The Role And Related Mechanism Of TRIM28 On The Regulation Of Metastasis In Colorectal Cancer

Objective: To improve our understanding of colorectal cancer metastasis,we explore the biological role of TRIM28 and related mechanism in colorectal cancer metastasis.Methods: 1.Cell Culture: HEK293 T cell,human normal mucosa cell NCM460 and human CRC cell lines Lo Vo,HCT116,SW48,DLD-1,HT-29 were obtained from American Type Culture Collection(ATCC).Cells were cultured in DMEM medium containing 10% fetal bovine serum.2.Plasmids Construction: The c DNA of indicated gene was amplified from c DNA library of HEK293 T cells and cloned into clone vector.Oligonucleotides specific sh RNAs against indicated genes were inserted into PLKO.1 vector.All plasmids were confirmed by DNA sequencing.3.Transfection and Small interfering RNAs: Transfection of si RNAs and plasmids into cells was performed with Lipofectamine 2000 according to the manufacturer’s protocols.4.Lentiviral Transduction: HEK293 T cells were transfected with indicated lentiviral plasmids.Virus-containing supernatant was collected at 48 h or 72 h post-transfection.To generate the stably transfected cell lines,Lo Vo,HCT116 and SW48 cells were infected with lentivirus in medium in the presence of polybrene(8 μg/m L)and then selected by puromycin(1 μg/m L)for one week.The overexpression or knockdown efficiency was identified using Western blot.5.Western Blot: Total protein was extracted by lysing the cells with RIPA buffer supplemented with protease inhibitors.The protein concentration was measured by the BCA kit.The cell lysate was incubated at 98℃ for 10 minutes,separated by SDS-PAGE and then transferred onto nitrocellulose(NC)membranes.After blocking with 5% skimmed milk,membranes were incubated with indicated antibodies.The signals were detected by chemiluminescence system.6.Immunoprecipitation and protein ubiquitination assay: For Immunoprecipitation and protein ubiquitination assay,cells were collected and lysed in immunoprecipitation lysis buffer supplemented with protease inhibitors.Cell extracts were incubated with indicated antibodies overnight at 4 °C.Then protein A/G plus-agarose beads were added and incubated for another 2 hours at 4 °C.The beads were washed three times using immunoprecipitation lysis buffer,boiled at 98 °C for 5 min in protein loading buffer and then subjected to western blot analysis.Antibodies used in the co-immunoprecipitation and protein ubiquitination assay were as follows: anti-Flag,anti-Myc,and anti-ubiquitin antibodies.7.Real Time PCR and Quantitative Real Time PCR: Total RNA was extracted with the Trizol reagent and reverse transcribed into c DNA using the M-MLV reverse transcriptase.Quantitative Real Time PCR was performed on Step One Plus TM Real-time PCR system using a standard SYBR Green PCR kit.Quantitation of the relative expression levels of each gene was performed in triplicate and calculated using the 2-△△CT method.8.Cell migration and invasion assay: The cell migration and invasion assay were performed using 24-well Boyden chambers.1×105 cells were seeded in the upper chamber.The lower chamber was added with 700μl DMEM medium containing 10% FBS.Cells on the upper side of the filter were removed after 12 h for Lo Vo cells,48 h for HCT116 cells.The migrated cells were stained with 0.1% crystal violet and counted under microscopy.Cellular invasion ability was measured with Matrigel matrix.1×105 cells were added to the matrigel-coated upper chamber and allowed to invade 24 h for Lo Vo cells,72 h for HCT116 cells.Invaded cells were stained with 0.1% crystal violet and counted under the microscope.9.Animal experiments: 2×106 SW48 cells(sh NC,sh TRIM28#1 and sh TRIM28#2)were injected into the tail vein of male Balb/c nude mice(n=5/each group,4–6 weeks).Five weeks later,mice were sacrificed,and lungs were fixed and performed with hematoxylin and eosin staining.The incidence and number of metastatic lesions were photographed and quantified.The mice in this study were maintained under specific pathogen-free(SPF)conditions.10.TOP-flash/FOP-flash reporter assay: TRIM28 stably knockdown Lo Vo and HCT116 cells were seeded into 24-well plates and transfected with TOP-flash or FOP-flash reporter plasmid for 48 h.The relative luciferase activities(firefly luciferase activity divided by Renilla activity)were measured by a dual-luciferase reporter assay system.The TOP/FOP ratio reflected the activity of the WNT/β-catenin pathway.All results were repeated at least three times.11.Immunohistochemistry: Immunohistochemical staining was performed by the streptavidin-biotin-peroxidase detection method.Briefly,CRC tissues and adjacent normal tissues were fixed with 4% formaldehyde and embedded with paraffin.After rehydration and microwave antigen retrieval,the sections were labeled with anti-TRIM28 antibody(proteintech,1:100)or anti-CARM1 antibody(proteintech,1:100)overnight and followed with the specific HRP-conjugate secondary antibody for 30 minutes.Staining was performed with DAB and counterstained with Mayer’s hematoxylin.12.Statistical Analysis: All analyses were performed with SPSS version 22 or Graphpad Prism 5.0.Results:1.TRIM28 knockdown promotes the metastasis of CRC in vitro and in vivo.Kaplan–Meier survival analysis revealed that TRIM28 expression was positively associated with improved OS(Overall Survival)and RFS(Relapse-free Survival)of CRC patients from TCGA and GSE39582 datasets(P<0.05).Knockdown of TRIM28 significantly promoted cell migration and invasion in Lo Vo,HCT116 and SW48 cells in vitro.SW48 cells with stably knockdown TRIM28(sh TRIM28)were injected into the tail vein of nude mice whose pulmonary metastasis were examined five weeks later.TRIM28 knockdown significantly enhanced the incidence and the number of pulmonary metastatic nodules.2.TRIM28 interacts with CARM1 and suppresses CRC metastasis in a CARM1-dependent manner.The interactions between TRIM28 and CARM1 were confirmed by reciprocal co-immunoprecipitation(Co-IP)experiments in HEK293 T cells and Lo Vo cells.We further found that the PHD/BRO domain of TRIM28 and EVH1 domain of CARM1 were necessary for their interaction.Kaplan-Meier survival analysis revealed that CRC patients with low levels of CARM1 had significantly poor outcomes in TCGA and GSE39582 datasets.Knockdown of CARM1 promoted the migration ability of Lo Vo and HCT116 cells.Depletion of CARM1 abolished the TRIM28-overexpression-induced suppression on CRC metastasis.Thus,TRIM28 inhibits CRC metastasis in a CARM1-dependent manner.3.TRIM28 protects CARM1 from proteasome-mediated degradation through physical protein-protein interaction.Knockdown of TRIM28 decreased CARM1 protein level without affecting its m RNA level.Moreover,depletion of CARM1 had no effect on TRIM28 protein levels.The cycloheximide chase assay showed that knockdown of TRIM28 significantly reduced CARM1 protein’s half-life in two CRC cell lines(Lo Vo and HCT116).Of note,when TRIM28 knockdown cells were treated with a proteasome inhibitor(MG132),CARM1 protein levels were increased,suggesting that TRIM28 protects CARM1 protein from proteasome-mediated degradation.The ubiquitination of CARM1 was enhanced by TRIM28 knockdown.Collectively,these results indicate that TRIM28 stabilizes CARM1 protein by inhibiting its ubiquitination.We further constructed TRIM28(C65/68A)RING mutant that lacked the ubiquitin ligase activity,and then performed ubiquitination assays and functional assays.Similar to wild type TRIM28,the inactive TRIM28(C65/68A)RING mutant and the TRIM28-△N mutant,containing the PHD/Bromo domain decreased the ubiquitination of CARM1.However,the TRIM28-N mutant lacking PHD/Bromo domain did not affect CARM1 ubiquitination.We further re-expressed empty vector,wild type TRIM28,the inactive TRIM28(C65/68A)RING mutant,TRIM28-N mutant,and TRIM28-ΔN mutant in TRIM28 stable knockdown Lo Vo and HCT116 cells.Western blot analysis showed that wild type TRIM28,the inactive TRIM28(C65/68A)RING mutant,and TRIM28-ΔN could partially restore CARM1 protein level that was decreased by TRIM28 knockdown.On the contrary,TRIM28-N failed to do so.Consistently,the migration assays showed that wild type TRIM28,the inactive TRIM28(C65/68A)RING mutant,and TRIM28-ΔN,but not TRIM28-N mutant,attenuated metastasis enhanced by TRIM28 knockdown in Lo Vo and HCT116 cells.Taken together,TRIM28 stabilizes CARM1 protein to suppress CRC metastasis independently of TRIM28 E3 ligase activity,but dependently on its physical interaction with CARM1.4.TRIM28 dampens WNT/β-catenin signaling to suppress CRC metastasis.GSE39582 and GSE21510 datasets showed a reverse correlation between TRIM28 and β-catenin in CRC.Consistently,TRIM28 negatively regulated the protein level of β-catenin in both Lo Vo and HCT116 cells.WNT-target genes(TCF4,C-Myc,Axin2)were obviously increased when TRIM28 was knockdown.The nuclear of β-catenin was increased in Lo Vo and HCT116 cells with TRIM28 stable knockdown.WNT signaling inhibitor XAV939 diminished β-catenin’s elevated transcriptional activity by TRIM28 knockdown.XAV-939 also suppressed the migration promoted by TRIM28 knockdown in Lo Vo and HCT116 cells.we further knockdown CARM1 in TRIM28 stably overexpressed Lo Vo and HCT116 cells.As expected,CARM1 knockdown increased the protein level of β-catenin,and reversed the inhibited migration phenotype in TRIM28 stably overexpressed cells.Notably,such reversal was abolished by XAV939.These results demonstrate that TRIM28 negatively regulates colorectal cancer metastasis through a TRIM28-CARM1-WNT/β-catenin axis.5.Low expression of TRIM28 is associated with poor prognosis in CRC.To assess the clinical significance of TRIM28 and CARM1 in human colorectal cancer,we investigated the expression of TRIM28 and CARM1 in 15 paired colorectal tumor tissues and adjacent normal tissues.Western blot showed that both TRIM28 and CARM1 were downregulated in tumor tissues compared to adjacent normal tissues in 66.7% of CRC patients,consistent with immunohistochemical staining of TRIM28 and CARM1.These results demonstrated that the expression of TRIM28 was positively correlated with CARM1,and both of them may act as an inhibitory factor in CRC progression.We divided CRC patients into low and high expression groups based on TRIM28 or CARM1 values in GSE39582 dataset.TRIM28 expression was associated with tumor pathologic stage(P<0.05).CARM1 expression was associated with the extent of the tumor and distant metastasis(P<0.05).Multivariate cox regression analysis revealed low TRIM28 expression was an independent prognostic factor for poor survival in CRC patients(HR 1.464,95% CI,1.047–2.045;P =0.026;).Univariate cox regression analysis demonstrated CRC patients with low CARM1 expression were with poor survival(P<0.05).However,Multivariate cox regression analysis revealed CARM1 expression was not an independent prognostic factor in CRC patients(P=0.079).Moreover,the patients with high levels of TRIM28 and CARM1(TRIM28highCARM1high)had the best RFS,whereas the TRIM28lowCARM1 low patients had the poorest outcomes.Taken together,TRIM28 and CARM1 in CRC are highly and clinically related in predicting survival of CRC patients.Conclusion: We display for the first time that TRIM28 and CARM1 suppress CRC metastasis.TRIM28 protects CARM1 protein from degradation by ubiquitin-proteasome system through the orchestrated interaction between TRIM28 and CARM1.TRIM28 can inactivate WNT/β-catenin pathway that may contribute to suppress CRC metastasis in CARM1 manner.The analysis of clinical tissues highlights the high clinical correlation between TRIM28 and CARM1 in CRC.Low expression of TRIM28 and CARM1 is associated with poor prognosis in CRC.