ERp57 Upregulation Promotes Progression Of Clear Cell Renal Cell Carcinoma By Initiating A STAT3/ILF3 Feedback Loop

Objective: Renal cancer is the second largest malignant tumor of the urinary system,accounting for about 2%-3% of adult tumors.Clear cell renal cell carcinoma(ccRCC)is the most common subtype of renal cancer.For the patients with metastatic disease or postoperative tumor recurrence and metastasis,there is no effective treatment at the moment.Therefore,it is necessary to further understand the occurrence and development mechanism of ccRCC and explore new effective therapeutic targets.ERp57 is an endoplasmic reticulum(ER)cavity glycoprotein-specific thiol oxidoreductase,which is involved in the glycoprotein folding process that promotes the formation of intramolecular and intermolecular disulfide bonds and the folding reaction.Under the induction of cell stress factors,ERp57 transfers from the cytoplasm to the nucleus,interacts with DNA or enhances the binding of signal transducer and activator of transcription 3(STAT3)to DNA by interacting with STAT3 to regulate gene transcription.ERp57 is dysregulated in many forms of cancer,moderate downstream genes such as DKC1 and EGFR to affect cell proliferation,take part in their progression and metastasis,which is associated with poor prognosis.However,the expression and function of ERp57 in ccRCC remains unclear.Interleukin enhancer-binding factor 3(ILF3),is a double-stranded RNA(ds RNA)binding protein.ILF3 forms complexes with other proteins,ds RNAs,small non-coding RNAs,or mRNAs to regulate gene expression or stabilize mRNAs.ILF3 can promote tumor cell proliferation.Knocking down the ILF3 protein may influence cell cycle progression to delay cell growth and inhibit cell proliferation by reducing mRNA stability.However,the role of ILF3 in ccRCC has not been fully elucidated.Objective: In his study,we intended to use human renal clear cellcarcinoma tissue,human renal clear cell carcinoma cell line and nude mouse renal clear cell carcinoma model as research objects,use immunohistochemistry and various molecular biological methods to observe the expression of ERp57,ILF3 and STAT3 in ccRCC,explore the role and relationship of the three in the development of ccRCC,and provide a theoretical basis for finding new targets in ccRCC clinical treatment.Methods:1.Collecting tumor tissues and adjacent tissues of 35 ccRCC patients,and the tissues were observed with HE stain routinely.Immunohistochemistry,western blot and qRT-PCR showed the expression levels of ERp57 and ILF3 in ccRCC cancer tissues and adjacent tissues.2.Through the Cancer Genome Atlas(TCGA)database,the expressions of ERp57 and ILF3 in ccRCC tissues and adjacent tissues were analyzed,and showed the effect on the overall survival rate of ccRCC patients.3.MTT was used to detect the effect of knocked down or overexpressed ERp57 on the proliferation of ccRCC.4.Colony formation method was used to detect the proliferation ability of ccRCC cells that knocked down ERp57 or overexpressed ILF3 in SW839 cells and overexpressed ERp57 or knocked down ILF3 in A498 cells.The effects of combined knockdown of ERp57 and overexpression of ILF3,combined knockdown of ERp57 and STAT3 in SW839 cells,and detection of combined overexpression of ERp57 and knockdown of ILF3,combined overexpression of ERp57 and knockdown of STAT3 in A498 cells on the proliferation of ccRCC cells were also examined.5.Brdu staining detected cell proliferation when ERp57?STAT3 or ILF3 was inhibited,or STAT3 and ILF3,STAT3 and ERp57 were simultaneously inhibited.6.Transwell migration assay was used to determine the effect of knockdown or overexpression of ERp57 on the migration ability of ccRCC cells.7.Immunofluorescence staining was used to detect the location betweenERp57 and ILF3 expression in ccRCC tissues.8.After using actinomycin D(Act D)to prevent transcription,qRT-PCR analysis was used to measure the effects of knockdown of ILF3 in SW839 cells and overexpression of ILF3 in A498 cells on changes in ERp57 mRNA levels over time.9.The STAT3 inhibitor nicotinamide was used to treat SW839 cell and A498 cells,and the expression of STAT3,p-STAT3 and Cyclin E1 was analyzed by knocking down ILF3 in SW839 cells and over expressing ILF3 in A498 cells.10.Western blot and real-time quantitative PCR(qRT-PCR)were used to measure the expression of ERp57,ILF3,STAT3,Cyclin E1,Cyclin D1 protein and mRNA in ccRCC tissues and cells.11.Co-immunoprecipitation(CoIP)detects protein-protein interactions between ERp57 and STAT3.12.Chromatin immunoprecipitation(ChIP)and promoter luciferase analysis were used to demonstrate the effect of STAT3-ERp57 complex on ILF3 transcription and ILF3 transcription factor activity.13.Ribonucleoprotein immunoprecipitation(RIP)and RNA biotin pulldown were used to detect the interaction of ILF3 with ERp57 mRNA.14.The ccRCC cell lines stably knocked down ERp57,ILF3 or knocked down both of them were builded,and nude mouse xenograft tumor models were constructed.Western blot detected the expression of p-STAT3,ERp57,ILF3 and Cyclin E1,Cleaned caspase-3 in xenograft tumors.Brd U staining was used to detect the proliferation ability of the xenograft tumor cells.Results:1.ERp57 expression in ccRCC tissues.1.1 Relationship between ERp57 expression and prognosis in ccRCC tissues.qRT-PCR,Western blot and immunohistochemistry showed that both mRNA and protein levels of ERp57 were increased in ccRCC tissues compared to adjacent tissues.Analysis of ERp57 mRNA expression level and clinicopathological characteristics of ccRCC patients showed that ERp57 mRNA expression level was positively correlated with tumor size.The cancer genome atlas(TCGA)database was used to analyze the expression of ERp57 in ccRCC tumor tissues and overall survival rate of ccRCC patients with different ERp57 expression levels.The results showed that the level of ERp57 was increased in ccRCC tumor tissuses,and the patients with higher ERp57 mRNA levels in ccRCC tissues were associated with poor overall survival.1.2 Correlation between ERp57 and ccRCC cell proliferation and metastasis.qRT-PCR analysis showed that the higher levels of ERp57 mRNA expression were observed in the SW839 cell line,while the lower in A498 cell lines among 6 different ccRCC cell lines.MTT and clone formation experiments showed that,compared with the empty vector,overexpression of ERp57 in A498 cells promoted cell proliferation,while knocked down ERp57 inhibited the proliferation of SW839 cells.In addition,Transwell migration experiments showed that overexpression of ERp57 induced migration of A498 cells,and knockdown of ERp57 in SW839 cells resulted in a significant reduction in cell migration ability.2.Regulatory relationship between ERp57 and ILF3 in ccRCC.Screening the previous report to find the gene ILF3 which were regulated by ERp57 and related to cell proliferation.Immunofluorescence results showed that high expression levels of ERp57 in cytoplasm were associated with high expression of ILF3 in nucleus in ccRCC tissues.qRT-PCR and Western blot results showed that ILF3 was significantly downregulated in ERp57-depleted cells,while was upregulated in ERp57 overexpressing cells.In addition,the results of qRT-PCR,Western blot and IHC showed that the ILF3 expression and level of mRNA in ccRCC tissue was significantly higher than that in adjacent tissues.At the same time,the analysis results of the TCGA database also confirmed that ILF3 expression is higher in ccRCC tissues,and higher expression of ILF3 was associated with poor prognosis.Additionally,analysis of the clinical features of ILF3 in ccRCC found that ILF3 mRNA level significantly associated with tumor size.Correlation analysis found that ERp57 was positively correlated with ILF3 inccRCC tissue.3.The role of ERp57/STAT3/ILF3 feedback pathway in ccRCC cell proliferation.3.1 STAT3 mediates the regulation of ILF3 by ERp57Western blot and qRT-PCR results showed that co-transfection of SW839 cells with sh STAT3 and sh ERp57 significantly enhanced the inhibitory effects on ILF3 compared with knockdown of ERp57 or STAT3 alone.In contrast,transfection of A498 cells with sh STAT3 reduced ILF3 levels,while this reduction effect could be reversed by co-transfection with ERp57 overexpression vector,which demonstrated that STAT3 mediates the relationship between ERp57 and ILF3.Depletion of STAT3 or ERp57 alone suppressed cell proliferation by using Brd U assay.And these suppression effects could be strengthened by knocking down STAT3 and ERp57 together.3.2 ERp57/STAT3 complex regulates the expression of ILF3 at transcription levelCo-immunoprecipitation(CoIP)further confirmed the protein-protein interactions between STAT3 and ERp57.ChIP-q PCR analysis showed that STAT3 and ERp57 protein can be combined with ILF3 promoter region.Luciferase reporter gene analysis found that overexpression of ERp57 markedly increased the activity of the luciferase vector present in the ILF3 promoter.However,the luciferase activity remained unchanged after knockdown of the endogenous STAT3 and transfected with the ERp57 overexpression vector.This indicated that STAT3 directly cross-linked with the ILF3 promoter and regulated ILF3 transcription.3.3 ILF3 regulates ERp57 expression in ccRCCqRT-PCR results found that overexpression of ILF3 promoted ERp57 mRNA levels,while knockdown of ILF3 inhibited ERp57 mRNA level in ccRCC cells.After blocking transcription with Actinomycin D(Act D),qRT-PCR analysis was used to measure changes in ERp57 mRNA levels over time.It was found that depletion of ILF3 inhibited the effect of Act D on the stability of ERp57 mRNA,and overexpression of ILF3 promoted the effect ofAct D.CoIP show that ILF3 antibody can pull down sufficient levels of endogenous ILF3 protein.Then RIP analysis showed that Cyclin E1 and ERp57 mRNA 3'UTR were present in the protein-RNA complex pulled down by ILF3 antibody.Consistently,the biotin oligonucleotide pulldown also revealed that precipitates from the ERp57 mRNA-3'UTR and positive control Cyclin E1-mRNA probes contained ILF3 protein.These results indicated that ILF3 can bind to ERp57 mRNA 3'UTR and enhance its mRNA stability.The results of colony forming assays showed that ccRCC cell proliferation was increased by ILF3 overexpression but decreased by depletion of ILF3.Moreover,overexpression of ILF3 combined with knockdown of ERp57 reversed the increase in cell proliferation induced by ILF3.However,the decrease in cell proliferation due to depletion of ILF3 could only be weakened by overexpression of ERp57.These findings supported that ILF3 was involved in the regulation of ccRCC cell proliferation by promoting ERp57 mRNA stability.3.4 Role of ILF3 / ERp57 / STAT3 feedback loop in ccRCC cell proliferation.SW839 and A498 cells treated with the STAT3 inhibitor,niclosamide.Western blot showed that decreased levels of phosphorylated STAT3(p-STAT3),Cyclin E1,and ILF3,but showed no effect on total STAT3 protein levels.This inhibitory effect can be enhanced by simultaneously knocking down ILF3 in SW839 cells and reversed by overexpressing ILF3 in A498 cells.The results of Brd U staining and clone formation test showed that inhibition of STAT3 or ILF3 can reduce the proliferation of SW839 cells,while the reduction effect can be enhanced by co-transfection of sh STAT3 and sh ILF3.The co-transfection of sh STAT3 and sh ERp57 in SW839 cells can enhance the inhibition of ccRCC cell growth,and the overexpression of ERp57 can reverse the growth inhibition of A498 cells caused by STAT3 knockdown.4.Role of ILF3 / ERp57 / STAT3 pathway in ccRCC cell proliferation in vivoSW839 cells with stably depleted ERp57 or ILF3 alone,or knockdown of both were implanted into nude mice.Compared with the vehicle control group,the tumor volumes were significantly decreased in the sh ERp57 and sh ILF3 groups.Furthermore,compared with depletion of either ERp57 or ILF3 alone,the tumor volume and average wet weight was much smaller in mice implanted with cells which were simultaneously knockdowned of ERp57 and ILF3.Western-blot analysis showed that silencing of either ERp57 or ILF3 alone significantly downregulated the levels of p-STAT3,ERp57,ILF3 and Cyclin E1,was accompanied by an increase in cleaved caspase-3 expression compared with vehicle control.These effects can be enhanced by the combined knockdown of ERp57 and ILF3.Brd U staining showed that knockdown of ERp57 or ILF3 inhibited the proliferation of tumor cells in xenograft tumors,and the combined suppression of ERp57 and ILF3 enhanced this effects.Conclusions:1.ERp57 and ILF3 are overexpressed in renal clear cell carcinoma,high levels of ERp57 and ILF3 are associated with poor patient prognosis.2.ILF3 binds to ERp57 and positively regulates its expression by enhancing its mRNA stability.3.Abnormal regulation of ERp57 by initiating STAT3/ILF3 feedback loop to promote the progress of ccRCC cell proliferation,which is related to the prognosis of ccRCC patients.These findings are the favorable evidence that ERp57 / STAT3 / ILF3 regulate tumor cell metabolism in ccRCC,it may provides a potential therapeutic target for ccRCC treatment.