A Novel Long Non-coding RNA LncRNA357 Functions As A Regulator Of LIF/LIFR Gene Expression To Promote Prostate Cancer Metastasis And Progression

Prostate cancer（PCa）,an emerging threat to the health of aging men,is the second most frequently diagnosed cancer in males worldwide.In the past decades,the incidence of PCa in China has been growing rapidly,especially among the population in some developed areas.The epidemiological data from Shanghai Center for Disease Control and Prevention shows that PCa has been the leading cancer in morbidity of male urinary malignancy for several years.Different from the situation in Europe and the United States,most of the patients with PCa in China are diagnosed when the cancers are already in the late stage.Some of the tumors have occurred local or distant metastasis.The molecular mechanism of metastatic PCa is not yet fully understood,which leads to the lack of biomarkers for high-grade PCa and target therapy drugs for metastatic PCa.Genomic studies on the mammalian genome transcription have shown that more than 98% of the noncoding RNA transcriptions are long noncoding RNA（lncRNA）.They have various biological functions,such as regulationg the genomic imprint,X chromosome silence,chromatin modification,nuclear transport and other important biological functions.Lots of studies have shown that lncRNA plays an important role in tumor development and progression.The reported lncRNAs which are related to PCa includes PCA3,PCGEM1,PRNCR,MALAT1,PlncRNA-1 and so on.These lncRNAs are involved in the development and progression of PCa by regulating AR transcriptional activity or post-transcriptional modification,affecting chromatin remodeling and inducing PTEN deletion.In previous research,we did RNA-Seq analysis of 65 pairs of PCa and its adjacent tissues in Chinese population,and identified 439 lncRNAs which were differentially expressed.According to the criteria that expression difference exists in at least 46 pairs of PCa and its adjacent tissues and the trends are consistent（Difference multiple ≥2,FDR ≤0.001）,we selected lncRNA NR₀46357.1（lncRNA357）as our research object.The qRT-PCR was used to detect the lncRNA357 expression levels in PCa tissue samples,and we found that average lncRNA357 level in metastatic PCa was significantly higher than that in non-metastatic samples.The full-length sequence（1867bp）of the lncRNA357 was successfully obtained by RACE experiment,and its subcellular localization（in both cytoplasm and nucleus while mainly in nucleus）was detected by both nuclear and cytoplasmic distribution experiment and RNA in situ hybridization experiment.In the study of biological phenotypes,the apply of interference siRNA and overexpression plasmid transfection achieved satisfied lncRNA357 expression knockdown and overexpression effects in PCa cell lines in vitro,finding out that lncRNA357 could enhance proliferation,migration,and invasion in PCa cells.These biological phenotypes were confirmed by the in vivo experiments using the established PCa cell lines with lncRNA357 stable knockdown or overexpression.By the qRT-PCR and immunofluorescence cell staining experiments,we proved that lncRNA357 could promote the epithelial-mesenchymal transition（EMT）process in PCa cells at both RNA and protein levels.In the study of molecular mechanism behind these phenotypes,the function of lncRNA357 in cytoplasm was studied firstly.The miRNAs targeting lncRNA357 were predicted by online database,then miR-637 was selected after the expression correlation between lncRNA357 and its potential targeting miRNAs was studied by qRT-PCR.Furthermore,the RIP and luciferase reporter assay identified lncRNA357 as competing endogenous RNA（ceRNA）to bind and then inhibit the activity of miR-637,enhance the miR-637 targeting gene leukemia inhibitory factor（LIF）,further promote migration,invasion and EMT in PCa cells.In addition,the function of lncRNA357 in nuclear was also studied.Firstly,the expression profile microarray detection was applied in the RNA samples from lncRNA357 knockdown and overexpression PCa cells.Based on the microarray data,gene function and signal pathway analyses were applied to find out the potential lncRNA357-regulated target genes and the signaling pathways lncRNA357 might participate in.Then qRT-PCR and western blot was done to confirm that lncRNA357 could further promote the distant metastastic process of PCa via LIFR/Jak1/STAT3 signal pathway.The subsequent RNA pull-down and RIP experiments were used to capture and confirm that lncRNA357 could bind to histone H1.2.Finally,the ChIP assay showed that lncRNA357 could promote the dissociation of histone H1.2 from the upstream sequence of LIFR transcription initiation site,thus promote the transcription of LIFR.