The Function And Mechanism Of LncRNA DLX6-AS1 In Esophageal Squamous Cell Carcinoma Progression

BackgroundEsophageal cancer(EC)is one of the most common malignant tumors in the world.According to the 2018 World Cancer Statistical report,its incidence ranks seventh among all malignant tumors and mortality ranks sixth.According to the 2015 Chinese Cancer Statistical report,the incidence of EC in China is significantly higher than that in western countries,and the mortality ranks fourth,which has become one of the diseases that seriously threaten public health Esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC)are the two main pathological types of EC.However,ESCC is still the most common type of EC in China and even in East Asia.90%of EC treated in China is squamous cell carcinoma.At present,although the treatment of ESCC,such as surgery,radiotherapy,chemotherapy and other treatment methods continue to develop,but the mortality rate of ESCC is still very high.According to a large number of epidemiological statistical analysis,because the most patients are already in the progressive stage,the prognosis is still not ideal,and the overall 5-year survival rate is less than 20%.Therefore,the treatment of ESCC has reached the bottleneck.At present,the clinicians are more and more aware that early prevention and early treatment should be done for the treatment of ESCC;another important aspect is that people do not fully understand the mechanism of occurrence and development of ESCC at the genetic and molecular level.Therefore,there is an urgent need to further study the pathogenesis of ESCC and find a new molecular target for diagnosis,treatment and prognosis has more important practical significance and theoretical value.Less than 2%of the nucleic acid sequences in the human genome can encode proteins,and the vast majority of genes are transcribed into non-coding RNA(ncRNA).Among many types of ncRNA,long-chain non-coding RNA(lncRNA)between the length 200-100000nt has been studied more and more deeply,and it has been found that lncRNA regulates a variety of biological processes of tumorigenesis,growth and metastasis through epigenetic,transcriptional and post-transcriptional mechanisms.Therefore,lncRNA may act as a tumor suppressor or oncogene to regulate the occurrence and development of a variety of cancers.In recent years,more and more evidence shows that lncRNA regulates tumor progression by competing with endogenous RNA.Therefore,we use biochip to analyze the significant differences between ESCC and adjacent tissues of LncRNA,for further screening and functional verification.So as to study the biological function of lncRNA DLX6-AS1 and its regulatory mechanism in ESCC,and the role and mechanism of lncRNA DLX6-AS1 in the occurrence and development of ESCC.It can more comprehensively understand the mechanism of ESCC and look for diagnostic markers and therapeutic targets of ESCC.In this study,we found that the expression of lncRNA DLX6-AS1 in ESCC was significantly higher than that in adjacent tissues,and negatively correlated with the prognosis.Then we revealed that lncRNA DLX6-AS1 can significantly promote the proliferation,invasion and metastasis of tumor cells and inhibit apoptosis.Through bioinformatics analysis and further experimental verification,we found that lncRNA DLX6-AS1 can block as a "molecular sponge" and combine with miR-100-5p,to negatively regulate the targeting of miR-100-5p and oncogene mTOR.In addition,we also found that knockout lncRNA DLX6-AS1 in subcutaneous tumors of nude mice could also inhibit the proliferation of tumors in vivo.To sum up,we proposed and verified the regulation of lncRNA DLX6-AS1/miR-100-5p/mTOR axis on a variety of biological behaviors of ESCC for the first time,which provides potential diagnostic markers and therapeutic targets for future diagnosis and treatment.Objective1.The differentially expressed lncRNA was found and verified by biochip analysis.2.To evaluate the abnormal expression of lncRNA DLX6-AS1 in ESCC and its correlation with clinicopathological features.3.To study the function of lncRNA DLX6-AS1 in ESCC cell line.4.Further explore the preliminary study on the mechanism of lncRNA DLX6-AS1.Methods1.Using the RNA sequencing data of 96 cases of ESCC and 13 cases of normal esophageal tissues downloaded by TCGA,44 cases of postoperative ESCC and adjacent tissues were collected by identifying differentially expressed lncRNAs;and the differentially expressed lncRNAs was detected by qRT-PCR.2.The effects of lncRNA DLX6-AS1 on ESCC in vitro and in vivo were detected by immunohistochemistry,CCK-8,flow cytometry,Western blotting,construction of low expression plasmid of lncRNA DLX6-AS1,migration and invasion,and tumor allotransplantation in nude mice.3.Western blotting,RNA fluorescence in situ hybridization,QRT-PCR and luciferase report were used to detect the mechanism of lncRNADLX6-AS1/miR-100-5p/mTOR signal pathway.Results1.Through the analysis and verification of TCGA database and clinical samples,the high expression of lncRNA DLX6-AS1 was screened in ESCC.The role and mechanism of lncRNA DLX6-AS1 in the occurrence and development of ESCC.2.Further experiments were showed that down-regulation of DLX6-AS1 could inhibit the growth of ESCC cells.In vitro,silencing DLX6-AS1 can inhibit the proliferation of ESCC cells and accelerate apoptosis.Subsequently,it was found that down-regulation of DLX6-AS1 inhibited cell migration.When DLX6-AS1 was down-regulated,the mammalian rapamycin target protein mTOR,apoptosis-related protein BCL-2 and invasion-related protein MMP-2 were also down-regulated.The results of experiment of transplanted tumor in nude mice showed down-regulation of DLX6-AS1 in vivo.3.The targeted binding of lncRNA DLX6-AS1 and miR-100-5p was determined by double luciferase experiment.Through cell function experiments,it is found that IncRNA DLX6-AS1 competitively suppresses miR-100-5p and regulate candidated target gene mTOR,to promote the growth and migration of ESCC.ConclusionlncRNA DLX6-AS1 is abnormally highly expressed in ESCC and can be used as a potential marker of ESCC.At the same time,lncRNA DLX6-AS1 can significantly promote the value-added ability,migration and invasion ability of ESCC cells.In addition,lncRNA DLX6-AS1 competitively suppresses miR-100 and regulate candidated target gene mTOR,to promote the growth and migration of ESCC,which provides a new research strategy for clinical treatment of ESCC.