SNRPD3 Affects Glucose Metabolism In Breast Cancer Cells By Regulating NAPRT Splicing And Exploring Its Mechanism

Breast cancer is a serious health problem worldwide.It is the number one cancer among women and the leading type of cancer among women.Breast cancer has overtaken lung cancer as the number one cancer in the world,according to new data released by the World Health Organization’s International Agency for Research on Cancer.Breast cancer is characterized by fast growth rate,poor prognosis,sensitivity to chemotherapy and lack of clinically accurate endocrine and targeted therapy.Therefore,it is particularly important to find a potential target for treatment.SNRPD3(small nuclear ribonucleoprotein D3 polypeptide)is the core protein of the spliceosome and plays an important regulatory role in the alternative splicing of RNA.However,the role of SNRPD3 in breast cancer is not known.Our previous study results(which have not been published yet)showed that SNRPD3 affects the proliferation,migration,invasion and clonogenesis of breast cancer cells,and knockdown SNRPD3 significantly inhibits the proliferation,migration,invasion and clonogenesis of breast cancer cells.In order to further explore the molecular mechanism of SNRPD3 affecting breast cancer cells,we extracted RNA from breast cancer cells(MDA-MB-231)of SNRPD3 knockdown group and control group for high-throughput sequencing,analyzed the sequencing results,and screened out genes with differential alternative splicing events.The proportion of SE(exon jumping)and RI(intron retention)splicing types is much higher than other differential splicing types.KEGG enrichment analysis and GO enrichment analysis were performed on the selected differential genes.It was found that most of these genes were enriched in metabolization-related pathways,and their biological functions were closely related to metabolism,choline metabolism,amino acid biosynthesis and other biological functions.The NAPRT gene was finally screened out by PCR verification of these genes,which were sorted from small to large according to p value.In the cells that knocked down SNRPD3,NAPRT-RI events(NAPRT-RI)significantly increased,while the normal NAPRT significantly decreased.We constructed NAPRT-RI and NAPRT overexpression plasmids,predicted their three-dimensional structures on SWISS-MODEL website according to the sequencing results,and analyzed and compared the NAPRT-RI and NAPRT domain.NAPRT(nicotinate phosphoribosyltransferase)is NAD synthesis(nicotinamide adenine dinucleotide)important speed limit of enzymes.Changes in the splicing of NAPRT will affect the expression of its protein and affect the biosynthesis and other biological functions of NAD.Warburg effect exists in tumor cells.Under aerobic conditions,glycolysis is still active,glucose is consumed in large quantities,and a large amount of lactic acid is produced,which is not conducive to the survival of immune cells.To explore the effect of NAPRT splice changes after knocking down SNRPD3 on breast cancer cells,we constructed MDA-MB-231 and BT549 stable cell lines in Ctrl group,sh3 group,sh3+NAPRT group and sh3+NAPRT-RI group.The effects of NAPRT splicing on proliferation,migration and clonogenesis of breast cancer cells after knocking down SNRPD3 were verified by cell phenotypic experiments.CCK-8 assay and plate cloning assay showed that cell proliferation and clonogenesis in sh3 group and sh3+NAPRT-RI group were inhibited,and cell proliferation and clonogenesis in sh3+NAPRT group were restored.Transwell assay and cell scratch assay showed that the migration ability of sh3 group and sh3+NAPRT-RI group was inhibited,while the migration ability of sh3+NAPRT group was restored.The contents of NAD,glucose and lactic acid in sh3 group and sh3+NAPRT-RI group were reduced,and the contents of sh3+NAPRT group returned to normal.The lactate dehydrogenase activity test showed that the lactate dehydrogenase activity of sh3 group and sh3+NAPRT-RI group was lower than that of the control group,but the lactate dehydrogenase activity of sh3+NAPRT group had no significant change.By analyzing the correlation between NAPRT and key glycolysis enzymes,combined with NAPRT,the m RNA and protein expression levels of key glycolysis enzymes were affected.The results showed that the expressions of key glycolysis enzymes GLUT1(glucose transporter 1),HK2(hexokinase 2),PFKM(phosphofructose kinase),PKM2(pyruvate kinase 2)in sh3 group and sh3+NAPRT-RI group were decreased.The expression of GLUT1,HK2,PFKM and PKM2 in sh3+NAPRT group recovered to normal.In order to further explore how SNRPD3 affects the expression of NAPRT through splicing regulation and thus the molecular mechanism of glucose metabolism,we conducted a series of experiments to study the relationship between SNRPD3,NAPRT and PI3K-Akt signaling pathway.The PI3K-Akt signaling pathway is widely activated in cancer cells and is closely related to several biological processes such as glucose metabolism,cell cycle and cell proliferation.GEPIA analysis showed that NAPRT was significantly correlated with genes related to PI3K-Akt signaling pathway.The results of q PCR showed that the expressions of AS160,PIP5 K,PTEN and PDK1 in sh3 group and sh3+ NAPRT-RI group were decreased,while those in sh3+NAPRT group were recovered.Western Blot analysis showed that the expression of phosphorylated Akt in sh3 group and sh3+ NAPRT-RI group decreased,while that in sh3+NAPRT group recovered to normal,suggesting that SNRPD3 affects glucose metabolism of breast cancer cells by regulating NAPRT splice-mediated PI3K-Akt signaling pathway.These results indicate that NAPRT is a potential splicing target of SNRPD3 and may provide a new effective target for the treatment of breast cancer.