RNF180-mediated DNMT3A Restores ADAMTS9 Expression Suppressing Lymph Node Metastasis In Gastric Cancer

BackgroundA disintegrin-like and metalloproteinase with thrombospondin motifs 9(ADAMTS9)which is the member of the ADAMTS family,has been reported to take part in multistep progression of tumor carcinogenesis and development.And even,the expression of ADAMTS9 is associated with patients’ survival outcomes in several types of cancer.In present study,we aimed to identify ADAMTS9 as the potential target for prediction of lymph node(LN)metastasis and prognosis in gastric cancer(GC)patients,and investigated the potential upstream and downstream molecular signal pathway.Methods1、16 pairs of gastric cancer tissue and matched adjacent non-tumor tissues which were randomly selected after gastrectomy between July 2018 and December 2018.Then,total RNA was extracted from these tissues.The m RNA expression level of ADAMTS9 was detected by Semi-quantitative reverse transcription and polymerase chain react(Sq RT-PCR).We retrospectively collected the clinicopathological data of 16 cases of GC and analyzed the relationship between the m RNA expression level and clinical parameters.2、150 pairs of gastric cancer tissue and matched adjacent non-tumor tissues were used for the preparation of the tissue microarrays(TMAs).Immunohistochemical staining was performed to detect the protein expression and histoscore system(H-score system)was adopted to assess the expression level of ADAMTTS9.Then,we analyzed the relationship between the protein expression level of ADAMTS9 and clinical characteristics and performed the survival analysis.3、The p CDNA3.1-ADAMTS9 plasmid were used to enhance the expression of ADAMTS9 in gastric cancer cell lines(AGS and BGC-823).The biological functional experiments were performed to detect the abilities of proliferation,migration and invasion.4、We performed genome-wide m RNA sequencing analysis to elucidate the downstream molecular mechanism of ADAMTS9.The potential downstream differentially expressed genes and potential signal pathways were further validated through using q PCR,Western Blot and immunohistochemical staining.5 、 After 5-Aza-2’-deoxycytidine(5Aza)treatment or transfection with sh DNMT1,sh DNMT3 A,sh DNMT3 B plasmid or RNF180-overexpressed plasmids,the expression of ADAMTS9 in gastric cancer cells were detected through using Sq RT-PCR and Western Blot.In addition,Mass ARRAY analysis was adopted to examine the methylation of ADAMTS9 promoter.6、Incubating the gastric cancer cells with cycloheximide(CHX)or MG132 proteasome inhibitor,we examined the potential substrate of RNF180(Ring finger protein 180,RNF180)to clarify the mechanism of inducing ADAMTS9 through Western Blot and Co-IP(Co-Immunoprecipitation,Co-IP).Results1、9/16 of gastric tumor tissues presented the lower m RNA expression level compared with that of adjacent non-tumor tissues.Patients with lower m RNA expression of ADAMTS9 had more advanced p N stage(P=0.044)and more metastatic LNs(P=0.024),compared with those with high m RNA expression2、With the immunohistochemistry staining,the H-Score of gastric tumor tissues was significantly lower than that of the adjacent non-tumor tissue(P=0.008).Patients with the lower ADAMTS9 expression presented more risks with more advanced p N stage(p=0.018)and more metastatic LNs(P=0.007).The survival analysis showed that the expression level of ADAMTS9 was identified as the independent risk factor to evaluate the prognosis of patients with lowest AIC(Akaike information criterion,AIC=71.55)and BIC(Bayesian Information Criterion,BIC=88.714).3 、 Results of biological functional experiments showed that ADAMTS9 attenuates viability and motile capacity of GC cells.And animal model showed ADAMTS9 inhibits tumor growth in vivo.4、With the m RNA sequencing and KEGG analysis to,CCL5 and CXCL11 were recognized as the key molecules to play its biological function.ADAMTS9 might suppress LN metastasis via CCL5/CXCL11-dependent pathway in GC.With q PCR and Western Blot results,ADAMTS9 downregulated the expression level of CCL5 and CXCL11.The immunohistochemistry staining of TMAs presented a significant negative relationship between ADAMTS9 and CCL5(Pearson r=-0.308,P=0.001),and between ADAMTS9 and CXCL11(Pearson r=-0.501,P<0.001).5、With Sq RT-PCR,Western Blot and Mass ARRAY analysis,we validated the hypermethylation of ADAMTS9 promoter in GC.DNMT1 and DNMT3 A may be the main methyltransferases to regulate the methylation level of ADAMTS9,especially for DNMT3 A.6、The IHC results of TMAs showed a significant positive correlation between RNF180 and ADAMTS9 expression,which was in agreement with the correlation analysis results from the GEPIA database(http://gepia.cancer-pku.cn/).The results of Sq RT-PCR,Western Blot and Mass ARRAY analysis showed that RNF180 enhances ADAMTS9 expression through demethylating ADAMTS9 promoter.Then,we incubated GC cells with CHX to block the protein synthesis or incubated GC cells with MG132 to inhibit protein degradation.The results showed RNF180 promotes the degradation of DNMT3 A protein and reduce their half-life.With Co-IP results,DNMT3 A was demonstrated as a substrate to directly interact with RNF180.Conclusions1、Patients with the lower ADAMTS9 expression presents more risks with more advanced p N stage,more metastatic LNs,and the poorer survival outcomes.ADAMTS9 may be identified as the potential target for prediction of LN metastasis and prognosis in gastric cancer2、ADAMTS9 attenuates viability and motile capacity of GC cells.3、ADAMTS9 suppresses LN metastasis via CCL5/CXCL11-dependent pathway in GC.4、DNMT1 and DNMT3 A are the main methyltransferases to upregulate the methylation level of ADAMTS9.5、DNMT3A is demonstrated as a substrate to directly interact with RNF180.RNF180 enhances ADAMTS9 expression through demethylating ADAMTS9 promoter.