Mechanistic Study Of M~6A Mediated YTHDC2 Regulation Of The ZNRD1-AS1/miR-942/TNS1 Axis In Cigarette Smoke Induced Lung Cancers

Background and Objective:Lung cancer is the most common cancer with high mortality in the world and a significant public health problem worldwide.Although in recent years clinical treatment technology has made some progress,the 5-year survival rate is still abysmal.Smoking is the first leading cause of lung carcinogenesis,and the incidence of lung cancer is much higher in smokers and reformed smokers.Our previous results demonstrated that long-term cigarette smoke exposure induced malignant transformation of human immortalized bronchial epithelial cells(BEAS-2B),manifested by the enhancement of cell proliferation and migration ability as well as apoptosis escape and the malignancy was also validated by subcutaneous tumorigenesis experiments in nude mice.Meanwhile,epigenetic indicators such as DNA methylation and histone modifications as well as miRNAs expression patterns are altered in malignantly transformed cells.Similar to DNA methylation,methylation modification also exists in RNAs,and its role in tumorigenesis and development is gaining more attention.This methylation modification at the RNA level is called"Epitranscriptomics".m6A modification is a highly dynamic and reversible process involving enzymes responsible for writing m6A known as "Writer",demethylated enzymes known as "Erasers",and recognition proteins known as "Reader"(collectively named"WER").These WERs synergistically maintain a homeostatic balance of intracellular m6A levels.In this study,we aimed to explore the expression of m6A associated WER as well as the changes of m6A modification levels and the related downstream regulatory mechanisms mediated by m6A during malignant transformation,focusing on function explore of smoking-related YTHDC2 in lung cancer and its target LncRNA ZNRD1-AS1.Meanwhile,multiple databases were combined to analyze the expression changes of YTHDC2 and ZNRD1-AS1 and the downstream pathway axis in lung cancer tissues as well as the clinical significance of both,which may provide the scientific basis to search for new therapeutic targets for lung cancer.Methods:(1)To explore the alteration of m6A modification patterns and associated WER expression in the malignant transformation of smoking-induced BEAS-2B cells and its significance.Long-term cigarette smoke-induced malignant transformation of human immortalized bronchial epithelial cells(BEAS-2B)were constructed in a previous study.Enzyme-linked immunosorbent assay(ELISA)was performed to measure global mRNA m6A levels in cigarette smoke-exposed cells.Fluorescent quantitative PCR(qPCR)was used to detect m6A related WER gene expression in cigarette smoke-exposed cells.Proteomic technologies were used for high-throughput analysis of differentially expressed proteins in cigarette smoke-exposed cells.Analysis of differentially m6A modified RNAs was conducted in smoke-induced malignant transformed cells using methylation RNA immunoprecipitation(meRIP)assay.(2)To explore the expression and clinical significance of m6A reader protein YTHDC2 in malignantly transformed cells and lung cancer.The expression changes of YTHDC2 in cigarette smoke-exposed cells were analyzed using qPCR and Western blot.The expression changes and copy number changes of YTHDC2 in lung cancer and its correlation with clinicopathological features were analyzed using TCGA and GEO database lung cancer-associated datasets.Kaplan-Meier plotter online tool was utilized to analyze the association between YTHDC2 mRNA expression and overall survival(OS)of lung cancer patients.Immunohistochemical analysis of YTHDC2 expression in a tissue microarray containing 70 lung cancer samples and paired normal tissues was carried out immunohistochemically and quantitatively using ImageJ.Copy number variation of YTHDC2 was determined by TaqMan assay in cigarette smoke-exposed cells.(3)In vitro and in vivo experiments to analyze the function of YTHDC2.YTHDC2 gene knockout cell lines were constructed using CRISPR-cas9 technology,and the differentially expressed proteins in the knockout cells were analyzed using proteomic technology.RNA immunoprecipitation(RIP)technology was used to pull down YTHDC2 interacted RNAs,which were further identified by high-throughput sequencing.Enrichment analysis of differentially expressed proteins and YTHDC2 interacted RNAs was performed using the DAVID online tool.To construct YTHDC2 overexpression plasmids,YTHDC2 was overexpressed and knocked down by the transfection of overexpression plasmids and siRNAs,respectively.We used flow cytometry to analyze the cell cycle after propidium iodide(PI)staining and EdU cell proliferation assay to analyze the proliferative ability of cells.We analyzed the migration ability of cells by wound healing and Transwell cell migration assays.In vitro tube formation assay of HUVEC cells was carried out to assess the proangiogenic ability of cells from each group.QPCR and western blot were used to analyze the proliferation and migration-related molecule expression of transfected cells,respectively.The protein levels of Ki-67 in cells were analyzed using the immunofluorescence assay.The effect of YTHDC2 on cell tumorigenic ability was explored by injecting H1299 cells stably overexpressing YTHDC2 and control H1299 cells into nude mice subcutaneously.Proliferation and migration-related protein expression in the tumor tissues was further analyzed using immunohistochemistry.(4)Screening of YTHDC2 downstream target LncRNAs and exploration of their functions and significance.The binding of YTHDC2 to ZNRD1-AS1 was validated by meRIP PCR and pull-down assays.RNA stability assay,that is,the regulation of ZNRD1AS1 stability by YTHDC2 was analyzed by qPCR analysis of intracellular RNA content after blocking the transcription in each group by actinomycin D.The expression of ZNRD1AS1 in GEO and TCGA databases and in cigarette smoke-exposed cells,as well as its immune and prognostic significance were studied in the second part.The function of ZNRD1-AS1 in vivo and in vitro was analyzed as in the third part.(5)Effect of m6A modification on ZNRD1-AS1 expression and the downstream ceRNA regulatory mechanism of ZNRD1-AS1.m6A sites in ZNRD1-AS1 were predicted using the SRAMP online tool,and these sites were validated by meRIP PCR/qPCR.Interfering with m6A modification level through treatment with methylation inhibitor 3deazaadenosine(3-DAA)or FTO inhibitor meclofenac(MA),the expression of ZNRD1AS1 in treated cells was detected by qPCR.Using the LncBase online tool as well as correlation analysi,the target miRNAs of ZNRD1-AS1 were predicted.The target genes of miR-942 were predicted based on 4 miRNA target gene prediction databases as well as correlation analysis.qPCR was used to assess the expression of miR-942 and TNS1 in cigarette smoke-exposed cells.Reporter gene plasmids were constructed and their targeting relationship was verified by dual-luciferase reporter assay.Rescue assay was used to validate the functional regulation between them,and the proliferation and migration functional analysis was shown in the third part.Similarly,the expression and prognostic significance of miR-942 and TNS1 were analyzed using TCGA and GEO databases.Results:(1)Cigarette smoke induces altered m6A modification patterns and associated WER expression in malignant transformation of human lung cells.Long term exposure to cigarette smoke induced reduced m6A modification in mRNA in the malignant transformed cells and H1299 cells compared with normal BEAS-2B cells."Eraser"protein ALKBH5 and "Reader" protein YTHDC2 also appeared to be aberrantly expressed in cigarette smoke-induced malignant transformed cells S30 and the lung cancer cell H1299.METTL3,FTO,YTHDC2,and YTHDF1 were significantly associated with patients’ smoking history in lung cancer patients.Furthermore,meRIP sequencing identified multiple mRNAs with aberrant m6A modification in S30 cells,and enrichment analysis revealed that these mRNAs were significantly enriched in multiple tumor related signaling pathways and biological processes.The intersection of differentially expressed proteins identified by proteomics and differentially m6A modified mRNAs identified by meRIP were found to be associated with tumor related biological functions,such as cell proliferation,adhesion and angiogenesis.(2)YTHDC2 copy number and expression are decreased in cigarette smokeexposed cell and lung cancer tissues and correlate with tumor stage,metastasis and immune cell infiltration,and patient prognosis.Significantly lower YTHDC2 expression was found in lung cancer tissues in smoking patients and in smoke-exposed cells.Pan-cancer analysis based on TCGA database showed that YTHDC2 was significantly downregulated in most cancer types.The immunohistochemistry results of tissue microarray indicated that YTHDC2 protein expression was downregulated in lung cancer tissues.The lower YTHDC2 expression was correlated with clinicopathological features including lung cancer stage,depth of invasion,distant metastasis and lymph node metastasis.Clinically,high YTHDC2 expressed lung cancer patients had a better prognosis and correlated with immune cell infiltration and immune checkpoint gene expression.In addition,YTHDC2 also showed a significant positive correlation with the expression of multiple tumor suppressor genes.Based on TCGA multi-omics data,we found that low expression of YTHDC2 was regulated through copy number deletion.(3)In vivo and in vitro study show that YTHDC2 can inhibit cell proliferation,migration and angiogenesis.Using the CRISPR-cas9 technology,YTHDC2 knockout cell lines were constructed in which differentially expressed proteins were identified by proteomic analysis.MRNAs and LncRNAs associated with YTHDC2 were identified by RIP-sequencing.Further enrichment analysis showed that the differentially expressed proteins in knockout cells and mRNA that can bind to YTHDC2 were significantly enriched in multiple tumor related signal pathways and biological processes,and were related to RNA processing and stability.In vitro cell assays showed that overexpression of YTHDC2 significantly inhibited the proliferation,migration,and angiogenic abilities of S30 and H1299 cells,whereas knocking down has the opposite effect.Nude mice tumorigenesis experiments also indicated that overexpression of YTHDC2 was able to suppress the tumorigenic capacity of H1299,and the expression of proliferation and migration related proteins was also decreased.(4)The target LncRNA ZNRDl-AS1 of YTHDC2 can inhibit cell proliferation and migration.RNA stability experiments showed that YTHDC2 overexpression significantly increased ZNRD1-AS1 stability.Bioinformatics analysis based on TCGA and GEO databases indicated that low ZNRD1-AS1 expression was correlated with smoking history,tumor stage and lymph node metastasis,as well as with immune cell infiltration and patient prognosis.ceRNAs of ZNRD1-AS1 were predicted using LncBase,and enrichment analysis showed that they were associated with cell cycle and apoptosis.FISH and nucleocytoplasmic fractionation assays confirmed that ZNRD1-AS1 was distributed in both the nucleus and cytoplasm,and were more abundant in the cytoplasm.Further in vitro and in vivo experiments confirmed that ZNRD1-AS1 overexpression could significantly inhibit the proliferation and migration of lung malignant cells as well as tumor growth.(5)m6A modified ZNRD1-AS1 regulates cell proliferation and migration through miR-942/TNS1 axis.ZNRD1-AS1 was found to contain 5 m6A sites within its full length with higher than moderate confidence as predicted by the SRAMP online tool,and meRIP assay validated the presence of significant m6A modification at one of these sites.Treatment of S30 and H1299 cells with 3-DAA significantly downregulated the expression of ZNRD1AS1,whereas MA treatment significantly upregulated its expression.Furthermore,YTHDC2 overexpression was able to significantly rescue ZNRD1-AS1 downregulation resulting from 3-DAA treatment.The database predicted and experimentally confirmed miR942 as a target RNA of ZNRD1-AS1,and TNS1 was also confirmed to be a downstream target gene of miR-942.Further dual-luciferase reporter gene experiments confirmed the targeted relationship between them.Furthermore,both miR-942 overexpression and TNS1 knockdown were able to significantly rescue the reduced proliferation and migration abilities caused by ZNRD1-AS 1 overexpression,further validating the ZNRD1-AS 1/miR-942/TNS 1 axis and the mechanism of smoking induced lung cancer.Conclusions:Long term cigarette smoke exposure can induce aberrant expression of demethylase ALKBH5 and reader protein YTHDC2 in lung cells,with the overall m6A modification level decreased.The copy number and expression of YTHDC2 are downregulated in cigarette smoke induced malignant transformed cells and lung cancer,and YTHDC2 reduction is related to tumor stage,metastasis,immune cell infiltration and prognosis.Functionally,YTHDC2 overexpression can inhibit cell proliferation,migration and angiogenesis.m6A modification can mediate the regulation of LncRNA ZNRD1-AS1 by YTHDC2,to regulate the proliferation and migration of lung malignant cells.YTHDC2 can regulate the migration and proliferation of lung malignant cells through the ZNRD1AS1/miR-942/TNS1 axis.