Function And Mechanism Of LncRNA CASC9 In ESCC Tumor Growth

Esophageal cancer is one of the most common malignant tumors in human beings.There is a high incidence of esophageal cancer in China,where the morbidity is the highest worldwide.The major histologic type of esophageal cancer in China is esophageal squamous cell carcinoma（ESCC）.Due to the lack of obvious clinical symptoms and effective screening methods in early esophageal cancer,most esophageal cancer patients are diagnosed at an advanced stage.Only 15-20% patients will survive for 5 years after operation.So it is of significant importance to explore the mechanism of esophageal cancer and find potential biomarkers for early diagnosis and clinical therapy.Long noncoding RNAs（lncRNA）are a class of transcripts over 200 nucleotides with no protein-coding capabilities,which are mostly transcribed by RNA polymerase II and have mRNA-like structures.Compared to mRNAs,lncRNAs are transcribed from universal genome and have more complex functions and mechanisms.Based on base pairing and stem-loop secondary structure,lncRNAs can interact with RNA,DNA and proteins,thus regulating genes from multiple aspects including epigenetic level,transcription level and post-transcription level.With the development of high-throughput gene chips and sequencing technology,an increasing number of cancer associated lncRNAs have been identified,such as PCA3,HEIH,MALAT1 and GAPLING,which are tissue-specific and often used as biomarkers for early diagnosis and prognosis of tumors.More and more attention has been paid to the role of lncRNAs in ESCC tumorigenesis.Numerous ESCC associated lncRNAs are emerging.The lncRNA POU3 F in plasma is reported to be used as a biomarker for early diagnosis of ESCC.A three-lncRNA signature（including ENST00000435885.1,XLOC₀13014 and ENS T00000547963.1）can predict ESCC prognosis.The functions and mechanisms of most lncRNAs in ESCC remain unclear,requiring further investigation.To further study the functional role of lncRNAs in ESCC tumorigenesis,we analyzed the lncRNA expression profile of ESCC using microarray and found the most upregulated lncRNA cancer susceptibility candidate 9（CASC9）in ESCC.Few studies reported CASC9,thus the function and mechanism of CASC9 in ESCC are needs to be further explored.The major results are as follow.1.lncRNA profile and qPCR analysis show that CASC9 is upregulated in ESCC tissues and cells.Its expression is positively associated with tumor size and TNM staging and can predict poor prognosis.Five pairs of ESCC and adjacent normal tissues were selected to perform lncRNAs and mRNAs expression profiling.qPCR was used to test the reliability of microarray result.lncRNA-mRNA co-expression network was conducted to predict function of the most upregulated lncRNA in microarray.Finally,the lncRNA expression was detected in 91 ESCC and 87 paired normal tissues and common cancer cells.Analyzed its clinical significance and specificity.CASC9 was the most upregulated lncRNA（355 fold）in ESCC microarray.Three upregulated lncRNAs（BC017398,RP5-907D15 and RP11-417E7）and three downregulated ones（CCAT1,LINC00443 and NR₀38940）were randomly selected from microarray.qPCR analysis of these lncRNAs expression was consistent with the microarray results,which confirmed the reliability of microarray results.lncRNA-mRNA co-expression network identified PLAUR,NR1H3 and CTLA4 as the molecules most closely related to CASC9.Analysis of bio-information and literatures demonstrated that these molecules were involved in cell growth from different aspects.Thus we speculated that CASC9 might have an effect on cell growth in ESCC.Moreover,CASC9 was upregulated in 90.8%（79/87）ESCC tissue.The expression of CASC9 was higher in tumor tissues with larger size and advanced stage,and predicted poor prognosis of ESCC,which indicated that CASC9 might be used as a prognostic marker.Compared to normal esophageal epithelial cell line Het-1A,the expression of CASC9 was higher in cancer cells and was highest in ESCC cells KYSE450 and KYSE150,which suggested the cell-specific of CASC9.The results of this part revealed that CASC9 is upregulated in ESCC tissues and cells.Its expression is positively associated with tumor size and TNM staging and can predict poor prognosis.Bio-information analysis predicted that CASC9 might have an effect on cell growth in ESCC.2.Interfering CASC9 inhibits ESCC growth in vivo and in vitro,indicating that CASC9 participates in the regulation of cell growth.To explore the biological role of CASC9,loss-of-function studies were conducted to investigate the effects of CASC9 on cell growth,cell proliferation,cell cycle and cell apoptosis.Western Blot was used to detect the expression of cell cycle regulation related proteins after interfering CASC9.In vivo and in vivo experiments showed that knockdown of CASC9 could inhibit cell growth and reduce the volume and weight of tumors transplanted in nude mice,suggesting that CASC9 participated in the regulation of ESCC growth.Subsequent studies revealed that CASC9 knockdown decreased cell proliferation by Edu assays and induced G1/S arrest by flow cytometry.Western Blot analysis of CDK4,CDK6,CCND1 and CCNE2 showed that their expression declined after CASC9 knockdown.But CASC9 made no difference to cell apoptosis.The results of this part revealed that CASC9 contributes to cell growth by regulating cell proliferation and cell cycle.3.CASC9 promotes cell growth by inversely negatively regulating PDCD4.PDCD4 expression predicts good prognosis of ESCC patients.To further investigate the mechanism CASC9 promoting cell growth,mRNA expression profiling in CASC9 knockdown KYSE450 cell was performed.After GO analysis of differentially expressed mRNAs,pathways associated with cell growth were selected.Tissue microarray and qPCR were used to find the genes regulated by CASC9,which was confirmed by recovery experiments.Analyzed the associations between target gene with other lncRNAs.GO analysis found that CASC9 could influence six cell growth associated pathways including cell cycle arrest and cell proliferation.Only VEGFC,PDCD4,PIK3 CA,TMX1 and PTHLH were correlated with CASC9 indicated by tissue microarray.Programmed cell death 4（PDCD4）was the only gene whose expression changed well along with the CASC9 expression,suggesting that PDCD4 was a direct target of CASC9.qPCR and Western Blot results found that PDCD4 was downregulated in ESCC tissues and its expression was further decreasing in larger and advanced tumors.PDCD4 predicted good prognosis of ESCC.The expression and clinical significance of PDCD4 was negatively associated CASC9,further indicating that PDCD4 was regulated by CASC9.Interfering PDCD4 could partly rescue the effect of CASC9 knockdown on cell cycle and CDK6,CCNE2 expression,proving that CASC9 functions in ESCC tumorigenesis dependent on PDCD4.qPCR analysis demonstrated that the expression of lncRNA CCAT1,LINC00443 and NR₀38940 was positively associated with PDCD4 but not CASC9,while the expression of lncRNA BC017398 and RP11-417E7 correlated with CASC9 but not PDCD4.No lncRNA correlated with CASC9 and PDCD4 at the same time in ESCC tissues,which suggested that CASC9 regulated PDCD4 in a specific way.The results of this part revealed that PDCD4 is regulated by CASC9 in a direct and specific way.The expression of PDCD4 is negatively associated CASC9 in ESCC tissues and predicts the opposite clinical characteristics indicated by CASC9.CASC9 promotes cell growth by inversely negatively regulating PDCD4.4.Nuclear CASC9 inhibits PDCD4 expression by recruiting EZH2 to PDCD4 promoter,increasing H3K27me3 signal in this region,thus inhibiting the transcription of PDCD4.To unveil the mechanism CASC9 regulating PDCD4,the possible regulation of PDCD4 was analyzed by bio-information.Then the mechanism was explored from three aspects including CASC9-protein interaction,subcellular location of CASC9 and the effect of CASC9 on PDCD4 transcription activity using RNA-protein pull down,RIP,ChIP and a series of assays.Bio-information analysis showed that PDCD4 promoter region was rich in Enhancer of zeste homolog 2（EZH2）binding sites and H3H27me3 signal.Interfering EZH2 increased both mRNA and protein expression of PDCD4,indicating PDCD4 under regulation of EZH2.RNA-protein pull down and RIP assays proved that CASC9 could bind EZH2,suggesting that CASC9 might regulate PDCD4 through EZH2.FISH and nucleus-cytoplasm fraction assays showed that CASC9 was distributed in both nucleus and cytoplasm,supporting the possibility from location side.ChIP assays found that CASC9 knockdown reduced EZH2 binding and H3K27me3 signal in the PDCD4 promoter region.Luciferase report experiments showed that overexpression of CASC9 inhibited the transcription activity of PDCD4.The results of this part revealed that CASC9 is distributed in both nucleus and cytoplasm.Nuclear CASC9 recruits EHZ2 to PDCD4 promoter and enhances the H3K27me3 signal of PDCD4 promoter,thus inhibiting the transcription activity of PDCD4 and decreasing PDCD4 expression.In conclusion,our study identifies CASC9 as the most upregulated lncRNA in ESCC by microarray.Overexpression of lncRNA CASC9 predicts poor prognosis of ESCC patients.LncRNA CASC9 promotes ESCC growth by negatively regulating PDCD4.CASC9 is distributed in both nucleus and cytoplasm.Nuclear CASC9 inhibits PDCD4 expression by recruiting EZH2 to PDCD4 promoter,increasing H3K27me3 signal in this region,thus inhibiting the transcription of PDCD4.Our findings prove that PDCD4 is regulated by lncRNAs and first clarify the regulatory mechanism of CASC9,which contributes to better understanding ESCC etiology and provides a potential biomarker for early diagnosis and prognosis of ESCC.