TRIM27 promotes the development of esophagus cancer via regulating PTEN/AKT signaling pathway

Esophageal squamous cell carcinoma tissues (n = 25) and adjacent-matched noncancerous samples (n = 18) were collected from The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China. All tissues were snap-frozen in liquid nitrogen, stored at − 80 °C.

The cell lines used in this research were TE-1, TE-11, ECA-109, KYSE150 and HEEC, which were purchased from Shanghai biology institute (Shanghai, China). 10% fetal bovine serum (GIBCO, USA) was added to culture media that containing 2 mM l-glutamine and 1% penicillin/streptomycin (Solarbio, China). Cells were grown in DMEM Medium (Trueline, USA) and maintained in a 5% CO₂, at 37 °C incubator. This study was in agreement with the Declaration of Helsinki. The AKT inhibitor LY294002 (25 μmol/L, S1105, Selleck, USA) and glycolytic inhibitor 3-BrPA (20 μmol/L, 1113-59-3, Sigma, USA) were dissolved in DMSO (D2650, Sigma, USA) and used to culture cells.

Total RNA from different samples were isolated by using TRIzol Reagent (Invitrogen, USA). Then, cDNA synthesis kit (Fermentas, Canada) was used to reverse transcribe RNA into complementary DNA (cDNA). The program of the real-time PCR reaction was listed as follows: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 45 s. GAPDH was used to normalize the gene expression. 2^−ΔΔCt method was applied to calculate the relative gene expression. All data represented the mean of three replicates. Primer sequences are provided in Additional file 1.

For silencing human TRIM27 (NM_006510.4), three shot interference RNAs (siRNA) that targeting TRIM27 were synthesized (Major, Shanghai, China) and subsequently transfected into the KYSE150 and TE-11 cells respectively by using Lipofectamine 2000 (Invitrogen, USA). Meanwhile, a nonspecific scrambled siRNA sequence was transfected into KYSE150 and TE-11 cells as negative control (siNC). The targeting locus and sequence of TRIM27 siRNA is provided in Additional file 1: Table S1.

As for overexpression of TRIM27, a lentiviral plasmid (pLVX-puro) containing the full-length human TRIM27 cDNA sequence and a mock plasmid (oeNC) were transiently transfected into TE-1 cells respectively.

RIPA lysis buffer (JRDUN, Shanghai, China) was used to extract protein as indicated. An enhanced BCA protein assay kit (Thermo Fisher, USA) was utilized to estimate the protein content. Total protein (25 μg) was fractionated by using 10% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, USA) for 2 h, which were probed at 4 °C for 12 h with the primary antibodies followed by incubation for 1 h at 37 °C with the secondary antibody (HRP-labeled goat anti rabbit IgG antibody; 1:1000, Beyotime, China). An enhanced chemiluminescence system (Tanon, China) was utilized to quantify the protein content. Each analysis was detected in triplicate. GAPDH was used as the internal reference. The crucial information of the primary antibodies was listed in Additional file 1: Table S2.

Cell counting kit-8 (CCK-8) assay kits (SAB, USA) was used to examine cell proliferation. All procedures were performed according to the instruction of the manufacture. In brief, cells were seeded in 96-well plates and incubated with CCK-8 solution (1:10) for 1 h. Then, OD450 value was examined by microplate reader (Pulangxin, China) at 12, 24 and 48 h after seeding. The experiment was independently repeated thrice at each time point.

Briefly, Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime, China) was used to examine the apoptosis rate of cells according to the instructions of the manufacturer. After 48 h of viral infection, flow cytometer (BD, USA) were utilized to determine cells. Three replications were needed for each sample.

In brief, the glucose analog 2-NBDG kits (Biovision, San Francisco, USA) were used as a fluorescent probe for determining the activity of Glucose transport. In order to examine the uptake of 2-NBDG, a total of 5 × 10⁵ cells from different groups were seeded in 6-well plates. Then, all cells were pre-incubated in Krebs–Ringer bicarbonate (KRB) buffer (glucose free) for 15 min after maintaining in a 5% CO₂ atmosphere at 37 °C for 24 h. After that, cells were incubated in fresh KRB buffer supplemented with 2-NBDG for 45 min at 37 °C, 5% CO₂. Flow cytometry using a GloMax^®-Multi + flow cytometer (Promega, USA) was used to quantitatively analyze the stained cells.

For IP, whole-cell extracts were prepared after transfection or stimulation with appropriate ligands, followed by incubation overnight with the appropriate antibodies plus Protein A/G beads (Santa Cruz Biotechnology, USA). Beads were washed five times and separated by SDS-PAGE. Western blot was performed by using the antibodies as indicated above.

KYSE150 cells were transfected with siNC or siTRIM27, the cells were lysed in 1% SDS-containing radio immunoprecipitation assay (RIPA) buffer by sonication on ice. Then, Lysates were treated by Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, USA) for 1 h. After that, each samples were incubated with the IgG (Proteintech, USA) overnight at 4 °C. Then, the nuclear pellet was collected by centrifugation at 3000 rpm for 5 min at 4 °C and subsequently washed by Protein A/G Plus-Agarose beads for four times. The purified proteins were separated by 4–20% gradient SDS-PAGE. Anti-TRIM27 antibody (12205-1-AP, Proteintech, USA), Anti-PTEN (ab32199, Abcam, UK) and anti-Ubiquitin antibody (ab7780, Abcam, UK) antibody were used for immunoblotting.

A total of 25 ESCC tissues and 18 adjacent-matched noncancerous samples were used to examine the mRNA level of TRIM27. Moreover, six pairs of ESCC tissues and matched noncancerous samples were utilized to quantify the protein content of TRIM27. Both the mRNA and protein level of TRIM27 were significantly increased in ESCC tissues compared with that of adjacent samples (Fig. 1a, b). Therefore, our results demonstrated that TRIM27 was upregulated in ESCC tissues.

Moreover, qRT-PCR and western blot were used to determine the mRNA and protein level of TRIM27 in four ESCC cell lines, including TE-1, TE-11, EAC-109 and KYSE150. The esophageal epithelial cells HECC were used as control. In the present study, the level of TRIM27 was also upregulated in ESCC cells compared with that of HECC, especially in TE-11 and KYSE150 cells (Fig. 1c, d). Therefore, knockdown of TRIM27 was induced in TE-11 and KYSE150 cells respectively. Meanwhile, TE-1 cells were chosen for overexpression of TRIM27.

To further examine the function of TRIM27 in ESCC cells, we synthesized three short interference RNAs (siRNAs) targeting human TRIM27 (siTRIM27-1, siTRIM27-2 and siTRIM27-3) and a nonspecific scrambled siRNA (siNC) and subsequently transfected into KYSE150 and TR-11 cells. The unprocessed cells were served as blank control (BLANK). All TRIM27-siRNAs significantly suppressed the expression of endogenous TRIM27. Cells transfected with siTRIM27-1 and siTRIM27-2 showed a stronger effect than that of siTRIM27-3 in ESCC cells. Therefore, siTRIM27-1 and siTRIM27-2 transfected cells were used for the following analyses (Fig. 2a, b).

Meanwhile, TE-1 cells were transfected with a plasmid for overexpressing TRIM27 (oeTRIM27). Both the mRNA and protein content of TRIM27 was significantly overexpressed in oeTRIM27-transfected cells (Fig. 2c, d). Therefore, the oeTRIM27 transfected cells were utilized for the following analyses.

Cell proliferation rate was examined by cell counting kit-8 (CCK-8). As shown in Fig. 3a, b, siTRIM27-1 and siTRIM27-2 were deeply suppressed the proliferation rate of ESCC cells in two cell lines. Therefore, TRIM27 presented the pro-proliferation property in ESCC cells. Moreover, we also analyzed the apoptosis profile of siTRIM27 transfected cells. Obviously, the apoptosis profile of siTRIM27-1 or siTRIM27-2 transfected cells was much higher than that of siNC transfected cells (Fig. 3c). These results elucidated the anti-apoptosis function of TRIM27 in ESCC cells.

The abnormal of energy metabolism is one of the biochemical characters for cancer cells, which leads to the acceleration of glycolysis activity [15]. In this study, we analyzed the activity of glycolysis by using the glucose analog 2-NBDG. Interestingly, the level of 2-NBDG was deeply suppressed in ESCC cells transfected with siTRIM27-1 or siTRIM27-2 (Fig. 3d). Therefore, TRIM27 might promote the proliferation of human ESCC cells through enhancing the glycolysis activity.

Previous reports have demonstrated that GLUT1 and HKII are positive correlative with the activity of glycolysis [16‐19]. To further examine the function of TRIM27 in glycolysis, we quantified the protein content of GLUT1 and HKII in different cells as indicated. The protein content of GLUT1 and HKII were significantly decreased in siTRIM27 transfected cells (Fig. 3e, f). Moreover, the protein level of cleaved caspased-3 was remarkably upregulated in siTRIM27-1 or siTRIM27-2 transfected cells. In addition, the phosphorylation of AKT (p-AKT) was also inhibited in siTRIM27-1 or siTRIM27-2 transfected cells.

In order to further explore the correlation between TRIM27 and AKT, the AKT inhibitor LY294002 (25 μmol/L) was used to culture cells transfected with oeTRIM27. Meanwhile, the oeTRIM27 transfected cells were also cultured in the present of the glycolytic inhibitor 3-BrPA (20 μmol/L).

The cell proliferation rate was significantly promoted in oeTRIM27 transfected cells (Fig. 4a). However, the AKT inhibitor LY294002 deeply suppressed the cell proliferation rate of oeNC and oeTRIM27 transfected cells. Meanwhile, the similar result was also obtained in 3-BrPA cultured cells. Moreover, the apoptosis rate was deeply inhibited in oeTRIM27 transfected cells. Interestingly, the inhibitor LY294002 and 3-BrPA remarkably promoted the cell apoptosis rate in oeNC or oeTRIM27 transfected cells (Fig. 4b).

In addition, we also analyzed the activity of glucose transport in different cells as indicated. It was clearly identified that overexpression of TRIM27 promoted the transport of glucose in cells transfected with oeTRIM27. Moreover, the inhibitor LY294002 and 3-BrPA deeply inhibited the glucose transportation in oeTRIM27 transfected cells (Fig. 4c). As shown in Fig. 4d, the protein content of GLUT1 and HKII were significantly increased in oeTRIM27 transfected cells. However, the AKT inhibitor LY294002 deeply inhibited the expression of GLUT1 and HKII in oeNC or oeTRIM27 transfected cells. Moreover, the protein level of cleaved caspase-3 showed the opposite results as that of GLUT1 and HKII in oeTRIM27 transfected cells. Importantly, the phosphorylation of AKT was positively correlated with TRIM27, which was deeply suppressed by the inhibitor LY294002 and 3-BrPA. Taken together, all these results demonstrated that the effect of TRIM27 was deeply abolished by the AKT inhibitor LY294002 and 3-BrPA on ESCC cells.

PTEN is identified as a critical component in PI3/AKT pathway [20]. PTEN/AKT pathway is the common abnormal signaling pathway in human cancers [21]. In order to further examine the function of TRIM27 in PI3/AKT, we analyzed the interaction between TRIM27 and PTEN by co-immunoprecipitation (Co-IP). As shown in Fig. 5a, it was easily identified that there was a stronger interaction between TRIM27 and PTEN. These results indicated that TRIM27 directly interacted with PTEN in ESCC cells.

Previous report has demonstrated that the ubiquitination of PTEN is critical for its stability and nuclear localization [22]. Enhancing the ubiquitination of PTEN has improved the activity of PI3/AKT pathway [23]. In this study, intracellular ubiquitination assays was established to examine whether TRIM27 affected the poly-ubiquitination of PTEN. As shown in Fig. 5b, knockdown of TRIM27 deeply reduced the level of PTEN poly-ubiquitination in ESCC cells. Therefore, TRIM27 not only interacted with PTEN but also promoted its poly-ubiquitination in ESCC cells.