The Functions And Mechanisms Of LncRNA LINC01980 Promoting The Development Of ESCC

Esophageal cancer is a malignant tumor that can seriously endanger human life and health.According to the latest global cancer statistics,there were 572,000 new cases of esophageal cancer and 590,000 deaths from esophageal cancer in 2018,ranking seventh and sixth in all malignant tumors,respectively.China has one of the highest incidences and mortality of esophageal cancer in the world,accounting for more than half of the global incidence and mortality of esophageal cancer.Esophageal squamous cell carcinoma(ESCC),one common type of pathological EC,causes more than 90% of the cases in China.Unfortunately,most ESCC cases are usually diagnosed at an advanced stage and are accompanied by local invasion and distant metastasis.Although great progress has been made in comprehensive treatment including surgery,radiotherapy,and chemotherapy,the prognosis of ESCC has remained poor with a 15%-25% 5-year survival rate for the past 20 years.At present,the pathogenesis of ESCC is not fully understood.Therefore,it is important to study the molecular mechanism underlying ESCC and explore the potential novel biomarkers and therapeutic targets to improve the prognosis of ESCC.Long noncoding RNAs(lncRNAs)are a class of RNA molecules more than 200 nt in length and no protein-coding function.lncRNAs have been shown to interact with DNA,RNA,and proteins through base-complementing and secondary-structure-forming stem loops to regulate gene expression at the epigenetic,transcriptional,and post-transcriptional levels and at the translational and post-translational levels.In-depth studies in recent years revealed that lncRNAs participate in many important regulatory processes such as X chromosome silencing,genomic imprinting,chromatin modification,transcriptional activation and inhibition,and intranuclear transport.Accumulating evidence has shown that lncRNAs play an important role in physiological activities,and imbalance in lncRNAs is associated with the oncogenesis and development of many cancers.In recent years,many cases of aberrant expression of lncRNAs in ESCC have been discovered using high-throughput microarray or RNA sequencing.Several lncRNAs have been shown to play a role in promoting and suppressing cancer during the development of ESCC.In addition,some lncRNAs have been functionally well-characterized in ESCC pathogenesis and development and may be used as biomarkers for diagnosis,treatment,and evaluation of prognosis of ESCC.Although our understanding of lncRNAs has made remarkable progress,the number,variety,structure,and mechanisms associated with lncRNA remain large,complex,and diverse.Differential expression of lncRNA in tumors is tissue-specific and cell-specific.Assessing the specific expression of lncRNAs in ESCC and exploring their molecular mechanisms are important to improving the prognosis of ESCC.We selected 5 pairs of ESCC tissues and adjacent tissues to profile lncRNA expression.The results showed that the level of LINC01980 expression(long intergenic non-protein coding RNA 1980)in ESCC tissues was more than 200 times higher than in adjacent tissues.Abnormal expression of LINC01980 was not observed in other tumors in search of the NCBI GEO database,suggesting that LINC01980 may be specifically expressed in ESCC.As far as we know,there is no research on LINC01980,so it is necessary to study the relationship between LINC01980 and clinical features of ESCC patients further,and its biological function and mechanism during the development of ESCC should be established.The major results are presented as follows.1.LINC01980 is upregulated in human ESCC tissues and it is correlated with poor prognosisReverse transcription-quantitative PCR(RT-qPCR)was used to detect the expression of LINC01980 in 74 pairs of ESCC tissues and adjacent tissues.The results showed that the expression of LINC01980 in 91.9%(68/74)ESCC tissues was significantly higher than in adjacent tissues(P < 0.01).Further analysis showed that the expression of LINC01980 was positively correlated with the depth of primary tumor invasion,lymph node metastasis,and TNM stage.Survival analysis showed the poor prognosis of ESCC patients with LINC01980 overexpression.These results suggest that LINC01980 plays a carcinogenic role in ESCC,which is related to the growth,metastasis,and prognosis of ESCC.LINC01980 may serve as a molecular marker for the prognosis of ESCC.2.LINC01980 promotes ESCC cell growth and proliferation in vitro and in vivoRT-qPCR showed the expression of LINC01980 in ESCC cells KYSE150 and KYSE450 to be significantly higher than normal esophageal epithelial cells Het-1A and ESCC cells EC109,suggesting that the expression of LINC01980 had cell-specificity.To investigate the biological role of LINC01980 in ESCC cell growth and cell proliferation,we performed knockdown of LINC01980 expression in KYSE150 and KYSE450 cells.The CCK-8 and Edu experiments showed that depletion of LINC01980 impacted growth and proliferation of both ESCC cell lines.In vivo experiments showed that knockdown of LINC01980 could reduce the volume and weight of tumors transplanted in nude mice.These findings indicated that LINC01980 promoted ESCC cell growth and proliferation.Flow cytometry was performed to assess the cell cycle and apoptosis after knockdown of LINC01980 expression in KYSE150 and KYSE450 cells.The results showed a significantly decreased proportion of S and G2/M phase in LINC01980-depleted cells than in control cells.Apoptosis was also elevated in the LINC01980-depleted cells.These results showed that LINC01980 promotes the growth and proliferation of ESCC cells in multiple ways,including accelerating cell cycle progression and preventing cell apoptosis.3.LINC01980 increases ESCC cell invasion and migrationTo explore the role of LINC01980 in cell invasion and migration,we performed knockdown of LINC01980 expression in KYSE150 and KYSE450 cells.In comparison to the control group,knockdown of LINC01980 significantly repressed the invasion and migration of both ESCC cell lines.In contrast,overexpression of LINC01980 promoted cell invasion and migration of EC109 cells.Similarly,the results of the wound healing assay showed that knockdown of LINC01980 decreased motility of KYSE150 and KYSE450 cells.These results suggested that LINC01980 could promote invasion,migration and motility of ESCC cells.4.LINC01980 promotes ESCC growth and migration by up-regulating the expression of GADD45 A and ETV5,respectivelyThe modes of action of lncRNAs are closely related to their cellular sublocalization.We first detected the intracellular distribution of LINC01980 by nucleus-cytoplasm separation assay and RNA FISH.The results showed that LINC01980 was abundantly expressed in both the nucleus and the cytoplasm,indicating that LINC01980 has the potential to perform regulatory roles through multiple mechanisms.To investigate the mechanism by which LINC01980 promotes ESCC cell growth and migration,microarray analysis was performed to detect gene expression profiles of KYSE450 cells transfected with control or LINC01980-targeting siRNAs.The results indicated that that LINC01980 participates in many biological processes and diseases,such as the MAPK signaling pathway,FoxO signaling pathway,and transcriptional misregulation in cancers.These pathways include many genes known to be associated with proliferation and metastasis(e.g.,TGFB2,CDC25 B,GADD45A,EP300,ETV5,BMP2 K,CCNT1,and ELK4).Further studies showed that growth arrest and DNA damage inducible alpha protein(GADD45A)and ETS variant 5(ETV5)were stably regulated by LINC01980,suggesting that GADD45 A and ETV5 may be target genes of LINC01980.We assessed GADD45 A and ETV5 expression in ESCC tissues and normal tissues.The results showed that GADD45 A was upregulated in 50%(11/22)of ESCC tissues,while ETV5 was upregulated in 72.7%(16/22)of ESCC tissues.Next,we explored the biological functions of GADD45 A and ETV5.Our results revealed that depletion of GADD45 A reduced cell growth in KYSE150 and KYSE450 cells.Similarly,knockdown of ETV5 significantly impaired cell migration of KYSE150 and KYSE450 cells.Rescue experiments found that overexpression of GADD45 A could partially attenuated LINC01980 knockdown-mediated growth suppression.Similarly,overexpression of ETV5 could partially reverse LINC01980 knockdown-mediated migration decrease.Hence,our results indicated that GADD45 A and ETV5 are target genes of LINC01980.These results showed that LINC01980 was distributed in the nucleus and cytoplasm of ESCC,and LINC01980 promoted the growth and migration of ESCC by up-regulating the expression of GADD45 A and ETV5,respectively.In conclusion,we here identified an important novel lncRNA,LINC01980,which was highly expressed in ESCC and associated with poor prognosis.LINC01980 promoted ESCC growth and migration by up-regulating the expression of GADD45 A and ETV5,respectively.Our findings revealed that LINC01980 can be a suitable biomarker for ESCC prognosis and therapeutic targeting.