The Role And Mechanism Of RNA-binding Protein NELFE In Gastric Cancer

RNA-binding proteins(RBPs)are a class of proteins that can bind to RNA to exert their biological activities and play an important role in post-transcriptional regulation.Abnormally expressed RNA-binding proteins are closely related to the occurrence and development of tumors.Abnormal expression of RNA-binding proteins in tumors is associated with genomic alterations,post-transcriptional regulation,and post-translational regulation.As upstream factors which regulate the expression,modification,stability of downstream RNAs,even very small changes can result in significant effects on downstream genes.At present,there are still many unknowns about the mechanism by which RNA-binding proteins regulate the occurrence and development of downstream RNAs in tumors,which deserves further study.NELFE is a member of the RNA-binding protein family,and there are few reports on its function in cancers.In previous studies in our laboratory,we found that NELFE showed an obvious tumor-promoting effect in liver cancer by regulating downstream RNAs.Therefore,we detected gastric cancer tissues and found that NELFE is also highly expressed in gastric cancer.After transfection of NELFE si RNA in gastric cancer cells,proliferation and metastasis were significantly inhibited,which suggested that NELFE may also play important roles in the occurrence and development of gastric cancer.In this experiment,we intend to study the mechanism of NELFE in the occurrence and development of gastric cancers to find new targets for the treatment of gastric cancer,and provide more theoretical basis for the clinical treatment of gastric cancer.Part Ⅰ:NELFE promotes proliferation and metastasis of gastric cancersThrough the detection of tissue samples,the expression of NELFE in gastric cancer and adjacent normal tissues were detected and the relationship between NELFE expression and clinicopathological parameters were analyzed.The effect of NELFE on the function of gastric cancer cells was explored in gastric cancer cells.The effect of tumorigenic and metastatic ability of NELFE on gastric cancer cells was verified in nude mice.Methods:1.The expression of NELFE in 32 pairs of fresh gastric cancer tissues and matched normal gastric mucosa was detected by real-time quantitative PCR(q RT-PCR)and western blot,and the relationship between NELFE and gastric cancer was preliminarily explored.Immunohistochemistry was used to detect the pathological sections of 224 pairs of gastric cancer patients.Immunohistochemical score was used to detect the expression intensity of NELFE to analyze the relationship between the expression of NELFE and clinicopathological parameters,and the relationship between the expression of NELFE and the prognosis of gastric cancer patients.2.Western blot was used to detect the expression of NELFE in gastric cancer cells and appropriate cells were selected for functional verification.NELFE interference lentivirus was constructed to establish gastric cancer cells with stable low expression of NELFE.We used gastric cancer with stable low expression of NELFE to perform functional experiments in vitro.MTT assays,clone formation assays,and transwell assays were conducted to verify the effect of knockdown of NELFE on cell proliferation and metastasis.3.The nude mouse model was constructed by using the cell line with low expression of NELFE.In the subcutaneous tumorigenesis experiment,the size and weight of the tumor were measured to evaluate the effect of NELFE on tumor growth.Lung metastasis was constructed by tail vein injection,and the effect of NELFE knockout on gastric cancer metastasis in vivo was observed.Results:1.In fresh gastric cancer tissue and paired normal gastric mucosa,the RNA and protein levels of NELFE were increased in gastric cancer tissue.The results of immunohistochemistry showed that the proportion of strong expression of NELFE in gastric cancer tissue was higher,and the proportion of strong expression of NELFE in normal gastric tissue was lower and the proportion of weak expression was higher.The expression of NELFE is related to the size and stage of the tumor.The larger the tumor and the more advanced the stage,the higher the expression of NELFE.Follow-up results showed that patients with higher NELFE expression had lower rates of both overall survival(OS)and progression-free survival(RFS).2.The expression of NELFE in six gastric cancer cell lines(BGC-823,AGS,SGC-7901,HGC-27,MKN-45 and MGC-803)was detected by western blot,and NELFE is highest expressed in BGC-823 and AGS.Immunofluorescence experiments showed that NELFE was distributed in both nucleus and cytoplasm.It is more distributed in the cytoplasm,which provides a basis for the later study of the mechanism of NELFE.MTT assay showed that the proliferation ability of gastric cancer cells was significantly weakened after reducing the expression of NELFE.The colony formation assay showed that the number of colonies formed by the cells decreased significantly after the expression of NELFE was reduced.Transwell assay showed that the invasive and metastatic ability of gastric cancer cells was weakened after the expression of NELFE was reduced.3.In vivo experiments,the subcutaneous tumorigenic experiments showed that the tumorigenic ability of gastric cancer cells was significantly weakened after knockdown of NELFE.Gastric cancer cells were injected into the tail vein,and the lung metastatic ability of gastric cancer cells was found to be attenuated after NELFE knockout.Conclusions:In this part,fresh specimens and pathological sections were used to find that NELFE was highly expressed in gastric cancer,and high expression of NELFE was associated with pathological features and prognosis.Both in vitro and in vivo functional experiments showed that knockdown of NELFE could inhibit the proliferation and metastasis of gastric cancer in vitro and in vivo,indicating that NELFE is a oncogene in gastric cancer and can promote the occurrence and development of gastric cancer.Part Ⅱ:NELFE promotes gastric cancer progression by stabilizing RNA of E2F2To explore the downstream regulatory genes of NELFE,we used RNA sequencing to search for changes in gene expression after knocking down NELFE.RNA decay assay,RIP assay,pulldown assay,and luciferase assay were used to reveal the regulation of NELFE on the downstream gene E2F2.Methods:1.BGC-823 and AGS cells with stable low expression of NELFE were sent to two companies for RNA-seq.The sequencing results of the two cells were intersected,and the downstream target genes were screened out.PCR and western blot were used to verify the RNA and protein levels of E2F2 in stable low-expressing cells.2.RNA decay assay was used to detect the effect of NELFE knockout on the RNA protection of E2F2.RNA-binding protein immunoprecipitation assay was used to verify the binding of NELFE to downstream genes;luciferase assay and pulldown assay were used to verify the specific binding of NELFE to the 3’UTR of downstream genes.3.Immunohistochemistry was used to detect the pathological sections of 20 pairs of gastric cancer patients.The expression of E2F2 in 32 pairs of fresh gastric cancer tissues and matched normal gastric mucosa was detected by real-time quantitative PCR to explore the correlation between NELFE and E2F2 expression in gastric cancer tissues.Results:1.Sequencing results showed that there were 23 genes whose expression decreased in BGC-823 and AGS cells after knocking down NELFE.Among them,the E2F2 gene drew our attention.The results of real-time fluorescence q PCR confirmed that the expression of E2F2 decreased significantly when NELFE was knocked down.The western blot results also confirmed that the protein expression of E2F2 decreased significantly when NELFE was knocked down.2.The m RNA decay rate of E2F2 decreased dramatically in both NELFE-knockdown cells.Depletion of NELFE significantly decreased the luciferase activity of the E2F2 3’UTR reporter.Moreover,the biotin-labeled E2F2 3’UTR could bind to the NELFE protein,while other regions and mutants labeled with biotin could not bind to the NELFE protein.In the RIP assay,the anti-NELFE antibody dramatically enriched E2F2 m RNA compared with the control Ig G.Thus,E2F2 is regulated by NELFE through post-transcriptional 3’UTR binding.Conclusions:In this part,the downstream gene E2F2 of NELFE was screened by RNA-seq.Mechanistically,it was confirmed that NELFE stabilizes its RNA by binding to the3’UTR of the E2F2 m RNA.Part Ⅲ:E2F2 promotes proliferation and metastasis of gastric cancersThe effect of E2F2 on the function of gastric cancer cells was explored in gastric cancer cells.The effect of tumorigenic and metastatic ability of E2F22 on gastric cancer cells was verified in nude mice.Methods:Western blot was used to detect the expression of E2F2 in gastric cancer cells and appropriate cells were selected for functional verification.We used gastric cancer with stable low expression of E2F2 to perform functional experiments in vitro.MTT assay,clone formation assay,and transwell assay were conducted to verify the effect of knockdown of E2F2 on cell proliferation and metastasis.2.The nude mouse model was constructed by using the cell line with low expression of E2F2.In the subcutaneous tumorigenesis experiment,the size and weight of the tumor were measured to evaluate the effect of E2F2 on tumor growth.Lung metastasis was constructed by tail vein injection,and the effect of E2F2 knockout on gastric cancer metastasis in vivo was observed.3.Rescue experiment: E2F2 was highly expressed in cells with stable low expression of NELFE,and the rescue of E2F2 on the proliferation and metastasis of cells after knockout of NELFE was verified by MTT assay,clone formation assay,and transwell assay.Results:1.The expression of E2F2 in six gastric cancer cell lines(BGC-823,AGS,SGC-7901,HGC-27,MKN-45 and MGC-803)was detected by western blot,and E2F2 is highest expressed in BGC-823 and AGS.MTT assay showed that the proliferation ability of gastric cancer cells was significantly weakened after reducing the expression of E2F2.The colony formation assay showed that the number of colonies formed by the cells decreased significantly after the expression of E2F2 was reduced.Transwell assay showed that the invasive and metastatic ability of gastric cancer cells was weakened after the expression of E2F2 was reduced.2.In vivo experiments,the subcutaneous tumorigenic experiments showed that the tumorigenic ability of gastric cancer cells was significantly weakened after knockdown of E2F2.Gastric cancer cells were injected into the tail vein,and the lung metastatic ability of gastric cancer cells was found to be attenuated after E2F2 knockout.3.Rescue experiment: knockdown of NELFE dramatically decreased the total number,viability,colony formation,migration and invasion of cells.In addition,these decreases were abated by the forced expression of E2F2.Conclusions:Independent functional experiments showed that knockdown of E2F2 could inhibit the proliferation and metastasis of gastric cancer in vitro and in vivo.Rescue experiment showed E2F2 partially rescues the inhibition of cellular function caused by NELFE knockdown.In conclusion,we concluded that NELFE has the function of promoting the proliferation and metastasis of gastric cancer,and its cancer-promoting effect is achieved by regulating the stability of E2F2 m RNA and promoting the up-expression of E2F2.