Mechanistic Study Of RBMS1 Regulates PD-L1 Expression And Melatonin Regulates RNA Alternative Splicing

Mechanistic study of RBMS1 Regulates PD-L1 Expression in Breast CancerBackground:Breast cancer is one of the most common cancers in women worldwide.Breast cancer is divided into four main subtypes:Luminal A,Luminal B,HER2-positive,and triple-negative breast cancer（TNBC）.Since triple-negative breast cancer lacks ER,PR and HER2 receptors,it has limited therapeutic approaches and poor therapeutic effect.Therefore,it is urgent to develop novel precise diagnosis and treatment strategy for triple-negative breast cancer.Tumor immunotherapy has been widely used in a variety of tumors.Specifically,immune checkpoint inhibitors and chimeric antigen receptor T cell therapy have been applied to clinic,and better clinical responses have been observed in many tumors.With the gradual development of anti-tumor immunotherapy,PD-L1inhibitor atezolizumab and PD-1 inhibitor pembrolizumab have been approved for the treatment of metastatic TNBC,improving anti-tumor immunity.Combination immunotherapy will provide a novel avenue for breast cancer therapeutics.Immune checkpoints（e.g.,PD-L1/PD-1）are regulated by factors stimulating or inhibiting the function of the immune system.Blocking immune checkpoints could offer new hope for treatment of multiple cancers.Programmed death ligand-1（PD-L1;also known as B7-H1 or CD274）is a 33 k Da typeⅠtransmembrane protein that interacts with programmed cell death protein-1（PD-1）,creating a major immune checkpoint,thereby inhibiting T cell proliferation and cytokine secretion,negatively regulating lymphocyte activation,and enabling cancer cells to evade T cell-mediated immune surveillance.PD-L1 expression is abnormally elevated in tumors,which in turn helps cancer cells evade immune checkpoints.The expression of PD-L1 in cancer cells is regulated by multiple pathways,including transcriptional and post-translational modifications and so on.For example,glycogen synthase kinase 3β（GSK3β）can directly phosphorylate the T180 and S184 sites of PD-L1,resulting in the polyubiquitination of PD-L1 by SCF^β-TRCPubiquitin ligase complex.In addition to ubiquitination,post-translational modifications of PD-L1also include N-linked glycosylation.Glycosylation of PD-L1 at three sites（N192,N200,and N219）can increase its protein stability and favor its binding to PD-1,leading to the occurrence of tumor immune escape.Therefore,studies on PD-L1 regulation can provide new potential molecular markers to improve the efficacy of blocking immunotherapy checkpoints.RNA-binding proteins are involved in many aspects of gene post-transcriptional regulation,such as alternative splicing,cellular localization,RNA stability,translation,etc.,and subsequently regulate various physiological functions such as cell proliferation,DNA damage and angiogenesis.Thus,RNA-binding proteins play important roles in the development of cancer.However,few studies have demonstrated the role of RNA-binding proteins in regulating immune checkpoints.Therefore,exploring the regulatory effect of RNA-binding proteins on immune checkpoints in breast cancer can provide a new research perspective for anti-tumor immunotherapy.Methods:1.（1）The protein levels of 82 RNA binding proteins in basal-like vs luminal A,luminal B,and HER2 subtypes of breast cancer were compared by a bioinformatic analysis of proteomic datasets from 108 patients with breast cancer in the TCGA database.（2）The RNA levels of 82 RNA binding proteins in FI subtype versus non-fully inflamed tumors（ID+MR+SR）were analyzed from thirty-eight TNBC patient samples from the Gene Expression Omnibus（GEO）database（GSE88847）.2.（1）Using sh RBP library to screen RNA-binding proteins that regulate PD-L1 in MDA-MB-231 triple-negative breast cancer cells.（2）The protein levels of RBMS1 in different breast cancer cells were detected by a western blot assay.（3）The effect of depleted RBMS1 on PD-L1 expression in MDA-MB-231,HCC1937,BT-549 and 4T1cells was detected by western blot.（4）The effect of overexpression of RBMS1 on PD-L1 expression in MDA-MB-231,HCC1937 and BT-549 cells was detected by a western blot.（5）The effect of RBMS1 depletion on tumor cell surface PD-L1 expression in MDA-MB-231,HCC1937,BT-549 and 4T1 cells was detected by flow cytometry.3.The protein levels of RBMS1 and PD-L1 in breast cancer tissues and normal breast tissues were analyzed by immunohistochemical staining,and the correlation between RBMS1 and PD-L1 expression levels were examined in breast cancer tissues.4.（1）The tumorigenic ability of mouse breast cancer cell line 4T1 with knockdown of RBMS1 was detected by nude mouse tumor-forming assay.（2）Using BALB/c mouse tumor-forming assay to detect the changes of tumorigenic ability of mouse breast cancer cell line 4T1 with depletion of RBMS1.（3）Flow cytometry was applied to examine the effects of depleted RBMS1 on the secretion of intracellular cytokines IFN-γand Gzm B in CD8⁺T cell populations of BALB/c mice.（4）The effect of RBMS1 knockdown on T cell-mediated tumor killing ability was detected by T cell-mediated in vitro tumor killing assay in MDA-MB-231,HCC1937,BT-549 and 4T1 cells.5.（1）The regulatory effect of RBMS1 knockdown on B4GALT1 m RNA level in MDA-MB-231 and BT-549 cells was detected by qPCR.（2）The regulation of RBMS1on m RNA levels of other members of B4GALT family in MDA-MB-231 cells was detected by qPCR assay.（3）The regulatory effect of depleted RBMS1 on the expression level of B4GALT1 in MDA-MB-231 and BT-549 cells was detected by western blot.（4）The regulatory effect of RBMS1 overexpression on B4GALT1 expression level in HCC1937 and BT-549 cells was detected by western blot.（5）The correlation of B4GALT1 and RBMS1 expression levels was analyzed in the TCGA TNBC dataset.（6）To determine the effect of knockdown of RBMS1 on the stability of B4GALT1 m RNA in MDA-MB-231 and BT549 cells the change of B4GALT1 m RNA degradation rate after actinomycin D treatment was detected.（7）The binding of RBMS1 to the 3’UTR region of B4GALT1 was detected by RIP assay.（8）The regulatory effect of depleted B4GALT1on PD-L1 expression in MDA-MB-231,HCC1937 and BT-549 cells was detected by western blotting.（9）The regulatory effect of B4GALT1 overexpression on PD-L1expression in MDA-MB-231 and HCC1937 cells was examined by western blot.（10）The interaction between B4GALT1 and PD-L1 was detected by IP assay.（11）The interaction between B4GALT1 and PD-L1 was determined by PLA approach.（12）In RBMS1knockdown cells re-expression of B4GALT1 to detect the effect of B4GALT1 on PD-L1expression.（13）Knockdown B4GALT1 in cells overexpressing RBMS1 to examine the effect of B4GALT1 on PD-L1 expression.6.（1）The rescue effect of B4GALT1 overexpression on T cell-mediated tumor killing ability was detected by T cell-mediated in vitro tumor killing assay in knockdown RBMS1 MDA-MB-231,HCC1937,BT-549 and 4T1 cells.（2）BALB/c tumor-forming assay was used to investigate the tumor-forming effect of B4GALT1 overexpression on4T1 breast cancer cells with RBMS1 knockdown,and the regulation of IFN-γand Gzm B secretion in Tumor CD8⁺T cells of BALB/c mice.7.Synergistic therapeutic effects of knockdown RBMS1 and CTLA-4 blocking therapy on tumor growth in mouse models and the regulation of anti-tumor immunity of T cells was analyzed.Results:1.（1）The bioinformatic analysis of proteomic datasets from breast cancer samples in the TCGA database showed that the expression of multiple RNA binding proteins,including DDX6,SND1,ACO1,and RBMS1,was increased in the basal subtype of breast cancer,as compared to other subtypes.（2）GEO database analysis showed that the m RNA expression level of RBMS1 was elevated and correlated with the non-fully inflamed phenotype.2.（1）Using the sh RBP library screening,we found that several RNA-binding proteins can regulate the protein level of PD-L1 in the triple-negative breast cancer cell line MDA-MB-231,and the deletion of RBMS1 has the most obvious down-regulation effect on the protein level of PD-L1.（2）Compared with different breast cancer cells,the expression level of RBMS1 was significantly increased in triple-negative breast cancer cell lines（such as MDA-MB-231 and HCC1937）.（3）Knockdown of RBMS1 in MDA-MB-231,HCC1937,BT-549 and 4T1 cells significantly reduced the expression level of PD-L1.（4）Overexpression of RBMS1 in MDA-MB-231,HCC1937 and BT-549 cells significantly increased the expression level of PD-L1.（5）Knockdown of RBMS1 in MDA-MB-231,HCC1937,BT-549 and 4T1 cells decreased the expression of PD-L1 on the tumor cell surface.3.More than 60%of breast cancer samples showed strong or extra-strong staining for both RBMS1 and PD-L1,while most normal breast tissues（～80%）showed weak positive/negative staining for RBMS1 and PD-L1,and RBMS1 positively correlated with PD-L1 expression in breast cancer patient samples.4.（1）Knockdown of RBMS1 had no significant effect on tumor growth of 4T1 cells in immunodeficient mice.（2）Knockdown of RBMS1 significantly inhibited the tumor growth of 4T1 cells in immunized normal mice.（3）There was no significant difference in the effector function of CD8⁺T cells in the spleen and lymph nodes of mice inoculated with knockdown RBMS1 4T1 cells or control cells,while knockdown of RBMS1significantly increased IFN-γsecretion and Gzm B release in tumors.（4）Knockdown of RBMS1 enhanced T cell-mediated tumor killing in vitro.5.（1）Knockdown of RBMS1 in MDA-MB-231 and BT-549 cells down-regulated B4GALT1 m RNA levels.（2）Knockdown of RBMS1 in MDA-MB-231 cells could decrease the m RNA level of B4GALT1.However,the expression levels of other members of the B4GALT family,such as B4GALT3,B4GALT4,and B4GALT7,were not affected by RBMS1.（3）Knockdown of RBMS1 in MDA-MB-231 and BT-549 cells down-regulated the expression of B4GALT1.（4）Overexpression of RBMS1 in HCC1937 and BT-549 cells up-regulated the expression of B4GALT1.（5）The expression levels of B4GALT1 and RBMS1 were positively correlated in the TCGA TNBC dataset.（6）Knockdown of RBMS1 in MDA-MB-231 and BT549 cells promoted B4GALT1 m RNA degradation.（7）RBMS1 interacts with the 3’UTR region of B4GALT1 in multiple cells.（8）Knockdown of B4GALT1 in MDA-MB-231,HCC1937and BT-549 cells reduced PD-L1 expression level.（9）Overexpression of B4GALT1 in MDA-MB-231 and HCC1937 cells up-regulated the expression level of PD-L1.（10）The exogenously expressed Flag-tagged PD-L1 was interacted with endogenous B4GALT1in an IP assay,and conversely,the exogenously expressed Flag-tagged B4GALT1 also interacted with exogenous PD-L1.（11）The interaction between endogenous B4GALT1and endogenous PD-L1 was detected by PLA assay in HCC1937 and MDA-MB-231 cells.（12）Overexpression of B4GALT1 reversed the expression of PD-L1 in RBMS1knockdown cells.（13）Depletion of B4GALT1 notably reduced the level of PD-L1 in breast cancer cells with overexpression of RBMS1.6.（1）Overexpression of B4GALT1 in RBMS1 knockdown MDA-MB-231,HCC1937,BT-549 and 4T1 cells restored T cell-mediated tumor killing.（2）Overexpression of B4GALT1 in RBMS1 knockdown 4T1 mouse breast cancer cells restored the tumorigenic ability of 4T1 cells and restored the secretion of intracellular factors IFN-γand Gzm B in the tumor CD8+T cell population of BALB/c mice.7.Knockdown of RBMS1 combined with CTLA-4 blockade therapy synergistically supressed tumor growth in a mouse model,and inhibited the secretion of intracellular factors IFN-γand Gzm B in the tumor CD8⁺T cell population of BALB/c mice.Conclusions:1.RBMS1 is highly expressed in TILs-deficient triple-negative breast cancer.2.Knockdown of RBMS1 significantly reduced the expression level of PD-L1 in triple-negative breast cancer.3.RBMS1 was positively correlated with PD-L1 expression in breast cancer clinical samples.4.Knockdown of RBMS1 enhanced T cell-mediated antitumor immunity.5.RBMS1 regulates the protein level of PD-L1 by regulating B4GALT1.6.B4GALT1 inhibits T cell-mediated antitumor immunity enhanced by knockdown RBMS1.7.Knockdown of RBMS1 and CTLA-4 blockade synergistically enhanced T cell antitumor immunity.Melatonin regulates RNA alternative splicing in hepatocellular carcinomaBackground:Liver cancer is the sixth most common cancer,and the third leading cause of cancer death all over the world.Due to the absence of specific symptoms in early stages and the lack of diagnostic markers,more than 70% of patients with hepatocellular carcinoma are often diagnosed in an advanced stage.Sorafenib is one of the most effective drugs for the advanced hepatocellular carcinoma.However,the treatment effect of sorafenib is also limited,which might lead to drug resistance and side effects as well.Therefore,explore new treatments for liver cancer is very urgent.Melatonin is a neuroendocrine hormone produced primarily by the pineal gland.It has various functions,including regulate circadian rhythm,promote sleep,participate in immune responses,and exert anti-cancer effect.Melatonin exerts oncostatic functions in numerous human malignancies,containing breast cancer,ovarian cancer,prostate cancer,skin cancer,and liver cancer,etc.Moreover,the tumor suppressing effect of melatonin is achieved by regulating various physiological functions,such as tumor proliferation,migration,invasion,angiogenesis,apoptosis,and cell cycle.However,there are still few studies on the mechanism of melatonin exerting its anticancer effect.RNA alternative splicing plays an important role in tumorigenesis and cancer progression.Aberrant splicing could induce the production of noncanonical and cancerspecific m RNA transcripts,causing the inactivation of tumor suppressors or the activation of oncogenes.For example,BIN1 generates a short isoform（BIN1-S,which lacks exon 12a）that exerts a tumor suppressing effect by inhibiting the binding of c-Myc to target gene promoter in the normal liver.In liver cancer,the expression of the DHX9–NONO–SFPQ complex is up-regulated,upregulated NONO helps the oncogenic splicing switch of BIN1 from BIN1-S to BIN1-L.BIN1-L plays a tumor-promoting role in liver cancer by binding to PLK1.Therefore,the study of molecular mechanisms of alternative splicing might provide novel therapeutics for liver cancer.However,whether alternative splicing is involved in melatonin-mediated inhibition of tumor progression is still largely unknown.Methods:1.（1）The IC50 of melatonin in HepG2 and Hep3 B cells was detected by CCK8.（2）HepG2 and Hep3 B cells were treated with different concentrations of melatonin,and the effect of melatonin on the proliferation of HepG2 and Hep3 B cells was detected by CCK8 and colony formation assay.2.（1）High-throughput m RNA sequencing was performed with HepG2 cells treated with 1 m M melatonin for 24 h and 48 h separately.Then gene ontology analysis and KEGG analysis were preformed to search for enriched functions and pathways.（2）The STRING database was used to analyze the interaction network of melatonin treatment induced genes in HepG2 cells.（3）Several target genes-induced by melatonin were validated with q-PCR.3.（1）Quantification of the different alternative splicing events affected by melatonin treatment for 24 h and 48 h.（2）We preformed gene ontology analysis and KEGG analysis to search for enriched functions and pathways of melatonin-induced splicing events.（3）The STRING database was used to analyze the interaction network of differential splicing events induced by melatonin treatment in HepG2 cells.（4）The splicing change induced by melatonin in HepG2 and Hep3 B cells were validated by the semi-quantitative RT-PCR assay.4.HepG2 cells with MT1 stable depletion and control HepG2 cells were treated with different concentrations of melatonin,and splicing switches were examined using semiquantitative RT-PCR.5.（1）We examined the splicing change of IKBKG upon melatonin treatment in NCIH1299 and A549 lung cancer cell lines,and human embryonic kidney cell line HEK-293T by semi-quantitative RT-PCR.（2）Western blot was used to detect the expression changes of NEMO long isoform and short isoform after melatonin treatment in HepG2 cells.（3）We stably overexpressed NEMO-L or NEMO-S in HepG2 cells respectively.The expression of NEMO-L and NEMO-S were verified at both RNA and protein levels using semi-quantitative RT-PCR and western blot assays.The regulation effect of NEMO-L on the proliferation of HepG2 cells was detected by CCK8 and colony formation assay.（4）The splicing of IKBKG in liver cancer samples and adjacent normal tissues were detected by semi-quantitative RT-PCR.Results: 1.（1）The IC50 of melatonin in HepG2 cells was 1.995 m M,and the IC50 of melatonin in Hep3 B cells was 1.409 m M.（2）The treatment of melatonin showed a significant inhibition of cell growth in HepG2 and Hep3 B cells.2.（1）These genes induced by melatonin are closely associated with DNA replication,DNA metabolic process,response to wounding,steroid metabolic process,extracellular matrix,calcium ion binding,cellular response to DNA damage stimulus,sulfur compound metabolic process and angiogenesis as judged by gene ontology analysis.KEGG analysis showed that melatonin-induced genes are enriched in homologous recombination,complement and coagulation cascades,steroid hormone biosynthesis,arachidonic acid metabolism,cell adhesion molecules,fatty acid biosynthesis,and PPAR signaling pathway.（2）Most of melatonin treatment for 24 h induced genes were functionally connected into a well linked interaction network that contains genes associated with steroid metabolic process,response to wounding and extracellular matrix,as judged by STRING database.Many of those genes induced by melatonin treatment for 48 h were functionally correlated into a network that includes genes correlated with DNA replication,response to wounding and extracellular matrix.（3）Melatonin up-regulated the expression of CRISPLD2,SESN3,EFR3 B,GPCPD1,CAPN5 and PPP1R36,and down-regulated the expression of CHST9 and UNC93 A in HepG2 cells.3.（1）We respectively identified 1670 and 2838 AS events with a significant change of percent-spliced-in（PSI）values（the change of PSI > 0.15）after 24 h and 48 h treatment with melatonin compared to controls.We found that melatonin can modulate various types of AS,including skipped exon（SE）,alternative 5′ ss exon（A5SS）,alternative 3′ ss exon（A3SS）,retained intron（RI）and mutually exclusive exons（MXE）.PSI of most of the SE events were increased by melatonin treatment.PSI of most of the RI events were decreased by melatonin treatment.（2）These splicing events are associated with mitotic cell cycle,microtubule-based process,kinase activity,DNA metabolic process,GTPase regulator activity,cellular response to DNA damage stimulus,histone modification,spindle,DNA repair and cell division as judged by gene ontology analysis.The alternative splicing events are abundantly enriched in PPAR signaling pathway,nucleotide metabolism,homologous recombination,peroxisome and other pathways as judged by KEGG analysis.（3）We performed STRING analysis and found that many melatoninregulated alternative splicing events were functionally connected into well linked interaction networks that contains genes associated with mitotic cell cycle,cellular response to DNA damage stimulus and kinase activity.（4）Melatonin stimulated the inclusion of exon 5 splicing of IKBKG,exon 6 of LPIN1,exon 25 of ITGA6,exon 7 of TERF1,exon 8 of KIF23 and exon 7 of PLEKHM2.Meanwhile,melatonin could induce the skipping of exon 10 of SIN3 B and exon 10 of ATXN2.4.We revealed that melatonin promoted the production of the long-isoforms of IKBKG,ITGA6,and PLEKHM2 in a dose-dependent manner,while the regulatory effect of melatonin was significantly attenuated in MT1-depleted HepG2 cells.5.（1）Melatonin could affect the splicing of IKBKG in multiple cell lines.（2）Melatonin could increase the protein level of long isoform of NEMO.（3）The expression of NEMO-L and NEMO-S were verified at both RNA and protein levels using semiquantitative RT-PCR and western blot assays.The expression of NEMO-L in HepG2 cells inhibited cell growth and colony formation.（4）PSI of IKBKG were significantly decreased in six liver cancer samples compared with paired normal tissuesConclusions:1.Melatonin inhibits hepatocellular carcinoma cell proliferation.2.In hepatocellular carcinoma,melatonin can regulate the expression of cancerrelated genes.3.In hepatocellular carcinoma,melatonin can induce splicing events involved in the regulation of multiple functions.4.The regulatory effect of melatonin on alternative splicing is partly dependent on MT1.5.IKBKG splicing switch participated in melatonin-induced HCC inhibition.