Effects And Molecular Mechanism Of SUMO Modification OfhnRNPK In Gastric Cancer Cells

Background Heterogeneous nuclear ribonucleoprotein K(hnRNP K)is a nucleic acid-binding protein,which exists in the nucleus and the cytoplasm at the same time.It can participate in chromatin remodeling,transcription,RNA alternative splicing,translation and other life process,and play an important role in the regulation of gene expression.In addition,it was found that hnRNP K was closely related to the development of tumors,and was highly expressed in many tumors.We demonstrated that hnRNP K can regulate the expression of c-myc in gastric cancer cells to promote the development of cancers.We found that hnRNP K mainly distributed in the nucleus in gastric cancer cells,which can regulate the expression of c-myc and promote the development of gastric cancer;and hnRNP K showed high sumo and low ubiquitination,which enhance its stability and play the role of promoting tumor proliferation process.Methods 1.Immunohistochemical method was used to detect the expression of hnRNP K in normal gastric epithelial tissues and gastric cancer tissues.The expression of hnRNP K protein in human normal gastric epithelial cells GES-1 and gastric cancer cells was detected by Western blot method.2.Cell immunofluorescence(CIF)was used to detect distribution of hnRNP K in GES-1,BGC823 and SGC7901 cells respectively;Nuclear and Cytoplasmic Protein Extraction Kit was used to detect hnRNP K protein distribution difference in normal gastric epithelial cell GES-1and gastric cancer cells BGC823,SGC7901.3 The c-myc protein level in GES-1,BGC823 and SGC7901 cells was detected by Western blot.The proliferation and tumorigenicity of BGC823 cells transfected with psin-vector and psin-Flag-hnRNP plasmids were detected by subcutaneous tumor xenograft in nude mice.4.The expression level of hnRNP K mRNA was detected by Real-time by PCR in normal gastric epithelial and gastric carcinoma tissues or cells;stability of hnRNP K was detected by using CHX blocking protein translation;using MG132 and CHX detected effect of ubiquitination modification on hnRNP K metabolism in GES-1 and BGC823 cells,the effect of autophagy hnRNP K metabolism was detected after treated with MG132 and CQ.5.Ub and sumo modification of hnRNP K were detected by using the technique of immune precipitation(IP)in normal gastric epithelial and gastric cancer tissues and cells;degradation pathway of Flag-hnRNP K and Flag-hnRNP K K422 R protein were dected by treated with MG132,CQ and CHX alone or combined use;the stability difference of Flag-hnRNP K protein and Flag-hnRNP K K422 R protein was detected by treated with CHX.6.The distribution of Flag-hnRNP K and Flag-hnRNP K K422 R was detected by CIF technique;the distribution of Flag-hnRNP K and Flag-hnRNP K K422 R were detected by the use of nuclear and cytoplasmic separation technique;the proliferation ability tumorigenicity of BGC823 cells by subcutaneous xenografted tumors assays after transfected with psin-vector,psin-Flag-hnRNP K and psin-Flag-hnRNP K K422 R plasmids.Results 1.The expression of hnRNP K was high in gastric cancer tissues,and the lower the degree of differentiation,the higher the expression of tissue.Compared with GES-1 cel s,the expression of hnRNP K protein was higher in gastric cancer cel s.2.Whether in normal gastric epithelial cells or gastric cancer cells,hnRNP K was mainly distributed in the nucleus;subcutaneous xenotransplanted tumors volume in nude mice was significantly increased in BGC823 cells transfected with Flag-hnRNP K plasmid compared to the vector group,and protein and mRNA levels of c-myc significantly increased in BGC823 cells after transfection of the Flag-hnRNP K plasmid.3.MG132 can antagonist CHX-mediated rapid degradation of hnRNP K to delay its metabolism process;while the use of CQ had no obvious effect on degradation of hnRNP K.4.In normal gastric epithelial and gastric cancer tissues or cells,there is no difference in levels of hnRNP K mRNA;and hnRNP K was mainly degradated through the proteasome pathway.5.Compared with normal gastric epithelial tissue,sumo modification was higher while ub modification was lower in cancer tissues;compared with normal gastric epithelium cells,sumo modification is higher while ub modification was lower in gastric cancer cells;compared with the Flag-hnRNP K K422 R,Flag-hnRNP K protein had higher stability and lower ub modification;Flag-hnRNP K and Flag-hnRNP K K422 R were both degradated by the proteasome pathway.6.Both Flag-hnRNP K and Flag-hnRNP K K422 R proteins were mainly distributed in the nucleus,and both of them could promote the tumor proliferation.Conclusions 1.Compared with normal gastric epithelium cells,hnRNP K show an overexpressed statement both in gastric cancer tissues and cells,which was distributed in the nucleus and overexpression of hnRNP K can promote tumor growth process;compared with the control group,xenograft tumor volume increased significantly.2.hnRNP K showed the high sumo modification and low ub modification state and mainly degraded by the ubiquitin proteasome pathway in gastric cancer cells;distribution of hnRNP K did not change after sumo site was mutated and played a role in promoting tumor development in a desumoylated stated.