| Objective:With economic development,people's lifestyle changes,environmental pollution,and poor dietary habits,the incidence of malignant tumors has increased year by year,threatening human health and affecting people's quality of life.At present,malignant tumors are still the leading cause of death for global people.The traditional treatment methods for malignant tumors include surgical treatment,radiation therapy,and chemical drug treatment.However;these treatments have limited effectiveness,risk of recurrence,and poor long-term prognosis.In recent years,the newly developed immunotherapy and gene therapy have the characteristics of high efficiency,strong specificity,and strong targeting.Therefore,it is of great significance to study the pathogenesis of malignant tumors in the molecular level and to find the targeted therapeutic molecules in order to achieve the best therapeutic effect.Programmed cell death factor 4(PDCD4)is a new molecule that involved in cell apoptosis and has a tumor suppressive effect.Studies have found that PDCD4 is downregulated or absent in many tumors such as esophageal cancer,gastric cancer,liver cancer,colon cancer,lung cancer,ovarian cancer,glioma,and melanoma.The rate of protein downregulation or loss in some tumors is higher than that of mRNA decrease or deletion in some tumors.The expression of PDCD4 was related to the degree of differentiation and malignancy of tumor cells in some tumors.The higher degree of differentiation and malignancy of tumors usually have the lower expression of PDCD4,suggesting that PDCD4 may be involved in the process of tumor development.The expression level of PDCD4 is also related to the sensitivity of tumor cells to chemotherapeutic drugs.Up-regulation of PDCD4 may increase the sensitivity of tumor cells to chemotherapeutic drugs.In addition,down-regulation or deletion of PDCD4 expression is also closely related to tumor cell migration,invasion,and poor prognosis.It has been reported that the down-regulation or deletion of PDCD4 at the mRNA level is mainly related to the degree of methylation of CpG islands,and the expression of PDCD4 at the protein level is mainly regulated by microRNAs,phosphorylation level and ubiquitination level of proteins.Given that PDCD4 plays an important role in the development of tumors,the mechanism underlying the down-regulation or deletion of PDCD4 in some tumors is not well understood.Therefore,exploring the expression regulation mechanism of PDCD4 is of great significance.TRIM27 is a member of the TRIM family.The distinctive feature of this family member is that it has three characteristic domains.From the N-terminus to the Cterminus,there is a RING finger domain,one or two B-box domain and a curly spiral domain.The zinc finger structure is also present in many other proteins.Functionally,the zinc finger domain can mediate the ubiquitin transfer between the proteins themselves or different substrates,so it is a characteristic marker of many E3 ubiquitin ligases.Since TRIM27 is a chaperonin of the proto-oncogene,it is also known as RFP.TRIM27 protein can be located in the nucleus,cytoplasm and nucleosomes of different cells,and its nuclear translocation is regulated by some signal pathways.Functionally,TRIM27 is involved in transcriptional regulation,cell apoptosis,cell differentiation,inflammatory response,and cell cycle.TRIM27 is closely related to the occurrence and development of tumors,and is highly expressed in many cancers such as lung cancer,breast cancer,ovarian cancer,and endometrial cancer.It has been found that TRIM27 can promote the migration and invasion of cancer cells.In some tumors,TRIM27 knockdown can increase the drug-induced apoptosis.In addition,TRIM27,as an E3 ubiquitin ligase,plays an important role in the regulation of antiviral natural immune responses,CD4+ T cell mediated immune responses,and the like.Based on background knowledge,we have known that the levels of PDCD4 and TRIM27 have a negative relationship in multiple tumors.In function,both PDCD4 and TRIM27 are involved in the process of tumorigenesis and progress,but the two proteins have opposite roles.Therefore,we speculate that TRIM27 may regulate the expression of PDCD4.In this study,we detected the expression of PDCD4 and TRIM27 in endometrial cancer cell lines,ovarian cancer cell lines,and 293T cell lines.The effect of TRIM27 overexpression and knockdown on PDCD4 expression and the effective mechanism of TRIM27 regulating PDCD4 were investigated in vitro.Methods1.The expression of PDCD4 and TRIM27 in endometrial cancer cell lines,ovarian cancer cell lines and 293T cell linesQRT-PCR was used to detect the mRNA expression of PDCD4 and TRIM27 in the endometrial cancer cell lines(Ishikawa,HEC-1-A,KLE,RL95-2),ovarian cancer cell line(A2780)and 293T cell line.The protein expression of PDCD4 and TRIM27 were detected by western blot in the above cell lines.2.The expression and distribution of PDCD4 and TRIM27 in endometrial cancer cell lines,ovarian cancer cell lines and 293T cell linesThe expression and distribution of PDCD4 and TRIM27 was detected by immunofluorescence double staining in the endometrial cancer cell lines(Ishikawa,HEC-1-A,KLE,RL95-2),ovarian cancer cell line(A2780)and 293T cell line.3.The effect of TRIM27 overexpression and knockdown on the mRNA expressionof PDCD4The specific siRNA of TRIM27 was transfected into Ishikawa,A2780 and 293T cells.After 24h of transfection,the interference efficiency of TRIM27 and the expression of PDCD4 were detected by qRT-PCR.TRIM27 expressive plasmid was transfected into Ishikawa and 293T cells.After 24h of transfection,the overexpression efficiency of TRIM27 and the expression of PDCD4 were detected by qRT-PCR.4.The effect of TRIM27 overexpression and knockdown on the protein expression of PDCD4The specific siRNA of TRIM27 was transfected into Ishikawa,A2780 and 293T cells.After transfection for 48h,the interference efficiency of TRIM27 and the expression of PDCD4 were detected by western blot.TRIM2 7 expressive plasmid was transfected into Ishikawa and 293T cells.After 48h of transfection,the overexpression efficiency of TRIM27 and the expression of PDCD4 were detected by western blot.5.Determine whether TRIM27 regulates PDCD4 expression by affecting the degradation of PDCD4TRIM27-specific siRNA and negative control siRNA were transfected into ovarian cancer cell line A2780 respectively,and the cell proteins were harvested at 24h after transfection.Two microliters of protein synthesis inhibitor CHX(lOmM)was added at 6h,4h,2h,and Oh before protein collection.After 6h,4h,2h and Oh of stimulation,the interference efficiency of TRIM27 and the expression of PDCD4 protein in the control and experimental groups were detected by western blot.TRIM27 expressive plasmid and empty plasmid were transfected into Ishikawa cells,and the cell proteins were harvested at 24h after transfection.Two microliters of CHX(10mM)was added at 6h,4h,2h,and Oh before protein harvest.After 6h,4h,2h and Oh of simulation,the expression of TRIM27 and PDCD4 protein levels in the control and experimental groups were detected by western blot.6.Determine whether the degradation of PDCD4 by TRIM27 is via the ubiquitin-proteasome pathwayTRIM27 expressive plasmid and empty plasmid were transfected into Ishikawa and 293T cell lines respectively.After 24h of transfection,the cell proteins were collected.Two microliters of proteasome inhibitor MG132(10mM)was added at 6h,4 h,and Oh before protein harvest.After 6h,4h,and Oh of stimulation,western blot was used to detect the overexpression efficiency of TRIM27 and the expression level of PDCD4 protein in the control and experimental groups.7.The effect of TRIM27 overexpression and knockdown on the ubiquitination levels of PDCD4TRIM27 specific siRNA and negative control siRNA were transfected into ovarian cancer cell line A2780.After 24h of cell culture,ubiquitin-HA plasmid was transfected in both control and experimental groups.The cell proteins were harvested after 24h of culture.Three microliters of MG132(lOmM)was added to the control and experimental groups at 6h before protein collection.The interference efficiency of TRIM27,the overexpression efficiency of ubiquitin molecules,and ubiquitination levels of PDCD4 were examined using co-immunoprecipitation(co-IP)and western blot.In the endometrial cancer cell line Ishikawa,TRIM27 expressive plasmid and ubiquitin-HA plasmid,empty plasmid and ubiquitin-HA plasmid were transfected respectively.The cell proteins were harvested after 24h of cell culture.Before cell proteins were collected,3?l of MG132(10mM)was added in the control group and experimental group.After 6h of stimulation,co-IP and western blot were used to detect the overexpression efficiency of TRIM27,the overexpression efficiency of ubiquitin molecules,and the change of PDCD4 ubiquitination levels.8.Detect ubiquitination site of PDCD4 by TRIM27TRIM27 expressive plasmid and K48-HA ubiquitin plasmid,empty plasmid and the K48-HA ubiquitin plasmid,as well as TRIM27 expressive plasmid and the K63-HA ubiquitin plasmid,empty plasmid and the K63-HA plasmid were co-transfected respectively in Ishikawa cells.The cells were harvested after 24h of culture,and stimulated with 3?l of MG 132(10mM)for 6h before the protein collection.For the collected cell proteins,the co-IP and western blot were used to detect the overexpression efficiency of TRIM27,the overexpression efficiency of ubiquitin molecules,and the change of ubiquitination levels of PDCD4 at different sites.9.Determine whether TRIM27 regulates PDCD4 expression through the RING finger domainThe expressive plasmid and control plasmid lacking the TRIM27 RING finger domain were transfected into the Ishikawa cells and 293T cells.The overexpression efficiency of the deleted plasmids and the change of PDCD4 at the protein level were detected by western blot.Results1.There is a significant negative correlation between the expression of TRIM27 and PDCD4 at the protein level in different cell lines,but there is no significant correlation at the mRNA level.The results of qRT-PCR showed that the expression of TRIM27 and PDCD4 had no significant correlation at the mRNA level in Ishikawa,HEC-1-A,KLE,RL95-2,A2780,and 293T cell lines.The results of western blot showed relatively low expression of TRIM27 and relatively high PDCD4 expression in Ishikawa,HEC-1-A,and 293T cell lines.In addition,there was relatively high expression of TRIM27 and relatively low PDCD4 expression in KLE,RL95-2 cell lines.Therefore,there is a significant negative correlation between the expression of TRIM27 and PDCD4 at the protein level.2.There is a phenomenon of co-localization at the distribution of TRIM27 and PDCD4 proteins in different cell lines.The expression of TRIM27 and PDCD4 in endometrial cancer cells and ovarian cancer cells was detected by double immunofluorescence staining.The results showed that PDCD4 protein was mainly distributed around the nucleus in Ishikawa,HEC-1-A,RL95-2,KLE and A2780 cells,and TRIM27 protein was in the nucleus and around the nucleus.Both PDCD4 and TRIM27 proteins mainly distribute around the nucleus in 293T cells.Therefore,there is a phenomenon of co-localization at the distribution of TRIM27 and PDCD4 proteins in different cell lines.3.TRIM27 had no significant effect on the expression of PDCD4 mRNA.TRIM27-specific siRNA was transfected into Ishikawa cells,293T cells,and A2780 cells,the results of qRT-PCR showed that PDCD4 has an upregulated tendency at the mRNA level after TRIM27 knockdown.TRIM27 expressive plasmid was transfected into Ishikawa cells and 293T cells,qRT-PCR results showed there was no significant change at the mRNA level of PDCD4 after TRIM27 overexpression.Therefore,TRIM27 had no significant effect on the expression of PDCD4 mRNA.4:TRIM27 has a significant negative effect on PDCD4 protein levelsThe specific siRNA of TRIM27 was transfected into Ishikawa cells,293T cells and A2780 cells.The results of western blot showed that the level of PDCD4 protein was significantly upregulated after TRIM27 knockdown.TRIM27 expressive plasmid was transfected into Ishikawa cells and 293T cells.Western blot results showed that PDCD4 protein levels were significantly downregulated after TRIM27 overexpression.Therefore,TRIM27 has a significant negative effect on the expression of PDCD4 protein.5.TRIM27 can promote the degradation of PDCD4 proteinA2780 cells were transfected with TRIM27-specific siRNA.Proteins were harvested 24h after transfection.CHX which can block the protein synthesis pathways was added to the cells at 6h,4h,2h,and Oh before protein collection.After 6h,4h,2h,and Oh of stimulation,the degradation levels of PDCD4 protein were observed.Western blot analysis showed that PDCD4 expression was significantly upregulated after TRIM27 knockdown.After the protein synthesis was blocked by CHX,the degradation rate of PDCD4 in TRIM27 knockdown groups was slower than that of control groups,indicating that TRIM27 can promote the degradation of PDCD4 protein.TRIM27 expressive plasmid was transfected into Ishikawa cells.Proteins were harvested 24h after transfection.Protein synthetic pathway was blocked by CHX at 6h,4h,2h,and Oh before protein collection.After 6h,4h,2h,and Oh of incubation,the degradation levels of PDCD4 protein were detected.Western blot results showed that the expression of PDCD4 was downregulated after TRIM27 overexpression.After the addition of CHX blocking protein synthesis,the degradation rate of PDCD4 was increased in TRIM27 overexpression groups compared with the control groups,indicating that TRIM27 can promote the degradation of PDCD4 protein.6.TRIM27 can promote the degradation of PDCD4 through the ubiquitin-proteasome pathwayTRIM27 expressive plasmid was used to transfect into Ishikawa and 293T cells respectively.After 24h of transfection,the cells were harvested.MG132 was added to block the degradation of proteasome pathway at 6h,4h,and Oh before protein collection.After 6h,4h,and Oh of incubation,the expression of PDCD4 protein was observed.Western blot results showed that PDCD4 expression was lower in TRIM27 overexpression group than that of the control group.After TRIM27 overexpression,the expression of PDCD4 was increased in MG132 stimulation group compared with the unstimulated group.Moreover,the expression of PDCD4 after MG132 treatment for 6h was higher than that after MG 132 stimulation for 4h.After the degradation pathway of proteasome was blocked,the decreased expression of PDCD4 induced by TRIM27 disappeared,indicating that TRIM27 regulates PDCD4 negatively by promoting the degradation of PDCD4 through the ubiquitin-proteasome pathway.7.TRIM27 can promote the ubiquitination of PDCD4A2780 cells were transfected with specific siRNA of TRIM27.After 24h,the cells were transfected with ubiquitin-HA expressive plasmid.The protein was harvested 24h later.The ubiquitination levels of PDCD4 were detected by co-IP.Western blot results showed that the level of ubiquitination of PDCD4 was downregulated after TRIM27 knockdown,indicating that TRIM27 can promote the ubiquitination of PDCD4.Ishikawa cells were transfected with TRIM27 expressive plasmid and ubiquitin-HA expressive plasmid.After 24h,the proteins were collected.The ubiquitination level of PDCD4 was detected by co-IP.Western blot results shown that the level of ubiquitination of PDCD4 was upregulated after TRIM27 overexpression,indicating that TRIM27 can promote the ubiquitination of PDCD4.8.TRIM27 can promote the ubiquitination of PDCD4 at the sites of K48 and K63TRIM27 expressive plasmid and K48-HA expressive plasmid,TRIM27 expressive plasmid and K63-HA expressive plasmid were transfected respectively into Ishikawa cells.The proteins were collected after 24h.The ubiquitination levels at K48 and K63 sites of PDCD4 were detected by co-IP.Western blot results showed that the ubiquitination levels of PDCD4 at the sites of K48 and K63 were upregulated after TRIM27 overexpression,indicating that TRIM27 can promote the ubiquitination of PDCD4 at the sites of K48 and K63.9.TIRM27 regulates PDCD4 expression through the RING finger domainThe expressive plasmid of lacking the TRIM27 RING finger domain was transfected into Ishikawa cells and 293T cells.The proteins were harvested after 24h.Western blot results showed that the expression of PDCD4 did not change because loss of RING finger domain,indicating TIRM27 regulates PDCD4 expression through the RING finger domain.Conclusions1.There is a negative correlation between TRIM27 and PDCD4 at the protein level in endometrial cancer cell lines and 293T cell lines.2.There is a phenomenon of co-localization at the distribution of TRIM27 and PDCD4 proteins in endometrial cancer cell lines,ovarian cancer cell lines and 293T cell lines.3.TRIM27 has no significant effect on the expression of PDCD4 mRNA,but TRIM27 has a significant negative effect on PDCD4 protein expression.4.TRIM27 can promote the degradation of PDCD4 through the ubiquitin-proteasome pathway.5.TRIM27 can promote the ubiquitination of PDCD4.6.TRIM27 can promote the ubiquitination of PDCD4 at the sites of K48 and K63.7.TIRM27 regulates PDCD4 expression through the RING finger domain.Innovations and significances1.In this study,we firstly explored the role of TRIM27 in the regulation of PDCD4 and found that TRIM27 regulated PDCD4 by promoting ubiquitination of PDCD4.2.Our findings may provide a new target for the treatment of tumors.Disadvantages of the paper1.In the present study about the ubiquitination sites of PDCD4,it was found that TRIM27 not only promoted the ubiquitination of the PDCD4 K48 site,but also promoted the ubiquitination of the PDCD4 K63 site.The role of ubiquitination of the K63 site needs to be further explored.2.The role of the TRIM27 RING finger domain in TRIM27 acted as E3 ubiquitin ligase needs further exploration. |
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