Alternative Splicing Analysis Of LACTB In Acute Myeloid Leukemia

Objective:Analyze the expression of alternative splicing of LACTB and its different transcripts in myeloid leukemia cell lines and peripheral blood of AML patients.Provide experimental basis for further research on the expression and function of LACTB in AML.Methods:1.Alternative splicing analysis and open reading frame analysis of LACTB:Use the NCBI website to download the full length of LACTB and different transcript sequences of LACTB,and perform alternative splicing analysis of LACTB.Use DNAstar software to analyze the open reading frame of each transcript of LACTB,and use Jaiview software to perform multiple sequence alignment of amino acids.2.To study the expression of LACTB in human myeloid leukemia cell lines:Extract RNA from human myeloid leukemia cell lines(THP-1,HL-60 and K562),amplify LACTB by RT-PCR,and visualize the amplified products by DNA agarose gel electrophoresis.At the same time,direct sequencing of PCR products and T-A cloning sequencing were used to detect the expression of different LACTB transcripts in myeloid leukemia cell lines,use Snap Gene software to read the sequencing results,and use the BLAST function of the NCBI website for sequence comparison.Real-time fluorescent quantitative PCR was used to analyze the expression levels of different LACTB transcripts in human myeloid leukemia cell lines,with donor bone marrow as a control.3.To study the expression of LACTB in AML peripheral blood:use the Oncomine database to analyze the expression of LACTB in AML,and use the UALCAN database to analyze the gender and age subgroups.The peripheral blood of30 healthy subjects and the peripheral blood of 30 AML patients with high white blood cell(WBC>50×10~9/L)(confirmed by bone marrow cytology combined with flow cytometry)were collected,and detected by real-time fluorescent quantitative PCR,the expression level of different transcripts of LACTB in the peripheral blood of AML patients,and analyze the age and gender subgroups.The receiver operating characteristic(ROC)curve was used to analyze the area under the curve(AUC),sensitivity,and specificity of LACTB in the diagnosis of AML,and the best cut-off value was determined.4.LACTB co-expressed gene enrichment analysis:Use Linked Omics database to analyze LACTB co-expressed genes in AML,use DAVID website to perform GO and KEGG enrichment analysis of co-expressed genes,use KEGG website to draw KEGG pathway diagram,use Gene MANIA website Analyze the interaction network between LACTB and enriched genes.Results:1.The analysis of LACTB alternative splicing shows that there are 5 different transcripts in LACTB,including 3 coding transcripts and 2 non-coding transcripts.PCR and sequencing results showed that LACTB has multiple splicing isoforms in myeloid leukemia cell lines,and different expression patterns exist in different cell lines,including XR1,V1,V2,and V3.At the same time,a new transcript was detected in HL-60 cells,which lacked the 5th and 6th exons.2.Through the open reading frame analysis of different transcripts of LACTB,it is found that the two non-coding transcripts and the new transcript can each encode at least one carbon-terminal truncated amino acid sequence,and each transcript has multiple different open reading frames,and there are multiple downstream promoters in the same open reading frame.3.Through bioinformatics analysis,it was found that the expression of LACTB in AML was lower than that of normal control,but the difference was not statistically significant.Among different types of leukemia,LACTB expression in AML is higher than other types of leukemia.Analysis of subgroups shows that LACTB expression in AML has nothing to do with gender and age.Enrichment analysis of co-expressed genes shows that LACTB co-expressed genes are mainly located in Cell lysosomes are related to lysosomal function.Through the interaction analysis of the enriched lysosomal function-related genes,the results show that there are complex interactions between the proteins of these target genes.4.Real-time fluorescent quantitative PCR was used to detect the total expression of LACTB in different myeloid leukemia cell lines and the expression of different transcripts.The results showed that compared with the donor bone marrow,the total expression of LACTB and each transcript in THP-1 cells The expression levels of all were reduced(P<0.05),and the total LACTB and transcript 1 expression levels in HL-60 cells were significantly higher than those in THP-1 and K562 cells(P<0.05).5.Real-time fluorescent quantitative PCR detected the expression of LACTB in AML,and the results showed that the total expression of LACTB and the expression of transcript 1 in the peripheral blood of AML patients were significantly lower than those of normal controls(P<0.001),regardless of gender and age(P>0.05);The expression levels of transcript 2 and transcript 3 in the peripheral blood of AML patients were significantly higher than that of normal controls(P<0.05),regardless of gender and age(P>0.05);ROC curve results showed that V2 and V3 has certain clinical value in the diagnosis of AML.Conclusions:1.In this study,a new transcript R-E5-E6 was found in the HL-60 cell line through PCR experiments and cloning and sequencing.This transcript lacks the 5th and 6th exons,and may encode a conservative nitrogen-terminal,A protein subtype with a truncated carbon end and a length of 352 amino acids.2.Transcript 2 and Transcript 3 of LACTB have the potential to become diagnostic markers of AML.3.Changes in the expression of different transcripts of LACTB in AML may affect its final biological function.More samples are needed to verify and study the expression of LACTB in AML,and it is necessary to explore the mechanism of LACTB in more depth.