The Difference Of BLCAP A-I RNA Editing Between Cervical Cancer-adj Cent Tissues And The Influence Of Editing In BLCAP’s Function

According to the World Health Organization(WHO)global cancer statistics,the occurrence of cervical cancer ranked fourth among femal cancer patients,and the incidence and mortality in women are olny next to breast cancer,colon&rectum cancer and lung&bronchus&trachea cancer worldwide.In developing countries,the incidence of cervical cancer ranked second and the mortality ranked third in women.Although high-risk human papillomavirus(HPV)is well established as the major environmental risk factor for the occurrence of cervical cancer,and the incidence of cervical cancer is greatly reduced as the result of HPV vaccine,the multiple processes of genetic and epigenetic alterations leading to the disease still need to be elucidated.For the past years,our laboratory has endeavored to the study of cervical cancer carcinogenesis when the novel down-expression BLCAP gene came to our attention.BLCAP contains two exons and an intron encoding a 10kDa protein,which was originally identified from invasive bladder carcinoma in 2002.We previously published data from in vizro and in vivo experiments indicated that BLCAP might be a potential anti-tumor gene in cervical cancer.In addition,BLCAP is also a novel ADAR-mediated editing substrate undergoes multiple A-to-I RNA editing events.Although the function of BLCAP has been examined in preliminarily studies,how BLCAP plays an anti-oncogenic role and edited BLCAP functions as a cancer driver still require further exploration.In our study,we analyzd the sequence of BLCAP in 35 paired cervical cancer samples through high-throughput sequencing to identify the relationship between BLCAP and A-to-I RNA editing in cervical cancer,and found that there were three novel editing sites in BLCAP coding sequence.Comparing the cancerous and adjacent tissues,we observed that the editing levels of three novel editing sites were statistically different.According to the results of our preliminary eperiment,we classified the high-throughput sequence database by eight cases,and found that editing of these three sites was closely correlated.In addition,through statistical correlation analysis and conducting cell function test,we found that ADAR1 rather than ADAR2 played a major role in BLCAP A-to-I RNA editing.Finally,we predicted using Eukaryotic Linear Motif(ELM)that two editing sites of BLCAP transcript coding region were in the key YXXQ motif which can bind to SH2 domain of STAT3.Signal transducer and activator of transcription 3(STAT3)is a transcription factor which regulates a variety of cellular physiological activities.In the canonical pathway,STAT3 is phosphorylated subsequent to Janus kinases(JAKs)activation by some key activators such as IL-6 family cytokines,and the activated STAT3 migrates to the nuclei to recognize the STAT3-specific DNA-binding elements.The main target genes of STAT3 include Bcl-2 family proteins,Cyclins and matrix metalloproteinases(MMPs),which are implicated in anti-apoptosis,pro-proliferation,induction of angiogenesis,promotion of metastasis and evasion of anti-tumor immunity.Constitutive activation of STAT3 is involved in a wide range of human cancers and associated with tumorigenesis,therefore,STAT3 is regarded as a promising target for cancer therapy.It has been reported that activated STAT3 was overexpressed in cervical cancer and could act as a predictor of poor prognosis,suggesting its potential role in progression of cervical.Depending on the results of ELM,we performed Co-IP assays to explore whether BLCAP interacted with STAT3.Results revealed that exogenous BLCAP was able to interact with exogenous STAT3 in 293T cells,and could also interact with endogenous STAT3 both in 293T cells and HeLa cells.Overexpression and RNA interference of BLCAP in HeLa and C33A cells showed that BLCAP inhibited STAT3 activation.Similarily,overepresion and RNA interference of BLCAP in HeLa cells inhibited the expression of STAT3 target genes such as Bcl-2,Mcl-1 and survivin as well.According to the results,we demonstrated that BLCAP inhibited STAT3 phosphorylation in cervical cancer cells lines.Given that A-to-I RNA editing substitutes two important amino acids in BLCAP YXXQ motif,we constructed two edited plasmids(CCLQ-FLAG and CCLR-FLAG)to further explore the biological significance of BLCAP editing.We first explored the interaction of these two mutants with STAT3.CCLQ-FLAG presented a notably weaker interaction compared with the wild type BLCAP,while CCLR-FLAG showed almost complete loss of STAT3 interaction.In the phosphorylation assay,the activation of STAT3 increased in CCLQ-FLAG and CCLR-FLAG transfected HeLa cells as well as C33A cells.As for the downstream genes,CCLQ-FLAG and CCLR-FLAG lost their ability to inhibit STAT3 target genes compared with wild type BLCAP.These results collectively suggest that A-to-I RNA editing transform BLCAP inhibition ability to STAT3 by editing the key adenosine in YXXQ motif.Our research delineate a new mechanism of BLCAP function as a tumor suppressor,and provide additional evidence that abnormal A-to-I RNA editing events alter the function of BLCAP by editing the critical sites and play a potential role in progression of cervical.