RBM4 Regulates Cellular Senescence Via MiR1244/SERPINE1 Axis

Background:Lung cancer is one of the most common cancers and a leading cause of cancer death,with an estimated 2.2 million new cancer cases and 1.8 million deaths by 2020.Treatment options for lung cancer include surgery,chemotherapy,radiotherapy,immunotherapy and targeted therapy.Despite there has been significant progress in the treatment of lung cancer,its incidence and mortality remain high.Thus,there is an urgent need to understand the molecular mechanisms of occurrence and development of lung cancer and to explore novel strategies for lung cancer.Cellular senescence is closely related to the occurrence and development of tumors,and has been included in the latest tumor markers.Cellular senescence is a state of irreversible cell cycle arrest initiated by various forms of cellular stress inducers,such as telomere shortening caused by serial passages,activation of oncogenes,and chemotherapy.Senescent cells often exhibit a variety of phenotypes,including morphological flattening,elevated senescence-associated β-galactosidase(SA-β-Gal)activity,and cell cycle arrest in G1/S phase.Cellular senescence is generally considered as an intrinsic inhibitory mechanism in the early stages of tumorigenesis.RNA binding proteins(RBPs)are a class of highly conserved proteins that involved in various aspects of the metabolism of RNA,including alternative splicing,polyadenylation,modification,stability and translation of RNA,as well as processing of miRNAs and circular RNA.Dysregulation or dysfunction of RBPs can lead to human diseases,including cancer.RBPs are involved in a variety of cancer-associated phenotypes,including proliferation,apoptosis,senescence,migration,invasion,and angiogenesis.As a member of the RBPs,RBM4 is a multifunctional RNA-binding protein,mainly involved in the regulation of alternative splicing and mRNA translation.RBM4 was generally considered as a potential tumor suppressor to inhibit cancer progression by modulating alternative splicing of cancer-related genes.In addition to splicing regulation,RBM4 can also trans locate to the cytoplasm and participate in the control of cap-dependent translation and mRNA turnover.Therefore,we want to investigate whether RBM4 can regulate cell senescence?And the functions and mechanisms involved in the senescence process of tumor cells?Materials and methods:1.The expression of RBPs during senescence was analyzed by the senescence-related data set GSE60340;RNA was extracted from three tissues of colon,liver,and small intestine from 2-month-old and 24-month-old C57BL/6 female mice,and the mRNA expression levels of RBM4 were determined by RT-qPCR;SA-β-Gal senescence staining assay to test whether knockdown of RBM4 induced cellular senescence;Flow cytometry was used to analyze the effect of RBM4 knockdown on cell cycle;The EdU cell proliferation assay examined the effect of knockdown of RBM4 on cell proliferation;The protein expression levels of senescence-related markers in H1299 and A549 cells with knockdown of RBM4 were measured by Western blot;The mRNA expression levels of senescence-associated pro-inflammatory genes after knocking down RBM4 were determined using RT-qPCR.2.Colony formation,soft agar clonal formation and CCK8 cell viability were performed on H1299 and A549 cells knocked down by RBM4 to detect the proliferation ability of lung cancer cells;SA-β-Gal senescence staining was used to detect the effect of RBM4 knockdown on HeyA8,HCT16,Hela and 786O tumor cells,and both colony formation assay and soft agar clone formation experiment were used to verify the growth and proliferation of RBM4.3.The effect of knockdown RBM4 on the growth of transplanted tumor in vivo was investigated by using doxycycline-induced stable H1299 cells in nude mice;Immunohistochemistry and SA-β-Gal senescence staining experiments were applied to test whether knockdown of RBM4 induced senescence.4.Transcriptome sequencing(RNA-seq)was performed on H1299 and A549 cell lines with stable knockdown of RBM4 to analyze the genes regulated by RBM4;GO analysis,KEGG analysis and STRING database analysis were performed to identify the enrichment of overlapping genes in H1299 and A549 cells;RT-qPCR was used to verify the expression changes of genes regulated by RBM4 knockdown in RNA-seq.5.Western blot analysis was performed to detect SERPINE1 protein levels in H1299 and A549 cells with knockdown RBM4,and to verify whether knockdown RBM4 affected SERPINE1 protein expression;SA-β-Gal senescence staining was performed to determine whether RBM4 depletion induced senescence by increasing SERPINE1 levels using tiplaxtinin treatment.6.RT-qPCR,western blot and luciferase reporter genes were applied to test whether miR-1244 could regulate SERPINE1 expression levels;Western blot and SA-β-Gal senescence staining were used to investigate whether miR-1244/SERPINEl was associated with RBM4-induced senescence;RT-qPCR and RNA Co-Immunoprecipitation assays were applied to investigate how RBM4 regulates miR-1244 expression.Results:1.Analysis of the senescence-related data set GSE60340 showed that RBM4 is a significantly down regulated RNA binding protein in senescent cells;RBM4 levels were significantly decreased in colon,liver,and small intestine tissues in 24-month-old mice compared with 2-month-old mice,suggesting that RBM4 may be involved in the regulation of cellular senescence;SA-β-Gal senescence staining assay showed that knockdown of RBM4 can promote senescence in normal and lung cancer cells;Flow cytometry analysis of knockdown of RBM4 blocks the cell cycle in Gi/S phase;The EdU cell proliferation assay demonstrated that knockdown of RBM4 inhibits tumor cell proliferation;Western blot results showed that knockdown of RBM4 resulted in changes in the expression of cell cycle related proteins;Knockdown of RBM4 can lead to increased mRNA levels of many senescence-associated proinflammatory genes.2.Colony formation,soft agar clone formation,and CCK8 cell viability assay,demonstrated that knockdown of RBM4 in lung cancer cells inhibited cell proliferation;SA-β-Gal senescence staining increased,cell cloning ability significantly decreased compared with the control group,and cell proliferation was inhibited,suggesting that knockdown RBM4 can induce senescence and tumor inhibition of different tumor cells.3.The growth rate of tumors in mice with doxycycline-induced knockdown of RBM4 was significantly inhibited compared with controls,indicating that knockdown of RBM4 significantly inhibited tumor growth in vivo;Both the results of immunohistochemistry and SA-β-Gal senescence staining assay indicated that knockdown of RBM4 was able to induce cellular senescence and thus inhibit tumor growth.4.Analyzing the RNA-seq sequencing results,we identified 1195 differentially expressed genes in A549 cells and 676 differentially expressed genes in H299,of which 206 genes were differentially expressed in both H1299 and A549;Through GO functional enrichment analysis,it was found that the genes regulated by down-regulated RBM4 were mainly enriched in ECM,cell migration,gene expression regulation,integrin-mediated cell adhesion,etc.KEGG enrichment pathway analysis indicates that genes regulated by knockdown RBM4 are mainly enriched in viral on cogenesis,proteoglycans in cancer,transcriptional deregulation in cancer,ECM receptor interactions,etc.STRING database analysis,genes regulated by RBM4 deletion were associated with genes in ECM tissue,regulation of gene expression,regulation of DNA replication and regulation of translation;The expression of SERPINE1 and other senescence-related genes was significantly increased after knocking down RBM4,suggesting that RBM4 may promote senescence through genes regulating these pathways.5.The SERPINE1 protein expression was enhanced after knockdown of RBM4,while verifying that the expression level of SERPINE1 was negatively correlated with the RBM4 expression lever;The SA-β-Gal senescence staining experiments showed that knockdown of RBM4 in lung cancer cells was partially reversed by tiplaxtinin(SERPINE1 inhibitor).6.In A549 and H1299 lung cancer cells,the expression level of SERPINE1 was significantly increased after knocking down RBM4,and the mature miR-1244 was down-regulated.;Target Scan analysis showed that 3’-UTR of SERPINE1 contains a potential binding site of miR-1244;luciferase reporter results indicated that miR-1244 directly binds 3’-UTR of SERPINE1 to inhibit SERPINE1 expression;SA-β-Gal senescence staining showed enhanced staining activity in cells treated with miR-1244 inhibitor,indicating that knockdown of RBM4 increased the level of SERPINE1 through down regulation of miR-1244;RT-qPCR results confirmed that knockdown of RBM4 promoted the degradation of pri-miR1244,and RNA Co-Immunoprecipitation assay confirmed that RBM4 could indeed interact with pri-miR1244.In conclusion,our study reveals a new mechanism by which RBM4 regulates cancer progression by modulating senescence,providing a new avenue for targeting RBM4 in cancer therapy.Conclusions:RBM4 is closely related with cell senescence,and knockdown of RBM4 can induce senescence in different types of cells;Knockdown of RBM4 inhibited the growth of tumor cells both in vitro and in vivo;Knockdown of RBM4 reduced miR-1244 levels by promoting the degradation of pri-miR1244,thereby increasing SERPINE1 expression and inducing subsequent senescence,inhibiting tumor cell growth.