Mechanism Underlying LARP7-mediated Gene Expression And Alternative Splicing In Regulating Non-small Cell Lung Cancer Progression

Background Lung cancer is one of the threatening cancers in the world.According to cancer statistics,lung cancer ranks second in the incidence of malignant tumors and the mortality rate ranks first in the world.In China,both the incidence and mortality rate of lung cancer are the first.Lung cancer is divided into non-small cell lung cancer(NSCLC)and small cell lung cancer,in which NSCLC accounts for the vast majority.Even though that much progress has been madded for lung cancer,such as early screening and diagnosis,progress in radiotherapy technology like intensity modulated radiotherapy and stereotactic radiotherapy,target therapy and immunotherapy,and improvement in treatment mode,treatment outcome were partially improved,however,the overall survival was still dismal,5-year survival rate was 19%.Therefore,it is crucial to identify molecular biomarkers and promising therapeutic strategies to improve effectiveness and prognosis of lung cancer.La ribonucleoprotein domain family member 7(LARP7)is an RNA-binding protein and belongs to the LARP family,which was reported to act as toumor suppressor in multiple cancers.LAPP7 was revealed to regulate the malignant biological behavior of A549 lung cancer cells by regulating the transcriptional extension process of genes and related factors in some signal transduction pathways.However,the role of LARP7 in lung cancer cells and underlying mechanisms were not well clarified.Based on the previous studies,we first used public databases to explore the expression of LARP7 in lung cancer,then in vivo and in vitro experiments were performed to uncover the effects of LARP7 on the proliferation,migration,invasion,apoptosis of lung cancer cells and tumor growth,and in-depth research was done to discuss the possible mechanisms by which LARP7 regulates lung cancer progression.The results will provide new insights and theoretical support for finding molecular targets for the diagnosis,prognosis and treatment of NSCLC.Methods 1)TCGA databases were utilized to explore the expression of LARP7 in NSCLC tumor tissues and normal tissues,and on-line analysis of the relationship between LARP7 expression level and prognosis was conducted by K-M plot.2)Tumor tissue samples of NSCLC patients from our hospital were collected to measure the expression of LARP7,and the relationship between LARP7 expression level and prognosis of NSCLC patients were analyzed.3)A549/ H1299 cells transfected with LARP7-expressing vectors(LAPR7)and blank vectors(Ctrl)were prepared to explore the role of LARP7 on lung cancer cells,q RT-PCR and western-blot were employed to detect overexpression efficiency.4)In vitro functional experiments were condutcted to detect the effects of LARP7 on the biological behaviors of lung cancer cell lines,including proliferation,migration,invasion and apoptosis.The CCK8 assay and plate clonal formation assay were utilized to evaluate lung cancer cell proliferation.In the CCK8 experiment,cell activities in LAPR7 and Ctrl groups were monitored for 5 consecutive days and the OD value was recorded.The larger the OD value,the more the number of cells and the stronger the cell proliferation ability.Cell scratch experiment and transwell chamber experiment were used to evaluate the effects of LARP7 on cell migration and invasion ability,respectively.Flow cytometry Annexin V-PE/7-AAD method was used to detect the apoptosis of lung cancer cells in LARP7 and Ctrl groups.5)In vivo experiments were performed to observe the effect of LARP7 on tumor growth: The A549 cells transfected with virus-packaged LARP7 plasmid(A549-OE-LARP7 group)or virus-packaged LARP7 plasmid vector(A549-Ctrl group)were planted in the armpits of two groups of nude mices,respectively.The body weight,tumor volume and tumor weight of the mices were monitored,then xenografts were subjected to immunohistochemical analysis.6)RNA sequencing was utilized to study the transcriptome and variable splicing levels of upregulated cells by LAPR7 and control cells,and differential expressed genes(DEGs)and alternative splicing events(ASEs)were identified.GO and KEGG analysis were performed to explore enriched biological function of DEGs and regulated alternative splicing genes(RASGs).7)Verification of the regulation of LARP7 on DEGs and alternative splicing: DEGs and RASGs that have significant changes and are related to lung cancer were selected,q RT-PCR was applied to verify differences of gene expression and gene alternative spliceosome between the LARP7 and Ctrl groups.Results 1)LARP7 is downregulated in lung cancer samples compared with normal tissues according to TCGA database,further survival analysis showed that decreased LARP7 expression was significantly associated with poorer survival of NSCLC patients(P = 0.00082,HR = 0.79).2)6-paired NSCLC tumor samples from our hospital were randomly collected to measure the expression of LARP7.Consistantly,the expression level of LARP7 in lung cancer tissuses was decreased compared to normal tissues.Immunohistochemistry test revealed the same resuls,namely,the expression of LARP7 in tumor tissues was significantly down-regulated.Survival analysis of 56 NSCLC patients from our hospital confirmed the findings from TCGA,revealed that patients with decresed LARP7 expression had significantly worse survival than those with high LARP7 expression.3)QRT-PCR determined significant gene overexpression of LARP7 in LAPR7 group cells,further western blot confirmed that LARP7 expression were significantly higher in LAPR7 group cells,which confirmed that LAPR7 overexpressed A549/ H1299 cells were successfully constructed.4)The CCK8 assay showed that the OD value of LARP7 cells was significantly decreased compared with Ctrl cells,indicting LARP7 overexpression inhibited the proliferation ability of lung cancer cells.Plate clonal formation assay showed that the number of colonies formed by LAPR7 cells were less than those of Ctrl cells,the difference was statistically significant(P < 0.001).The results demonstrated that high expression of LARP7 inhibit the proliferation of lung cancer cells in vitro.5)Cell scratch experiments showed that the migration ability of LARP7 cells was inhibited.Transwell chamber experiments revealed that after overexpression of LARP7,the number of A549/ H1299 cells passing through the chamber decreased,indicating that the invasive ability was weakened.Transwell experiments and scratch experiments demonstrated that overexpression of LARP7 could inhibit the migration and invasion ability of lung cancer cells in vitro,respectively..6)The apoptosis of LARP7 and Ctrl group cells was detected by flow cytometry Annexin V-PE/7-AAD method.It showed that the incidence of apoptosis(early apoptosis + late apoptosis)of A549 and H1299 cells after overexpression of LARP7 was significantly increased compared with the Ctrl group cells(P < 0.001).The results proved that overexpression of LARP7 can induce the apoptosis of lung cancer cells in vitro.7)The size of the tumors in the A549-OE-LARP7 group was significantly smaller than that in the A549-Ctrl group since the 19 th day,indicating that the overexpression of LARP7 can inhibit the growth of lung cancer in vivo.The xenografts were harvested,the final tumor volume and weight were measured to be significantly smaller and lower in A549-OE-LARP7 group than those in the A549-Ctrl group,and the difference was statistically significant(P < 0.01).Subsequently,the xenografts were subjected to immunohistochemical analysis.The immunohistochemical results showed that in the tumor tissue with low expression of LARP7,Ki67 was highly expressed,indicating that the tumor tissue has strong proliferation ability.The above results demonstrated the inhibitory effect of LARP7 on lung cancer tissue in vivo.8)RNA-seq analysis revealed that LARP7 overexpressed caused a range of DEGs and alternative splicing events.DEGs were mainly enriched in immune-inflammation-related pathways,and RASGs were mainly enriched in cell proliferation and apoptosis-related biological processes.The intersection analysis of DEGs and RASGs found 143 overlapping genes,in which two genes closely related to lung cancer.The expression levels of FN1 and ADAR in the LARP7 group were significantly downregulated and the occurrence of alterantive splicing was significantly higher compared with those in the Ctrl group.9)Genes that have significant changes including ADAR,FN1,IFI6,IFI27,B2 M,BST2,HIST3H2 A,SLC45A1 and ARHGAP42 were selected,and q RT-PCR was applied to verify the differential gene expression between the LARP7 and Ctrl groups.The results suggested that the expression of most genes in the LARP7 group was significantly downregulated,which is consistent with RNA-seq results.The overlap analysis of DEGs and RASGs revealed that gene expression and alternative splicing of FN1 and ADAR genes were both significantly different between the LARP7 and the Ctrl group,moreover,FN1 and ADAR were reported to be closely related to lung cancer.QRT-PCR verified that more alternative splicng events occurred in the FN1 and ADAR genes after overexpression of LARP7.Conclusion We identified LARP7,an RNA-binding protein closely associated with the prognosis of lung cancer progression,through public database and validated it in our single-center database.Moreover,we have explored the intrinsic mechanism of LARP7 in lung cancer.We found that LARP7 showed low expression in lung cancer tumor tissues,and in invtro and in vivo experiments revealed that overexpression of LARP7 significantly inhibited proliferation,migration and invasion of lung cancer cells,promoted apoptosis of lung cancer cells and inhibited tumor growth in vivo,while similar effects were not observed in control group.Mechanistically,LARP7 overexpression caused significant differential gene expression and variable splicing,and these genes were mainly enriched in tumor-related functional pathways such as immune inflammation and apoptosis,suggesting that LARP7 may regulate lung cancer progression by causing differential expression and variable splicing of a wide range of genes.Notably,gene expression and alternative splicing of FN1 and ADAR genes were both significantly different between the LARP7 and the Ctrl group,moreover,FN1 and ADAR were reported to be closely related to lung cancer.LARP7,as an RNA-binding protein,may bind immature m RNA precursors such as nucleolar small molecule RNA(sno RNA),leading to variable shear events that affect the differential expression of transcripts of genes such as FN1 and ADAR,which in turn cause altered malignant tumor behavior.The results provide new ideas for relevant drug development.