The Association And Mechanism Study Of Cancer/Testis Gene LIN28B In Prognosis Of Gastric Cancer

Background:Gastric canceris one of the most common malignant gastrointestinal carcinomas,which forming a threat to human health worldwide.By2012,there had been more than 720,000 cases dead from gastric cancer in the world per year.Remarkably,the dead cases from China accounts for nearly half of the world,indicating that China is a focus country of international protection against gastric cancer.Lack of early,specific and sensitive biomarkers for screening and diagnosis,most gastric cancer patients were detected at advanced stage.Meanwhile,owing to lack of effective medical treatment,the prognosis of gastric cancer patients tendto be serious with 5-year overall survival merely equal to 25%.Hence,it is profound to explore the reliable and predictable indicators for the prognosis of gastric cancer and identify the molecular targets for clinical therapy,which would promote the treatment and healthcare of gastric cancer.Over the past decade,next generation sequencing has revolutionalized our understanding of cancer pathogenesis.Previous sequencing researches in gastric cancer population has preliminary identified some significant mutated genes（SMGs）,the mutations of which would inactivate tumor suppressor genes（TSGs）or activate oncogenes to promote the development of gastric cancer.These functional mutations provide a theoretical basis for the molecular mechanism of gastric cancer.Several mutated oncogenes are promising to become the clinical targets in gastric cancer therapy,such as EGFR,KRAS and PIK3CA.But the fact is that the mutation frequency of these oncogenes for targeted medicine in gastric cancer is very low,and even many individuals with gastric cancer don’t carry these mutations,which indicated that mutated oncogenes could only account for the genetic mechanism of a portion of gastric cancer patients.So far,epigenetic-drivers（epi-drivers）have been considered as the important driving factors of tumors.Tumorigenesis is usually accompanied by universal epigenetic alternations,which can lead to aberrant expression patterns.In turn,aberrant expression pattern caused by abnormal epigenetic alternations is an important characteristic of epi-drivers.Epi-driver genes can play important roles in tumorous formation and progression,which probably involve the pathogenesis of gastric cancer patients without significant mutated genes.In addition,abnormal epigenetic alternations usually take place at the early stage of tumorigenesis.So,identification of the tumor specific epi-drivers could improve our understanding of epigenetic mechanisms of carcinogenesis and conduce to early diagnose,prognostic predicition and even clinical therapy.Epigenomics includes DNA methylation,histone modification,non-coding RNA regulation,chromosome remodeling,and nucleosome positioning,among which DNA methylation alternation at genomic regulatory regions is acknowledged as one of the key biological processes responding to extracellular environment.Promoter region is usually enriched with Cp G islands,the methylation of which plays an important role in regulation of gene expression.So far,previous researches on gastric cancer have identified a stock of oncogenes by promoter demethylation.These genes are usually abnormal expressed but barely mutated,which provides evidence of the epigenetic mechanism in gastric cancer patients.However,the discovery of these genes in previous studies is simply a tip of the iceberg,and function of most genes remained unclear as well.With the development and improvement of high-throughput techniques and public databases,the exploratory researches on such genes in gastric cancer need to be further conducted.CT genes are restricted to express in normal testis,but aberrantly activated in several tumor tissues.Ectopic activation of CT genes may involve the carcinogenesis process,the feature of which conforms with epi-drivers,indicating that they are important cancer genes.Recently,our group identified a series of cancer/testis（CT）genes in multiple tumors and revealed that these genes shared the characteristic of epi-driver genes,which could be regarded as the important candidates of epi-drivers.In our previous study,we systematically identified 876 CT genes in 19 cancer types and explored their enriched characteristics by integrating multiple public databases.We determined that these CT genes could be activated by epigenetic modification and have a promoting effect on formation and progression of malignant tumors.Besides,promoter demethylation was one of the potential activation mechanisms of these CT genes.However,limited to the small sample size of gastric cancer（<100）,we didn’t identify CT genes in gastric cancer.With the accumulation of The Cancer Genome Atlas（TCGA）,the sample size of gastric cancer has met our analysis requirement.Therefore,based on the previous research,we identified CT genes in gastric cancer and revealed CT gene Lin-28 homolog B（LIN28B）was an impotant oncogene asscociated with the poor prognosis of gastric cancer.Finally,we investigated the function of LIN28B and its activation mechanism in gastric cancer.Methods:Out of 1319 testis-specific genes,we determined 605 CT genes activated in gastric cancer on the basis of transcriptome data from TCGA project.By integrating the multiple profiles of gastric cance in TCGA database,the correlation of these genes with prognosis,and their potential activation mechanism was further investigated.The select strategy and relevant statistical methods were briefly stated as follows:transformed t test was adopted to analysis the difference of genes expression in cancer tissues and adjacent tissues;multiple Cox regression was applied to analyze the correlation between gene expression or methylation of Cp G sits and prognosis;Spearman’s rank test was used to analyze the correlation between methylation of Cp G sits and gene expression.The consequence of statistical analysis was controlled with a false discover rate（FDR）of 0.20.We conducted further functional assays according to the consequence of selection above.We determined the neoplastic phenotypic of proliferation,migration and invasion in gastric cancer cell lines via gain-and loss-of-function analysis.Further,in order to explore the potential biological mechanism of LIN28B in gastric cancer,we performed enrichment analysis on the basis of TCGA project with the Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway module on DAVID website.Finally,we introduced CRISPR/d Cas9 technique to alter the targeted methylation and identified the effect of targeted methylation on transcription by bisulfite sequence PCR（BSP）and reverse transcription and quantitative real-time PCR（RT-q PCR）.Result:According to the above bioinformatics selection based on TCGA datasets,we revealed the activation of 10 CT genes is significantly associated with the poor prognosis of gastric cancer out of 605 CT genes(P<0.05,P_FDR<0.20),among which the promoter methylation of 3 CT genes（LIN28B,NUP210L and SPINLW1）is negatively correlated with their expression(P<0.05,P_FDR<0.20).Of the 3 genes,LIN28B promoter methylation shows a significant correlation with the prognosis of gastric cancer（P=0.020）.The consequence of screening indicates that LIN28B acts as a potentially functional CT gene in gastric cancer.TCGA shows LIN28B is hardly expressed in adjacent tissues but significantly activated in partial gastric cancer tissues（P<0.001）.After adjustment for age,gender and clinical stage,we reveal that the elevated expression of LIN28B significantly shortens the survival time of gastric cancer patients（Adjusted HR=2.13,95%CI:1.25-3.62,Log-rank P=0.005）.To further confirm the effect of LIN28B on tumorigenic and metastatic ability of gastric cancer,we determine its function in vitro by Koch’s postulates.Knocking out LIN28B in HGC27 cells delays tumor growth and inhibits the ability of migration and invasion according to CCK-8,colony formation and transwell assays.To the contrast,delivery of LIN28B-expression plasmid in gastric cancercells increases the ability of migration and invasion compared with those transfected with empty vector.Furthermore,we explore the potential mechanism of LIN28B in gastric cancer using the KEGG pathway analysis.Pathway analysis reveals LIN28B could involve Fox O signaling pathway,PPAR signaling pathway,cell cycle and base excision repair（BER）,which are reported to be remarkly associated with tumorigenesis.We identified LIN28B was activated by promoter demethylation according to the further bioinformatic and functional analysis.A negative correlation between promoter hypomethylation（cg11044575,cg17119521,cg17933583,cg19702779）and transcriptional expression of LIN28Bwas identified(P_cg11044575=7.4×10^-4,P_cg17119521=2×10^-3,P_cg17933583=5.8×10^-4,P_cg19702779=0.01).Cox regression showed that hopymethylation of cg17933583 significantly shortens the survival of gastric cancer patients after adjustment for age,gender and clinical stage（Adjusted HR=1.31,95%CI:1.04-1.65,Log-rank P=0.020）.Finally,we verified whether the abnormal promoter demethylation（cg11044575,cg17119521,cg17933583,cg19702779）could lead to the activation of LIN28B in gastric cancer by introducing the CRISPR/d Cas9system into HGC27 cells.The methylation level of each 4 Cp G sits located region was measured by BSP while LIN28B m RNA expression was determined by RT-q PCR.As is expected,we observed LIN28B m RNA expression increased while promoter methylation decreased,indicating that promoter demethylation could activated LIN28B directly in gastric cancer.Conclusion:In this study,we identified LIN28B was an important CT gene correlated with the prognosis of gastric cancer through a systematic screening out of the CT genes in gastric cancer based on the multiple profiles from TCGA project.Then,we verified the function in vitro by gain-and loss-of-function assays.Finally,we demonstrated alternation of the promote methylation was an important mechanism of LIN28B activation in gastric cancer.Taken together,we identified a novel epi-driver gene in gastric cancer,which provided a molecular evidence and theoretical basis for prognosis prediction and individualized treatment among gastric cancer patients.In addition,our research introduced CRISPR/d Cas9 technique to study the effect of targeted methylation on gene expression,which provided an actual reference example for subsequent similar studies.