The Function Of LncRNA-J23 In Esophageal Squamous Cell Carcinoma

China has the highest incidence and mortality of esophageal cancer in the world.According to histopathological classification,esophageal carcinoma can be classified into two types:esophageal adenocarcinoma(EAC)and esophageal squamous cell carcinoma(ESCC).The majority of esophageal cancer in Europe and America are esophageal adenocarcinoma,while the majority of Chinese patients with esophageal cancer are esophageal squamous cell carcinoma.ESCC is malignant,with the onset of concealment,fast progress,and poor prognosis.The patient usually diagnosed with stagⅢor IV.Lacking of clinical targeted drugs is a current problem,so the early diagnosis and treatment of ESCC is a difficulty.Compared with lung cancer,colorectal cancer or breast cancer,ESCC related basic research and drug development is relatively lagging.It has an important significance to find biomarkers in early diagnosis of ESCC and explore the pathogenesis and therapeutic targets.In recent years with the help of next generation sequencing technology,especially the RNA-seq technology rapid development,people have more understanding for non-coding RNA,including the long non-coding RNA(lncRNA).LncRNA is a kind of transcripts with a length of ＞ 200nt and no protein coding ability.LncRNA with a large number,long length,rich secondary structure and strong tissue specificity.More and more IncRNA have been found to have important functions.LncRNA abnormal expression is closely related to many diseases,many studies have shown that IncRNA play an important role in cancer,IncRNA has a potential in as biomarkers or therapeutic targets in cancer.Our laboratory performed RNA-seq for 91 ESCC tissues and corresponding adjacent normal tissues.The data show that IncRNA lnc-J23 has higher expression level in ESCC tissues.We first identified the basic information of lnc-J23 by querying the database,and predicted that it did not have the protein coding capability.Then we determined the foll length sequence of lnc-J23 through the RACE and Northern bolt.We verified the up-regulated expression of lnc-J23 in ESCC in other 95 patients.Cell functional experiments showed that lnc-J23 promote the invasion,migration and ability of cloning of ESCC cells,but it had no significant effect on proliferation ability of ESCC cells.Experiments in nude mice showed that lnc-J23 has the ability to promote the subcutaneous blastoma and metastasis ability of ESCC cells in vivo.Because the function of IncRNA is directly related to its localization,we determined that lnc-J23 is mainly in the cytoplasm through FISH and separation experiments.In order to find the protein that binds with lnc-J23,we hybridized the protein chip after transcript-synthesis of lnc-J23 in vitro,and found the MSI2 can bind with lnc-J23.MSI2 belongs to the RNA binding protein,MSI2 proteins overexpressed in multiple cancers and have a correlation with poor prognosis,invasion and metastasis of lymph nodes.Research reveals MSI2 overexpressed in hepatocellular carcinoma(HCC)with poor prognosis,knocking down MSI2 can inhibit the liver cancer cell invasion ability and EMT.MSI2 overexpressed in non-small cell lung cancer increased,and by promoting the expression of TGFBR1 and SMAD3,promote tumor cell invasion and migration.KLF4 was found in pancreatic cancer as MSI2 transcription inhibiting factor,the lack of KLF4 will result in the overexpression of MSI2 and promote the development and metastasis of pancreatic cancer.Further study found that the protein expression level of MSI2 decreased after knockdown lnc-J23,and the mRNA level did not significantly change.The degradation of MSI2 was detected by CHX treatment to inhibit cell protein synthesis after Inc-J23 knockdown.We found that the half-life of MSI2 protein was shorter after Inc-J23 knockdown,but there was no significant change in mRNA level.However,knockdown and overexpression of MSI2 had no significant effect on the expression of Inc-j23.Overexpression of MSI2 can restore the cell phenotype of lnc-J23 knockdown.The results show that lnc-J23 binds to MSI2,enhancing its protein stability.We will further explored the specific molecular mechanism of Inc-J23 and MSI2.Cyclin B1 is an important regular protein involved in mitosis,which is expressed predominantly during G2/M phase of the cell cycle.In the early stage of mitosis,Cyclin B1 and p34(Cdk1)form the maturation-promoting factor(MPF)and involved in the process of chromosome condensation,nuclear envelope breakdown,and spindle pole assembly.CyclinB1 is mainly present in cytoplasm prior to mitosis,and relocates to the nucleus in late prophase.At the end of mitosis,cyclin B1 is targeted for degradation by the APC.Deregulation of cyclin B1 will lead to abnormal cell cycle,several studies have found that cyclin B1 have abnormally high expression in breast cancer,cervical cancer,gastric cancer,colorectal cancer,non-small cell lung cancer,colon cancer,prostate cancer,and esophageal cancer.Cyclin B1 will promote the proliferation of cancer cells and also associated with invasion and metastasis.In addition,the expression of cyclin B1 level is closely related to the prognosis of patients.The initiation of translation usually need eukaryotic translation initiation factor 4E(eIF4E)identify mRNA 5’ cap structure,then recruit other eukaryotic translation initiation factor to enter the ribosome.Internal Ribosome Entry Site(IRES)can initiate translation without 5’cap structure of mRNA.It is a non-canonical mechanism active under stress conditions,such as nutritional deprivation,DNA damage and hypoxia.IRES are usually located in mRNA 5’UTR region,the IRES structure can enter the 40 S subunit of ribosome directly.Many studies reveal that proteins associate with cancer,such as p53,c-myc,have IRES.Further studies confirm that initiation of translation through IRES leads to abnormally high expression of genes involved in cancer,this led to the progress of cancer.Our early study found cyclin B1 overexpression in esophageal squamous cell carcinoma and associated with poor prognosis.We have a hypothesis that the cyclin B1 may have the IRES mechanism,so we construct a bicistronic fluorescence reporter plasmid contains 5’UTR region of cyclin B1,the reset show that cyclin B1 have IRES and have more translation in nutrition deficiency and hypoxia conditions.RNA pull down show that PTBP1,as the Interacting trans-acting factors(ITAFs),binding cyclin B1 5’UTR region,promote the cyclin B1 IRES mechanism.We also found that PTBP1 have the ability to promote the esophageal squamous carcinoma malignant ability.We believe the IRES translation mechanism may be the one of the important reasons for cyclin B1 overexpression in tumor.IRES is a non-canonical translation mechanism,therefore as therapeutic targets maybe have the curative effect at the same time has less side effects.