Research On The Mechanism Of Gene Copy Number Amplification-driven RNA Methyltransferase KIAA1429 In Lung Adenocarcinoma Tumorigenesis

Background:Lung cancer is one of the malignant tumors with the highest morbidity and mortality worldwide.Lung cancer is mainly divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC)according to pathological types.NSCLC is the main pathogenic type,including two pathological subtypes of lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC),the most common type being LUAD.According to the data released by the World Health Organization’s International Agency for Research on Cancer(IARC),there will be 2.21 million new cases of lung cancer worldwide in 2020;the proportion of deaths due to lung cancer will account for 18%.In recent years,with the emergence of problems such as increasing tobacco consumption,serious environmental pollution,and accelerated aging of the population,lung cancer has become an important public health problem endangering national health.In 2020,the number of new cases and deaths from lung cancer will be approximately 815,000 and 715,000,ranking first among all malignant tumors in China.Therefore,based on the population level,in-depth research on the molecular mechanism of the occurrence and development of lung cancer and the discovery of efficient and specific candidate target molecules will help to provide a theoretical basis for the early diagnosis and treatment of lung cancer.The regulation mechanism of gene expression is multi-layered,and epigenetic modification,as one of the important mechanisms to regulate gene expression,has always been a research hotspot.Epigenetic modification refers to the stable inheritance of changes in gene expression or function by regulating the interaction between the genome and the environment without changing the DNA sequence of the gene,mainly including histone modification,DNA methylation,RNA modification and chromatin remodeling,etc.Numerous studies have shown that epigenetic changes can be involved in a variety of biological processes in the body,and abnormal modifications can be involved in the occurrence of various diseases including tumors.At present,people know more about DNA methylation and histone modification.In recent years,RNA modification,as an important epigenetic modification method,has attracted extensive attention and made remarkable progress in many research fields.RNA modification is a new field in the development of "RNA epigenetics".Among them,RNA methylation modification represented by N6-methyladenosine(m6A)is the most common,and it is the most important post-transcriptional modification of mRNA in eukaryotic cells.The unique motif of m6A is RRACH(R:A/G,H:A/C/U),which is highly conserved and mainly enriched in the stop codon.3’UTR and exon regions of mRNA.M6A modifications play important roles in RNA translation,stability and alternative splicing.M6A modification is mainly regulated by three types of proteins,including the most important "writer(RNA methyltransferase)" responsible for catalyzing m6A modification of adenylate on mRNA,"eraser(demethylase)" and "reader(m6A recognition protein)" are responsible for demethylation and recognition of methylated sites,respectively.Studies have found that m6A modification abnormalities are closely related to tumorigenesis,and m6A modification-related enzyme genes,especially "writer",regulate gene expression by writing m6A methylation modifications,and their dysregulated expression can play an important role in a variety of tumorigenesis.For example,the highly expressed RNA methyltransferase METTL3 regulates ATAD2 expression through RNA methylation modification to promote osteosarcoma growth and invasion.The RNA methyltransferase WTAP is significantly activated in liver cancer and promotes the development of liver cancer by regulating the HuR-ETS1-p21/p27 axis.However,less attention has been paid to the causes of dysregulated RNA methyltransferase gene expression,especially at the genomic level.In recent years,with the continuous development of sequencing technology,whole-genome sequencing(WGS)has been widely used in the research of various diseases,especially tumors.Tumor WGS can detect point mutations in coding and non-coding regions of the whole genome,and can also detect copy number variation(CNV)of genes.At the same time,combined with transcriptome sequencing,it can systematically study the relationship between genome changes and gene expression,which is conducive to the systematic screening of effective potential targets.This study was based on whole-genome and transcriptome sequencing data from pre-laboratory LUAD samples,combined with The Cancer Genome Atlas(TCGA)data,to perform population-level systematic copy number variation analysis of major RNA methyltransferase genes and further study the role and molecular mechanism of RNA methyltransferase gene in the occurrence and development of LUAD.Part Ⅰ Analysis of copy number variation of RNA methyltransferase gene in LUADMethods:Based on the whole-genome sequencing data of 55 LUAD patient samples in the Nanjing Lung Cancer Cohort(NJLCC),combined with the data of 511 LUAD in the TCGA database,the GATK process was used to identify the major RNA methyltransferase genes(METTL3,METTL14,WTAP and KIAA1429)were subjected to comprehensive copy number variation analysis to screen for RNA methyltransferase genes with copy number amplification,and to detect the amplification levels of candidate RNA methyltransferases in various LUAD cells.Then,further combined with NJLCC and TCGA transcriptome data,Spearman rank correlation coefficient was used to analyze the correlation between copy number amplification and expression of candidate RNA methyltransferases.In addition,we also used T test and Cox proportional hazards regression model to analyze the differential expression of candidate RNA methyltransferase genes in LUAD and paracancerous tissue samples and their correlation with the prognosis of LUAD patients.Results:Among the 4 major RNA methyltransferase genes(METTL3,METTL14,WTAP,and KIAA1429)in the NJLCC and TCGA LUAD data,KIAA1429 had the highest copy amplification rate in LUAD samples,and was also amplified in various LUAD cells.KIAA1429 copy number amplification was positively correlated with its expression level.Compared with paracancerous tissues,KIAA1429 showed significantly higher expression in LUAD tissues,and high expression of KIAA1429 could indicate poor prognosis of LUAD patients.Conclusion:Through population data analysis,it was found that copy number amplification can drive the activation of KIAA1429 in LUAD and suggest a poor prognosis,which may play an important role in the occurrence and development of LUAD.Part Ⅱ RNA methyltransferase KIAA1429 regulates the proliferation and migration of LUADMethods:In this study,multiple LUAD cell lines were used.We designed and synthesized small interfering siRNA and constructed an overexpression plasmid for KIAA1429.After knockdown and overexpression of KIAA1429 in LUAD cell lines,experiments such as MTT,clone formation,EdU,Transwell,cell scratch and apoptosis detection were performed to explore the effect of KIAA1429 on the proliferation and metastasis of LUAD in vitro.A KIAA1429 knockdown lentiviral vector was constructed,and the effect of KIAA1429 on the proliferation and metastasis of LUAD cells in vivo was detected by subcutaneous tumor-bearing and tail vein transfer experiments in nude mice.Results:Knockdown of KIAA1429 can significantly inhibit the cell viability,cell proliferation,clone formation and cell metastasis of LUAD cells,while overexpression of KIAA1429 can promote the proliferation and metastasis of LUAD cells.Flow cytometry analysis found that knockdown of KIAA1429 induced apoptosis in LUAD cells.In vivo animal experiments found that after knockdown of KIAA1429,the subcutaneous tumorigenic ability of LUAD cells in nude mice was weakened,and the lung metastasis ability of LUAD cells was also significantly weakened.Conclusion:Both in vitro cellular level and in vivo animal level results confirmed that RNA methyltransferase gene KIAA1429 could regulate the proliferation and metastasis of LUAD.Part Ⅲ The mechanism of RNA methyltransferase KIAA1429 in regulating the proliferation and metastasis of LUADMethods:After knockdown of KIAA1429 in LUAD cell lines,the downstream target genes regulated by KIAA1429 were screened by transcriptome sequencing,followed by GO and GSEA analysis,and the sequencing results were verified by qRT-PCR.Using MeRIP-seq,methylated RNA Immunoprecipitation(meRIP),mRNA stability analysis,and RNA co-immunoprecipitation(RIP)experiments,it was confirmed that KIAA1429 modifies m6A in a YTHDF2-dependent manner to regulate the expression of target genes.Results:The study found that BTG2 can be a target gene regulated downstream of KIAA1429,and KIAA1429 can regulate the expression of BTG2 to promote the proliferation and metastasis of LUAD cells.Moreover,KIAA1429 recognizes BTG2 in a YTHDF2-dependent manner,regulates the level of BTG2 mRNA m6A,and inhibits the mRNA stability of BTG2.Conclusion:By studying the downstream regulatory mechanism of KIAA1429,it was found that KIAA1429 regulates the level of BTG2 m6A modification in a YTHDF2-dependent manner,affects the stability of BTG2 mRNA,and promotes the occurrence and development of LUAD.