| BackgroundGastric cancer is a malignant tumor derived from gastric mucosal epithelium.In recent years,the incidence of gastric cancer has declined globally.However,the incidence and mortality of gastric cancer in China rank the second and that remains a major threat to human healthy.The occurrence and development of gastric cancer is a complex and multi-factor involved process,and the abnormal regulation of gene expression plays a key role in the occurrence of gastric cancer.The regulation mainly occurs at the level of transcription and translation,and the proteins encoded by genes can directly or indirectly act on signal pathways that regulate cell proliferation,invasion and migration.At present,the regulation of protein translation levels,especially the study of the translation initial step,has demonstrated that eukaryotic initiation factors(eIFs)play a key role in protein translation,cell cycle regulation and the development of tumors.The eukaryotic translation initiation factor eIF3 is the most complex and most important multi-subunit complex in eIFs and consists of 13 subunits(eIF-3a,-3b,-3c...-3m).eIF3 binds to 40S ribosomal subunit through the interactions with other translation initiation factors,which promotes the formation of 43 S initiation complex precursors,further binds to mRNA,recognizes the initiation codon AUG,and plays a key role in translation initiation.This function is mainly based on the subunits of eIF3,which contain eIF3a,eIF3b,eIF3e,eIF3f,eIF3h and eIF3i.The study found that these 6 subunits are abnormally expressed in many tumors,which are closely related to the progression of tumor.EIF3b is considered to be the main scaffolding subunit in the eIF3 complex.Human eIF3b is a protein with an RNA recognition sequence(RRM,located at the N-terminus).The RRM domain can provide a specific site to interact with eIF3J.At present,a large number of studies suggest that eIF3b is involved in protein translation,cell cycle regulation and tumor development.The eIF3b subunit has been shown to be overexpressed in prostate and bladder cancers,and the overexpression is associated with cancer prognosis;silencing of eIF3b can inhibit the proliferation of colon cancer cells and glioblastoma cells;eIF3b are abnormally expressed and play an important role in the invasion and migration of cancer cells both in osteosarcoma,esophagus squamous cell carcinoma and renal cell carcinoma.However,the expression of eIF3b in gastric cancer and its role in the development of gastric cancer have not yet been reported.Therefore,this study investigated the expression of eIF3b in gastric cancer and its role in the development of gastric cancer.AimsTo investigate the expression of eIF3b in gastric cancer cells and tissues and its effect on the proliferation,invasion and migration of gastric cancer cells,and to explore the potential value of eIF3b as a diagnostic marker and therapeutic target of gastric cancer.Experimental Methods1.The effect of eIF3b on proliferation,invasion and migration of gastric cancer cellsQRT-PCR was used to detect the expression of eIF3b mRNA in different gastric cancer cells(SGC7901,BGC823,MGC803,AGS and HGC27).The small interfering RNA of eIF3b was transfected into SGC7901 and MGC803,the interference effect of eIF3b was detected by QRT-PCR and Western blot.The effect of downregulating eIF3b on proliferation of gastric cancer cells was determined by colony formation assay and CCK8 assay.The effect of eIF3b on the migration of gastric cancer cells was detected by Transwell assay and cell scratch assay.The effect of eIF3b on the invasion of gastric cancer cells was detected by Transwell matrigel invasion assay.2.Animal experiments to detect the effect of eIF3b on proliferation and migration of gastric cancer cells in vivoGastric cancer cells SGC7901 were infected with eIF3b interference Lentivirus vector which can effectively integrate eIF3b siRNA sequence into the host chromosome,thereby effectively reducing the expression of eIF3b,and the interference effect was detected by QRT-PCR.Nude mice were divided into two groups,control group and the interference group.Control group and the interference group were further divided into subcutaneous injection group and tail vein injection group.SGC7901 cells treated by lentiviral-negative control or lentiviral-eIF3b were injected subcutaneously or into caudal veins of nude mice.Then we observed the subcutaneous tumorigenesis and tumor metastasis in nude mice,and the effect of eIF3b on proliferation,invasion and migration of gastric cancer cells.3.The regulation of eIF3b on the expression of key genes in the signaling pathways related to gastric cancerGastric cancer cells SGC7901 and MGC803 were infected lentivirus vectors carrying eIF3b interference sequence,and Western blot and ELISA were used to detect the expression of important genes in the signaling pathways involved in proliferation,invasion,and migration of gastric cancer cells,including P53,p27,E2F1,AKT,IL-8,Vimentin,CyclinE,CyclinD,β-catenin and so on.Further more,we selected the molecule with more obvious changes,constructed highly expressed plasmid,and used rescuing experiments including clone formation and Transwell experiments confirming that the down-regulation of eIF3b can directly or indirectly regulate the expression of important genes in the signaling pathways related to gastric cancer,thus affecting the proliferation and metastasis of gastric cancer cells.4.Expression of eIF3b in human chronic gastritis and gastric cancer tissue specimensQRT-PCR was used to detect the expression of eIF3b mRNA in 39 specimens of chronic gastritis and normal gastric mucosa,and 46 pairs specimens of gastric cancer and adjacent normal tissues.Immunohistochemistry was used to detect the protein level of eIF3b in gastric cancer tissue specimens.The correlation between the expression of eIF3b and the related pathological parameters in gastric cancer patients was analyzed to assess whether it could be used as a potential diagnostic marker for gastric cancer.5.The effect of Helicobacter pylori on regulation of eIF3b in gastric cancer cellsAGS cells were infected with Hp11637 and Hp26695 according to MOI 1:100,and PBS was added to the control group.The cells were harvested on 6h,12h and 24h after infection,and QRT-PCR was performed to detect the expression of eIF3b mRNA.AGS cells were infected with Hp11637 and Hp26695 according with MOI 1:50,1:100 and 1:150 respectively.The cells were collected after 12 hours,and the mRNA and protein levels of eIF3b in each group were detected by QRT-PCR and Western blot.Results1.Downregulation of eIF3b inhibits proliferation,invasion and migration of gastric cancer cellsThe results of QRT-PCR showed that the expression of eIF3b mRNA was significantly higher in gastric cancer cells SGC7901 and MGC803 than in other gastric cancer cells.Gastric cancer cells SGC7901 and MGC803 were transfected with specific siRNA-eIF3b.Compared with control group,the expression of eIF3b mRNA and protein in the experimental group was significantly decreased.Colony formation assay showed that downregulating eIF3b significantly inhibited the colony formation of gastric cancer cells SGC7901 and MGC803.CCK8 assay showed that downregulating eIF3b significantly inhibited the proliferation of SGC7901 and MGC803.Transwell assay and cell scratch assay showed that downregulating eIF3b significantly inhibited the migration of gastric cancer cells SGC7901 and MGC803.Transwell invasion assay showed that downregulating eIF3b significantly inhibited the invasion of gastric cancer cell SGC7901 and MGC803.2.Downregulation of eIF3b inhibits proliferation and metastasis of gastriccancer cells in vivoLentivirus-eIF3b can effectively downregulate the expression of eIF3b mRNA in SGC7901 cells,which was detected by QRT-PCR and Western blot.Nude mice were injected with gastric cancer cells subcutaneously,after 5 days,tiny tumors were observed in the control group.After 9 days,the size of tumors were measured,and after 19 days,the nude mice were sacrificed.The growth curve of tumors showed that downregulation of eIF3b expression inhibited the proliferation of tumor in nude mice compared with the control group.Nude mice injected by tail vein were sacrificed two months later,the lung tissues were taken out.The control group showed more severe lung metastasis than the experimental group by using the small animal optical imager.The number of tumor nodules in the control group was more than that in the experimental group and the lung volume was also larger in the control group.HE staining showed that the infiltration of cancer cells in control group was significantly more than that of the experimental group in the lungs of nude mice.3.eIF3b regulates the expression of key genes in the signaling pathways related to gastric cancerWestern blot results showed that down-regulation of eIF3b inhibited the expression of E2F1,CyclinE,CyclinD,Vinentin,β-catenin and increased the expression of tumor suppressor gene P27 in SGC7901 and MGC803 cells.ELISA results showed that down-regulating the expression of eIF3b inhibited the expression of IL-8 in SGC7901 cells and increased the expression of IL-8 in MGC803 cells,which were directly or indirectly involved in the proliferation and metastasis of gastric cancer cells.The results of rescuing experiments showed that up-regulation of E2F1 could antagonize the role of eIF3b in the proliferation and migration of SGC7901 and MGC803 cells.4.Overexpression of eIF3b in human chronic gastritis and gastric cancer tissue specimensQRT-PCR results showed that eIF3b mRNA expression levels were higher in human chronic gastritis and gastric cancer tissues than in normal gastric tissues.Immunohistochemistry results showed that eIF3b protein expression was higher in gastric cancer tissues than in adjacent normal tissues and eIF3b was mainly expressed in the cytoplasm.And higher eIF3b expression was correlated with advanced stages of gastric cancer.5.Hp11637 and Hp26695 can upregulate eIF3b expression in gastric cancer cellsQRT-PCR results showed that eIF3b expression level in AGS cells was upregulated by Hp11637 and Hp26695 infection,especially at 12h after infection.Then AGS cells were infected by Hp11637 and Hp26695 with MOI 1:50,1:100 and 1:150 for 12h respectively,and the mRNA and protein levels of eIF3b were found to be upregulated gradually.Conclusion1.EIF3b promotes the proliferation,migration and invasion of gastric cancer cells by regulating cancer-related genes in vitro.2.EIF3b promotes the proliferation and metastasis of gastric cancer cells in vivo.3.The expression of eIF3b in human chronic gastritis and gastric cancer tissues is elevated and correlates with the staging of gastric cancer,suggesting that eIF3b may serve as a new diagnostic marker for gastric cancer.4.Helicobacter pylori infection can upregulate the expression of eIF3b in gastric cancer cells,suggesting that eIF3b may be involved in the carcinogenic process of Helicobacter pylori. |
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