Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and high- throughput sequencing for RNA (RNA-seq) were performed on hASCs. Osteogenic ability was detected by alkaline phosphatase (ALP) staining, Alizarin Red S(ARS) staining, ALP quantification and Quantitative real-time polymerase chain reaction analysis (qRT-PCR). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis predicted the osteogenesis-related pathways enriched for the lncRNAs and identified the target lncRNAs. After overexpression and knockdown of METTL3, methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) and qRT-PCR were used to detect the levels of m6A modification and the expression of the target lncRNA, and the binding of both was confirmed by RNA binding protein immunoprecipitation (RIP) assay. The effects of lncRNA and METTL3 on phosphorylation of the key proteins of the pathway were detected by western blot analysis. In vitro experiments showed that METTL3 can promote osteogenic differentiation and that its expression level is upregulated. KEGG pathway analysis predicted that lncRNAs with differentially upregulated methylated peaks were enriched mostly in the mitogen-activated protein kinase (MAPK) signaling pathway, in which Serine/threonine protein kinase 3 (STK3) was the predicted target gene of the lncRNA RP11-44N12.5. The m6A modification and expression of RP11-44N12.5 were both regulated by METTL3. Subsequently, lncRNA RP11-44N12.5 and METTL3 were found to regulate the phosphorylation levels of three key proteins in the MAPK signaling pathway, ERK, JNK and p38. This study shows, for the first time, that METTL3 can activate the MAPK signaling pathway by regulating the m6A modification and expression of a lncRNA, thereby enhancing the osteogenic differentiation of hASCs.

Epithelial-mesenchymal transition (EMT) is a key process contributing to cervical cancer (CC) metastasis, and microRNAs (miRNAs) modulate the expression of genes implicated in EMT. However, the accurate role of miR-361 in CC-associated EMT and the mechanisms underlying its function in CC remains largely unknown. The functional roles of miR-361 in CC cells were explored by a series of cell functional assays. Luciferase reporter assays were used to demonstrate the potential interaction between miR-361, HSP90, and long non-coding RNA (lncRNA) NEAT1. We detected a reduction of miR-361 expression in CC tissues compared with normal tissues, and miR-361 overexpression inhibited invasion and EMT phenotypes of CC cells by directly targeting a key EMT activator HSP90. Additionally, we detected significantly higher levels of HSP90 in CC tissues compared with normal tissues, and high expression of HSP90 predicted a poorer prognosis. We further identified NEAT1 as a significantly upregulated lncRNA in CC tissues and high expression of NEAT1 was associated with worse survival in CC patients. NEAT1 directly repressed miR-361 expression and played an oncogenic role in CC cell invasion and sphere formation. Conclusions: These results demonstrated that miR-361 directly targets HSP90 to inhibit the invasion and EMT features, and NEAT1 functions as an oncogenic lncRNA that suppresses miR-361 expression and induces EMT and sphere formation in CC cells, thus providing critical insights into the molecular pathways operating in this malignancy.

Background: Human dental pulp stem cells (hDPSCs) play an important role in endodontic regeneration. N6-methyladenosine (m6A) is the most common RNA modification, and noncoding RNAs have also been demonstrated to have regulatory roles in the expression of m6A regulatory proteins. However, the study on m6A modification in hDPSCs has not yet been conducted. Methods: Single base site PCR (MazF) was used to detect the m6A modification site of lncSNHG7 before and after mineralization of hDPSCs to screen the target m6A modification protein, and bioinformatics analysis was used to analyze the related pathways rich in lncSNHG7. After knockdown and overexpression of lncSNHG7 and METTL3, the osteogenic/odontogenic ability was detected. After METTL3 knockdown, the m6A modification level and its expression of lncSNHG7 were detected by MazF, and their binding was confirmed. Finally, the effects of lncSNHG7 and METTL3 on the Wnt/β-catenin pathway were detected. Results: MazF experiments revealed that lncSNHG7 had a m6A modification before and after mineralization of hDPSCs, and the occurrence site was 2081. METTL3 was most significantly upregulated after mineralization of hDPSCs. Knockdown/ overexpression of lncSNHG7 and METTL3 inhibited/promoted the osteogenic/odontogenic differentiation of hDPSCs. The m6A modification and expression of lncSNHG7 were both regulated by METTL3. Subsequently, lncSNHG7 and METTL3 were found to regulate the Wnt/β-catenin signaling pathway. Conclusion: These results revealed that METTL3 can activate the Wnt/β-catenin signaling pathway by regulating the m6A modification and expression of lncSNHG7 in hDPSCs to enhance the osteogenic/odontogenic differentiation of hDPSCs. Our study provides new insight into stem cell-based tissue engineering.

<div>Abstract<p>Cervical cancer severely affects women’s health with increased incidence and poor survival for patients with metastasis. Our study aims to investigate the mechanism by which lncRNA <i>LRRC75A-AS1</i> regulates the epithelial–mesenchymal transition (EMT) of cervical cancer through modulating m6A and ubiquitination modification. In this study, tumor tissues were collected from patients to analyze the expression of LRRC75A-AS1 and SYVN1. Migratory and invasive capacities of HeLa and CaSki cells were evaluated with wound healing and transwell assays. CCK-8 and EdU incor-poration assays were employed to examine cell proliferation. The interaction between LRRC75A-AS1, IGF2BP1, SYVN1, and NLRP3 was evaluated through RNA immunoprecipitation, RNA pull-down, FISH, and coimmunoprecipitation assays, respectively. MeRIP-qPCR was applied to analyze the m6A modification of <i>SYVN1</i> mRNA. A subcutaneous tumor model of cervical cancer was established. We showed LRRC75A-AS1 was upregulated in tumor tissues, and LRRC75A-AS1 enhanced EMT through activating NLRP3/IL1β/Smad2/3 signaling in cervical cancer. Furthermore, LRRC75A-AS1 inhibited SYVN1-mediated NLRP3 ubiquitination by destabilizing SYVN1 mRNA. LRRC75A-AS1 competitively bound to IGF2BP1 protein and subsequently impaired the m6A modification of SYVN1 mRNA and its stability. Knockdown of LRRC75A-AS1 repressed EMT and tumor growth via inhibiting NLRP3/IL-1β/Smad2/3 signaling in mice. In conclusion, LRRC75A-AS1 competitively binds to IGF2BP1 protein to destabilize SYVN1 mRNA, subsequently suppresses SYVN1-mediated NLRP3 ubiquitination degradation and activates IL1β/Smad2/3 signaling, thus promoting EMT in cervical cancer.</p><p><b>Implication:</b> LRRC75A-AS1 promotes cervical cancer progression, and this study suggests LRRC75A-AS1 as a new therapeutic target for cervical cancer.</p></div>

Cervical cancer severely affects women's health with increased incidence and poor survival for patients with metastasis. Our study aims to investigate the mechanism by which lncRNA LRRC75A-AS1 regulates the epithelial-mesenchymal transition (EMT) of cervical cancer through modulating m6A and ubiquitination modification. In this study, tumor tissues were collected from patients to analyze the expression of LRRC75A-AS1 and SYVN1. Migratory and invasive capacities of HeLa and CaSki cells were evaluated with wound healing and transwell assays. CCK-8 and EdU incor-poration assays were employed to examine cell proliferation. The interaction between LRRC75A-AS1, IGF2BP1, SYVN1, and NLRP3 was evaluated through RNA immunoprecipitation, RNA pull-down, FISH, and coimmunoprecipitation assays, respectively. MeRIP-qPCR was applied to analyze the m6A modification of SYVN1 mRNA. A subcutaneous tumor model of cervical cancer was established. We showed LRRC75A-AS1 was upregulated in tumor tissues, and LRRC75A-AS1 enhanced EMT through activating NLRP3/IL1β/Smad2/3 signaling in cervical cancer. Furthermore, LRRC75A-AS1 inhibited SYVN1-mediated NLRP3 ubiquitination by destabilizing SYVN1 mRNA. LRRC75A-AS1 competitively bound to IGF2BP1 protein and subsequently impaired the m6A modification of SYVN1 mRNA and its stability. Knockdown of LRRC75A-AS1 repressed EMT and tumor growth via inhibiting NLRP3/IL-1β/Smad2/3 signaling in mice. In conclusion, LRRC75A-AS1 competitively binds to IGF2BP1 protein to destabilize SYVN1 mRNA, subsequently suppresses SYVN1-mediated NLRP3 ubiquitination degradation and activates IL1β/Smad2/3 signaling, thus promoting EMT in cervical cancer. Implication: LRRC75A-AS1 promotes cervical cancer progression, and this study suggests LRRC75A-AS1 as a new therapeutic target for cervical cancer.