Investigation Of The Mechanism Of LncRNA DAPK1-215 Inhibiting DAPK1 Expression And Promoting The Invasion And Metastasis Of Liver Cancer

Liver cancer is the sixth most common cancer in the world.Metastasis and recurrence are the main reasons for the high mortality rate of liver cancer.The identification of genes related to liver cancer metastasis is of great significance in clinical application.Death Associated Protein Kinase 1(DAPK1)is a calmodulin-regulated serine/threonine-protein kinase,a positive regulator of apoptosis.DAPK1 is involved in the regulation of apoptosis,proliferation,autophagy,migration,and other physiological processes,and its expression is inhibited in a variety of tumors.To detect the expression of DAPK1 in liver cancer,firstly,the expression of DAPK1 in liver cancer was detected by GEO(Gene Expression Omnibus)database and liver cancer tissue.The results showed that the expression of DAPK1 was significantly inhibited in liver cancer tissues,and the low expression of DAPK1 m RNA was an unfavorable prognostic factor for liver cancer patients.Immunohistochemical experiments showed that the expression of DAPK1 protein was significantly lower than that in adjacent tissues,and the low expression of DAPK1 protein was an unfavorable prognostic factor in patients with liver cancer.Next,the correlation between DAPK1 expression and its promoter methylation and mi RNA expression were analyzed using TCGA(The Cancer Genome Atlas)liver cancer data.The results showed that there was no significant negative correlation between the expression of DAPK1 in liver cancer tissues and its 24 methylation sites and mi RNA expression in the TCGA database.Previous studies have shown that a s-DAPK-1 protein generated by the alternative splicing of Pre-DAPK1 can mediate the lysosomal degradation of DAPK1 protein.In this study,we investigated whether 11 alternatively spliced lnc RNAs of DAPK1 were involved in the regulation of DAPK1 expression in hepatocellular carcinoma by western blot.The results showed that DAPK1-215 down-regulated DAPK1 protein most obviously,and RT-PCR showed that DAPK1-215 was expressed in hepatoma cells.Therefore,in this study,the molecular mechanism of DAPK1-215 regulating DAPK1 expression was investigated.Through western blot and RT-PCR experiments,we found that DAPK1-215 can not only inhibit the expression of exogenous HA-DAPK1 m RNA and protein,but also inhibit the expression of endogenous DAPK1 m RNA and protein.It indicates that DAPK1-215 may be involved in the regulation of DAPK1 m RNA expression,and this regulation does not depend on the UTR sequence of DAPK1 m RNA.Through m RNA stability experiments and truncation experiments,we found that DAPK1-215 can down-regulate the stability of DAPK1 m RNA,and its regulatory region for DAPK1 is located in its kinase region(1-364aa).Using RNA pull-down technology and CLIP-Seq data,we identified two DAPK1-215 binding proteins DDX3X(DEAD(Asp-Glu-Ala-Asp)box helicase 3)and SF3B1(splicing factor 3b,subunit 1).SF3B1 is an alternative splicing-related protein involved in the alternative splicing of genes.When SF3B1 was knocked down,the expression of DAPK1-215 was significantly down-regulated,indicating that SF3B1 was involved in the formation of DAPK1-215 from Pre-DAPK1 via alternative splicing.CLIP-Seq data showed that DDX3 X could also bind to the m RNA sequence of DAPK1(1-364aa),and overexpression of DDX3 X could enhance DAPK1 m RNA stability and m RNA expression.Overexpression of DDX3 X in DAPK1-215 stably transfected cells could inhibit the inhibitory effect of DAPK1-215 on DAPK1 m RNA.Based on the above data,this study speculates that SF3B1 promotes alternative splicing of pre-DAPK1 m RNA to generate DAPK1-215 in liver cancer,and DDX3 X protein can improve DAPK1 m RNA stability.DAPK1-215 competes with DAPK1 m RNA for binding to DDX3 X,thereby inhibiting DAPK1 m RNA stability.Functional experiments showed that overexpression of DAPK1-215 could promote the invasion and migration of liver cancer cells,and the effect of DAPK1-215 on the function of liver cancer cells depended on DAPK1.In summary,the main results of this study are presented as follows:1)The m RNA and protein expression of DAPK1 is significantly down-regulated in liver cancer,and the low expression of DAPK1 m RNA and protein is an unfavorable prognostic factor for liver cancer patients;2)The non-coding spliceosome DAPK1-215 of DAPK1 can regulate the stability of DAPK1 m RNA in liver cancer,enriching the research that the non-coding spliceosome of gene can regulate the expression of its own coding gene;3)Revealed the potential molecular mechanism of DAPK1-215 regulating DAPK1.SF3B1 may promote alternative splicing of pre-DAPK1 m RNA to generate DAPK1-215,and DDX3 X protein can improve the stability of DAPK1 m RNA.The regulation of DAPK1 by DAPK1-215 is dependent on DDX3X;4)DAPK1-215 can promote the invasion and migration of liver cancer cells,and its effect on the function of liver cancer cells depends on DAPK1.So far,there is no research report analyzing the expression and function of DAPK1-215.This study found that the long non-coding RNA spliceosome DAPK1-215 regulates the expression of the coding gene DAPK1 in liver cancer,which is innovative.At the same time,we preliminarily clarified that DAPK1-215 affects the process of liver cancer invasion and metastasis by regulating the expression of DAPK1.This study will help to further understand the mechanism of liver cancer metastasis and lay a theoretical foundation for finding new targets for liver cancer treatment and exploring more effective liver cancer treatment methods.