The Regulatory Roles Of NUDT21 In SCLC Cell Growth And Metastasis And Its Mechanisms

Small cell lung cancer(SCLC)is a specific type of lung cancer in the bronchial mucosa.SCLC cells are characterized by rapid cell proliferation,high degree of malignancy and early metastasis,which leads to a low cure rate of SCLC.The diagnosis and treatment of SCLC still face many difficult problems,patients lack effective therapeutic approaches,and the most commonly used chemotherapy or radiotherapy show limited efficiency in SCLC therapy.Therefore,it is particularly important to further study the pathophysiological mechanism of SCLC and identify novel targets.Nudix hydroxylase(NUDTs)is a widespread superfamily of viruses,bacteria,and eukaryotes.NUDTs catalyze a variety of reactions such alternative splicing,alternative polyadenylation,ADP-ribose response transcriptional regulation,and ADP-ribose response calcium channel formation.NUDTs can also "clean up the nucleotide library" to prevent non-classical base incorporation into DNA.NUDTs has 24 members in the human genome and plays an important role in cell metabolism and reprogramming.NUDT21,also known as CFIm25 or CPSF5,is a member of the NUDTs family.NUDT21 has a quaternary structure of homodimer.NUDT21 is the main regulator of 3'UTR shortening,and it plays the function of changing alternative polyadenylation and alternative splicing by adjusting the length of 3'UTR.The alternative polyadenylation regulated by NUDT21 plays an important role in cell proliferation and differentiation.NUDT21 can participate in tumor glutamine metabolism by regulating mitochondrial glutaminase 1(GLS1).There are two subtypes of GLS1: hepatic glutaminase C(GAC)and renal glutaminase(KGA).GAC has high catalytic activity and plays a dominant role in many types of tumors.The abnormal changes of GAC/KGA ratio can reflect the metabolic characteristics of some diseases.Studies have confirmed that NUDT21 can regulate protein formation at post-transcriptional levels and is closely related to a variety of diseases,including hematology,immune diseases,endocrine diseases and tumors.These diseases are involved in inducing abnormal glutamine metabolism.This study elucidates the expression of NUDT21 in SCLC samples and the influence of NUDT21 on the internal and external biological characteristics of SCLC cells,and reveals the mechanism of NUDT21 participating in glutamine metabolism by regulating the alternative splicing of mitochondrial glutaminase 1.1.Low expression of NUDT21 in SCLC and is influenced by hypoxiaFirst,immunohistochemical staining of SCLC patient samples revealed down-regulated NUDT21 expression.Analysis of SCLC TCGA data set found that NUDT21 was closely related to the survival rate of clinical patients.Low expression of NUDT21 in SCLC reduces short survival.Therefore,we infer that NUDT21 expression is closely related to survival in SCLC patients.Secondly,the relationship between NUDT21 and tumor hypoxic microenvironment was verified.The expression of HIFs and NUDT21 in A549 cells in hypoxic microenvironment was changed and time-dependent.HIFs expression was up-regulated with time.NUDT21 expression is up-regulated with time extension in short time and down-regulated with time extension after long time.On this basis,NUDT21 expression was monitored 48 hours after the intervention of HIFs by transduction.There was no significant difference in NUDT21 expression after HIF-2a intervention.We designed and constructed the recombinant lentivirus vector PLKO.1-Sh NUDT21 and the control recombinant lentivirus vector sh-scramble.The recombinant lentivirus was prepared by lentivirus packaging method for interference with NUDT21 expression in A549 cells.2.The influence of NUDT21 on biological characteristics of SCLC cells in vitroThen,the role of NUDT21 in SCLC cell proliferation,apoptosis,cycle,migration,clone formation and other biological characteristics was determined.Results PLKO.1-sh NUDT21-mediated NUDT21 silencing promoted A549 cell proliferation and inhibited apoptosis.At the same time,the A549 cell cycle further showed a significant increase in the G2 and M stage after NUDT21 silencing,compared with the plko-1-shscramble control group of A549.The results showed that the number of cell clones in PLKO.1-sh NUDT21 group was significantly higher than that in PLKO.1-shscramble control group.As the expression of NUDT21 was down-regulated after hypoxia for a long time,we found that A549 had a stronger ability to transfer under hypoxia through trans-well experiment.It is suggested that NUDT21 plays an anticancer gene role in SCLC and may be related to hypoxic regulation.3.NUDT21 silence promotes SCLC cell proliferation in vivoBased on the results of in vitro biological effects of interference NUDT21 and high expression NUDT21 on lung cancer A549 cells,the effects of silent NUDT21 and high expression NUDT21 on tumor formation in mice were further studied by xenograft model in mice.The results showed that the tumor growth was the fastest in the silent NUDT21 group,and the tumor growth in the highly expressed NUDT21 group was lower than that in the silent NUDT21 group.There was no significant change in body weight in mice.After the experiment,the mice were euthanized and the tumors were taken out for further analysis.The results showed that the tumor weight of the silent NUDT21 group was significantly higher than that of the control group,and the high expression of NUDT21 was lower than that of the control group.The differences in tumor volume were also observed.Then the expression of NUDT21 in all groups was analyzed by immunohistochemistry.The expression of NUDT21 in the silent NUDT21 group was significantly lower than that in the control group,while that in the high-expression group was higher than that in the control group.Therefore,the role of NUDT21 silencing in promoting SCLC cell proliferation was also verified in vivo.4.NUDT21 regulates glutamine metabolism in SCLC cells through GLS1 ASTo reveal the mechanism of NUDT21 regulating glutamine metabolism in SCLC cells by variable splicing and the inhibition of SCLC metastasis by NUDT21.The SCLC cell line A549 cells were transduced by NUDT21 interference and highly expressed NUDT21 lentivirus,and A549 was cultured in normal conditions and hypoxic microenvironment for 48 hours,and then glucose metabolism and glutamine metabolism were measured.Results Glut1 and Glut3 were up-regulated in hypoxic microenvironment,and interference NUDT21 significantly increased Glut1 expression in A549 cells.RT-PCR and western blot were used to identify the expression of GLS1 and two types of the same GAC and KGA.The GAC/KGA ratio goes down.It has been previously demonstrated that SCLC cells are up-regulated by HIF1 a and down-regulated by NUDT21 in a hypoxic microenvironment.Subsequently,western blot also showed that HIF1 a was up-regulated and NUDT21 was down-regulated in hypoxic microenvironment.Meanwhile,it was also found that NUDT21 could down-regulate E-cadherin and up-regulate Vimentin and A549 cells after interference with NUDT21,and the results were also verified by high expression of NUDT21 in A549 cells.To reveal that NUDT21 can inhibit EMT in SCLC.These results indicate that NUDT21 regulates the growth and metabolism of SCLC cells and also affects SCLC metastasis.In conclusion,we reveal the important influence of NUDT21 on the survival and cell growth of SCLC patients and the important role of NUDT21 in SCLC metabolism.This study innovatively elucidates the pathophysiological role of NUDT21 in the occurrence and development of SCLC,providing new ideas for promoting NUDT21 gene targeted therapy for SCLC and new strategies for promoting accurate treatment.