| Research objectiveHepatocellular carcinoma(HCC)is a highly malignant primary liver cancer on account of its undetectable onset and rapid progression,which contributes HCC to be the second leading cause of cancer death worldwide.Although various therapies including surgical resection,percutaneous chemoembolism,and radiofrequency ablation have relived the misfortune of some HCC patients,prognosis of the disease is still poor due to high recurrence and early metastasis.Over the years,molecular machnism of HCC has been extensively studied,but is still not fully illuminated.Revealing the molecular pathogenesis and finding the key molecules involved in the process will be critical for upgrading therapeutic targets and treatment strategies.Tripartite motif containing 7(TRIM7)is a new member protein of TRIM family first identified in 2002 as glycogenin interacting protein(GNIP),serving as a promotor in glycogen biosynthesis which could combine and activate glycogen protein.TRIM7 possesses highly conserved RBCC structures comprising of an N-terminal RING(really interesting new gene)domain,a B-box domain(zinc-finger motif),a coiled-coil domain,and a C-terminal B30.2/SPRY domain,which indicates its role as a RING-type E3 ubiquitin ligase to directly bind with target proteins for ubiquitination.Current studies about TRIM7 were limited and its biological function need to be further addressed.Moreover,the expression and biological functions of TRIM7 in the carcinogenesis of HCC are unclear till date.This study explored the role of TRIM7 in HCC progression and try to clarify the involved molecular mechanism through clinical liver cancer specimens,HCC cellular models and in vivo xenograft tumor models,which provided a therapeutic strategy for the improvement of clinical treatment strategies.Methods1.Investigation of TRIM7 expression in HCC tissues and corresponding non-cancerous liver tissues1.1 Detection of the location and expression of TRIM7 in HCC tissues The location and expression of TRIM7 were detected by immunohistochemistry(IHC)staining in HCC tissues and corresponding non-cancerous liver tissues from 80 clinical HCC patients.Statistical analysis of TRIM7 expression by IHC assay from the investigated HCC patients was conducted.1.2 Detection of protein level of TRIM7 in HCC tissues The protein level of TRIM7 was detected by western blot in HCC tissues and corresponding non-cancerous liver tissues from 52 clinical HCC patients.Statistical analysis of TRIM7 protein level by western blot assay from the investigated HCC patients was conducted.1.3 Detection of mRNA level of TRIM7 in HCC tissues The mRNA level of TRIM7 was detected by real-time polymerase chain reaction(real-time PCR)in HCC tissues and corresponding non-cancerous liver tissues from 64 clinical HCC patients.Statistical analysis of TRIM7 mRNA level by real-time PCR assay from the investigated HCC patients was conducted.2.Investigation of the biological functions of TRIM7 in HCC cells2.1 Construction of gain-of-function HCC cellular models2.1.1 Construction of transient TRIM7-overexpressing HCC cellular models HepG2 and Huh cells were transiently transfected with Flag-tagged TRIM7 plasmid(Flag-TRIM7)or mock control plasmid.The successful overexpression of TRIM7 was validated by western blot assay after transfection for 24 h.2.1.2 Generation of a stable TRIM7-overexpressing HCC cell line To construct stable TRIM7-overexpressing HCC cells,a Flag-TRIM7 plasmid was transfected into Huh7 cells using Lipofectamine 2000.The successfully transfected cells were selected by complete medium containing 2 μg/mL of puromycin at 48 h after the transfection.The surviving colonies of transfected cells were further amplified,followed by validation by western blot.2.2 Effect of TRIM7 overexpression on the malignant behaviors of HCC cells The malignant behaviors of the gain-of-function HCC cells,including the proliferation,invasion,and colony formation were further detected and analyzed.2.3 Construction of loss-of-function HCC cellular models2.3.1 Construction of Si-TRIM7-transiently transfected HCC cellular models HepG2 and Huh7 cells were transiently transfected with Si-NC,Si-TRIM7-1 or Si-TRIM7-2.The successful knockdown of TRIM7 was validated by western blot assay after transfection for 48 h.2.3.2 Generation of a lentivirus-mediated Sh-TRIM7-stably transfected HCC cell lineTo generate a stable TRIM7-knockdown cell line,lentivirus carrying TRIM7-RNA interference sequence(Sh-TRIM7-1 and Sh-TRIM7-2)or its mock control(Sh-NC)were transfected into Huh7 cells using lentivirus according to the manufacturer’s protocol.The transfected cells were further selected with puromycin(2 μg/mL)and stable TRIM7 knockdown clones were amplified and validated by western blot.2.3.3 Generation of a TRIM7-knockout cell line based on the CRISPR/Cas9 systemTRIM7-knockout cells were constructed with the CRISPR/Cas9 system.A single-guide RNA(sgRNA)targeting TRIM7 was transfected into Huh7 cells using the pLentiCRISPRv2 system.The sgRNA-transfected cells and the pLenti-V2-transfected mock control cells were further selected with puromycin(2μg/mL)and validated by western blot.The selected positive clones were cultured for further experiments.2.4 Effect of TRIM7 knockdown on the malignant behaviors of HCC cells The malignant behaviors of the loss-of-function HCC cells,including the proliferation,invasion,and colony formation were further detected and analyzed.3.The molecular target of TRIM7 to exert anti-tumor effect in HCC cells In order to explore the molecular targets of TRIM7 to exert anti-tumor effect in HCC cells,Co-immunoprecipitation(Co-IP)assay was used to screen the tumor promotors or suppressors that bound to TRM7,including Src,p53,TSC2,HIF-la,SNAIL,MMP-9 and Rac1.The results suggest that TRIM7 and Src were directly interacted with each other,indicating that Src might be a molecular target of TRIM7 to exert anti-tumor effect in HCC cells.3.1 Detection of the binding effect between TRIM7 and Src3.1.1 Detection of the binding effect between exogenous TRIM7 and exogenous SrcFlag-TRIM7 plasmid and HA-tagged Src plasmid(HA-Src)were co-transfected into HEK293T and Huh7 cells,while Flag-pENTER and HA-Src plasmids were co-transfected into the mock control cells.The binding effect between exogenous TRIM7 and exogenous Src was detected by Co-IP assay.3.1.2 Detection of the binding effect between exogenous TRIM7 and endogenous SrcFlag-TRIM7 plasmid was transfected into HEK293T and Huh7 cells,while Flag-pENTER plasmid was transfected into the mock control cells.The binding effect between exogenous TRIM7 and endogenous Src was detected by Co-IP assay.3.1.3 Detection of the binding effect between endogenous TRIM7 and endogenous SrcThe binding effect between endogenous TRIM7 and endogenous Src was detected by Co-IP analysis in Huh7 cells.3.1.4 Detection of the binding effect between TRIM7 and Src in vitro Flag-TRIM7 and HA-Src proteins were obtained from an in vitro transcription and translation system and further Co-IP analysis explored whether TRIM7 could directly interact with Src protein in vitro.3.2 Detection of the binding domains responsible for the interaction between TRIM7 and Src3.2.1 The domain of TRIM7 responsible for the interaction between TRIM7 and SrcA series of domain-deleted mutants of TRIM7 were generated including TRIM7△RINQ TRIM7△Poly-Ala,TRIM7△B-box,TRIM7ACoiled-coil,and TRIM7△B30.2/SPRY.Flag-TRIM7 truncation mutant plasmids together with HA-Src plasmid were co-transfected into HEK293T cells,and Co-IP assay was performed to detected the interaction between Src and TRIM7 truncation mutants.3.2.2 The domain of Src responsible for the interaction between TRIM7 and SrcA series of domain-deleted mutants of Src were generated including Src△SH3,Src△ASH2,and SrcAprotein kinase.HA-Src truncation mutant plasmids together with Flag-TRIM7 plasmid were co-transfected into HEK293T cells and Co-IP assay was performed to detected the interaction between TRIM7 and Src truncation mutants.4.The regulatory effect of TRIM7 on Src4.1 The effect of TRIM7 on the Src and p-Src levels4.1.1 The effect of TRIM7 overexpression on the Src and p-Src levelsThe Src and p-Src levels were detected by western blot assay in the transient TRIM7-overexpressing HepG2 and Huh7 cells and stable TRIM7-overexpressing Huh7 cells as well as these corresponding mock control cells.4.1.2 The effect of TRIM7 knockdown or knockout on the Src and p-Src levels The Src and p-Src levels were detected by western blot assay in the Si-TRIM7-transiently transfected HepG2 and Huh7 cells,lentivirus-mediated Sh-TRIM7-stably transfected Huh7 cells,and TRIM7-knockout Huh7 cells based on the CRISPR/Cas9 system as well as these corresponding mock control cells.4.2 The effect of TRIM7 on the mRNA level of Src4.2.1 The effect of TRIM7 overexpression on the mRNA level of SrcThe mRNA level of Src was detected by real-time PCR in the transient TRIM7-overexpressing HepG2 and Huh7 cells as well as these corresponding mock control cells.4.2.2 The effect of TRIM7 knockdown on the mRNA level of Src The mRNA level of Src was detected by real-time PCR in the Si-TRIM7-transiently transfected HepG2 cells and mock control cells.4.3 The correlation of Src and TRIM7 levels in HCC tissues The expression of Src in HCC tissues was detected by IHC and the correlation of Src and TRIM7 levels was further analyzed.4.4 The regulatory effect of TRIM7 on the stability of Src protein HepG2 and Huh7 cells were transfected with Flag-TRIM7 plasmid or mock control plasmid and were cultured for 24 h before being further incubated with CHX(1μM)for 0h,3 h,6 h and 9 h.The Src protein level of the transfected cells was detected by western blot assay.4.5 The pathway of TRIM7-mediated Src protein degradation Proteasome pathway and lysosomal pathway are major pathways for protein degradation in eukaryotic cells.HepG2 cells were transfected with Flag-TRIM7 plasmid or mock control plasmid and were cultured for 24 h before being further incubated with MG132(10 μM)for 4 h or chloroquine(25 μM)for 6 h.The Src and p-Src protein levels in the transfected cells were detected by western blot assay.5.Investigation of the ubiquitination on Src mediated by TRIM75.1 Detection of the ubiquitination on Src mediated by TRIM75.1.1 Detection of the ubiquitination on Src mediated by exogenous TRIM7 HEK293T cells were co-transfected with HA-tagged UB plasmid(HA-UB),Flag-TRIM7,and HA-Src plasmid,while the mock control cells were co-transfected with HA-UB,Flag-pENTER,and HA-Src plasmids.The ubiquitination of Src mediated by exogenous TRIM7 was detected by Co-IP assay.5.1.2 Detection of the ubiquitination on endogenous Src mediated by exogenous TRIM7 HEK293T cells were co-transfected with HA-UB and Flag-TRIM7 plasmid,while the mock control cells were transfected with Flag-pENTER,HA-UB or Flag-TRIM7 plasmid,respectively.The ubiquitination of endogenous Src mediated by exogenous TRIM7 was detected by Co-IP assay.5.1.3 Detection of the ubiquitination on Src by TRIM7 in vitroFlag-TRIM7 and HA-Src proteins were obtained from an in vitro transcription and translation system.The ubiquitination of Src in the presence of in-vitro-translated Flag-TRIM7 and HA-Src,ubiquitin,El,and UbcH5a was detected in an in vitro ubiquitination system by western blot.5.2 The type of TRIM7-mediated ubiquitination on SrcK11(Lys11),K48(Lys48)and K63(Lys63)are common sites of ubiquitination.The HA-UB-K11,HA-UB-K48,and HA-UB-K63 plasmids are mutants of HA-UB,in which all other lysine residues are replaced by arginine except for the lysine residues at sites 11,48,or 63,respecti-vely.HEK293T cells were co-transfected with Flag-TRIM7,HA-Src,and HA-UB-K11 or HA-UB-K48 or HA-UB-K63 plasmids,while the mock control cells were co-transfected with Flag-pENTER,HA-Src,and HA-UB-K11 or HA-UB-K48 or HA-UB-K63 plasmids.The ubiquitination of Src was detected by Co-IP assay.5.3 The role of the RING domain in TRIM7-mediated ubiquitination on SrcFlag-TRIM7 plasmid or Flag-TRIM7 truncation mutant plasmid(Flag-TRIM7△RING)as well as HA-Src plasmid and HA-UB plasmid were co-transfected into HEK293T cells.The ubiquitination of Src was detected by Co-IP assay.6.The signal pathway of TRIM7 to exert tumor suppressive effect in HCC cellsIn order to explore the signal pathways of TRIM7 to exert tumor suppressive effect in HCC cells,the signal pathways that might be regulated by TRM7 were screened,including mTOR,AMPK,AKT,NF-Kappa B and MAPK pathways.The results suggest that TRIM7 significantly suppressed the mTORCl-S6K1 pathway.6.1 The regulatory effect of TRIM7 on the mTORC1-S6K1 pathway6.1.1 The effect of TRIM7 overexpression on the mTORCl-S6K1 pathwayThe Src,p-mTOR,p-S6K1,p-S6,and p-4E-BP1 levels were detected by western blot assay in the transient TRIM7 overexpressing HepG2 and Huh7 cells and stable TRIM7 overexpressing Huh7 cells as well as these corresponding mock control cells.6.1.2 The effect of TRIM7 knockdown or knockout on the mTORCl-S6K1 pathwayThe Src,p-mTOR,p-S6K1,p-S6,and p-4E-BP1 levels were detected by western blot assay in the Si-TRIM7-transiently transfected HepG2 and Huh7 cells,lentivirus-mediated Sh-TRIM7-stably transfected Huh7 cells,and TRIM7-knockout Huh7 cells based on the CRISPR/Cas9 system as well as these corresponding mock control cells.6.2 The role of Src in TRIM7-mediated inhibition of mTORCl-S6K1 signaling pathwayHuh7 cells were co-transfected with Flag-TRIM7 plasmid and HA-Src plasmid-The Src-mTORC1-S6K1 signaling were detected by western blot assay.Huh7 cells were transfected with Si-NC,Si-TRIM7-1 or Si-TRIM7-2 before treatment with rapamycin(100 nM)for 24 h.The Src-mTORC1-S6K1 signaling were detected by western blot assay.6.3 Detecting whether TRIM7 exerted tumor suppressive effect in HCC cells by regulating Src-mTORCl-S6K1 signaling pathway6.3.1 Detecting whether TRIM7 exerted tumor suppressive effect in HCC cells by regulating SrcHepG2 and Huh7 cells were co-transfected with Flag-TRIM7 plasmid and HA-Src plasmid,and successful overexpression of TRIM7 and Src in the transfected cells was detected by western blot.The proliferation,invasion,and colony formation of the transfected HCC cells were further detected and analyzed.6.3.2 Detecting whether TRIM7 exerted tumor suppressive effect in HCC cells by regulating mTORCl-S6K1 signaling pathwayHepG2 cells were transfected with Si-NC,Si-TRIM7-1 or Si-TRIM7-2 before treatment with rapamycin(100 nM)for 24 h,and p-S6K1,p-S6,Src and p-Src levelsin the transfected cells were detected by western blot.The proliferation,invasion,and colony formation of the transfected cells were further detected and analyzed.7.The functional domains of TRIM7 to exert anti-tumor effect in HCC cellsOur study suggested that TRIM7 directly bound to Src through its B30.2/SPRY domain,and induced Lys48-linked polyubiquitination of Src through its RING domain.Thus,the RING domain and B30.2/SPRY domain were essential for TRIM7 to exert its functions.We will further explore whether TRIM7 exerted the suppressing effect on Src-mTORC1-S6K1 axis and HCC progression through its RING domain and B30.2/SPRY domain.7.1 The functional domains of TRIM7 in regulating the molecular target and signaling pathwayHepG2 and Huh7 cells were transfected with Flag-TRIM7 plasmid or TRIM7 truncation mutantplasmids(TRIM7△RING or TRIM7AB30.2/SPRY),and successful overexpression of TRIM7,TRIM7△RING,or TRIM7AB30.2/SPRY in the transfected cells was detected by western blot.The Src-mTORCl-S6K1 signaling was detected by western blot.7.2 Detecting whether TRIM7 exerted a tumor suppressor effect in HCC cells through its functional domainsHepG2 and Huh7 cells were transfected with Flag-TRIM7 plasmid or TRIM7 truncation mutantplasmids(TRIM7ARING or TRIM7△B30.2/SPRY),and successful overexpression of TRIM7,TRIM7ARING,or TRIM7AB30.2/SPRY in the transfected cells was detected by western blot.The proliferation,invasion,and colony formation of these transfected HCC cells were further detected and analyzed.8.The effect of TRIM7 on HCC in xenograft tumor models8.1 The effect of TRIM7 on HCC in the TRIM7-exogenous overexpressing xenograft tumor modelHCC cells were injected into both flanks of the male BALB/c nude mice(5 weeks).After visible tumors appeared,we injected Flag-TRIM7 plasmid into tumors in the left flanks and mock control plasmid into tumors in the right flanks of the mice every other day before the mice were sacrificed for analysis.The growth status of the formed tumors was monitored every other day before the mice were sacrificed and the tumor growth curve was drawn.After the nude mice were sacrificed,the tumors were separated,measured and weighed,and the differences between the TRIM7 exogenous overexpressing group and the mock control group were compared.The protein in tumor tissues was extracted,and the expression of TRIM7,its molecular target and signal pathway in tumor tissues were detected by western blot.8.2 The effect of TRIM7 on HCC in the TRIM7-stably overexpressing xenograft tumor modelThe TRIM7-stably overexpressing xenograft tumor model was constructed by subcutaneously injecting TRIM7-stably overexpressing Huh7 cells into one flank and mock control cells into the other flank of nude mice.The growth status of the formed tumors was monitored every other day before the mice were sacrificed and the tumor growth curve was drawn.After the nude mice were sacrificed,the tumors were separated,measured and weighed,and the differences between the TRIM7-stably overexpressing group and the mock control group were compared.The protein in tumor tissues was extracted,and the expression of TRIM7,its molecular target and signal pathway in tumor tissues were detected by western blot-8.3 The effect of TRIM7 on HCC in the TRIM7-knockout xenograft tumor modelThe TRIM7-knockout xenograft tumor model was constructed by injecting TRIM7-knockout Huh7 cells generated by the CRISPR/Cas9 system into one flank of nude mice and the mock control cells into the other flank of nude mice.The growth status of the formed tumors was monitored every other day before the mice were sacrificed and the tumor growth curve was drawn.After the nude mice were sacrificed,the tumors were separated,measured and weighed,and the differences between the TRIM7-knockout group and the mock control group were compared.The protein in tumor tissue was extracted,and the expression of TRIM7,its molecular target and signal pathway in tumor tissues were detected by western blot.Results1.The TRIM7 expression in HCC tissues was significantly downregulated compared with that in the corresponding distal noncancerous liver tissues1.1 The TRIM7 expression in HCC tissues was significantly downregulatedThe location and expression of TRIM7 were detected by IHC in paired tissues from 80 HCC patients.The IHC data showed that TRIM7 was mainly expressed in the cytoplasm of HCC cells and hepatocytes,and the expression level of TRIM7 in HCC tissues was significantly downregulated compared with that in the corresponding distal noncancerous liver tissues.1.2 The protein level of TRIM7 in HCC tissues was significantly downregulated The protein level of TRIM7 was measured by western blot in paired tissues from 52 HCC patients.The results showed that TRIM7 protein level in HCC tissues were significantly decreased compared with that in the corresponding distal noncancerous liver tissues.1.3 The mRNA level of TRIM7 in HCC tissues was significantly downregulated The mRNA level of TRIM7 was measured by real-time PCR assay in paired tissues from 64 HCC patients.The results showed that TRIM7 mRNA level in HCC tissues was significantly decreased compared with that in the corresponding distal noncancerous liver tissues.2.TRIM7 acted as a tumor suppressor and inhibited malignant behaviors of HCC cells2.1 The malignant behaviors of HCC cells were inhibited in the gain-of-function HCC cellular modelsConstructing of gain-of-function HCC cellular models including transient TRIM7-overexpressing HepG2 and Huh7 cellular models and a stable TRIM7-overexpressing Huh7 cell line.The malignant behaviors of the gain-of-function HCC cells,including the proliferation,invasion,and colony formation were detected and analyzed.The results showed that in HCC cells transiently or stably overexpressing TRIM7,the proliferation,invasion,and colony formation were significantly inhibited.2.2 The malignant behaviors of HCC cells were promoted in the loss-of-function HCC cellular modelsConstructing of loss-of-function HCC cellular models including Si-TRIM7-transiently transfected HepG2 and Huh7 cellular models,a lentivirus-mediated Sh-TRIM7-stably transfected Huh7 cell line,and a TRIM7-knockout Huh7 cell line based on the CRISPR/Cas9 system.The malignant behaviors of the loss-of-function HCC cells,including the proliferation,invasion,and colony formation were detected and analyzed.The results showed that in the TRIM7-knockdown and knockout HCC cells,the proliferation,invasion and colony formation were all significantly promoted.3.TRIM7 directly interacted with Src3.1 TRIM7 interacted with Src3.1.1 Exogenous TRIM7 interacted with exogenous SrcFlag-TRIM7 and HA-Src plasmids were co-transfected into HEK293T and Huh7 cells,while Flag-pENTER and HA-Src plasmids were co-transfected into the mock control cells,and further detected by Co-IP assay.Our data showed that exogenous TRIM7 could interact with exogenous Src.3.1.2 Exogenous TRIM7 interacted with endogenous Src Flag-TRIM7 plasmid was transfected into HEK293T and Huh7 cells,while Flag-pENTER plasmid was transfected into the mock control cells,and further detected by Co-IP assay.Our data showed that exogenous TRIM7 could interact with endogenous Src.3.1.3 Endogenous TRIM7 interacted with endogenous SrcThe binding effect between endogenous TRIM7 and endogenous Src was detected by Co-IP analysis in Huh7 cells.The results showed that endogenous TRIM7 could bind to endogenous Src,which indicated their interaction in natural conditions.3.1.4 TRIM7 directly interacted with Src in vitroFlag-TRIM7 and HA-Src proteins were obtained from an in vitro transcription and translation system and further Co-IP analysis demonstrated that TRIM7 could directly interact with Src in vitro.3.2 The B30.2/SPRY domain of TRIM7 and the SH2 domain of Src were required for the interaction between TRIM7 and Src3.2.1 TRIM7 bound to Src via its B30.2/SPRY domain Flag-TRIM7 truncation mutant plasmids together with HA-Src plasmid were co-transfected into HEK293T cells and further Co-IP analysis demonstrated that the B30.2/SPRY domain of TRIM7 was required for the interaction between TRIM7 and Src.3.2.2 Src bound to TRIM7 via its SH2 domain HA-Src truncation mutant plasmids together with Flag-TRIM7 plasmid were co-transfected into HEK293T cells and further Co-IP analysis demonstrated that the SH2 domain of Src was required for the interaction between TRIM7 and Src.4.TRIM7 reduced the abundance of Src protein4.1 TRIM7 reduced the Src and p-Src levels in HCC cells4.1.1 TRIM7 overexpression reduced the Src and p-Src levels The Src and p-Src levels were detected by western blot assay in the transient TRIM7-overexpressing HepG2 and Huh7 cells and stable TRIM7-overexpressing Huh7 cells as well as these corresponding mock control cells.Our data showed that after successful overexpression of TRIM7 by both transient and stable transfection of the TRIM7 plasmid,the protein levels of both Src and p-Src were significantly reduced.4.1.2 TRIM7 knockdown or knockout increased the Src and p-Src levelsThe Src and p-Src levels were detected by western blot assay in the Si-TRIM7-transiently transfected HepG2 and Huh7 cells,lentivirus-mediated Sh-TRIM7-stably transfected Huh7 cells,and TRIM7-knockout Huh7 cells based on the CRISPR/Cas9 system as well as these corresponding mock control cells.Our data showed that in all of these cell models of TRIM7 inhibition,the protein levels of both Src and p-Src were significantly increased.4.2 TRIM7 had no significant effect on the mRNA level of Src4.2.1 TRIM7 overexpression had no significant effect on the mRNA level of SrcThe mRNA level of Src was detected by real-time PCR in the transient TRIM7 overexpressing HepG2 and Huh7 cells as well as mock control cells.Our data showed that after successful overexpression of TRIM7,the mRNA level of Src had no significant change.4.2.2 TRIM7 knockdown had no significant effect on the mRNA level of SrcThe mRNA level of Src was detected by real-time PCR in the Si-TRIM7-transiently transfected HepG2 cells and mock control cells.Our data showed that after successful knockdown of TRIM7,the mRNA level of Src had no significant change.4.3 The Src level was significantly inversely correlated with TRIM7 expression in HCC tissuesThe expression of Src in HCC tissues was detected by IHC.The results showed that Src protein accumulated significantly in HCC tissues with lower expression of TRIM7,and statistical analysis showed that TRIM7 expression was significantly inversely correlated with the abundance of Src,which verified the TRIM7-mediated negative regulation of Src in clinical HCC patients.4.4 TRIM7 facilitated the decrease of Src protein level in HCC cellsHepG2 and Huh7 cells were transfected with Flag-TRIM7 plasmid or mock control plasmid and were cultured for 24 h before being further incubated with CHX for 0h,3 h,6 h and 9 h.Further western blot assay demonstrated that TRIM7 significantly facilitated the decrease of Src protein level.4.5 TRIM7 reduced Src protein level via proteasome-mediated degradation in HCC cellsHepG2 cells were transfected with Flag-TRIM7 plasmid or mock control plasmid and were cultured for 24 h before being further incubated with MG132(10 μM)for 4 h or chloroquine(25 μM)for 6 h.Further western blot assay suggested that the TRIM7-mediated negative regulation of Src could be significantly rescued by the proteasome inhibitor MG 132,which indicated that Src was degraded by TRIM7 through proteasome-mediated degradation.5.TRIM7 induced Lys48-linked polyubiquitination of Src via its RING domain 5.1 TRIM7 induced polyubiquitination of Src5.1.1 Exogenous TRIM7 induced polyubiquitination of SrcHEK293T cells were co-transfected with HA-UB,Flag-TRIM7,and HA-Src plasmid,while the mock control cells were co-transfected with HA-UB,Flag-pENTER,and HA-Src plasmid.Further Co-IP assay showed that exogenous TRIM7 was able to attach a polyubiquitin chain to Src protein.5.1.2 Exogenous TRIM7 induced polyubiquitination of endogenous SrcHEK293T cells were co-transfected with HA-UB and Flag-TRIM7 plasmid,while the mock control cells were transfected with Flag-pENTER,HA-UB or Flag-TRIM7 plasmid,respectively.Further Co-IP assay showed that exogenous TRIM7 was able to attach a polyubiquitin chain to endogenous Src protein.5.1.3 TRIM7 induced polyubiquitination of Src in vitroFlag-TRIM7 and HA-Src proteins were obtained from an in vitro transcription and translation system.The ubiquitination of Src in the presence of in-vitro-translated Flag-TRIM7 and HA-Src,ubiquitin,E1,and UbcH5a was detected in an in vitro ubiquitination system by western blot.The results showed that the polyubiquitin chain could be attached to Src protein by TRIM7 in an in vitro ubiquitination system-5.2 TRIM7 induced Lys48-linked polyubiquitination of SrcHEK293T cells were co-transfected with Flag-TRIM7,HA-Src,and HA-UB-K11 or HA-UB-K48 or HA-UB-K63 plasmid,while the mock control cells were co-transfected with Flag-pENTER,HA-Src,and HA-UB-K11 or HA-UB-K48 or HA-UB-K63 plasmid.Further Co-IP assay revealed that TRIM7 could attach a Lys48-linked but not Lys1 1-linked or Lys63-linked polyubiquitin chain to Src protein,which implied that TRIM7 could induce Lys48-linked polyubiquitination of Src.5.3 TRIM7 induced polyubiquitination of Src via its RING domainFlag-TRIM7 plasmid or Flag-TRIM7△RING plasmid as well as HA-Src plasmid and HA-UB plasmid were co-transfected into HEK293T cells and further Co-IP assay showed that the polyubiquitination of Src in TRIM7△RING plasmid transfected group was significantly reduced in comparison with that of the wild type TRIM7 plasmid transfected group.Thus,it indicated that TRIM7 induced ubiquitination of Src via its RING domain.6.TRIM7 acted as a tumor suppressor through the suppression of the Src-mTORCl-S6K1 axis in HCC cells6.1 TRIM7 suppressed the mTORCl-S6K1 pathway6.1.1 TRIM7 overexpression suppressed the mTORCl-S6K1 pathwayThe Src,p-mTOR,p-S6K1,p-S6,and p-4E-BP1 levels were detected by western blot assay in the transient TRIM7 overexpressing HepG2 and Huh7 cells and stable TRIM7 overexpressing Huh7 cells as well as these corresponding mock control cells.Our data showed that both transient and stable overexpression of TRIM7 efficiently reduced the protein levels of p-mTOR,p-S6K1,p-S6,and p-4E-BP1,which indicated its inhibitory effect on the mTORCl pathway.6.1.2 TRIM7 knockdown or knockout promoted the mTORCl-S6K1 pathwayThe Src,p-mTOR,p-S6K1,p-S6,and p-4E-BP1 levels were detected by western blot assay in the Si-TRIM7-transiently transfected HepG2 and Huh cells,lentivirus-mediated Sh-TRIM7-stably transfected Huh7 cells,and TRIM7-knockout Huh7 cells based on the CRISPR/Cas9 system as well as these corresponding mock control cells.Our data showed that in TRIM7 knockdown and knockout cells,the protein levels of p-mTOR,p-S6K1,p-S6,and p-4E-BP1 were significantly upregulated.6.2 TRIM7 suppressed the mTORCl-S6K1 pathway through negative regulation of SrcHuh7 cells were co-transfected with Flag-TRIM7 plasmid and HA-Src plasmid,and further western blot assay showed that overexpression of Src could rescue the TRIM7-mediated suppression of the mTORCl-S6K1 signaling pathway.Huh7 cells were transfected with Si-NC,Si-TRIM7-1 or Si-TRIM7-2 before treatment with rapamycin(100 nM)for 24 h,and further western blot assay showed that the mTORC1 pathway inhibitor rapamycin significantly abolished the increased protein levels of p-S6K1 and p-S6 in Si-TRIM7-transfected HCC cells,but did not change the protein levels of either Src or p-Src.In summary,our data indicated that mTORC1-S6K1 signaling was downstream of the TRIM7-mediated negative regulation of Src,and TRIM7 inhibited Src protein as well as its downstream mTORC1-S6K1 signaling in HCC cells.6.3 TRIM7 suppressed HCC through the inhibition of the Src-mTORCl-S6K1 axis6.3.1 TRIM7 suppressed HCC through negative regulation of the SrcHepG2 and Huh7 cells were co-transfected with Flag-TRIM7 plasmid and HA-Src plasmid,and successful overexpression of TRIM7 and Src in the transfected cells was detected by western blot.The proliferation,invasion,and colony formation of the transfected HCC cells were further detected and analyzed and the results showed that exogenous overexpression of Src significantly rescued the TRIM7-mediated suppression of the proliferation,invasion,and colony formation of HCC cells.These data verified that TRIM7 exerted its anti-tumor effect through its negative regulation of Src.6.3.2 TRIM7 suppressed HCC through negative regulation of the mTORCl-S6K1 pathwayHepG2 cells were transfected with Si-NC,Si-TRIM7-1 or Si-TRIM7-2 before treatment with rapamycin(100 nM)for 24 h,and p-S6K1,p-S6,Src and p-Src levels in the transfected cells were detected by western blot.The proliferation,invasion,and colony formation of the transfected HepG2 cells were further detected and analyzed.Our data showed that treatment with the mTORC1 inhibitor rapamycin significantly abolished the increased protein levels of p-S6K1 and p-S6 in Si-TRIM7-transfected HCC cells,and the proliferation,invasion,and colony formation of Si-TRIM7-transfected cells were strikingly suppressed by rapamycin.These data verified that TRIM7 exerted its anti-tumor effect through its negative regulation of mTORC1-S6K1 pathway.Altogether,these data confirmed that TRIM7 exerted its anti-tumor effect through its suppression of the Src-mTORC1-S6K1 axis.7.TRIM7 played a tumor suppressor role in HCC through its functional domains 37 Our study suggested that TRIM7 directly bound to Src through its B30.2/SPRY domain,and induced Lys48-linked polyubiquitination of Src through its RING domain.Thus,the RING domain and B30.2/SPRY domain were essential for TRIM7 to exert its functions.7.1 TRIM7 suppressed the Src-mTORCl-S6K1 axis through its functional domainsHepG2 and Huh7 cells were transfected with Flag-TRIM7,Flag-TRIM7△RING or Flag-TRIM7△B30.2/SPRY plasmids,and further western blot assay showed that deletion of either the B30.2/SPRY domain or the RING domain in TRIM7 efficiently rescued the TRIM7-mediated inhibition of the Src-mTORCl-S6K1 axis,which indicated that TRIM7 suppressed the Src-mTORCl-S6K1 axis thr... |
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