Prominin 2 decreases cisplatin sensitivity in non-small cell lung cancer and is modulated by CTCC binding factor

Our study has been authorized by the Ethics Committee of the Third Affiliated Hospital of ZunYi Medical University (The First People's Hospital of ZunYi). A total of 35 NSCLC and adjacent non-tumor tissues were collected and stored in −80°C. The patient characteristics was listed in Table 2. All procedures performed in studies involving human participants were in accordance with the standards upheld by the Ethics Committee of the Third Affiliated Hospital of ZunYi Medical University (The First People's Hospital of ZunYi) and with those of the 1964 Helsinki Declaration and its later amendments for ethical research involving human subjects.

All animal experiments were approved by the Ethics Committee of the Third Affiliated Hospital of ZunYi Medical University (The First People's Hospital of ZunYi) for the use of animals and conducted in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines.

The animal experiment complies with the ARRIVE guidelines and in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).

Human normal lung epithelial cells (BEAS-2B) and lung cancer cells (NCI-H1650, A549, NCI-H1299, PC-9) were purchased from American Type Culture Collection (ATCC, MA, VA, USA), cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, USA) and incubated at 37°C in 5% CO₂.

For transfection, the A549 and PC-9 cells were transfected with transfecting plasmid or Lentivirus to overexpress or knock down PROM2 using Lipofectamine®3000 (In vitrogen, Carlsbad, CA, USA) reagent.¹⁶ For co-transfection, the A549/DPP cells were co-transfected with small interfering RNA of CTCF and/or plasmid of PROM2, and then the cell viability and proliferation were examined.

Resistant NSCLC cells were established by continuously exposing A549 and PC-9 cells to cisplatin in a series of concentration gradients (0.1 μM to 6 μM). Cells that survived in cell medium with 6 μM cisplatin were identified as cisplatin resistant cells (A549/DDP, PC-9/DDP). Thereafter, the parental cells or drug-resistant cells were treated with different concentrations of cisplatin (0, 1.0 μM, 10 μM, 50 μM, 100 μM, 200 μM). CCK-8 was performed to measure cell viability and calculated half maximal inhibitory concentration (IC₅₀).

The tissues were fixed in 4% paraformaldehyde, embedded with paraffin, sectioned at 4 μm, dewaxed in xylene, soaked in 3% hydrogen peroxide solution to eliminate endogenous catalase, and repaired in citrate solution pH 6.0 at high temperature. Primary antibody PROM2 (Abcam, Cambridge, UK; ab118492; 1:100) was added after serum blocking. After overnight at 4°C, the sections were developed by DAB, counterstained with hematoxylin for observation.

Clinical tissue samples or cells of each group were collected, and RIPA lysis buffer was added to extract total protein. Appropriate amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the protein was transferred to PVDF membrane, blocked in 5% skim milk for 1 h at 25°C, added with primary antibody, and incubated overnight at 4°C on a shaker.¹⁷ Specific primary antibodies are as follows: PROM2 (Abcam, ab74997, 1:1000), CTCF (Abcam, ab128873), β-actin (Abcam, ab8226). After incubation with secondary antibodies, electrochemical luminescence reagent was added without light. Image J software was used to analyze the gray value of the strips.

5 × 10³ cells/mL cells were seeded into a 96-well plate (100 μl/well) and then cultured for 0, 1, 2 and 3 days, respectively. CCK-8 solution (Beyotime, Shanghai, China; 10 μl) was added to each well, and the culture was continued for 2 h.¹⁸ The absorbance value (OD value) at 450 nm was detected by microplate reader.

The cells after transfection were seeded in 6-well plates and cultured for 14 days. When visible clones appeared, the colonies were stained with gentian violet (Goodbio Technology, Wuhan, China) for 30 min. The proliferation of cells was observed under a microscope.

For cell migration assay, 100 μl cell suspension was added to the upper chamber, and 600 μl RPMI-1640 containing 10% FBS was added to the lower chamber. The cells were cultured for 24 h at 37°C in a 5% CO₂ incubator. For cell invasion assays, Transwell chambers coated with extracellular matrix gel were used, and the rest of the procedure was the same as for cell migration assays. At the end of culture, the upper chamber was removed and the cells on the inner surface of the filtration membrane of the chamber were wiped off. The cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, observed by microscope, counted and photographed. Five fields of view were randomly selected and averaged.

Cells (2 × 10⁶ cells/mL) were seeded into 96-well cell culture plate and incubated at room temperature for 24, 48 and 72 h. The cells were treated according to Annexin V-FITC/PI apoptosis kit (Beyotime) instructions. The cells were resuspended in 300 μl PBS. The cells were stained with Annexin V (5 μl) and PI (5 μl) for 15 min at room temperature, and the apoptosis rate was detected by flow cytometry within 4 h.¹⁹

A549 cells were transfected with pcDNA-CTCF or CTCF small interfering RNA and cultured in an incubator containing 5% CO₂ at 37°C for 24 h. After removal of the medium, the cells were fixed with 16% paraformaldehyde. They were divided into IgG+siNC group, CTCF+siNC group, IgG+siCTCF group and CTCF+siCTCF group. CTCF-bound DNA was captured using antibodies according to the ChIP kit (Cell signaling Technology, Boston, MA, USA) instructions. PCR was used to verify the capture of CTCF gene promoter DNA, and ChIP-PCR was used for quantitative analysis. Immunoprecipitation efficiency was calculated using input sample percentage method.

Our study was approved by the Animal Ethics Committee of the Third Affiliated Hospital of ZunYi Medical University (The First People's Hospital of ZunYi). BALB/c nude mice were subcutaneously injected with PROM2 knockdown stable A549/DDP cells (5 × 10⁶). One week later, cisplatin (4 mg/kg) was injected into the peritoneum every 3 days. All nude mice were divided into 5 groups: control group, shNC group, cisplatin +shNC group, shPROM2#1 group and cisplatin +shPROM2#1 group, with 5 mice in each group. On the 25th day, the nude mice were sacrificed under anesthesia. The weight of the tumors was weighed and the volume of the tumors was measured. The tumors were collected for subsequent experiments.

Graphpad 7.0 software was used for data analysis. Data were expressed as mean ± standard deviation, and comparison between two groups was performed by t test. One-way analysis of variance analysis of variance was used to compare the differences between more than two groups. P < 0.05 was considered statistically significant. IC50 value was calculated by Graphpad according to the results of CCK-8 assay.

To explore the pathogenesis of NSCLC, we analyzed the data GEO database data (GSE32863) confirmed to compare the expression of differential genes in NSCLC tissues and adjacent normal tissues and the results showed that PROM2 was up-regulated in NSCLC compared to adjacent normal tissues (Figure 1A). Interestingly, GEO microarray data GSE32863 confirmed the differential genes (DEGs) in lung cancer tissues and adjacent normal tissues. Notably, PROM2 expression was increased in lung cancer tissues (Figure 1B–1D). More importantly, online platform GEPIA data showed that both PROM2 transcript and expression levels were promoted in LUSC and LUAD patients compared to normal subjects (Figure 1E and 1F). Similarly, high expression of PROM2 predicted poor prognosis (Figure 1G). Representative images of IHC confirmed that PROM2 was highly expressed in NLCSC patient tissues compared to adjacent tissues (Figure 1H). Consistently, PROM2 expression was significantly enhanced in lung cancer cells (NCI-H1650, A549, NCI-H1299, PC-9) compared to BEAS-2B, especially in PC-9 and A549 cells (Figure 1J). Thus, the PC-9 and A549 cells was selected for subsequent experiments. These results indicated that the expression levels of PROM2 were up-regulated in NSCLC.

FIGURE 1.

To explore the role of PROM2 in NSCLC, PROM2 was overexpressed or knocked down in A549 and PC-9 cells. As expected, PROM2 was efficiently over-expressed or knocked down (Figure 2A). Then, the effects of altered PROM2 on NSCLC viability and motility were examined. As shown in Figure 2B, A549 and PC-9 cell viability was remarkably increased after PROM2 overexpression, and was decreased by knockdown of PROM2. In addition, knockdown of PROM2 reduced the number of colonies, whereas overexpression of PROM2 exhibited opposite effect (Figure 2C). Furthermore, knockdown of PROM2 observably inhibited the number of migrated and invaded cells, while over-expression of PROM2 increased the number of migrated and invaded cells (Figure 2D). These findings indicated that PROM2 can promote the cell viability, proliferation, migration and invasion of lung cancer cells.

FIGURE 2.

Subsequently, to investigate the effect of PROM2 on cisplatin sensitivity in NSCLC, we constructed drug-resistant cell lines (A549/DDP and PC-9/DDP). CCK8 assay results showed that both A549/DDP and PC-9/DDP cells had a lower cisplatin sensitivity than A549 (IC₅₀ 84.55 vs. 15.69) and PC-9 cells (IC₅₀ 72.28 vs. 10.10) (Figure 3A). Moreover, PROM2 expression level was significantly higher in resistant cells than in parental cells (Figure 3B). To investigate the role of PROM2 in drug-resistant cells, PROM2 was down-regulated or over-expressed in A549/DDP and PC-9/DDP cells (Figure 3C). Subsequently, CCK-8 assay implied that knockdown of PROM2 enhanced cisplatin sensitivity in A549/DPP (IC₅₀ 28.76 vs. 79.28) and PC-9/DDP (IC₅₀ 27.02 vs. 70.43) cells, while over-expression of PROM2 inhibited cisplatin sensitivity in A549/DPP (IC₅₀ 112.40 vs. 81.80) and PC-9/DDP (IC₅₀ 98.37 vs. 67.66) cells (Figure 3D). Moreover, the colony formation results indicated that knockdown of PROM2 reduced cell proliferation but overexpression of PROM2 promoted cell proliferation of A549/DPP and PC-9/DDP cells (Figure 3E). Consistently, flow cytometry analysis manifested that downregulation of PROM2 increased apoptosis, while overexpression of PROM2 decreased apoptosis of A549/DPP and PC-9/DDP cells (Figure 3F). These results unfolded that PROM2 reduced cisplatin sensitivity through reducing apoptosis and promoting proliferation.

FIGURE 3.

BALB/c nude mice were subcutaneously injected with PROM2-knockdown A549/DDP cells to investigate the effect of PROM2 in vivo. The data indicated that cisplatin treatment reduced the volume and weight of tumors, while knockdown of PROM2 further inhibited the tumor growth (Figure 4A). More importantly, cisplatin treatment increased PROM2 expression, which was decreased by shPROM2 (Figure 4B). All these results suggested that PROM2 knockdown suppressed tumor growth through enhancing the cisplatin sensitivity.

FIGURE 4.

By analyzing Chip ChIP-seq data, it was found that CTCF, REST, MAFK, and TEAD4 can bind to PROM2 promoter, especially CTCF (Table 1). In addition, TIMER 2.0 data showed that CTCF was overexpressed in lung cancer (Figure 5A). Besides, GEPIA platform analysis revealed a positive correlation between CTCF and PROM2 in LUAD and LUSC (Figure 5B). Furthermore, overexpression of CTCF significantly promoted PROM2 expression, while knockdown of CTCF reduced PROM2 expression in A549 cells (Figure 5C). Interestingly, ChIP-PCR result unfolded that the CTCF distinctly bound to PROM2 promoter (Figure 5D). These findings concluded that CTCF promoted PROM2 expression via directly binding to its promoter.

FIGURE 5.

To explore the effect of CTCF on cisplatin sensitivity in NSCLC, CTCF was knocked down in A549/DDP cells. As shown in Figure 6A, siCTCF decreased PROM2 expression, which was increased by overexpression of PROM2. Moreover, CCK-8 assay showed that siCTCF promoted cisplatin sensitivity (IC₅₀ 29.26 vs. 89.51), while overexpression of PROM2 attenuated this effect (Figure 6B). Furthermore, knockdown of CTCF reduced the number of colony formation of A549/DDP cells, which was reversed by overexpression of PROM2 (Figure 6C). Consistently, the apoptosis rate was increased by knockdown of CTCF in A549/DDP cells, which was decreased by PROM2 overexpressed (Figure 6D). Thus, these findings revealed that knockdown of CTCF can increase the sensitivity of lung cancer cells to cisplatin through down-regulating PROM2.

FIGURE 6.