Introduction

The incidence and mortality in colorectal carcinoma (CRC) are 6.1% and 9.2%, respectively, which is influenced by the dietary patterns, obesity, and lifestyles [1]. Based on the effective therapy and management, the incidence of CRC in transitioned countries is lower than transitioning countries [1]. The early screening for colorectal carcinoma could improve survival and diagnosis delay [2]. Thus, exploring the molecular mechanisms underlying CRC oncogenesis and progression is crucial for the CRC diagnose and therapy.

Tripartite motif (TRIM) belongs to E3 ligase family, which involves in more than 80 members in human. TRIM participates in multiple cell functions such as proliferation, migration, and invasion in human carcinomas [3, 4]. Unlike the other members of TRIM, tripartite motif containing 44 (TRIM44) is a deubiquitinase instead of an ubiquitinase. Researches indicate functions of TRIM44 in different cancers. For example, overexpression of TRIM44 promotes cell proliferation and migration in renal cell carcinoma, lung cancer, glioblastoma, and esophageal cancer [3, 5,6,7]. Besides, clinical researches find that TRIM44 can be a potential biomarker for cervical cancer and skin squamous cell carcinoma [8, 9]. Only handful of publications (a total of 2) revealed the correlation between TRIM44 and CRC. The following mechanisms are rarely investigated.

Chemokine and the corresponding receptors have been revealed to be associated with tumor growth, invasion, and metastasis. CXCL12/CXCR4 axis can mediate cancer metastasis [10, 11]. TNF-α, an inflammatory factor, upregulates CXCR4 expression to facilitate the chemotaxis of Myeloid-derived suppressor cells to tumors [12]. Various proteins including TCF12, ZEB1, and C/EBPα p42 bind to promoter region of CXCR4 [13,14,15]. Besides, CXCR4 binds to the NF-κB binding site in its promoter region, convincing the direct upregulation of CXCR4 by NF-κB in breast cancer and pancreatic cancer cell lines [16, 17]. Also, CXCR4 positive cells is more observed in liver metastases than in primary colorectal tumors [18]. Increasing expression of CXCR4 in cancer enhances the levels of matrix metalloproteinases (MMPs) and promotes epithelial-mesenchymal transition to induce invasion and migration in tumors [19, 20]. Previous investigations indicated that TRIM44 modulates NF-κB in breast cancer, hepatocellular carcinoma, and lung cancer [21,22,23]. In summary, TRIM44 might regulate NF-κB/CXCR4 to promote cancer progression. Here, we analyzed the expression of TRIM44 and assessed its role in proliferation, migration, and invasion in CRC.

Materials and Methods

Cell Culture and Transfection

Human CRC cell lines (SW620, SW480, HT-29, and HCT-116) were obtained from Shanghai Institute of Biochemistry and Cell biology (Shanghai, China) and cultured in DMEM (Gibco, Thermo Fisher Science, USA) supplemented with fetal bovine serum (FBS; Gibco, Thermo Fisher Science, USA). The fetus-sourced normal colorectal mucosa cell line (NCM460) was obtained from Gaining Biological technology (Shanghai, China) and cultured in RPMI 1640 (Gibco) supplemented with 10% FBS. Cells were incubated at 37 °C with 5% CO2. Human TRIM44 cDNA was amplified by polymerase chain reaction (PCR) with primers. SW480 cells were transfected with TRIM44‐shRNA (shRNA-NC), pGMLV‐SC5‐Puromycin‐EGFP‐shRNA‐TRIM44, pGMLV‐SC5‐Puromycin vectors (NC), and pGMLV‐PE3‐RFP‐TRIM44 lentiviral vectors (Shanghai Genomeditech company, Shanghai, China). The shRNA-NC and pGMLV‐SC5‐Puromycin vectors were used as controls. The SW480 cells (5 × 103 cells/well) were planted into 96-well plates. After TRIM44 was inhibited or overexpressed in SW480 cells, cell viability was detected by CCK-8 assay kit (ApexBio Technology, USA) according to manual’s protocol.

NUclear and Cytosolic Fractionation and Western Blot

The total proteins from cells were isolated by RIPA lysis buffer (Thermo Fisher Scientific, USA). To detect the translocation of NF-κB protein in cells, proteins from the nucleus and cytoplasm were fractionated using Qproteome Nuclear Protein Kit (Qiagen). Western blotting analyses were performed as previously described. LaminB1 was used as the inner control of nuclear protein and GAPDH as the inner control of cytoplasm or total protein.

Real-time Quantitative-PCR

TriZol was used to extract RNA in cells. For quantifying mRNA expression of TRIM44, NF-κB, and CXCR4 in cell lines, RT-qPCR was conducted using Fast SYBR Green Master Mix (4385610, Thermo Fisher Scientific, USA) according to manufacturer’s instruction, and GAPDH was used to normalize the CT values. The thermal cycling steps contained polymerase activation (95 °C, 10 min), denaturation (95 °C, 15 s for 40 cycles) and extension (60 °C, 1 min for 40 cycles). The gene expression was assessed by a previously described method [24].

CHromatin Immunoprecipitation Assay

High-sensitivity chromatin immunoprecipitation (ChIP) kit purchased from Millipore (USA) was used to detect the binding of NF-κB to CXCR4 gene promoter following manufactures’ instruction. Rabbit anti-NF-κB (the same as western blotting) was diluted with 1:100 in bovine serum albumin (BSA; Merck, Germany), and the rabbit anti-IgG (#3900, Cell signaling Technology, USA) was diluted referring to NF-κB. PCR was performed to detect the CXCR4 promoter. The primers of CXCR4 promoter: forward 5'-CTTAGGAAATGCCTCTGGGAGGTC-3' and reverse 5'-GCAGACGCGAGGAAGGAG-3'.

CEll Migration and Invasion

Cell migration was determined by Scratch assay. The cells were planted at the density of 5 × 105 cells/well in 6-well plates. After cells reached 100% confluence, the monomer confluence was scratched. The cells were incubated in serum-free DMEM medium for 24 h. The migratory distance of cells was observed under light microscope (Olympus, Japan) and analyzed by Image J software.

Cell invasion was detected by Transwell assay. The upper chamber with Matrigel coating (8.0 μm PET membrane, 24 well plate, Corning, USA) was prepared. The cells were placed into upper chamber with 1 × 104 cells/well and cultured in 400 μL serum-free DMEM medium. The lower chamber was added with 600 μL DMEM containing 10% FBS. After incubation for 24 h, cells on the bottom of the upper chamber was fixed with 90% ethanol solution for 30 min. 0.1% crystal violet was used to stain the cells for 10 min. At last, the invasive cells were observed by the light microscope.

Statistics

All the statistical analyses were calculated via GraphPad Prism 6.0 software. All the data are presented as the means ± SD. All experiments were repeated for 3 times. Groups were compared by one-way ANOWA. It was believed as statistically different when P value < 0.05.

Results

TRIM44/NF-κB/CXCR4 Biological Axis was Activated in CRC

A research shows that TRIM44 modulates NF-κB signaling in breast cancer [21]. Herein, we found that the protein and mRNA expression of TRIM44, NF-κB, and CXCR4 was dramatically enhanced in CRC cell lines (SW620, SW480, HT-29, and HCT-116), especially in SW480 cells, compared with colorectal cells (NCM460) (Fig. 1). To investigate the effects of TRIM44 in CRC, TRIM44 was knocked down and overexpressed, separately, in SW480 cells (Fig. 2a). Results showed that TRIM44 positively regulated the expression of CXCR4 in SW480 cells (Fig. 2b). Furthermore, data indicated that nuclear p65 expression was upregulated while cytoplasmic p65 expression was downregulated under overexpression of TRIM44 in SW480 cells (Fig. 3a, b). However, this phenomenon was reversed by NF-κB inhibitor, BAY11-7082. Whether the regulation of CXCR4 by TRIM44 in SW480 cells was due to activation of NF-κB was explored by ChIP assay. After TRIM44 were overexpressed or BAY11-7082 was added, the NF-κB binding to the CXCR4 promoter was investigated. The CXCR4 gene promoter was amplified by PCR. The results demonstrated that and TRIM44 enhanced the NF-κB activation to enhance its binding to CXCR4 promoter (Fig. 3c).

Fig. 1
figure 1

Expression of TRIM44, NF-κB, and CXCR4 in CRC cells. The protein (a) or mRNA (b) expression of TRIM44, NF-κB, and CXCR4 in CRC cell lines compared with normal colorectal cells. (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001 compared with NCM460

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Fig. 2
figure 2

TRIM44 regulates CXCR4 expression in SW480 cells. a TRIM44 was overexpressed or silenced in SW480 cells. b The expression of CXCR4 after overexpression or inhibition of TRIM44. (n = 3) *p < 0.05, **p < 0.01, ***p < 0.001 compared with control; #p < 0.05, ##p < 0.01 compared with NC; ΔΔp < 0.01, ΔΔΔp < 0.001 compared with shRNA-NC group

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Fig. 3
figure 3

TRIM44 induces the activation of NF-κB and enhances the binding of NF-κB to the CXCR4 promoter in SW480 cells. a The fractions of NF-κB/p65 protein in the cell nuclei and cytoplasm after overexpression of TRIM44 or cotreatment with NF-κB/p65 inhibitor, BAY11-7802, were assessed by western blotting. b ChIP assays were performed to investigate the activation of CXCR4 promoter by NF-κB in different groups. (n = 3) **p < 0.01, ***p < 0.001 compared with control; ##p < 0.01 compared with NC; ΔΔp < 0.01, ΔΔΔp < 0.001 compared with TRIM44 group

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TRIM44 Regulated CRC Viability, Migration and Invasion

CCK-8 assay discovered that cell viability was reduced in SW480 cells with silenced TRIM44 (Fig. 4a). CXCR4, as a chemokine receptor, has been shown to regulate proliferation and invasion in colon cancer [25]. Therefore, we investigated the migration and invasion ability in TRIM44-silenced SW480 cells. The wound-healing and Transwell assays manifested that knockdown of TRIM44 inhibited the cell migration and invasion, respectively (Fig. 4b, c). Furthermore, the expression levels of MMP2 and MMP9 were significantly downregulated after TRIM44 downregulation (Fig. 4d).

Fig. 4
figure 4

TRIM44 regulates cell viability, migration, and invasion in SW480 cells. a CCK-8 was performed to investigate cell viability after TRIM44 silence. Wound-healing (b) and Transwell (c) assays were used to assess cell migration and invasion, respectively. d Western blotting analysis was performed to investigate the expression of MMP2 and MMP9. (n = 3) ***p < 0.001 compared with control; ###p < 0.001 compared with shRNA-NC

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Cell Viability, Migration, and Invasion were Regulated by TRIM44/NF-κB/CXCR4 Biological Axis

To further investigate whether TRIM44 nodulated cell viability, migration, and invasion by activating NF-κB/CXCR4, the NF-κB inhibitor (BAY11-7082) and CXCR4 inhibitor (AMD3100) were used. Data showed that inhibition of NF-κB or CXCR4 significantly reduced TRIM44 elevation-triggered cell viability, migration, and invasion (Fig. 5a–e). Besides, levels of MMP2 and MMP9 enhanced by overexpression of TRIM44 were dramatically suppressed by treatment of NF-κB or CXCR4 inhibitor (Fig. 5f).

Fig. 5
figure 5

The effects of TRIM44 in SW480 cells were mediated by activation of NF-κB/CXCR4. Wound-healing (a, c) and Transwell (b, d) experiments were performed to evaluate cell migration and invasion after cells were overexpressed TRIM44 and treated with NF-κB inhibitor (BAY11-7802, 10 μM) or CXCR4 inhibitor (AMD3100, 100 μM). e CCK-8 was performed to assess cell viability. f The expression of MMP2 and MMP9 was determined by immune-blotting. (n = 3) ***p < 0.001 compared with control; ###p < 0.001 compared with NC; Δp < 0.05, ΔΔp < 0.01, ΔΔΔp < 0.001 compared with TRIM44 group

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Discussion

In the current investigation, our data implied that TRIM44 was a key point for the migration and invasion of CRC cells in vitro. TRIM44 not only activates Akt/mTOR signaling pathway in CRC cell proliferation, migration, and invasion [26], but also activated NF/κB and further promoted CXCR4 activation in CRC cancer, as the current work revealed. Besides, TRIM44 enhanced the expression of MMP2 and MMP9, indicating its potential effects on tumor microenvironment restoration.

TRIM44 has been reported to modulate multiple cellular functions, proliferation, metastasis, migration, and invasion included [3, 5, 27]. Here, we unveiled that the level of TRIM44 was upregulated in CRC cell lines, similar with other cancers. Besides, TRIM44 regulated CRC viability, migration, and invasion. TRIM44 plays an important regulatory role in a wide range of biological processes, including cell proliferation, innate immunity, viral infection, and tumor development. Here, we firstly showed that the down-regulation of TRIM44 inhibited the cell viability, migration, and invasion in SW-480 cells which were similar to previous studies in other cancers [28, 29].

Moreover, TRIM44 has been indicated as a modulator of NF-κB signaling in breast cancer to promote cell proliferation and migration [21]. An important finding in this investigation is that TRIM44 activates NF-κB/CXCR4 axis to boost cell viability, migration and invasion. A previous investigation has proved that NF-κB activates the CXCR4 promoter region [30]. In the present investigation, ChIP assays affirmed that NF-κB interacted with CXCR4 gene promoter. Regulation of CXCR4 affects the expression of MMP-2 and MMP-9 in endometrial cancer cells [31]. MMPs including MMP2 and MMP9 are closely associated with cancer cell migration and invasion [32]. Extracellular matrix degradation is crucial for cancer progression. MMPs are involved in the degradation and subsequent rearrangement of extracellular matrix during the invasion and migration of cancer cells [33]. In terms of MMP2, MMP2 was found to function as an early response protein in ovarian cancer metastasis, and the initial steps of ovarian cancer cell metastasis are mediated by MMP2 cleavage of vitronectin and fibronectin [34]. In the current paper, an NF-κB inhibitor or CXCR4 inhibitor hindered the TRIM44 overexpression-induced cell viability, migration, and invasion abilities. Therefore, the promotion of cell viability, migration, and invasion by TRIM44 might be partly attributed to activation of NF-κB/CXCR4 axis.

Also, TRIM44 serves as a biomarker for diagnosis and prognosis. For instance, TRIM44 has been identified and validated as a clinical molecular biomarker for esophageal adenocarcinoma [35]. High TRIM44 expression might be a potential biomarker for cervical cancer diagnosis [8]. Thus, TRIM44 can be a valuable oncogene to be investigated in CRC progression, which requires further exploration. Based on the previous investigations and our present preliminary data, TRIM44 could be implied as a molecular target for CRC therapy and diagnosis through activating NF-κB/CXCR4 biological axis.