Downregulation of transcription factor EB inhibits the growth and metastasis of colorectal carcinomas

Patient and tissue specimens

Cancer tissues and paired non-cancerous tissues were obtained from 40 patients who were diagnosed with CRC at Yantaishan Hospital from 2015 to 2017. The 40 CRC cases were pathologically confirmed and histologically graded in accordance with the World Health Organization classification. Exclusion criteria were as follows: two or more different malignancies and patients who have received hormonal therapy, preoperative radiotherapy, or chemotherapy. The patients included were 18 males and 22 females and aged between 36–67 years with an average age of 52 years. All patients signed informed consent before the use of the samples. Fresh tissues were obtained during surgical resection without prior radiotherapy or chemotherapy. All samples were immediately frozen in liquid nitrogen and stored at −80°C until processed. The paired non-cancerous tissues were dissected away from the tumor tissues and confirmed lacking cancer cells by microscope. This study followed institutional ethical guidelines which were reviewed and approved by the Ethic Committee of Yuhuangding Hospital affiliated to Qingdao University.

Cell culture and transfection

All human colon cancer cell lines (HT-29, HCT-8, C2BBe1) and normal cell line CCD-18Co were purchased from ATCC, USA. Cell lines were cultured in RPMI 1640 medium (Solarbio, Beijing) complemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and 100 μg/mL streptomycin, incubated in a humidified atmosphere containing 5% CO₂ at 37°C, and the culture medium was replaced with fresh medium every 2 days, and logarithmic growth cells were harvested for the following experiment.

Cells were digested by 1 mL trypsin, added 2 × 10⁵ cells in six-well plates and cultured in 0.5 mL of serum-contained, antibiotic-free growth medium. Each colon cancer cell line or normal cell line was divided into three groups: small interference RNA (siRNA) group, negative control (NC) group, and control group. The TFEB-siRNA (5’-CAGGCUGUCAUGCAUUACATT-3’) in the siRNA group and scrambled TFEB-siRNA-A (5’-ACGAGAUUAGGCAUCACAAUA-3’) in the NC group were obtained from GenePharma. To develop the TFEB-siRNA vectors, the sequence of TFEB-siRNA and TFEB-siRNA-A were inserted into pcDNA3.1/Myc-His(-) B vector, then 2 μg of vectors were diluted with 50 μL of serum-free RPMI 1640 medium, dissolved in Lipofectamine 2000 reagent (Invitrogen, USA) and added to the each well. The transfected cells were incubated at 37°C in a 5% CO₂ incubator (Thermo, USA) for 48 h.

Quantitative real-time polymerase chain reaction

Total RNAs from colon cancer cells or tissues were extracted with TRIzol kit (Invitrogen, USA). RNAs were subjected to reverse transcription reactions using SuperScript III Reverse Transcriptase (No.18080044, Thermo Fisher Scientific, USA). Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out using the Master ep realplex2 (Eppendorf, Hamburg, Germany) under conditions: 94°C for 3 min, 94°C for 30 s, 60°C for 30 s, 35 cycles. The results were calculated by 2^−ΔΔCt(Livak and Schmittgen 2001), normalized to internal control β-actin mRNA. The primer sequences were as follows: TFEB sense 5’-CCAG-AAGCGAGAGCTCACAGAT-3’ and anti-sense 5’-TGTGATTGTCTTTCTTCTGCCG-3’; β-actin sense 5’-TCTACAATGAGCTGCGTGTG-3’ and anti-sense 5’-GGTGAGGATCTTCATGAGGT-3’.

Transwell assay

Precooled RPMI 1640 medium and Matrigel (Solarbio, Beijing, China) were mixed at the ratio of 1 and spread evenly on the bottom of the upper chamber (Thermo, Germany) with 100 μL per well and incubated at 37°C for 4 h. A total of 5 × 10⁴ cells in 200 μL medium without FBS from each group were seeded into the upper chamber. In the lower chamber, 500 μL of RPMI 1640 medium containing 10% FBS was added and incubated in a 37°C incubator. After 72 h, the chambers were washed twice by PBS, wiped with a cotton swab, then fixed in 5% methyl alcohol at room temperature, stained with 0.5% crystal violet solution (Solarbio, Beijing, China) for 30 min and washed twice with PBS. Five random visual fields were selected, and the cell numbers in each field were counted for each well using an inverted microscope (Olympus, Japan) to calculate the average value. Relative cell numbers were used for drawing bar graph.

Wound-healing assay

The stably-infected cells were plated in 24-well plates until full confluence. Cell layers were scratched using sterile 10 μL pipette tips and washed three times with serum-free RPMI 1640 medium to carefully remove the excised cell debris, then incubated in serum-free RPMI 1640 medium for 72 h. The cell-free wound area was photographed with an inverted microscope (Olympus, Japan). The results were analyzed by software ImageJ64. Eight horizontal lines were randomly drawn to measure the distance between cells, and the values were calculated for average. Thus, the relative migration rates were calculated as: (original scratch width–new scratch width)/original scratch width × 100%.

Western blot

Cellular proteins from cells and tissues were extracted using RIPA lysis buffer (No. R0020, Beijing Solarbio Science & Technology Co, Ltd, China). The concentration of protein was measured by BCA kit (No. PC0020, Beijing Solarbio Science & Technology Co., Ltd, China). A protein sample (4 μL) was mixed with 5× sample loading buffer, and the mixture was loaded on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. After the samples were separated by molecular weight, they were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck, Darmstadt, Germany). The membranes were washed and blocked with 5% skimmed milk for 2 h. Primary antibodies rabbit anti-TFEB antibody (1: 2000, ab96834, Abcam, UK) and rabbit anti-β-actin antibody (1: 1000, ab8226, Abcam, UK) were added and incubated overnight at 4°C. After 24 h, the membranes were washed and incubated with goat anti-rabbit IgG-HRP (1: 1000, MBS435036, MyBioSource, USA) for 1 h after rewarming. The results were observed and recorded with a Roche Elecsys-2010 (Roche, Switzerland). Protein expression levels were normalized to β-actin and quantified by Image J (National Institutes of Health (NIH)) software.

Xenograft model in nude mice

A total of 60 Balb/c nude mice (6–8 weeks, 20 ± 2 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co, Ltd (Animal Production License No.: SCXK 20140007). After acclimatization, 40 healthy mice were selected and randomly divided into two groups (the ratio of male/female is 1, n = 20/group).

HT-29 cells transfected with TFEB-siRNA (si-TFEB group), TFEB-siRNA-A (NC group) were harvested and resuspended in PBS. Later, 100 μL, 1 × 10⁶ cells in PBS mixture were injected subcutaneously into the back shoulder swollen of each mouse. Tumor size was measured once a week using the following equation: volume (mm³) = (length × width²)/2. All mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium at a dose of 40 mg/kg and sacrificed using cervical dislocation method.

In the end of sixth week after the initiation of experiment, the tumor cells were excised and weighed immediately.

Statistical analysis

SPSS, version 19.0, statistical software was used to analyze the monitoring data. The data analysis results were presented as the mean values ± SD. The t-test was used to analyze the data between the two groups. One-way analysis of variance (ANOVA) was used for multi-group comparisons followed with least significant difference (LSD) test. P < 0.05 was considered statistically significant.

Up-regulation of TFEB expression in colorectal carcinomas tissues

To determine expression of human TFEB in colorectal carcinomas, we performed qRT-PCR and Western blot analyses in patient tissues, respectively. Compared with normal colon tissues, TFEB mRNA level was significantly increased in colorectal carcinomas. Furthermore, we compared the TFEB protein levels in cancer and non-cancerous tissues, confirming that the expression of TFEB protein in colorectal carcinomas tissue was also significantly increased in comparison with normal tissues in two representative cases (Figure 1(b), P < 0.05).

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Figure 1.

The expressions of TFEB mRNA and protein were upregulated in colorectal cancer tissues: (a) The relative TFEB expression was determined using quantitative real-time PCR. β-actin was used as an internal control. (b) Protein expression was detected by Western blot. β-actin was used as an internal control. Data were represented as the mean ± SD. *P < 0.05, compared with normal tissue.

High expression of TFEB in colon cancer cells

To examine mRNA expression of human TFEB in CRC cell lines, we also conducted qRT-PCR for HT-29, HCT-8, and C2BBe1 cells. Our results showed that the mRNA expression of TFEB was significantly increased (P < 0.05) in the three types of CRC cell lines when comparing with normal cell line CCD-18Co (Figure 2). The mRNA expression was increased by 89% in HT-29 cells, 102% in HCT-8 cells, and 86% in C2BBe1 cells, respectively. In addition, there was no significant difference between HT-29, HCT-8, and C2BBe1 cells.

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Figure 2.

Higher level of TFEB mRNAs in human colon cancer cell lines (HT-29, HCT-8, and C2BBe1 cells) measured by quantitative RT-PCR. β-actin was used as an internal control. Data were represented as the mean ± SD. #P < 0.05, compared with normal cell line.

TFEB silencing downregulated expression of TFEB protein in CRC cells

We transfected TFEB-siRNA into HT-29, HCT-8, and C2BBe1 colon cancer cells to detect the potential roles of TFEB. The expression of TFEB protein in these cells was confirmed by Western blot (Figure 3). Our results showed that the expression of TFEB protein was significantly decreased (P < 0.05) after siRNA transfection. We also transfected TFEB-siRNA-A into carcinomas cell lines as NC group. In contrast, siRNA-A had no effect in TFEB protein compared with control group.

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Figure 3.

TFEB-siRNA transfection reduced expression of TFEB. (a) Expression of TFEB proteins in colorectal cancer cells measured by western blot. The protein levels were normalized to β-actin. Control group, no siRNA transfected; NC group, TFEB-siRNA-A transfected as negative control; siRNA group, TFEB-specific siRNA transfected. (b) The bar graph of relative western blots expressions of TFEB protein in three cancer cell lines. Data were represented as the mean ± SD. Compared with control group, #P < 0.05; compared with NC group, &P < 0.05.

TFEB silencing hindered CRC cells migration and invasion

Next, we detected the functional roles of TFEB in colon cancer cell lines. HT-29, HCT-8 and C2BBe1 cells were transfected with TFEB siRNA or siRNA-A followed by exploring cell migration and invasion ability that contribute to cancer progression. Through the photos, we could intuitively observe the decreased cell number of migration and invasion after knockdown of TFEB mRNA as compared to control and NC groups (Figure 4(a) and (b)). In Figure 4(c), we employed the percentage of migration rate to show the migration abilities of HT-29, HCT-8 and C2BBe1 cells. We found that the migration rate of the colon cancer cell was significantly reduced after TFEB-siRNA interference (P < 0.05). Figure 4(d) showed the quantified cell number of invasion, indicating that TFEB siRNA transfection could markedly decrease the invasion ability of HT-29, HCT-8 and C2BBe1 cells (P < 0.05).

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Figure 4.

TFEB mRNA silencing inhibited cell migration and invasion. (a) Cell migration was evaluated by wound-healing assays. Microscopic observations were recorded at 0, 72 h after scratching the cell surface. Cells were counted through selecting five random fields, and the results were represented as the average number of cells per field of view. (b) Invasion ability was evaluated using transwell assay. After 72 h, the tumor cells in bottom chamber were stained by 0.5% crystal violet for 30 min. Representative photographs of stained HT-29, HCT-8 and C2BBe1 cells were taken by inverted microscopy. (c) and (d) The statistical plots of wound-healing rate (%) and the relative number of invasion cells in each group, respectively. The data were mean ± SD. Compared with control group, #P < 0.05; compared with NC group, &P < 0.05.

TFEB silencing inhibited xenograft tumor growth in nude mice

To study the effects of TFEB on tumorigenicity, we constructed a xenograft tumor model. Since TFEB-siRNA showed the same effect in HT-29, HCT-8, and C2BBe1 CRC cell lines, the TFEB-siRNA and TFEB-siRNA-A transfected HT-29 cells were injected intraperitoneally into nude mice to form tumors. In the end of each time point, the tumor tissues were excised and the volumes of the tumors were measured every week (Figure 5(a)). The growth of tumors in the TFEB-siRNA group was significantly smaller than those in the NC group (P < 0.05). After 6 weeks, the nude mice were executed and the tumor weight was weighed. We found that the tumor excised from mice in siRNA group was lighter than NC group, indicating the better antitumor effect in vivo (Figure 5(b)).

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Figure 5.

TFEB silencing inhibited the tumorigenicity of HT-29 cells in vivo (n = 20). (a) Tumor growth curve of HT-29 tumors transfected by TFEB-siRNA in nude mice. The tumor size was monitored once a week. Compared with NC group, &P < 0.05. (b) A statistical plot of tumor weight in the animal model in NC and siRNA groups. The mean weight of tumors was weighed immediately after execution at sixth week. Compared with NC group, &P < 0.05. NC group, cells transfected with TFEB-siRNA-A; siRNA group, cells transfected with TFEB-siRNA.