The Function And Molecular Mechanism Of NELFA MRNA In ESCC

Esophagus cancer is considered to be one of the most common solid cancer across the world which could be divided into two pathological types,Esophageal Squamous Cell Carcinoma(ESCC)and Esophageal Adenocarcinoma(EAC).The incidence rate of esophagus cancer ranked in the top fifth and its mortality rate was the fourth in China.As we all know,the DNA damage repair response is regulated by an extremely complicated network,during which the DNA damage checkpoint proteins play an important role in the biological process of cell cycle and considered to be vital for the maintenance of genomic integrity.The critical role of DNA damage checkpoint proteins in the cell cycle regulation heavily relies on multiple elements which consists of sensors including Rad17,Rad9-Rad1-Hus1,ATM,ATR and so on,adapters like BRCA1,CLASPIN,Chk1,Chk2 and also target proteins like Cdc25 family proteins,P53,P21.At the early stage of DNA damage repair response,Rad17 could combine with RFC proteins to finally form an unique DNA-binding checkpoint complex,which is vital for Rad17 to preserve genomic integrity.Afterwards,Rad 17-RFC complex and Rad9-Rad1-Hus1complex connects to the DNA damage spot and thus initiating the process of DNA damage repair.Long non-coding RNAs(lincRNAs)are known to be single-strand RNA molecules with more than 200 nucleotides in length,most of which could not translate into proteins and few of them could encode oligopeptides according to the latest researches.Multiple studies of lincRNAs proved that they extensively took part in the regulation of many biological processes like cell growth,cell differentiation and metabolism.A large amount of research programs demonstrated that the abnormal expression of lincRNAs were closely related with multiple kinds of diseases including cancers,diabetes,autoimmune diseases,neurodegenerative diseases and so on.In our study,we utilized RNA immunoprecipitation assays to extract the RNA molecules that possibly interacting with Rad 17 protein.Subsequently,the immunoprecipitated and also enriched RNAs in the previous step were subjected to high throughput and deep sequencing technic.Those results were finally verified by real-time fluorescence quantitative PCR(polymerase chain reaction,PCR).Above all,we confirmed that NELFA mRNA could actually combine with Rad17 protein which was further verified by RNA pull-down assays.In order to further explore how the interaction between NELFA mRNA and Rad17 protein participated in the regulation of esophageal squamous cell carcinoma onset and progression and its specific molecular mechanisms implicated in the above biological process,we firstly detected the expression levels of NELFA mRNA and Rad 17 protein in ESCC cell line as well as normal immortalized epithelial cells.It turned out that the mRNA and protein expression levels of Rad17 were both significantly highly expressed in ESCC cell line compared with normal immortalized epithelial cells,while for NELFA mRNA,only its mRNA expression level was elevated with comparable protein expression level.During the downstream regulatory mechanism study of Rad 17 and NELFA,we found that the genomic amplification of USF2 in ESCC lead to the overexpression of itself and also promote the overexpression of Rad 17 and NELFA in the downstream.Consistently,in our functional assays,when we downregulated the expression level of NELFA mRNA by ASO in ESCC cell line,the proliferation and colony formation were obvious inhibited as anticipated.We further demonstrated that downregulation of NELFA mRNA also prohibited DNA damage repair response and promote cell apoptosis.In order to elucidate the implyling molecular mechanisms involved in the effects of NELFA mRNA on ESCC cell line,we implemented a series of experiments and found that downregulation of NELFA mRNA by ASO interrupted the combination between Rad17 and RFC2-5 complex and thus subsequently inhibited the phosphorylation of CHK1?CHK2 and BRCA1.Finally,in order to explore the clinical significancy of NELFA mRNA in ESCC,the RNAscope ISH(in situ hybridization,ISH)assays were applied to detect the NELFA mRNA expression through tissue microarrays from oesophageal squamous cell cancer and para-carcinoma tissue.As a result,we found that NELFA mRNA expression was significantly up-regulated in oesophageal squamous cell cancer compared with para-carcinoma tissue and closely related with overall survival and prognosis of ESCC patients.To sum up,NELFA mRNA overexpression might be a vital factor in ESCC progression and prognosis.Taken together,the abnormal expression of NELFA mRNA in ESCC played an important role in ESCC onset and progress.