1.ZEB1 Induced MiR-99b/let-7e/miR-125a Cluster Promotes Invasion And Metastasis In Esophageal Squamous Cell Carcinoma 2.USP39 Promotes The Growth Of Esophageal Squamous Cell Carcinoma And Predicts Poor Prognosis

Esophageal cancer is one of the most common digestive tract tumors in the world.Esophageal squamous cell cancer(ESCC)accounts for 90%of esophageal cancer diagnosed,with approximately 70%of global esophageal cancer occurring in China.ESCC is the sixth most lethal cancer worldwide and the fourth most lethal cancer in China.The overall 5-year survival rate is less than 25%mainly due to diagnosis in later stages frequently accompanied with local invasion and distant metastasis.Therefore,to improve early diagnosis of ESCC and inhibit tumor metastasis has become the top priority of the current work.microRNAs(miRNAs)are a class of small non-coding RNAs that regulate gene expression through target mRNA destabilization or translational inhibition.miRNAs play crucial roles in various physiological and pathological processes.Recent studies demonstrate that miRNAs are involved in the initiation and progression of ESCC through different pathways.In this study,we aimed to screen miRNAs that promote invasion and metastasis of ESCC from a Lentivirus Library containing approximate 1000 pre-miRNAs using an in vitro Transwell mode.As a result,a miRNA cluster,termed miR-99b/let-7e/miR-125a emerged from the candidates.Overexpression of miR-99b/let-7e/miR-125a cluster promoted ESCC cell migration and invasion in vitro and induced an experimental metastasis in vivo.Meanwhile,they had no effect on cell proliferation or cell cycle.The high expression of miR-99b/let-7e/miR-125a was correlated with lymph node metastasis in ESCC tissue samples.Through bioinformatics analysis,ZEB1 was predicted binding to the promoter region of miR-99b/let-7e/miR-125a cluster,which was further confirmed by ChIP assay.Moreover,ZEB1 regulated the expression of miR-99b/let-7e/miR-125a at transcriptional level Knockdown of ZEB1 resulted in a decrease of both mature and primary miRNAs.To investigate the potential target genes of miR-99b/let-7e/miR-125a cluster in ESCC,we combined microarray-based expression profiling and publicly available algorithms.Subsequent research revealed AT-rich interaction domain 3A(ARID3A)as a direct target of miR-99b/let-7e/miR-125a cluster.Reduced expression of ARID3A phenocopied miR-99b/let-7e/miR-125a overexpression,and elevated ARID3A counteracted the pro-metastasis effect of miR-99b/let-7e/miR-125a cluster.Furthermore,ARID3A was downregulated by ZEB1 in a miR-99b/let-7e/miR-125a dependent manner.Three independent cohorts from GEO Datasets show that ARID3A is inversely correlated with ZEB1 in ESCC specimens.In conclusion,miR-99b/let-7e/miR-125a cluster acts as a driver in ESCC metastasis.ZEB1 modulates the expression of miR-99b/let-7e/miR-125a at transcriptional level.As a direct target,ARID3A plays an inhibitory role in tumor cell motility and is downregulated by ZEB1 in a miRNA dependent manner.We found that the expression of miR-99b/let-7e/miR-125a was positively correlated with lymph node metastasis through analysis of clinical information.Our study sheds light on the essential role of miR-99b/let-7e/miR-125a cluster in ESCC metastasis.Ubiquitin-proteasome system is a widespread protein regulatory system in eukaryotic cells,participating in the degradation of more than 80%proteins in cells.In this system,there exists a reversible process where deubiquitinases(DUBs)play an important role.At present,there are 6 different types of DUBs.They are able to hydrolyze the ubiquitin chain from the labeled substrate protein,further preventing protein from degradation.The abnormal expression and dysfunction of some DUBs are directly correlated to metabolic disorders and the development of neurological diseases or tumors.To identify DUBs associated with tumor development,we carried out a systematic screening employing a siRNA library which targets 98 known human DUBs.Cells were seeded and transfected with each DUB siRNA.Cell viability was assessed 48 hours after transfection by counting using SRB staining.After a simple pre-experimental detection of the preliminary screening results,we found USP39 had a significant effect.Then we decided to develop a further study on the function and mechanism of USP39 in esophageal carcinoma.First,we demonstrated that USP39 could promote cell proliferation.KYSE450 cells with USP39 knockdown showed strong inhibition of cell proliferation,reduced single cell colony formation and impaired growth of xenograft in nude mice.In contrast,the esophageal cancer cells overexpressing USP39 exhibit an opposite effect by promoting cell proliferation,enhancing cell colony formation ability and accelerating the growth of xenograft in nude mice.Next,we detected the role of USP39 in cell apoptosis.Knockdown of USP39 did not induce apoptosis directly,however,it enhanced the cytotoxicity of DDP on esophageal cancer cells substantially.Further mechanistic study shows that knockdown of USP39 promotes the cleavage of the PARP protein,which improves the instability of cells and lays the foundation for apoptosis.Knockdown of USP39 also promoted the expression of Bak,inhibited the expression of Bcl-2,which are closely related to the apoptosis.Overexpression of USP39 in KYSE30 cells could protect cells from the injury of DDP,contributing to cleavage inhibition of PARP and Caspase 3.Therefore,we suggest that knockdown of USP39 has a potential clinical value and may increase the sensitivity of patients with esophageal cancer to DDP(and even other chemotherapeutic drugs).Furthermore,we confirmed the pro-migration ability of USP39 through an in vitro transwell assay.Knockdown of USP39 exhibited an opposite effect with inhibitory mgration We elucidated that the effect of USP39 on cell migration is independent of cell proliferation or apoptosis.Moreover,we employed an IP-MS method in order to thoroughly figure out the fundamental mechanism of USP39.As a result,numerous proteins are supposed to interact with USP39 in esophageal cancer cells,most of which are small nucleic ribonucleoproteins(snRNPs)that constitute spliceosome complex or ribosome complex,such as the well-known EFTUD2,PRPF3,PRPF31 and PRP6.After GO analysis for the IP-MS result,the most frequently enriched biological process is "spliceosome induced mRNA splicing"(36.2%),followed by "RNA splicing"(27.7%)and "RNA processing"(25.5%).Analysis by KEGG resulted in two pathways:spliceosome pathway and ribosome pathway.According to public reports,we speculate that USP39 is directly correlated to the stability of spliceosome or ribosome,thereby affecting pre-mRNA maturation,protein translation,and eventually regulating the growth and survival of tumor cells.However,more data are needed to sustain this idea.Finally,in order to tell the clinical significance of USP39,we detected the expression of USP39 in 199 cases of esophageal cancer tissue microarray by means of IHC.The results showed that patients with high expression of USP39 tended to have poor prognosis and the 5-year overall survival rate was about 25%.In contrast,patients with low expression of USP39 had a longer overall survival and disease-free survival,and the 5-year overall survival rate was more than 50%.Collectively,our results demonstrate that USP39 promotes the growth of esophageal carcinoma.Knockdown of USP39 could inhibit cell proliferation in vitro,reduce colony formation,slow down the growth of xenograft in nude mice,and enhance the cytotoxicity of DDP on esophageal cancer cells.USP39 might interact with snRNPs in esophageal cancer cells,suggesting that USP39 participates in the regulation of spliceosome or ribosome related pathways.Clinically,patients with high expression of USP39 have a short survival.Therefore,USP39 could be a potential prognostic marker.