Whole Exome Sequencing Identified LncRNAGAS8-AS1 As Papillary Thyroid Carcinoma Suppressor Gene And Its Mechanism Research

The causes of cancer are very complicated,it’s closely related to the living environmental and genetic background factors.Therefore,it is necessary to study the somatic mutation and mechanism of cancer.This study is divided into two parts:in the first part,we performed whole-exome sequencing to paint the somatic mutation spectra of papillary thyroid carcinoma(PTC)of Chinese population,identified frequently altered gene noncoding RNA(lncRNA)GAS8-AS1 from Chinese population;in the second part,we evaluated the mechanism of GAS8-AS1 and its critical role in occurrence and development of hepatocellular carcinoma.With quickly increased incidence,thyroid carcinoma become the most common endocrine malignancy in latest decade.However,the mutational spectrum in the Chinese population is absence.Whole exome sequencing can provide a plenty of mutational information of cancer.Thus,this study identified the somatic mutations of Chinese thyroid carcinoma patients using 402 tumor-normal pairs samples.Discovery team 91 pairs perform exome sequencing;validation team 311 pairs perform Sanger sequencing.We painted the somatic mutational spectrum and landscape of PTCs,and observed three distinct mutational signatures,significantly different from the two mutational signatures among Caucasian PTCs.In the other words,they are the specific mutational signatures of Chinese PTCs.We found 23 genes with>3 somatic mutations,among them BRAF V600E was a mutational hot-point.It was present in 59.0%patients,more frequently observed in patients with lymph node metastasis(P ＝1.6 × 10-4).Thus,BRAFwas regard as an important driver gene of PTC.Notably,we observed that a IncRNA gene GAS8-AS1 is the second most frequently altered gene except BRAF.There were two mutation site on GAS8-AS1 c.713A>G/714T>C.They always mutated together,more frequently observed in advanced patients.According to cell experiments,the wild-type IncRNA GAS8-AS1(A713 T714)and mutated IncRNA(G713C714)could inhibit cancer cell growth,however the mutated type showed consistently lower capability to inhibit cancer cell growth compared to the wild type.Lysophosphatidic acid receptor 4(LPAR4)was another important mutated gene in Chinese PTC patients,with only one mutation pattern(c.872T>G,p.Ile291Ser).LPAR4 was a G protein-coupled receptor with seven times transmembrane domain.As an oncogene,LPAR4 play crucial role in PI3K-AKT pathway.Thus,both protein-coding gene and protein-noncoding gene were involved in PTC etiology.Mutated genes were enriched in BRAF signal pathway and PI3K signal pathway.The role of long noncoding RNA in cancer is increasingly important.Worldwide,China bears more than fifty percentages of the whole burden,and IncRNA plays a very important role in liver cancer.According to the first research part,lncRNA GAS8-AS1 located in chromosome 16 the intron 2 of GAS8 producing a 994nt long noncoding RNA.However,the foundational mechanism is still unknown.In the second research part,we evaluated its critical role in regulating chromatin remodeling and gene expression.We observed that GAS8-AS1 and its host gene GAS8 act as tumor suppressors.Analyzing the expression of GAS8-AS1 or GAS8 in HCC tissues,significantly decreased exists and is associated with poor disease prognosis.Using cell experiments,we discovered that GAS8-AS1 and GAS8 have the ability to suppress cell growth,induce apoptosis without any effect to cell cycle,sensitize them to sorafenib and inhibit migration and invasion.All above indicated the cancer suppressor gene nature of GAS8-AS1 and GAS8.Noteworthly,we found significantly positive correlation between GAS8-AS1 and GAS8 expression in tissue,which demonstrated that the relation of expressional regulation between GAS8-AS1 and GAS8.Although there is a CpG island in GAS8 promoter,lncRNA GAS8-AS1 didn’t impact DNAmethylation of GAS8 promoter.But,we successfully annotated two GAS8-AS1 interacting proteins,Mixed Lineage Leukemia 1(MLL1)and its partner WD-40 Repeat Protein 5(WDR5).Chromatin immunoprecipitation and RNA immunoprecipitation assays elucidate that GAS8-AS1 is required to maintain the GAS8 promoter in an open chromatin state via recruiting MLL1/WDR5 complex to this region and enhancing functions of RNA polymoerase II and GAS8 transcription.This study firstly painted the somatic mutational spectrum and landscape of Chinese PTCs,discovered a number of mutated genes and pathway related to cancer.Our results highlight that we identified a InCRNA GAS8-AS1 plays an important anti-cancer role in papillary thyroid cancer and hepatocellular carcinoma.GAS8-AS1 regulate neighboring gene expression of gene GAS8 via epigenetic modifications(H3K4me3).This study proved the important role of long-chain non-coding RNA in cancer,enriched the mechanism of epigenetic gene expression regulation of IncRNA,provide an important theoretical basis for clinical diagnosis and treatment.