| Objective:1.Screening and identifying theRNA-binding proteins which is closely related to HCC metastasis,and examining its expression levels in clinical HCC tissues.2.Investigating the role of RPS7,a kind ofRNA binding protein,on HCC cells’ cell-matrix adhesion,migration and invasion and metastasis in vitro and in vivo.3.Analyzing the molecular mechanism by which RPS7 promotes HCC progression.4.Evaluating the function of LOXL2 in RPS7-induced HCC cells migration and invasion.Methods:1.RNA-seq analyzed the differentially expressedRNA-binding protein(RBP)genes in extrahepatic metastatic HCC tissues(EHMH)compared to metastasis-free HCC tissues(MFH).GO analysis,TCGA datasets and KM plotter were performed to screen the candidate genes.2.The mRNA and protein levels and localization of RPS7 in HCC clinical samples were respectively detected by q RT-PCR,Western blot and IHC assays.3.The effects of RPS7 knockdown/knockout on cell proliferation,cell-matrix adhesion,migration and invasion of HCC cells were evaluated by CCK-8 assay,colony formation assay,cell-matrix adhesion assay,wound-healing assay and Transwell assay,respectively.The orthotopic mouse model and lung metastasis mouse model was respectively constructed to evaluate the effect of RPS7 knockout on lung metastasis.4.The effects of overexpressed RPS7 on cell proliferation,cell-matrix adhesion,migration and invasion of HCC cells were evaluated by CCK-8 assay,colony formation assay,cell-matrix adhesion assay,wound-healing assay and Transwell assay,respectively.5.RNA-seq screened the differentially expressed downstream genes in RPS7-knockdown MHCC97 H cell.KEGG and cat RAPID datasets were performed to screen the downstream targets of RPS7 that simultaneously potentially binds to RPS7.6.The most significantly differentially expressed candidates of down-regulated genes were tested by q RT-PCR.The effects of RPS7 on expression levels of LOXL2 were detected by q RT-PCR and Western blot,respectively.7.The dual luciferase reporter assay system was utilized to evaluate the effect of RPS7 on the promoter activity of LOXL2.The nascentRNA capture assay was performed to evaluate the effect of RPS7 on nascentRNA expression levels of LOXL2.RNA decay assay was utilized to test the effect of RPS7 on LOXL2 mRNA stability.8.RIP assay was used to confirm the binding interaction between RPS7 and LOXL2 mRNA.The combination ofRNA pull-down assay with truncated mutations of LOXL2 were used to investigate the specific binding sequence of LOXL2 mRNA with RPS7.9.The relative mRNA levels of LOXL2 in HCC samples were determined by q RT-PCR assay.The protein levels of LOXL2 in human HCC cell lines were determined by Western blot assay.510.MHCC97 H cells were transfected with small interferingRNA targeting LOXL2(si LOXL2)for 48 h.The effects of LOXL2 silencing on cell-matrix adhesion,migration and invasion,respectively,were assessed.11.Plasmids encoding LOXL2 were constructed and transfected to Huh7 cells.The alteration of cell-matrix adhesion,migration and invasion were evaluated,respectively.12.RPS7-knockdown MHCC97 H cells were transfected with plasmids encoding LOXL2,the alteration of cell-matrix adhesion,migration and invasion were evaluated.13.RPS7-overexpressed Huh7 cells were transfected with si LOXL2 for 48 h,the alteration of cell-matrix adhesion,migration and invasion were evaluated.14.Spearman correlation analysis was performed to evaluate the relationship between RPS7 levels and LOXL2 levels by using TCGA datasets and 60 HCC samples,respectively.Results:1.A total of 102 HCC metastasis associated differentially expressed RBP genes were found out.Seven candidates belonging to Focal Adhesion module was screened by GO analysis.After using TCGA datasets and KM plotter,RPS7 was finally identified.2.The mRNA and protein levels of RPS7 in HCC tissues were significantly higher than those in adjacent normal liver tissues.Additionally,the mRNA and protein levels of RPS7 in EHMH group were significantly upregulated compared to those in MFH group.High expression of RPS7 was markedly positively associated with poor outcomes of HCC patients.3.Compared to control group,RPS7 knockdown or knockout apparently decreased cell proliferation,cell-matrix adhesion as well as migration and invasion.Results from two mouse models mentioned above showed that RPS7 knockout inhibited the tumor size in situ and lung metastasis.4.Overexpression of RPS7 increased cell proliferation,cell-matrix adhesion as well as migration and invasion.5.RNA-seq data of RPS7-knockdown MHCC97 H cells screened out the downstream genes.KEGG analysis found that the down-regulated genes were mainly enriched in the signaling pathways related to cell invasion and metastasis.6.The q RT-PCR found that the mRNA levels of LOXL2 from the Top12 candidate genes were most significantly down-regulated upon RPS7 knockdown.Results of western blot analysis showed that the protein levels of LOXL2 were down-regulated after RPS7 knockdown.7.Double luciferase report showed that RPS7 had no effect on the promoter activity of LOXL2.NascentRNA capture assay showed that RPS7 had no effect on the levels of LOXL2 nascentRNA.However,RNA decay assay revealed that RPS7 silencing shortened the half-life of LOXL2 mRNA,while RPS7 overexpressing showed the opposite effect.8.The binding relationship between RPS7 and LOXL2 mRNA was confirmed by RIP assay.TheRNA pull-down combined with truncation mutation revealed that RPS7 mostly binds to the two consecutive AUUUA motifs in 3190-3259 sequence of the 3’ UTR of LOXL2 mRNA.9.q RT-PCR analysis displayed a higher mRNA levels of LOXL2 in HCC tissues compared to those in adjacent normal liver tissues.Additionally,compared to primary human hepatocytes,the levels of LOXL2 were highly expressed in human HCC cell lines.10.Gene knockdown by small interferingRNA targeting LOXL2 decreased cell-matrix adhesion,migration and invasion of MHCC97 H cells.11.Huh7 cells transfected with LOXL2 showed significantly enhanced cell-matrix adhesion,migration and invasion compared to control cells.12.Overexpression of LOXL2 partly enhanced the decreased cell-matrix adhesion capacity and migration and invasion ability that induced by RPS7-knockdown in MHCC97 H cells.13.Silencing LOXL2 apparently weakened the enhanced cell-matrix adhesion capacity,migration and invasion ability that induced by RPS7 overexpression in Huh7 cells14.There was a significant positive relationship between RPS7 levels and LOXL2 levels in human HCC according to analysis using TCGA datasets and 60 HCC sample.Conclusion:RPS7 was generally highly expressed in HCC tissues and the levels of RPS7 were higher in metastatic HCC tissues than those in metastasis-free HCC tissues.Highly expressed RPS7 was closely related to the worse prognosis of HCC patients.RPS7 acted as a pro-invasion and pro-metastasis factor in HCC cells.Mechanically,RPS7 maintained the LOXL2 mRNA stability via binding to the two consecutive AUUUA motifs in the 3’ UTR of LOXL2 mRNA,thus upregulated the expression of LOXL2.LOXL2 also showed pro-invasion functions in HCC.Moreover,LOXL2 was essential for maintaining the RPS7-induced HCC cells adhesion,migration and invasion.Collectively,RPS7 plays a tumor-promoting role in HCC progression and metastasis process via regulating LOXL2 expression. |
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