| mRNA alternative splicing plays an important role in the development of tumor,its function disorder is closely related to the occurrence of tumor development;mRNA alternative splicing also plays a pivotal role in tumor evolution and to a large extent affects the clinical prognosis and therapeutic efficacy.However,the mRNA alternative splicing mechanism in the process of tumor evolution has not been fully elucidated until now.BUD31 has been reported to play a role in intron splicing and mRNA maturation of yeast for the first time.Its dysfunction leads to impaired mRNA maturation and decreased gene expression of specific genes in yeast,which affects the budding and proliferation of yeast and yeast cell morphology.Another literature reported that BUD31 regulated the levels of multiple gene’ expression in c-MYC-driven breast cancer cell lines through intron splicing.The up-regulation of transcription level caused by c-MYC activation depended on the function of the spliceosome.Functional inhibition of BUD31,one of the spliceosome components,led to a decrease in the expression level of multiple genes,and increased the tumor cell lethal synthesis caused by c-MYC knockdown,suggesting that BUD31 played an important role in regulating gene networks through intron splicing in eukaryotic cells.According to our previous experimental results,we found that compared with normal tissues,BUD31 was up-regulated in tumor tissues,suggesting that its mechanism may also be mediated by mRNA spliceosome.Given that the mRNA intron splicing and mRNA alternative splicing exist similarity functions and a wide range of inter-section,so we hypothesize that BUD31 may regulate tumor oncogenes not only by intron splicing,but also by mRNA alternative splicing or by the both mechanisms in regulating tumor gene networks to promote tumor characteristics.This study focuses on the study of BUD31 regulating tumor genes by mRNA alternative splicing or by mRNA intron splicing combined with mRNA alternative splicing to promote the development of tumor characteristics.First of all,we analysis the BUD31 mRNA expression level through our lab’s 94 pair esophageal squamous carcinoma RNA-seq and TCGA esophageal RNA-seq data.Compared with normal tissues,the level of BUD 31 gene expression in esophageal tumor tissues is significantly up-regulated.We also further analysis TCGA pan-caner RNA-seq data.At the same time,we also analysis the correlation of the BUD31 gene expression levels with clinical outcomes for tumor patients.The results show that BUD31 gene expression levels are significantly correlated with overall survival in patients in multiple tumors,such as head and neck squamous cell carcinomas,malignant glioma,endometrial carcinoma.The higher level of BUD31 gene expression is correlated with worse clinical prognosis in patients,which suggests that BUD31 may play an important role in tumor cancer characteristics.Next,we confirm that BUD31 knockdown leads to the reduction of malignant phenotypes of tumor cells by cell phenotype experiments,such as cell proliferation,tumor cell migration,cell invasion and cell clone formation.On the contrary,the overexpression of BUD 31 gene leads to the increase of malignant phenotype in tumor cells.Given that BUD31 gene is significantly up-regualted in multiple tumors,such as esophageal cancer and breast cancer,the mechanism of BUD31 gene in tumors has large similarity,so we download the published RNA-seq data of BUD31 instantaneous knock-down in breast cancer.We analyze the differentially expressed genes and acquire the 750 up-regualted genes and the 940 down-regulated genes.Then we respectively acquire 113 and 242 genes in our laboratory 94 paire esophageal squamous carcinoma RNA-seq data and TCGA esophageal RNA-seq data by gene expression correlation analysis,81 of which coexist in the both datasets.The top 10 positively and negatively correlated genes are obtained in the both datasets,including MCM7 and CPSF4 genes.Given MCM7 and CPSF4 genes play an oncogene role in the tumor characteristics and both genes have multiple mRNA transcript variants and protein isoforms,so we choose MCM7 and CPSF4 two genes as a typical,in order to explore BUD31 mechanisms by mRNA alternative splicing or at the same time through mRNA alternative splicing and intron splicing to play an oncogene role in promoting tumor characteristics.In order to confirm that BUD31 regulates gene expression level through intron splicing,we instantaneously knock-down the expression level of BUD31 gene.The qRT-PCR results indicate that the decreased expression level of BUD31 leads to increased intron retention and decreased gene expression levels of multiple genes,which is also consistent with previous literature reports.Surprisely,qRT-PCR and WB experiments confirm that the decreased expression level of BUD31 gene leads to the imbalance of the ratio of MCM7 transcript variant 1 and MCM7 transcript variant 2 and the subsequent imbalance of the ratio of MCM7 protein isoform 1 and MCM7 protein isoform 2.On the contrary,the overexpression of BUD31 gene leads to the opposite MCM7 mRNA and protein changes.Next,we also obtain the similar experimental results of the CPSF4 gene.These results confirm for the first time the mechanism of BUD31 gene in mRNA alternative splicing and the simultaneous regulation of gene expression levels through alternative splicing of mRNA and intron splicing of mRNA to promote the development of tumor characteristics.In addition,we also find that BUD31 regulates the expression levels of two classical oncogenes,PIK3CA and c-MYC genes,but no significant changes in intron retention rates of these two genes are detected,which may be related to the increased expression levels of CPSF4 and MCM7.We also confirm that MCM7 and CPSF4 play an oncogene role in tumors through gene knockdown experiments of MCM7 and CPSF4.In addition,we find that the expression of MCM7 protein isoform 1 is significantly up-regulated in esophageal tumor tissues compared with normal tissues,while the change of MCM7 protein isomform 2 is not significantly up-regulated.Similar results are obtained in cell lines,suggesting that MCM7 protein isoform 1 plays an oncogene role,which is consistent with BUD31’s role in promoting carcinogenesis by upregulating MCM7 protein isoform 1.In order to furtherly confirm that the mechanism of BUD31 gene has a wide range of tumor characteristics,we select other tumor cell lines for verification,and the experimental results are totally consistent with the results found in esophageal cancer cells,suggesting that the mechanism of BUD31 gene has a wide range of tumor characteristics.To sum up,we firstly report BUD31 gene plays a vital role in tumor characteristics by mRNA alternative splicing regulation of MCM7 and CPSF4 mRNA transcripts and subsequent protein isomforms ratio change to promote carcinogenesis.We confirm that BUD31 gene plays an oncogene role by mRNA intron splicing and mRNA alternative splicing in the regulation MCM7 and CPSF4 expression level and the change of transcriptional rate.In addition,we verify the new mechanism of BUD31 gene through a variety of tumor cell lines,indicating that the mechanism of BUD31 gene has a wide range of tumor characteristics.We believe that B UD31 gene is a candidate target gene for tumor therapy and has a wide range of tumor application value.Meanwhile,it also highlights the important role of mRNA alternative splicing in tumor characteristics.In the current tumor immunotherapy,PD-1/PD-L1 immune checkpoint blocker as the representative immunotherapy have achieved promising therapeutic effects.However,the objective tumor response rate was only about 40%,and some tumor patients had poor response to PD-1/PD-L1 blockers and had poor clinical efficacy.Tumor infiltrating lymphocytes were detected in tumor tissue in some part of patients,but the expression of tumor tissue PD-L1 was not detected,which prompts that the tumor tissue may not negatively regulate tumor immunity through the PD-1/PD-L1 pathways,affect the tumor microenvironment,and in turn evade immune system attack through the other immune checkpoints pathway.Therefore,it is of great importance to find new immune checkpoints for the patients with poor efficacy for PD-1/PD-L1 immune checkpoint blockers and to develop new tumor immune methods.This study attempts to find a new tumor immune checkpoint to explore a new negative regulatory mechanism of tumor immunity and elaborate the tumor immune microenvironment.Firstly,we analyze the differentially expressed genes based on our lab 94 esophageal squamous cell carcinoma RNA-seq data and TCGA esophageal carcinoma RNA-seq data.The results indicate that the expression level of LAMP3,lysosomal associated membrane protein 3,a member of LAMP protein family protein,is significantly up-regulated in tumor tissues compared with normal tissues.LAMP3 has been reported to be expressed during the maturation of dendritic cells,suggesting that LAMP3 may be involved in the regulation of normal immunity,especially in the negative regulation of normal immunity to prevent the overreaction of immune systme.Subsequently,we analyze the data of LAMP3 expression in HPA database,and the results show that LAMP3 is rarely expressed in normal tissues,except for the lungs,appendix,and testes.LAMP3 is widely expressed in different types of immune cells in blood and various tumor tissues,suggesting that LAMP3 may play a vital role in the immune regulation of immune cells and tumor cells,and that the immune regulation mechanism involved in LAMP3 may be widely involved in the immune system and tumor tissues.In addition,gene expression analysis of TCGA pan-cancer RNA-seq data suggests that LAMP3 is significantly up-regulated in tumor tissues,such as breast carcinoma,colon carcinoma,stomach carcinoma,and head and neck squamous cell carcinoma,compared with normal tissues.Subsequent survival correlation analysis indicates that LAMP3 expression in tumor tissue levels is significantly associated with clinical overall survival in patients,and the patients with high LAMP3 expression have shorter clinical overall survival in several cancers,such as breast cancer,endometrial cancer and kidney cancer.The results suggest that LAMP3 may play an important role in tumor immune regulation and affect the clinical prognosis through the mechanism of direct tumor immune regulation.Next,we further analyze the correlation of the gene expression levels in our lab RNA-seq data and TCGA RNA-seq data in esophageal squamous cell carcinoma.The results indicate that the expression levels of LAMP3 in tumor tissues are significantly correlated with the expression levels of various immune marker genes,such as CD3E,GZMA,GZMB,GNLY,PRF-1,IFNG,etc.In addition,we also confirm the correlation analysis of gene expression levels in TCGA breast cancer and melanoma RNA-seq,and the results are consistent with the new findings in esophageal cancer,suggesting that LAMPS is significantly correlated with immune function in tumor tissues and may be widely involved in tumor immune regulation.We further analyze the correlation of LAMP3 expression level and PD-L1 expression level and find that there is little correlation between LAMPS and PD-L1 expression level,and the proportion of LAMP3 and PD-L1 both high expression at the same time is about 5%in tumor tissue.The protein BLAST results also show that the both don’t have any protein similarity or homology,which indictes that LAMP3 may regulate tumor immune through the new mechanisms completely different from PD-L1 regulation immune mechanism.The analysis of immune marker genes’ expression levels show that compared to normal tissues,the levels of immune marker gene expression in tumor tissue are significantly increased,such as GZMA,GZMB,GNLY and IFNG,suggesting the levels of immune system appeared to be enhanced in tumor tissues.However,the tumor has not been effectively controlled,which indicates that the tumor may escape the immune cells attack through other immune negative regulatory mechanisms and in-turn has tumor immune evasion.Finally,we do the correlation analysis in single-cell RNA-seq data from 64 cases of esophageal cancer tissues in our lab.The result indicates that CD3E expression of T lymphocytes and LAMP3 positive tumor cells proportion have significantly positive correlation.The IL-2 expression of effector T lymphocytes and LAMP3 expression on tumor epithelial have significantly negative correlation,suggesting LA MP3 may be directly associated with tumor immune negative regulation.According to our previous experiment results,it is suggested that LAMP3 may be directly involved in the negative regulation of tumor immunity,thus causing the escape of immune system attack.First,we validate the LAMP3 expression in several cell lines and find that LAMP3 is widely expressed in a variety of tumor cell lines and normal cell lines,suggesting that LAMP3 may have a broad function in tumor cells.Next,we explore whether LAMPS protein is located in tumor cell membrane by ICF experiments,and the results show that LAMP3 protein is not only expressed in tumor cell cytoplasm,but also expressed in tumor cell membrane,suggesting that the the LAMP3 protein of tumor cell membranes may have tumor immune negative regulation through direct interaction,and it needs further immune function experiments to validate.We have successfully constructed the stable LAMPS knock-down and stable LAMP3 overexpressed KYSE450 cell lines.Cell phenotypic experiment results show that LAMP3 has no significant effect on cell proliferation,but has significant effect on cell migration and invasion,suggesting that LAMP3 plays a vital role in tumor characteristics through tumor cell metastasis and invasion.Subsequent T lymphocyte killing assays confirm that the stable knockdown cell lines of LAMPS are more sensitive to T lymphocyte killing than the control group.Compared with the control group,the cell lines with stable overexpression of LAMP3 are insensitive to the killing of T lymphocytes,suggesting that LAMP3 in tumor cells directly decreases the killing ability of T lymphocytes.The further co-culture experiment of immune cells and tumor cells shows that the expression levels of multiple immune marker gene of PBMC co-cultured with LAMP3 stable knockdown cell line are up-regualted,such as GZMA,GZMB,GNLY,PRF-1 and IFNG,suggesting that LAMP3 in the tumor cells negatively regulates the immune function of T lymphocytes.In addition,we also find that compared with the control group,the LAMPS expression level in T lymphocyte is significantly increased when PBMC is co-cultured with stable knock-down LAMPS cell lines and T lymphocyte function is enhanced,which is consistent with previous literature report about the significant increase of LAMP3 expression exactly on the activation of T cells,suggesting LAMPS on T lymphocyte may have directly negative regulation of T lymphocyte function and a new candidate immune checkpoint.In conclusion,the level of LAMP3 expression in tumor tissues is widely up-regulated,and is significantly correlated with the prognosis of patients.LAMP3 has significant effects on cell migration and metastasis,but has no effect on cell proliferation and cell colony formation.Tumor cells LAMPS has directly negative regulation of T lymphocyte functions and killing ability.Considering the significant increase of LAMP3 expression on T lymphocyte,LAMPS is a new tumor immune negative regulatory molecule and likely to be a new candidate immune checkpoint. |
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