RNF128 Promotes The Invasion And Metastasis Of Hepatocellular Carcinoma Via EGFR/MEK/ERK Signaling Pathway

Background: The ubiquitin-proteasome system(UPS)plays an important role in maintaining and regulating cell homeostasis and participates in a variety of intracellular pathological and physiological processes.As the core component of the ubiquitinproteasome system to recognize substrates,E3 ubiquitin ligase participating in the occurrence and development of a variety of tumors has attracted much attention.The RING-finger protein family is the largest family of E3 ubiquitin ligases,which contain an RNF domain.Recently,the RING-finger protein family has been found to play a very important role in tumorigenesis and progression.Hepatocellular carcinoma(HCC),as one of the most common malignant tumors,has very poor prognosis due to its high ability to invade and metastasize.In addition to surgical treatment,there is still a lack of other effective treatments.Therefore,it is of great clinical significance to explore the potential molecular mechanism of HCC and find the promising therapeutic targets from the RNF family.Materials and Methods: First,we determined the expression differences of RNF128 protein and mRNA levels in HCC tissues and normal tissues adjacent to cancer by Western blot and quantitative real time polymerase chain reaction(q RT-PCR).Then,we performed immunohistochemical staining on tissue microarrays of 171 HCC patients to analyze correlation between the expression of RNF128 and clinical outcomes in HCC.Furthermore,we knocked down and overexpressed RNF128 in HCC cell lines with different metastatic potentials by transfecting lentivirus.After selecting with puromycin,the transfection efficiency was detected by Western blot to establish a stable transfected cell line.We detected the impact of RNF128 on cell proliferation by CCK-8 and clone formation assay.Wound-healing test and Transwell test were used to detect its effect on cell invasion and migration.Flow cytometry was used to detect its effect on cell apoptosis.We then established transplanted tumor models by subcutaneously injecting stable transfected cell lines into nude mice,and tested the effect of RNF128 on the growth of transplanted tumors in vivo.Finally,a bioinformatics platform(Gene Expression Profiling Interactive Analysis,GEPAI)was used to identify EGFR / MEK / ERK as the possible targeting pathway for impacts of RNF128 on HCC.The EGFR pathway inhibitor gefitinib was used to further clarify that effects of RNF128 on HCC is induced via activating the EFGR/MEK/ERK pathway.Results: RNF128 expression was found to be remarkably elevated in HCC tissues compared with adjacent normal tissues,both in the protein and mRNA levels.In addition,the high expression of RNF128 is significantly correlated to clinical pathological characteristics such as tumor size,tumor encapsulation,Edmondson-Steiner Grade and advanced TNM stage.Kaplan-Meier analysis revealed that HCC patients with high RNF128 expression tended to herald a shorter survival time and a higher recurrence rate compared with low RNF128 expression.Univariate and multivariate analyses showed that RNF128 expression level is an independent risk factor for HCC prognosis.Overexpression of RNF128 enhanced the proliferation,migration,invasion and apoptosis resistance of HCC cells in vitro and in vivo,at the same time increased the phosphorylation levels of EGFR,MEK and ERK,and vice versa.We further found that Gefitinib can inhibit the expression levels of p-EGFR,p-MEK and p-ERK up-regulated by RNF128.In addition,the inhibitory effect of gefitinib on EGFR / MEK / ERK signaling counteracts the enhancement on migration,invasion and proliferation caused by RNF128 overexpression.Conclusion: RNF128 promotes HCC progression by activating EGFR / MEK / ERK signaling pathway and is correlated with the poor prognosis of HCC patients,which might function as a novel prognostic molecular signature with the potential to be a candidate therapeutic target for HCC patients.