Overexpression of TRIM28 predicts an unfavorable prognosis and promotes the proliferation and migration of hepatocellular carcinoma

Objectives: Previous studies have shown that tripartite motif-containing 28 (TRIM28) might be a latent target for cancer therapy. However, the detailed roles and mechanisms of TRIM28 in hepatocellular carcinoma (HCC) remain ambiguous. Methods: We systematically analyzed TRIM28 mRNA expression and protein levels in HCC tissues based on large-scale data and publicly available immunohistochemistry images. We estimated the prognostic capacity of TRIM28 in HCC. Additionally, we performed gene enrichment, immune in ﬁ l-tration, and drug sensitivity analyses to further explore the roles of TRIM28 in HCC. To determine the e ﬀ ect of TRIM28 expression on HCC cell proliferation and migration, successful transfection of siRNAs was conducted in MHCC97-L and Huh7 cells, followed by cell functional assays. Results: We veri ﬁ ed the overexpression of TRIM28 in HCC at the mRNA and protein levels. The summary receiver operating characteristics curve with the area under curve of 0.84 (95 % CI: 0.81 – 0.87) indicated the high accuracy of increasing TRIM28 expression for discriminating HCC from non-HCC tissues. According to The Cancer Genome Atlas datasets, TRIM28 mRNA expression was signi ﬁ cantly related to age, grade, stage, and pathologic T (p<0.05). Increased TRIM28 expression levels were signi ﬁ cant correlated to poor survival in HCC patients. An enrichment analysis suggested that TRIM28-reated genes primarily participated in the spliceosome signaling pathway, with hub genes including SNRPA1, SNRPF, SNRPD1, SF3B2, SNRPB, SNRPE, and EFTUD2. TRIM28 expression was correlated with the in ﬁ ltration of ﬁ ve immune cells. Higher TRIM28 expression was linked to better sensitivity of tumor cells to pluripotin. Molecular docking showed that pluripotin could bind to TRIM28. Further, knockdown of TRIM28 inhibited the proliferation and migration of HCC cells. Conclusions: TRIM28 is highly expressed in HCC and contribute to the proliferation and migration of HCC cells, leading to unfavorable outcomes. These ﬁ ndings indicate TRIM28 promise as a novel prognostic indicator.


Introduction
As one of the most frequent neoplasms worldwide, hepatocellular carcinoma (HCC) is characterized by a high rate of metastasis and recurrence, and its mortality rate is still increasing.The present five-year survival rate of HCC is 21 %, second only to pancreatic cancer [1].HCC typically arises from factors such as cirrhosis, chronic hepatitis, alcohol consumption, and other metabolic diseases [2].In recent years, although therapeutic strategies for treating HCC have constantly improved, many HCC patients still experience unsatisfactory clinical outcomes.Hence, further exploration of new therapeutic targets and molecular mechanisms that involved in the pathogenesis of HCC is urgently needed.
Many investigators have reported that TRIM28, also known as KAP1, TIF1β or KRAB-associated protein 1, is a key transcriptional regulator responsible for regulating multiple biological processes, which include promoting cell proliferation, inducing anti-proliferative activities, regulating the epithelial-mesenchymal transition (EMT), inhibiting and degrading p53 tumor suppressor, as well as mediating autophagy [3][4][5][6].Several studies have reported a link between elevated TRIM28 and worse clinical outcomes across distinct types of cancers, including lung adenocarcinoma, ovarian cancer, and prostate cancer [7][8][9].Similarly, TRIM28 was found to be significantly overexpressed in HCC patients as opposed to noncancerous tissues.Increased TRIM28 expression was also correlated with poor survival in HCC patients [10].Hence, TRIM28 might be a promising biomarker for cancer therapy.However, the specific roles and molecular mechanisms of TRIM28 in HCC have not been fully elucidated.
In this research, we aimed to determine the level of TRIM28 expression in HCC and to analyze its relationship with clinical characteristics of HCC cases.In addition, we sought to knock down TRIM28 expression in MHCC97-L and Huh7 cells to explore its function, thereby expanding the current knowledge on the potential mechanisms underlying HCC.

Mining high-throughput data sets
We mined HCC-correlated microarray and RNA-sequencing (RNA-seq) data in several databases, including Sequence Read Archive (SRA), ArrayExpress, and Gene Expression Omnibus (GEO).The search terms were as follows: (hepatocellular OR HCC OR hepatic OR liver) AND (tumor OR tumour OR carcinoma OR cancer OR neoplas*OR malignan*).We screened eligible studies according to the following criteria: (1) the subjects were Homo sapiens; (2) studies involving HCC and non-cancerous liver tissues; and (3) essential clinical information was provided.Further, RNA-seq data of HCC and non-HCC samples were downloaded from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases.We classified the datasets included in the present study into 40 merged datasets according to the platform type using the "sva" algorithm in R. Log(x+1) conversion was applied to normalize the TRIM28 expression data.

Single-cell RNA-seq (scRNA-seq) analysis
ScRNA-seq data of 17,164 liver cancer cells and 35,625 non-cancerous cells were obtained from the GSE151530 dataset to assess gene expression patterns in cancerous and various non-cancerous cells.Then, t-distributed stochastic neighbor embedding (t-SNE) analysis was used for the visualization of high-dimensional data in two dimensions, and t-SNE-1 and t-SNE-2 were both used to demonstrate cell clustering.The t-SNE plots were taken from the Single-cell Atlas in Liver Cancer (scAtlasLC, https://scatlaslc.ccr.cancer.gov)[11].

Collecting TRIM28 protein expression data from public database
After identifying differentially expressed genes (DEGs) at the mRNA level, immunohistochemical staining (IHC) results of TRIM28 were obtained from the Human Protein Atlas (HPA) database (http://www.proteinatlas.org/).The link for downloading proteomics data was https://pdc.cancer.gov[12].

Statistical analysis
Comparisons between two groups were assessed using the student's t-test, and Mann-Whitney-Wilcoxon test.The statistical results were exhibited in the form of mean(M) ± standard deviation (SD).p-values (p) less than 0.05 were considered significant.The standard mean difference (SMD) was calculated using Stata 12.0 (College Station, TX, USA).IF p<0.05 or I 2 >50 %, a random-effect model was calculated; otherwise, a fixed-effects model was selected.Begg's test was performed to evaluate the publication bias.
To assess the potential of TRIM28 for distinguishing between the HCC and non-HCC groups, a summary receiver operating characteristic curve (sROC) was created in Stata 12.0 (College Station, TX, USA).The area under curve (AUC) of sROC was used to determine the capacity of TRIM28 for discriminating HCC from non-HCC.HCC cases with follow-up times no fewer than 30 days were employed to carry out a survival analysis using the "survival" package in R. Kaplan-Meier (K-M) curves with a log-rank test were generated to evaluate the overall survival (OS) status of HCCs with different TRIM28 levels.The Hazard ratio (HR) was used to assess the prognostic value of TRIM28 for the HCC group.The pooled HR was calculated using Stata 12.0 (College Station, TX, USA).

Screening for latent TRIM28-related dysregulated target genes
As mentioned above, TRIM28 is a transcription factor that is associated with immune infiltration in multiple tumors.We retrieved the chromatin-immunoprecipitation coupled with sequencing (ChIP-seq) data of TRIM28 and collected latent target genes and potential binding position from the Cistrome DB database (http://cistrome.org/db/#/).Scores>1.0were used to filter the TRIM28 putative target genes.Subsequently, differentially expressed genes (DEGs) in the mRNA datasets were calculated via the "limma" package of R. DEGs with a SMD>0 and a lower 95 % confidence interval (CI) >0 were identified as upregulated DEGs (Up-DEGs) of HCC.In addition, we performed Pearson's correlation analysis to examine the co-expressed genes derived from HCC-related datasets.Genes were considered positively co-expressed with HCC in a minimum of 15 mRNA datasets when the correlation coefficient (r) was greater than or equal to 0.4 and the p was less than 0.05.The latent target genes, Up-DEGs and the positively co-expressed genes were intersected, with genes that appeared three times selected as the candidate target genes for downstream analysis.

Potential molecular mechanism of TRIM28 in HCC
To explore the underlying signaling pathways of TRIM28 potential target genes in HCC, we utilized the "clusterProfiler" package to analyzed and visualized these genes [13].If adjusted p-value<0.05,the items from the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were identified.Additionally, protein-protein interaction (PPI) networks were constructed utilizing the Search Tool for the Retrieval of Interacting Genes (STRING) database (http://string-db.org/).

The relationship between TRIM28 and immune infiltration levels in HCC
A previous study revealed that immune cell infiltration was closely related to tumor progression [14].Therefore, we leveraged the TIMER algorithm to calculate patients' immune scores based on TCGA data.The violin plots were popped out to present the infiltration scores of six types of immune cells in the high-TRIM28 and low-TRIM28 expression groups.We performed the Spearman correlation to investigate the immune correlation of TRIM28 in HCC.Scatter plots were drawn to show the correlation between TRIM28 expression and immune infiltration levels employing the SangerBox platform (http://vip.sangerbox.com/),which is a publicly available website for analyzing TCGA data [15].

Drug sensitivity analysis
Gene expression and drug sensitivity data were obtained from the CellMiner database (http://discover.nci.nih.gov/cellminer/)[16].When drugs without clinical trials or FDA approval were excluded, we utilized the Pearson algorithm in R to evaluate the associations between TRIM28 expression and drug sensitivity IC50 values, and p<0.05 was the threshold for significance.The "impute", "limma", and "ggpubr" algorithms in R were employed for data processing.

Molecular docking
The three-dimensional structure of TRIM28 (database ID: 2RO1) was downloaded from the RCSB Protein Data Bank (http://www.rcsb.org/).The structure of the drug ligand was retrieved from PubChem database.AutoDock Tools 1.5.6 (Scripps Research Institute, USA) were used for the docking analysis.Finally, visualization was performed using PyMOL software 2.2.0.The active ligand on the target was denoted by a threshold lower than −6.0 kcal/mol.
siRNA preparation and transfection: The sequences of short interfering RNAs (siRNAs) targeting TRIM28 are presented in Table 1.These siRNAs were synthesized and ordered from Sangon Biotech (Shanghai, China).Cells were transfected with siRNAs according to the manufacturer's instructions.
Quantitative real-time PCR and cell functional assays: The primers used for detecting TRIM28 mRNA are displayed in Table 2. GAPDH served as the endogenous reference gene.The relative mRNA expression levels of TRIM28 were determined by the algorithm 2 −ΔΔCt .CCK8 cell viability and transwell migration experiments were performed following the standard protocol when the TRIM28 gene in MHCC97-L and Huh7 cells was knocked out successfully.Cell Counting Kit-8 reagent (Vazyme Biotech, Nanjing, China) and 96-well plates were utilized for detecting the cell growth rate.A Multiscan MS spectrophotometer (Thermo Fisher Scientific, USA) was used for reading the absorbance of the wells at 450 nm.A transwell migration experiment was conducted to check cell migration capability.

Results
Up-regulated TRIM28 expression at both the mRNA and protein levels in HCC Of the 40 merged datasets, 26 showed that the expression of TRIM28 in the HCC group was higher than in the non-HCC group (p<0.05).Representative cohorts were GPL570, GSE25097-GPL10687, and TCGA_GTEx.Additionally, the findings of the integrative analysis utilizing the random effects model provided further evidence of up-regulated expression of TRIM28 in HCC.(pooled SMD=0.97,95 %CI: 0.83-1.12, Figure 1A).Based on the funnel plot, no significant publication bias was observed (p=0.258, Figure 1B).The sensitive analysis revealed no significant differences among the enrolled datasets.
The scRNA-seq analysis showed the broad existence of up-regulation of TRIM28 in the HCC cells, whereas TRIM28 overexpression rarely occurred in the non-malignant cells except for T cells (Figure 2A).It was further verified that highly expressed TRIM28 in HCC indeed came from the malignant cells.A proteomics analysis indicated that TRIM28 protein expression was highly expressed in the tumor compared to the control (0.2642 ± 0.4597 vs. −0.2652± 0.1237,  p<0.0001).Based on publicly available immunohistochemical staining, we found that positive TRIM28 staining was conspicuous in the HCC samples but not in the non-HCC samples (Figure 2B).Therefore, TRIM28 was observed to exhibit higher expression levels at both the mRNA and protein levels in HCC.

Clinical potential of TRIM28 in HCC
We generated an sROC curve with an AUC of 0.84 (95 % CI: 0.81-0.87),sensitivity of 0.64 (95 % CI: 0.58-0.70),and specificity of 0.88 (95 % CI: 0.84-0.92),demonstrating the high accuracy of increased TRIM28 expression in discriminating HCC from non-HCC tissues (Figure 3A).We estimated the prognostic capacity of TRIM28 and found that the low TRIM28 group exhibited superior OS compared to the high TRIM28 group for HCC patients based on three microarrays, TCGA, and the proteomics cohort (all p<0.05).Additionally, the increased TRIM28 mRNA levels corresponded to the shortened OS in HCC, as confirmed by the online web-tool, Gene Expression Profiling Interactive Analysis (GEPIA2) (http://gepia.cancer-pku.cn/)(Figure 3B, p<0.05).A forest plot with a pooled HR of 2.36 (95 % CI: 1.81-3.09)demonstrated that high expression of TRIM28 may be a risk factor of HCC (Figure 3C).Additionally, by utilizing the TCGA dataset, we examined the association between TRIM28 mRNA levels and clinicopathological features in individuals with HCC.Notably, as shown in Table 3, TRIM28 mRNA expression showed a significant correlation with age (p=0.0306),grade (p<0.0001),stage (p=0.0002)and pathologic T (p=0.0002), while it did not show significant relevance to other clinical parameters.

Enrichment analysis
Through the Cistrome DB database, we obtained five TRIM28 ChIP-seq datasets, which revealed 4,063 TRIM28 putative targets that were found in a minimum of three datasets.By investigating the links between all genes and TRIM28 across all data sources, including TCGA and gene chips, we identified 8676 Up-DEGs and 1754 positively co-expressed genes.These genes were then overlapped to obtain 629 potential dysregulated target genes related to TRIM28.Subsequently, we conducted GO and KEGG enrichment analyses using these overlapping genes.
The KEGG pathway analysis showed enrichment in the spliceosome, RNA transport, and cell cycle pathways (Figure 4A).The results of the GO analysis suggested that TRIM28 played a role in HCC by regulating biological processes including the cell cycle, cell cycle process, as well as chromosome organization.The most significantly enriched GO cell components were nuclear protein containing complex, chromosome, and the catalytic complex.In terms of molecular function, the most enriched GO terms were RNA binding, enzyme binding, and hydrolase activity acting on acid anhydrides (Figure 4B-D).Additionally, to further explore the correlation among these proteins, we constructed PPI networks based on the related genes enriched in the top three clustered pathways (Figure 4E-G).Using Cytoscape 3.7.2,we identified seven hub genes, including SNRPA1, SNRPF, SNRPD1, SF3B2, SNRPB, SNRPE, and EFTUD2.Further analysis using the Cistrome DB database revealed binding peaks of these hub genes at the transcription initiation sites of TRIM28 (Figure 5 and S1).

Correlation between TRIM28 and drug sensitivity
As shown in Table 4, 24 anticancer drugs that showed a significant correlation with TRIM28 expression were extracted.Surprisingly, we observed that TRIM28 expression was highly negatively correlated with the IC50 values of pluripotin (r = −0.309;p=0.018),where lower IC50 values indicated higher drug sensitivity.Further, the results showed that higher TRIM28 expression was linked to enhanced sensitivity of tumor cells to pluripotin.

Molecule dynamics simulation
To investigate the potential of pluripotin as a target for TRIM28, we performed molecular docking between pluripotin and TRIM28.As shown in Figure 7, the docking analysis revealed stable interactions between the target molecules of pluripotin and TRIM28, with a binding energy of −10.46 kcal/ mol.This value that was within the activity threshold of less than −6.0 kcal/mol indicating the presence of robust binding forces.These findings suggest that pluripotin holds promise as a potential agent for targeting TRIM28.

TRIM28 contributes to cell proliferation and migration in HCC
We used siRNA to knock down TRIM28 expression in MHCC97-L and Huh7 cells.The RT-qPCR results suggested that TRIM28 expression was down-regulated by TRIM28-siRNA (Figure 8A and B).As expected, the CCK-8 and transwell migration experiments showed that down-regulation of TRIM28 suppressed the proliferation and migration of liver cancer cells (Figure 8C-F).

Discussion
With a high mortality rate, HCC is the most frequent type of visceral neoplasm [17].Elucidating the molecular mechanisms involved in HCC carcinogenesis is crucial for identifying effective therapeutic targets and potential molecular biomarkers.In recent years, there has been significant attention focused on the role of TRIM28 in HCC.Studies have revealed that TRIM28 is overexpressed in HCC, and its expression serves as an independent predictor of survival in HCC patients [10].Further, TRIM28 has been found to regulate the development of HCC in mice models by engaging in physical and functional interactions with TRIM24 and TRIM33 [18].However, the precise mechanisms underlying the involvement of TRIM28 in HCC remain poorly understood.This study presented a comprehensive analysis of the expression and clinical value of TRIM28 in HCC by integrating microarray and RNA sequencing data from various sources worldwide.Our findings demonstrated a significant increase in TRIM28 expression at both the mRNA and protein levels in cancerous samples compared to non-cancerous samples.Importantly, scRNA-seq analysis further confirmed that the elevated expression of TRIM28 in HCC predominantly originates from malignant cells.Notably, our study highlighted the predictive potential of TRIM28, as indicated by the sROC curve, which yielded an AUC of 0.84, suggesting its excellent discriminatory ability.Further, we established a correlation between TRIM28 expression and prognosis at both the mRNA and protein levels.The K-M analysis revealed that high TRIM28 is associated with unfavorable survival outcomes in HCC patients.Additionally, elevated TRIM28 expression exhibits a significant correlation with age, grade, stage, and pathologic T status based on the TCGA dataset.The combined HR of 2.36 further supports TRIM28 overexpression as a risk factor for poor outcomes in HCC, consistent with previous research [10].In summary, we confirmed the differential expression of TRIM28 in HCC and found that up-regulated TRIM28 may function as a prognostic biomarker for HCC.
However, the molecular mechanisms of TRIM28 in HCC remain unclear.Therefore, we conducted a functional enrichment analysis to investigate the function of TRIM28 in HCC.Our results revealed that candidate downstream genes of TRIM28 mainly participated in the spliceosome signaling pathway.Numerous studies have demonstrated that spliceosome signaling has a vital impact on tumorigenesis and progression in various malignancies [19,20].Notably, several spliceosome-related genes, including SF3B2, SNRPD1,  SNRPB, SNRPE, SNRPF, SNRPA1, and EFTUD2, were found to be significantly up-regulated in HCC compared to normal tissue samples [21][22][23][24].Previous studies have shown that upregulation of EFTUD2 and SNRPA1 is associated with tumor propagation and poor survival outcomes in HCC [23,24].Similarly, over-activation of SNRPB, mediated by c-MYC, is closely correlated with unfavorable survival in HCC [25].These findings suggest that TRIM28 may function as an oncogene, promoting the genesis and progression of HCC through the spliceosome signaling pathway.Tumor-infiltrating lymphocytes (TILs) are critical components of the tumor microenvironment and have important implications in the development, prognosis, and antitumor therapy of HCC [26].In this study, we investigated the correlation between TRIM28 expression and immune cell infiltration, revealing a positive association between TRIM28 expression and the infiltration levels of B cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells, supporting the findings of Han et al. [27].The results from the TIMER algorithm also demonstrated that the high-TRIM28 group had higher immune scores compared to low-TRIM28 group for six immune cell types, including CD8+T cells, in HCC patients.While CD8+T cells have been shown to exert a protective effect in HCC [28], a study by Wolf et al. revealed that a high fraction of CD8+T cells is involved in liver damage and the carcinogenic process through interactions with natural killer T cells [29].Some researchers have suggested that high CD8+T cell levels are valuable for predicting a high relapse rate and a poor prognosis [30], which contradicts the notion cytotoxic T lymphocytes' antitumor effects.It has been reported that macrophages interact with infiltrating T lymphocytes, thereby promoting HCC progression [31].Additionally, recent research published in 2021 [32] implicated neutrophils, as an important component of the immune system, in the elimination of tumor cells.Collectively, this evidence highlights a significant relationship between TRIM28 and the immune response in HCC.
Further, we used the CellMiner database to investigate drugs related to TRIM28 in HCC.Our analysis revealed a significant negative correlation between TRIM28 expression and pluripotin IC50 values, and molecular docking indicated that pluripotin has the ability to bind to the molecular target of TRIM28.These findings provide compelling evidence that TRIM28 could serve as a promising therapeutic target for HCC.
Accumulating evidence suggests that TRIM28 is pivotal in promoting cell proliferation and metastasis in multiple cancer types [3,33].In the current study, we demonstrated that specific down-regulation of TRIM28 expression via siRNA resulted in a reduction in the proliferative capacity of HCC, consistent with previous research [10].Moreover, we observed a significant inhibition of HCC cell migration upon TRIM28 knockdown.As far as we know, no previous study has explored the influence of TRIM28 on the migratory capabilities of HCC cells.Our in vitro experiments suggested that knockdown of TRIM28 can disrupt malignant behaviors of HCC cells and may contributed to HCC carcinogenesis.
There are certain limitations in our research that should be acknowledged.First, our study primarily relied on cell lines to investigate the functions of TRIM28.To establish a more robust foundation for our findings, further research involving animal models is necessary.Second, it is crucial to validate the specific regulatory mechanisms of TRIM28-related hub genes through a comprehensive series of cell and animal experiments.More scientific investigations are required to address these limitations.
Overall, our findings indicate that elevated levels of TRIM28 promote both the proliferation and migration of HCC cells, which are associated with an unfavorable prognosis.Therefore, TRIM28 has potential as a novel predictive prognostic factor for HCC.The findings of this research may facilitate the development of innovative therapeutic strategies supporting the treatment of HCC.

Figure 1 :
Figure 1: Comprehensive analysis of TRIM28 expression level in HCC and non-HCC groups.(A) Forest plot of pooled standard mean deviation values for TRIM28 in HCC.(B) Begg's funnel plot of the enrolled studies.No significant publication bias exists (p=0.258).

Figure 2 :
Figure 2: (A) Single-cell transcriptomic atlas of malignant cells and various non-malignant cells in liver cancer.(B) The IHC-based protein expression of TRIM28 in HCC tissues and normal liver tissues.All the IHC staining images were obtained from the HPA database (https://www.proteinatlas.org/).IHC, immunohistochemistry.

Figure 3 :
Figure 3: The clinical significance of TRIM28 expression in liver cancer.(A) SROC curve demonstrating performance of TRIM28 in discriminating HCC from non-HCC tissues.(B) The GEPIA database was used to demonstrate the impact of high TRIM28 expression on the prognosis of overall survival in HCC patients.(C) Pooled HR based on five datasets.SROC, summary receiver operating characteristic; AUC, area under the curve; HR, hazard ratio.

Figure 4 :
Figure 4: Enrichment analyses.(A) The KEGG enrichment analysis.(B) GO enrichment analysis in terms of biological process.(C) GO enrichment analysis in terms of cellular component.(D) GO enrichment analysis in terms of molecular function.(E) PPI in the spliceosome pathway, which is the most significantly clustered KEGG pathway.Seven hub genes (SNRPA1, SNRPF, SNRPD1, SF3B2, SNRPB, SNRPE, and EFTUD2) were identified.(F) PPI in the RNA transport pathway.(G) PPI in the cell cycle pathway.KEGG, Kyoto encyclopedia of genes and genomes; GO, gene ontology; PPI, protein-to-protein internet.

Figure 6 :
Figure 6: (A) The relationship between TRIM28 expression and infiltration levels of immune cells; the blue "violin" refers to the low-TRIM28 expression group, while the red "violin" refers to the high-TRIM28 expression group.(B) Relevance between TRIM28 expression with infiltration levels of all the six immune cells.

Figure 8 :
Figure 8: TRIM28 in human liver cancer cell lines.(A) RT-qPCR verification of the TRIM28 siRNAs transfection efficiency in MHCC97-L cells.(B) RT-qPCR verification of the TRIM28 siRNAs transfection efficiency in Huh7 cells.(C) Line chart of OD450 values for different groups of MHCC97-L cells.(D) Line chart of OD450 values for different groups of Huh7 cells.(E) The effect of TRIM28 knockdown on the migration ability of MHCC97-L cells detected by transwell migration assay.(F) The effect of TRIM28 knockdown on the migration ability of Huh7 cells detected by transwell migration assay.NC, negative control; OD, optical density.*p<0.05,**p<0.01,***p<0.001.

Table  :
The sequences of short interfering RNAs (siRNAs) targeting TRIM.

Table  :
The primers used for RT-qPCR experiment.

Table  :
Clinical and pathological features of TRIM in HCCs.

Table  :
Correlation of TRIM with anticancer drug sensitivity.