RNF180 Inhibits The Proliferation And Metastasis Of Gastric Cancer Through The Ubiquitination Of DNMT1

Backgrounds:Gastric cancer represents a global health-care challenge.At present,due to the lack of effective screening for the early detection of gastric cancer,and the mild symptoms usually exhibited by these patients,most are initially diagnosed with advanced gastric cancer.While the overall incidence and mortality of gastric cancer has declined over the past several decades and progress has been achieved in diagnostic and therapeutic strategies,prognosis has only incrementally improved,even for patients with localized disease at diagnosis.Therefore,a deeper understanding of the development and progression of gastric cancer and expounding the possible molecular mechanisms involved in malignant biological behaviors of cancer cells may reveal more opportunities for early diagnosis,drug research and development and precise targeted therapeutics.A fundamental trait of virtually all gastrointestinal cancers is genomic and epigenomic DNA alterations.Cancer cells acquire genetic and epigenetic alterations that drive the initiation and progression of the cancers by altering the molecular and cell biological processes of the cells.The discovery of hypermethylation in the promoter regions of TSGs suggests that epimutation may function as a driver of oncogenesis.Altered activity of key regulatory proteins is the driver of tumorigenesis and progression and the consequential acquisition of the malignant phenotypes.Posttranslational modifications(PTMs)are essential mechanisms that dynamically regulate protein functional activity.A key mechanism of post-translational modification is ubiquitination by the ubiquitin-proteasome system(UPS)and E3 s are the most heterogeneous class of enzymes in the ubiquitination pathway,as they mediate substrate specificity.Currently,E3 ligases can regulate numerous cellular processes,including homeostasis,metabolism and cell cycle progression.Consequently,the stability and/or activity of E3 substrates are also altered,in some cases leading to downregulation of tumor-suppressor activities and upregulation of oncogenic activities.Therefore,a deeper,context-dependent understanding of the role for E3 ligases in tumorigenesis and their underlying mechanisms might contribute to find new biomarkers and precise treatment.As an important human tumor suppressor gene family,RNF(Ring Finger Protein)family genes involve a variety of biological processes,including DNA repair,gene transcription and cell apoptosis.RNF180,RING finger motif proteins,is located on chromosome 5(5q12.3)and displays structural hallmarks of a RING finger ubiquitin ligase.Moreover,it can regulate substrate protein stability by inducing K48-linked polyubiquitination and proteasome-dependent degradation.We have previously shown that RNF180 is a novel potential tumor suppressor in gastric carcinogenesis and has potential clinical utility as a biomarker for gastric cancer patients.However,the mechanisms underlying the methylation of RNF180 and DNA methylation-mediated gastric carcinogenesis and progression still remain obscure.Aims:1.To reveal the relationship between RNF180 expression and the development of gastric cancer.2.To validate the role of RNF180 in gastric cancer cell proliferation and metastasis.3.To elucidate the molecular mechanisms underlying how RNF180 inhibits gastric cancer proliferation and metastasis.4.To explore a new therapeutic targeting RNF180 DNA methylation for GC patients.Methods:1.The expression of RNF180 in gastric cancer was detected and the relationship between its expression and the clinicopathological characteristics and prognosis of GC patients was also evaluated.Gene expression profiles and clinical information of GC patients extracted from public databases were used to analyze the expression levels of RNF180 in cancer and matched normal tissues and further assess the association of RNF180 expression with the clinicopathological characteristics and prognosis in patients with GC.The expression level of RNF180 in human gastric cancer,paired normal tissue samples and gastric cancer cells were was detected using Western blotting,Immunohistochemistry and RT-q PCR assays.And the correlation of RNF180 expression with clinicopathologic parameters in patients with GC in IHC database was validated.2.The role of RFN180 in GC proliferation and metastasis is evaluated.The RNF180 overexpression and sh RNA expression lentiviral vector was constructed.To obtain loss-offunction and gain-of-function cell models,we transfected gastric cancer cells with relevant RNF180 sh RNA or specific over-expressing lentivirus,respectively.Cell Counting Kit-8(CCK8)and clone formation assay was used to measure cell proliferation ability and the apoptosis and cell cycle distribution were detected by flow cytometry.To evaluate the effect of RNF180 on cancer cell proliferation in vivo,xenograft experiments in nude mice were performed.The migration and invasion ability were assessed using the transwell assays.Further,we used nude mouse tail vein metastasis model to access the metastatic ability of the HCC cells in vivo.The “tail vein–lung metastasis” nude mice model was constructed to access the metastatic ability of the GC cells in vivo.3.The key molecules that mediate aberrant DNA methylation of RNF180 is identified.Gene expression profiles,DNA methylation and clinical information of GC patients extracted from public databases were used to analyze the methylation levels of RNF180 in tumor tissues,matched normal tissues and gastric cancer cells.The correlation between RNF180 expression level with DNA methyltransferases(DNMTs)expression was analyzed.To obtain loss-of-function cell models,we transfected gastric cancer cells with relevant DNMT1,DNMT3 A and DNMT3 B sh RNA lentivirus,respectively.The DNA methylation level of RNF180 after DNMTs knockdown was measured by NGS sequencing.Expression levels of RNF180 proteins after DNMTs knockdown were estimated by Western blot and RT-q PCR.4.The molecular mechanisms underlying the inhibition of cell proliferation and metastasis by RNF180 were explored.i TRAQ proteomic screening was used to identify potential RNF180 interacting proteins.To obtain loss-of-function and gain-of-function cell models,we transfected gastric cancer cells with relevant RNF180 sh RNA or specific overexpressing lentivirus,respectively.The DNMT1 m RNA and protein expression levels after RNF180 overexpression or knockdown were verified by Western blot and RT-q PCR.The rescue experiments were further carried out to further characterize the regulatory effects of RNF180 upon DNMT1.The subsequent Immunofluorescence staining and Co-IP experiments further validated the direct binding between RNF180 and DNMT1.The subsequent Co-IP in combination with Western blot experiments were further performed to assess the ubiquitination level of DNMT1 after RNF180 knockdown.Using IHC on gastric cancer tissue microarrays,we evaluated the protein expression of RNF180 and DNMT1 in human gastric cancer and normal tissue samples and analyzed their clinical and prognostic relevance.xenograft experiments in nude mice were performed to further validate the ability of tumor proliferation after RNF180 overexpression and cooverexpression of RNF180 and DNMT1.5.To explore a new therapeutic targeting RNF180 DNA methylation for GC patients.CCK8,RT-q PCR,Western blot,ubiquitination IP experiments and other technologies were used to investigate the effect of decitabine on the proliferation of gastric cancer cells and the action mechanisms.Results:1.The expression levels of RNF180 were decreased in GC tissues,compared with para-cancer normal tissues,and the correlation analysis suggested that the low expression level of RNF180 in gastric cancer tissues was associated with the malignant clinicopathological characteristics and poor prognosis of gastric cancer patients.2.The results show RNF180 overexpression can could effectively impede gastric cell proliferation,induce phase cell cycle arrest and promote cell apoptosis in vitro.Furthermore,the overexpression of RNF180 was shown to cause significant inhibition of cell migration and invasion in gastric cancer cells.The xenograft nude mouse model and“tail vein–lung metastasis” nude mouse model experiments the overexpression of RNF180 can suppress proliferation and metastasis of gastric cancer in vivo.3.Bioinformatics analysis combined with WB,q PCR and NGS methylation sequencing showed that DNA methylation of RNF180 is directly regulated by DNMT1.The results showed that downregulation of RFN180 due to DNA hypermethylation was mainly mediated by DNMT1.4.The results showed that RNF180 could positively regulates DNMT1 ubiquitination and promote its proteasome-dependent degradation.These subsequently resulted in a decrease of DNMT1 and potentially affect tumor development.5.The in vivo and vitro experiments revealed that low-dose decitabine could impede gastric cancer cell proliferation.Further experiments showed that decitabine treatment induced the increased RNF180 expression by inhibition of DNA methylation and further promote DNMT1 ubiquitination and degradation.Conclusion:RNF180 was lowly expressed in GC tissues,which was closely associated with clinical characteristics and survival outcomes in GC patients.Additionally,RNF180 as a new diagnostic marker for gastric cancer,can promote DNMT1 ubiquitination and degradation and inhibit DNMT1 as well as DNA methylation,which in turn can affect gastric tumorigenesis and development.Pharmacologic disruption of the RNF180-DNMT1 interaction shed important new light on gastric cancer clinical treatment.