lncRNA TUSC7 regulates oxidative stress level by targeting miR-23b in colorectal cancer and thus inhibits cell proliferation, migration and invasion

Animal modeling

Male BALB/c mice (purchased from Jackson Laboratory) and colon adenocarcinoma CT26 cell lines (Procell) were used. CT26 cells were routinely cultured with Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% fetal bovine serum (FBS). The cells in the exponential growth phase were collected, digested, and pipetted into single-cell suspension, then centrifuged at 1,000 r/min for 5 min. After the supernatant was discarded, the cell concentration was adjusted to 1 × 10⁶ cells/mL using an appropriate amount of serum-free RPMI-1640 medium. Finally, 0.2 mL of CT26 cell suspension (1 × 10⁶/mL) was slowly injected into the right of midline of the lower abdomen of BALB/c mice. The abdominal circumference of mice was then observed and measured.

Flow cytometry

Tissues aseptically harvested were washed with D-phosphate buffered saline (PBS) to remove blood stains, and cut into 1 × 3 mm blocks with sterile scissors. The tissue blocks were stirred in a solution containing 0.2% collagenase type IV (Sigma_Aldrich, USA) and 0.25% trypsin (Thermo Fisher, USA) for 5 min at 37°C [19]. After termination of digestion, the supernatant was harvested, filtered through a 200-micron mesh, resuscitated with staining buffer (1 × PBS containing 1% BSA), and stored at 4°C. Cell staining and flow sorting were then conducted. After the cell density was adjusted to 1 × 10⁶/100 μL, the cells were stained with CD68 Monoclonal Antibody (Ki-M7), FITC, Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody and Alexa Fluor™ 594 away from light for 30 min. Then the cells were analyzed and sorted with a BD FACSAria III sorting flow cytometer (BD Biosciences, USA), and the sorted macrophages were used in subsequent assays.

Immunofluorescence staining

The prepared sample was taken out, cooled to room temperature, and washed three times with PBS for 5 min each time. Then, the sample was soaked in the citric acid buffer, and repaired with low heat in the microwave oven. After cooling, the sample was washed three times with PBS for 5 min each time and blocked for 20 min. The first antibody was the diluted to the target ratio with antibody diluent at 4°C overnight. After rewarmed for 30 min, the fist antibody was rinsed three times with PBS for 5 min each time. The fluorescent secondary antibody was diluted with antibody diluent, incubated for 2 h at room temperature away from light. Then it was eluted with PBS three times for 5 min each time. Then, the nucleus was stained with anti-fluorescence quenching blocking solution and the tablet was sealed and kept at 4°C away from light. Last, the fluorescent image was captured after the fluorescence microscope observation.

Cell transfection and culture

The overexpression (OE) vector pE-TUSC7 and blank vector pEGFP-N1 (Genescript, Nanjing, China) were used. To inhibit TUSC7, 4 small interfering RNAs (siRNAs) (TUSC7-siRNA1, TUSC7-siRNA2, TUSC7-siRNA3 and TUSC7-siRNA4; Gene Pharmaceuticals, Shanghai, China) were synthesized and optimized. Upon reaching approximately 60–70% confluency, the cells were transfected with Lipofectamine™ 3000 reagent. Then the cells were divided into negative control (NC) group, TUSC7 OE group and TUSC7 KD group. Mouse macrophages were cultured with Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, 100 U/mL streptococci, non-essential amino acids (NEAA), and 100 U/mL streptomycin in a 5% CO₂ humid chamber at 37°C, and then incubated with LPS (1 μg/mL) overnight at 37°C. Finally, the supernatants and cell lysates were collected and stored at −70°C for further analysis.

Real-time quantitative polymerase chain reaction (RT-qPCR)

To quantify TUSC7 and miR-26b, total RNA was extracted using TRIzol and reversely transcribed into cDNA using the Reverse Transcription Kit (TaKaRa, Bio, USA). Then real-time quantitative PCR was performed using the SYBR-Green PCR Master Mix Kit (TaKaRa Bio, USA). The gene expression level was calculated using the 2^−ΔΔCt method. The primer sequences are as follows: TUSC7 (F: 5′-TTTATGCTTGAGCCTTGA-3′; R: 5′-CTTGCCTGAAATACTTGC-3′), miR-26b (F: 5′-GCGGTCATCACATTGCCAG-3′; R: 5′-GCGGTCATCACATTGCCAG-3′), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F: 5′-TGAAGGGTGGAGCCAAAAG-3′; R:5′-AGTCTTCTGGGTGGCAGTGAT-3′).

Dual luciferase assay

A double luciferase reporter plasmid was constructed using Genescript (Nanjing, China), including pmiR-TUSC7-wt (containing miR-23b target binding sequence in TUSC7) and pmiR-MACC1-mut (containing mutational binding sequence). The dual luciferase assay is performed by co-transfection of pmiR-TUSC7-wt or pmiR-MACC1-mut and agomir-23b or agomir-NC into peritoneal macrophages. Luciferase activity after 48 h of co-transfection of peritoneal macrophages was detected with the Dual Luciferase Reporter Detection System (Promega, USA) in accordance with the manufacturer’s protocol.

Western blotting

The protein was extracted from cells using lysis buffer (Solarbio, Beijing, China). By 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA), and then the membranes were incubated with primary antibodies against P22, P47, gp91, phosphorylated (p)-SHP2, t-SHP2, p-STAT3, p-STAT6, interleukin-4 (IL-4), IL-10, matrix metalloproteinase-2 (MMP2), MMP9 and GAPDH overnight at 4°C. After washed with TBST, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies at room temperature for 2 h, and detected using an enhanced chemiluminescence detection system (Bio-Rad, USA) according to the manufacturer’s protocol.

Cell co-culture

Peritoneal macrophages in each group were seeded into cross-well chambers and cultured for 24 h. CT26 cells and CMT93 cells were seeded in culture plates and cultured for 24 h. After incubation overnight, the medium in the chambers and plates was removed and a new culture medium was added to the lower chamber. Then, the Transwell chamber was placed in the culture plate for 48 h, and then new medium was added for detection. The cross-well chamber was removed and the medium was aspirated from the culture plate. Finally, the cells were fixed with 4% paraformaldehyde and washed twice with PBS.

Transwell assay

The migration and invasion capacity of cells was detected in vitro using Transwell chambers with polycarbonate membranes (251.87 mm in diameter, a pore size of 6 μm) (Costar Corning, USA). In the migration assay, the transfected cells were resuspended in 10 μL of serum-free medium at a density of 100 × 5/mL and added to the upper chamber. Next, 12 μL of DMEM supplemented with 10% FBS was added to the lower chamber. After incubation for 20 h, the cells on the upper membrane surface were removed, and the migrating cells were fixed with methanol and stained with 80% Giemsa stain solution. Finally, the stained cells were counted microscopically in five randomly selected fields, and the average was taken. In the invasion assay, the Transwell membranes were applied to 500 μL of Matrigel solution (37 ng/μL, BD Biosciences, USA), and the cells were incubated at 37°C for 4 h. The remaining steps were the same as those in the migration assay.

Monoclonal formation assay

After 48 h of transfection, the cells were collected, digested with trypsin and counted. They were cultured in an incubator at 37°C until visible cell colonies were formed. Then the medium was discarded, and the cells were washed with PBS 3 times. Afterwards, the cells were fixed with methanol for 15 min, then dried and stained with crystal violet for 30 min. After drying, the cells were scanned and photographed, and the visible colonies were counted.

Cell counting kit-8 (CCK8) assay

After 48 h of culture, 10 μL of CCK8 reagent was added to each well and incubated for 2 h. Then the optical density (OD) at 450 nm was measured with a microplate reader to determine the cell viability.

Data analysis

Statistical analysis was performed using SPSS 22.0 software and processed with GraphPad Prism 5.0. Measurement data of normal distribution were expressed by mean ± standard deviation ($\overline{x}\pm \text{s}$). Independent-sample t-test and one-way analysis of variance were used for comparison between two groups and among multiple groups, respectively. P < 0.05 or p < 0.01 was considered statistically significant.

The metastasis in cancer is stronger than the tissues around cancer

By immunofluorescence staining, we found that the relative fluorescence intensity of MMP9 in human cancer was significantly stronger than that in the peritoneal tissues. It indicated that the metastatic ability in cancer was stronger than that in the tissues around cancer. Therefore, the study on the cancer itself is more convincing than that on the cancerous tissues that surround it (Figure 1).

Figure 1. The relative fluorescence intensity of MMP9 in cancer and in peritoneal tissues was detected by immunofluorescence staining. (A) The graph of immunofluorescence results; (B) Statistics for relative fluorescence intensity of MMP9 in cancer and in its peritoneal tissues. ^**p < 0.01.

TUSC7 can inhibit the metastasis of CRC cells to the peritoneum

By measuring the abdominal circumference of mice in each group, we found that the abdominal circumference of mice in the TUSC7 KD group increased significantly compared with the NC group. The abdominal circumference of mice in the TUSC7 OE group was significantly reduced. It shows that TUSC7 could restrain the metastasis of CRC (Figure 2).

Figure 2. TUSC7 can inhibit the transfer of CRCs. (A) Mouse CRC peritoneal transfer results plot. (B) Statistics of mouse abdominal circumference measurement data. ^**p < 0.01.

TUSC7 can inhibit the expression of miR-23b

Bioinformatics analysis was used to search for potentially targeted microRNAs for TUSC7, such as Targetcan, Starbase. A miR-23b binding site (1125-1140 bp) was identified in the transcript of TUSC7, the predictor gene for miR-23b. The double luciferase reporter gene showed that by enhancing miR-23b expression, the luciferase activity of wild-type vector-transfected peritoneal macrophages was relatively reduced, and the Mu vector restored the above inhibition. These results imply that TUSC7 is a specific target gene for miR-23b (Figure 3).

Figure 3. TUSC7 specifically inhibits miR-23b. (A) Putative binding sites of miR-23b within TUSC7 mRNA, and the sequences of wild-type and mutant-type vectors. (B) In peritoneal macrophages co-transfected with wild-type vectors and pagomir-23b, relative luciferase activity was inhibited rather than co-transfected with mutant vectors. Firefly luciferase activity normalized to renilla luciferase. ^**p < 0.01.

The results of real-time quantitative PCR showed that compared with the NC group, the expression level of TUSC7 in the TUSC7-OE group was significantly increased, the expression level of miR-23b was significantly reduced (p < 0.01), however, the expression level of TUSC7 in the TUSC7-KD group was significantly reduced, and the expression level of miR-23b was significantly increased, indicating that TUSC7 could inhibit the expression of miR-23b (Figure 4A, 4B).

Figure 4. Transfection efficiency detected by real-time quantitative PCR, oxidative stress and SHP2 phosphorylation levels in peritoneal macrophages. (A) Data statistics of TUSC7 expression in peritoneal macrophages in each group. (B) Data statistics of miR-23b expression in peritoneal macrophages in each group. (C) Protein bands of expressions of oxidative stress-related proteins P22, P47 and gp91 in peritoneal macrophages. (D) Data statistics of SHP2 phosphorylation level in peritoneal macrophages. ^**p < 0.01.

Transfection efficiency and effects of TUSC7 on oxidative stress and SHP2 phosphorylation in peritoneal macrophages

The results of real-time quantitative PCR revealed that in TUSC7 OE group, TUSC7 had a significantly higher expression and miR-23b had a markedly lower expression than NC group (p < 0.01), while in TUSC7 KD group, TUSC7 had a significantly lower expression and miR-23b had a significantly higher expression than NC group (p < 0.01), indicating a successful transfection. Furthermore, the results of Western blotting showed that compared with NC group, the expression of P22, P47 and gp91 in TUSC7 OE group was significantly increased, and the expression of p-SHP2 was significantly reduced. Meanwhile, the expression of P22, P47 and gp91 in the TUSC7 KD group decreased significantly, and the expression of p-SHP2 was significantly increased (p < 0.01). It can be inferred that TUSC7 can enhance oxidative stress in CRC cells and reduce phosphorylation of SHP2 (Figure 4C, 4D).

TUSC7 could promote M2 polarization of macrophages

The results of Western blotting showed that TUSC7 OE group exhibited significantly higher expressions of STAT6, STAT3, IL-4 and IL-10 and significantly lower expressions of MMP2 and MMP9 in comparison to NC group (p < 0.01), whereas TUSC7 KD group had the opposite results (p < 0.01). These results manifested that TUSC7 could promote the M2 polarization of macrophages and suppress the MMP expressions through the STAT3/STAT6 signaling pathway (Figure 5).

Figure 5. Expressions of STAT3/STAT6 signaling pathway-related proteins in peritoneal macrophages. (A) Protein bands of STAT3, STAT6, IL-4, IL-10, MMP2, and MMP9. (B) Data statistics of protein expressions of STAT3, STAT6, IL-4, IL-10, MMP2, and MMP9. ^**p < 0.01.

TUSC7 could suppress the migration and invasion of CRC cells

In the Transwell assay, there were significantly more migrating and invading cells in TUSC7 OE group and obviously fewer migrating and invading cells in TUSC7 KD group than NC group (p < 0.01), indicating that TUSC7 can inhibit both migration and invasion of CRC cells (Figure 6).

Figure 6. Migration and invasion of CT26 cells detected by Transwell assay. (A) Results of migration and invasion assays. (B) Data statistics of migration and invasion assays. ^**p < 0.01.

TUSC7 could suppresses the proliferation of CRC cells

The results of monoclonal formation assay showed that the number of colonies was significantly raised in TUSC7 OE group but evidently reduced in TUSC7 KD group compared with that in NC group (p < 0.01). In CCK8 assay, the OD values at 24 h and 48 h were significantly higher in TUSC7 OE group than those in NC group, but they were significantly lower in TUSC7 KD group than those in NC group (p < 0.01) (Figure 7).

Figure 7. Cell proliferation was detected by monoclonal formation assay and CCK8 assay. (A) Results of monoclonal formation assay. (B) Data statistics of monoclonal formation assay. (C) Data statistics of CCK8 assay. ^**p < 0.01.