RNAi mediated silencing of Nanog expression suppresses the growth of human colorectal cancer stem cells

Background: Colorectal cancer (CRC) is the third most common cancer in the world known for its poor recurrence-free prognosis. Previous studies have shown that it is closely linked with cancer stem cells (CSCs), which have self-renewal potential and the capacity to differentiate into diverse populations. Nanog is an important transcription factor that functions to maintain the self-renewal and proliferation of embryonic stem cells; however, many recent studies have shown that Nanog is also highly expressed in many cancer stem cells. Methods: To investigate whether Nanog plays a crucial role in maintaining the stemness of colorectal CSCs (CCSCs), RNA interference was used to downregulate Nanog expression in the CRC stem cell line, EpCAM + CD44 + HCT-116. We examined the anti-tumor function of Nanog in vitro and in vivo, using small interfering RNA. Results: Our results revealed that the Nanog mRNA expression level in CCSCs was higher than that in HCT-116 cells. We found that the depletion of Nanog inhibited proliferation and promoted apoptosis in EpCAM + CD44 + HCT-116 cells. In addition, the invasive ability of EpCAM + CD44 + HCT-116 cells was markedly restricted when Nanog was silenced by small interfering RNA. Furthermore, we found that the silencing of Nanog decreased tumor size and weight and improved the survival rate of tumor-bearing mice. Conclusions: In conclusion, these �ndings collectively demonstrate that Nanog, which is highly expressed in CRC stem cells, is a key factor in the development of tumor growth, and it may serve as a potential marker of prognosis and a novel and effective therapeutic target for the treatment of CRC.


Background
Colorectal cancer (CRC) is one of the most common malignant neoplasms with poor prognosis and high frequency of recurrence [1], accounting for 600,000 mortalities per year worldwide.Tumor recurrence and metastasis to distant organs are the main factors for the high mortality and low survival rates [2].
Although the incidence of CRC has decreased, current treatments have serious side effects, with a recurrence rate of more than 50%, mainly due to resistance to conventional chemotherapy drugs [3,4].
Recent studies have shown that cancer stem cells (CSCs), which are present in many tumors, are a subset of cancer cells with the ability to self-renew.CSCs are primarily implicated in tumor initiation, progression, metastasis, and relapse after therapy [5][6][7][8].The ineffectiveness of current cancer treatments may be the result of increased resistance of CSCs [9].Thus, it is vital to improve the current therapeutic strategies for CRC and nd novel treatments to eradicate cancer stem cells.
Nanog is a unique homeobox transcription factor required to maintain the self-renewal and pluripotency of embryonic stem cells (ESCs).Recently, emerging evidences have demonstrated that Nanog is expressed in a variety of cancer cell lines and tissues, and is associated with aggressive tumors [10,11].
A number of studies have revealed that the Nanog is a biomarker of CSCs, regulating cancer progression [12], as well as playing an important role in proliferation, apoptosis, differentiation, and stress response in CSCs in many cancers such as cervical, breast, and bladder cancers [12][13][14][15][16]. Several lines of evidence have suggested that the expression of Nanog is closely related to tumorigenesis, tumor metastasis, and distant recurrence after treatment [17].Nanog plays key role in maintaining CSC status and evasive resistance to conventional chemotherapy in bladder cancer stem cells and lung cancer stem cells [18,19].
However, the potential role of Nanog in CSC in CRC remains to be elucidated.
In our previous study, we found that Nanog mRNA expression in colon cancer stem cells (CCSCs) was higher than that in the total CRC cells.In the present study, we used RNA interference technology to silence Nanog mRNA and to examine the effect of Nanog on CCSCs.The results showed that silencing of Nanog suppressed proliferation, invasion, and tumorigenesis, as well as induced apoptosis of CCSCs, thus laying a foundation for further studies on the biological characteristics of CCSCs.Our ndings also suggest that Nanog could be a novel therapeutic target in CRC.

Animals
A total of 35 6-week-old female athymic BALB/c nude mice (20±2 g) were purchased from Beijing HFK Bioscience Co., Ltd. and housed in the Laboratory Animal Center at Jilin University.All animals bred in speci c pathogen-free conditions, with 25±2℃ temperature, 55±5% humidity and a 12-h light/ dark cycle as the same conditions previously described [20].Animals had ad libitum access to water and mouse chow diet.An acclimation period of at least 1 week was implemented for all mice prior to use in experiments.All the procedures were approved by Animal Care Committee of Jilin University (No. 2019-0046) and performed according to the Jilin University Guidelines for Animal Research.

Magnetic activated cell sorting (MACS)
As we previously described, the experiment of MACS was performed using a CELLection™ Biotin Binder kit following the manufacturer's instructions [6].In brief, HCT-116 cells were collected and incubated with anti-human EpCAM-biotin at 4°C for 10 min.Subsequently, the cells were labeled with dynabeads (500 µl) for 20 min at 4°C, and a magnet was used to obtain the labeled cells (EpCAM + cells).EpCAM + cells were then incubated in releasing buffer (100 µg/mL Dnase I) for 15 min at room temperature (RT) and collected.EpCAM + cells were incubated with anti-human CD44-biotin for 10 min, and subsequently with dynabeads (50 µl) for 20 min.Target cells (EPCAM + CD44 + CCSCs) were separated using a magnet after adding releasing buffer (100 µg/mL Dnase I) into the cells and incubating for 15 min at RT. EpCAM + /CD44 + CCSCs were freshly prepared for use.

Silencing by siRNA transfection
To inhibit Nanog expression in the EpCAM+ CD44+ HCT-116 cells (CCSCs), silencing by small interfering RNAs (siRNAs) was performed using riboFECT TM CP Transfection Kit (Ribobio, Guangzhou, China) according to the manufacturer's instructions.Nanog siRNA and its negative siRNA control were designed and synthesized by Riobobio (Guangzhou, China).Nanog siRNA sequence is 5'-AACTATCCATCCTTGCAAA-3'. The CCSCs were rst cultured in 6-well plates (10 6 cells/well) in DMEM/F12 medium for 48 h, and then transfected with 20 µM Nanog siRNA or negative siRNA control.Cells that had not been transfected served as controls.After transfection for 48 h, the cells were cultured for further evaluation.

Flow cytometry
The cells (10 6 /tube) were washed twice with phosphate-buffered saline (PBS) and incubated with PEanti-human CD44 and FITC-anti-human EpCAM (appropriate dilution per antibody) at 4°C for 20 min.
Subsequently, the cells were washed with PBS twice.The labeled cells were analyzed using a ow cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Serum-induced differentiation
To induce differentiation, CCSCs were cultured in DMEM/F12 medium supplemented with 10% FBS for 3 days.The results were observed using an inverted microscope (Olympus IX71, Tokyo, Japan).

Single-cell colony formation
EpCAM + CD44 + HCT-116 cells were seeded in DMEM/F12 medium at a density of 200 cells per well on 6well plates and cultured at 37°C for 3 weeks.The medium was replaced every 2 to 3 days.Plates were photographed with an optical inverted microscope (Olympus IX71, Tokyo, Japan).

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total cellular RNA was extracted using TRIzol reagent (Invitrogen, USA); reverse transcription was performed from 1 µg of the total RNA using PrimeScript RT Master Mix (TaKaRa, Dalian, China), according to the manufacturer's instructions.qRT-PCR was performed with TransStart Top Green qPCR SuperMix (TransGen, Beijing, China) using a real-time PCR system (PikoReal 96, ThermoFisher Scienti c, USA) with the following program: 94°C for 30 sec, followed by 40 cycles of ampli cation (94°C for 5 sec, 60°C for 15 sec, and 72°C for 1 sec).The primer sequences (GeneCreate, Wuhan, China) used for quantitative real-time PCR are shown in Table 1.GAPDH was used as an endogenous control.The relative expression levels of mRNA transcripts were analyzed by the 2 −ΔΔCt method.All the experiments were performed in triplicate.

Annexin V analysis
Annexin V analysis was performed using the -Annexin V-FITC kit (KeyGEN, Nanjing, Jiangsu, China) according to the manufacturer's instructions.Brie y, after 48 hours' transfection of CCSC with Nanog siRNA or negative siRNA control, they were harvested and washed twice with PBS and then re-suspended in binding buffer at a density of 1×10 6 cells/ml.Subsequently, Annexin V-FITC (5µl) and propidium iodide (PI) (5µl) were added to the cells (500 µl).After being incubated for 15 min in the dark at RT, the cells were analyzed using a ow cytometer (BD Biosciences, USA).Annexin V -/PI -cells present cell survival, Annexin V + /PI -cells were shown cells in early apoptosis, and Annexin V + /PI + cells were in late apoptosis or necrotic.The experiments were repeated independently three times.

JC-1 assay
Alterations in mitochondrial membrane potential were measured by ow cytometry using the JC-1 kit (KeyGEN, Nanjing, Jiangsu, China) according to the manufacturer's instructions.Brie y, after 48 hours' transfection of CCSC with Nanog siRNA or negative siRNA control, the cells were harvested and washed twice with PBS and re-suspended in 500 µl incubation buffer containing the JC-1 dye (1 µl) at a density of 1×10 6 cells/ml.After being incubated for 15 min at 37°C, 5% CO 2 , the cells were collected and washed twice with the incubation buffer.Subsequently, cells were re-suspended in 500 µl incubation buffer and analyzed using a ow cytometer (BD Biosciences, USA).

Transwell invasion assay
The invasion assays were performed using 6.5-mm diameter Transwell plates (8 µm pore size, Corning, Steuben County, NY, USA) coated with a thin layer of Matrigel (BD Biosciences, San Diego, CA, USA), through which invading cells could migrate and eventually attach to the bottom of the polycarbonate layer.The CCSCs were resuspended in serum free DMEM/F12 at a concentration of 1×10 6 /ml.The upper chamber was loaded with 100 µl of cell suspension and the lower chamber was loaded with 500 µl of DMEM/F12 medium supplemented with 10% FBS as the chemoattractant.After incubation for 24 h at 37°C and 5% CO 2 , non-invading cells in the upper chamber were removed with a PBS-soaked cotton swab and the cells that had invaded the membranes were stained with 0.5% crystal violet and counted under a light microscope.Each assay was replicated 3 times.The invaded cells were counted under the microscope in ve random elds in each chamber.The assay was performed in triplicate.

In vivo tumor xenograft assay
To generate tumor xenografts, EpCAM + CD44 + HCT-116 cells (5×10 5 ) were injected subcutaneously into the right ank of each mouse.When the mice attained a tumor volume of 40-60 mm 3 , they were randomly divided into 3 groups (n = 10) and treated with Nanog siRNA, mock, or negative siRNA control.Nanog siRNA or negative siRNA control was injected intratumorally twice a week for 3 weeks.The tumor size was measured every other day using a vernier caliper and calculated as (a×b 2 )/2, where a is the tumor length and b the width.At the end of the experiment, mice were euthanized by carbon dioxide asphyxiation for approximately 6 min (air displacement rate: 20%/min; carbon dioxide ow rate: 1.7 L/min; the mortality was ensured by cervical dislocation) and tumors were excised and weighed.

Statistical analysis
All data are presented as the mean ± standard deviation (SD) of at least three repeat experiments.
Student's t-test and one-way ANOVA analysis were used to analyze the variances between groups.The log-rank test was used to compare the survival rates in different groups.Signi cant differences were considered when P values were less than 0.05.

Results
Screening and identi cation of CCSCs from HCT-116 CRC cell lines EpCAM and CD44 have been previously identi ed as surface markers for CCSCs and used for isolating CCSCs from CRC cells [21,22].In our study, the proportion of the EpCAM + CD44 + subpopulation of HCT-116 cells isolated by MACS accounted for < 2.00%, and the CCSCs formed spheres after being cultured for 7 days in serum-free medium (Fig. 1A).The expressions of EpCAM and CD44 were evaluated by ow cytometry and the percentage of the EpCAM + CD44 + HCT-116 cells in the sorted cells were signi cantly higher than that in unsorted HCT-116 cells (Fig. 1B).Subsequently, CCSC spheres were observed over 21 days by the single-cell colony formation assay (Fig. 1C).CCSC spheroid cells became re-adherent and differentiated after serum was added to the medium (Fig. 1D).

High expression level of Nanog in CCSCs, and siRNA-mediated knockdown of Nanog
To address whether Nanog can serve as a novel therapeutic target for CRC, the relative mRNAs expression of Nanog is detected in colon cancer cell lines (HCT-116, SW480 and LoVo), the results suggested that there is higher-expression of Nanog in HCT-116 compared with SW480 or LoVo (Fig. 2A).Then the relative mRNAs expression of Nanog, Sox-2, Oct-4, and C-myc as putative stem cell markers, were analyzed by real-time PCR in HCT-116 and EpCAM + CD44 + HCT-116 cells.EpCAM + CD44 + HCT-116 cells exhibited signi cantly higher relative Nanog and Oct-4 mRNAs level (7.03±0.12 and 4.37±0.18,)than HCT-116 cells (1.00±0.066and 1.00±0.17) in Figure .2B.The protein expression of Nanog was also measured by western blot (Fig. 2C and 2D), the result showed that it was signi cantly higher expression in EpCAM + CD44 + HCT-116 cells than HCT-116 cells.

Down-regulation of Nanog inhibits CCSC proliferation and promotes apoptosis
To investigate the effect of Nanog on self-renewal of CCSCs, the MTS cell proliferation assay was performed.Proliferation ratio of CCSC-siNanog group (54.71±8.01%) was signi cantly inhibited compared with that of mock (100.00±3.12%) or negative control groups (96.73±5.72 %) in Figure .3A.
To further examine the effects of Nanog on CCSCs apoptosis, Annexin V and JC-1 assays were performed after Nanog siRNA transfection.The Annexin V/PI assay revealed that compared with Mock (4.16%) or NC siRNA (10.02%) groups, Nanog-siRNA (68.47%) transfection signi cantly increased the ratio of Annexin V-positive cells (Fig. 3C and D), at the meanwhile, the results of JC-1 staining assay indicated that the percentage of cells undergoing a loss of mitochondrial membrane potential increased signi cantly following Nanog siRNA (32.31%) transfection compared with Mock (4.94%) or NC siRNA (8.54%) groups (Fig. 3E and F).These results suggest that silencing of Nanog promotes CCSC apoptosis.In addition, the expression of B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), and caspase-3 were evaluated using RT-qPCR and western blot analysis.After treatment with Nanog siRNA for 48 h, the mRNA and protein expression of Bcl-2 (mRNA 0.33±0.027,protein 0.29±0.036)was signi cantly suppressed while the mRNA and protein expressions of Bax (mRNA 6.07±0.82,protein 5.50±0.041)and cleaved-caspase-3 (mRNA 2.68±0.32,protein 2.07±0.039)were signi cantly up-regulated (Fig. 3B, G and  H).Taken together, these results demonstrated that silencing of Nanog inhibited the proliferation of CCSCs and promoted signi cant apoptosis.

Nanog silencing decreases the invasive ability of CCSCs in vitro
In order to investigate the effect of Nanog on the invasive ability of CRC stem cells, which is often representative of the metastatic potential, the Transwell invasion assay was performed after the silencing of Nanog.Nanog siRNA (62±5) signi cantly decreased the number of CCSCs that passed through the matrigel compared to those of mock (143±3) or negative siRNA control (140±4) (Fig. 4A and B).
Nanog siRNA treatment suppressed tumor growth in vivo and increased mice survival rate Based on the in vitro studies described above, we further investigated the effect of Nanog siRNA on tumor growth in vivo.A total of 35 of 6-week-old female athymic BALB/c nude mice (20±2 g) were randomly divided into 3 groups (n = 10, note: 5 of mice were excluded because they were no obvious tumors after injected EpCAM + CD44 + HCT-116 cells for 2 weeks) and treated with Nanog siRNA, mock, or negative siRNA control.After treatment with Nanog siRNA or negative siRNA control (injected intratumorally twice a week for 3 weeks), we found Nanog siRNA treatment strongly decreased tumor size and weight compared with negative siRNA control or mock treatments (Fig. 6A and B).Meanwhile, in comparison with the mock (0/5 mice were alive at day 48, n=5) or control groups (0/5 mice were alive at day 48, n=5), Nanog siRNA treatment (4/5 mice were alive at day 48, n=5) signi cantly improved the survival rate of tumor-bearing mice (Fig. 6C).Taken together, these results demonstrate that Nanog siRNA treatment suppresses colorectal tumor growth in vivo and improves survival, implying that Nanog may serve as a novel therapeutic target for CRC treatment.For survival analysis, mice were sacri ced when tumors measured > 2000 mm 3 or the tumor diameter exceeded 2.0 cm.All of the treatments with mice are according to the protocol of the Animal Care Committee of Jilin University as previously described [20].

Discussion
In recent years, CRC has been ranked as the third leading cause of cancer-related mortalities among malignant tumors worldwide [23].Advances in the investigation of cancer stem cells have revealed that CSCs have the ability to self-renewal and maintain tumor growth, and therefore play a crucial role in tumorigenesis, development, metastasis, and recurrence [24,25].Nanog has been established as an important transcriptional factor required for maintaining self-renewal and pluripotency of embryonic stem cells [23].Previous studies have shown that Nanog is overexpressed in various types of CSCs [12][13][14] including CRC stem cells [19].It is consistent with previous studies [26,27], the CCSCs in the present study displayed relatively higher expression levels of Nanog compared with that in unsorted HCT-116 cells.M. Zhang et al. illustrated that Nanog siRNA signi cantly inhibited colony formation, suggesting the indispensable role of Nanog in colon tumor-repopulating cells growth[28].X. Wang et al. provides cancerassociated mutations of SPOP or the mutation of Nanog at S68Y abrogates the SPOP-mediated Nanog degradation lead to elevated prostate cancer stemness and poor prognosis [29].Furthermore, emerging evidence has suggested that Nanog plays a vital role during self-renewal of CSCs, and knockdown or silencing of Nanog suppresses CSCs growth and development [30].These results suggest that Nanog may be associated with tumor initiation, development, and therapeutic resistance.However, the role of Nanog in CRC stem cells has not been investigated.
To understand the functional role of Nanog in CCSCs, we used the RNA interference approach to knockdown Nanog expression in EpCAM + CD44 + HCT-116 cells, and assessed its effect on suppressing growth and promoting apoptosis of CCSCs in vitro.Nanog is a transcription factor involved in the regulation of pluripotency and stemness.The functional paralog of Nanog, NanogP8, differs from Nanog in only three amino acids and exhibits similar reprogramming activity.E. Mikulenkova et al. investigated the intriguing extranuclear localization of Nanog and demonstrated that a substantial pool of Nanog/NanogP8 is localized at the centrosome [31].NanogP8 is the main regulator of gastric cancer stem cells.It is closely associated with EMT, stemness, and CSC marker as well as Wnt signal pathway.NanogP8 is correlated with cell proliferation, migration, invasion, clonogenic capacity, beta-catenin accumulation in nucleus, and chemoresistance in gastric cancer [32].B. Liu et al. demonstrated that transgenic overexpression of NanogP8 in the mouse prostate is insu cient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model [33].The Nanog siRNA used in this study targets both Nanog and NanogP8.Our results, which revealed that Nanog silencing not only signi cantly suppressed CCSC proliferation but also markedly induced CCSC apoptosis, ware similar to those of studies reported for pancreatic and breast cancer stem cells [16,34].There are two main pathways associated with apoptosis: a pathway mediated by a cell death receptor and that mediated by mitochondrion, both of which lead to the activation of the caspase cascade.Mitochondrial membrane potential is regulated by a complex network of signaling pathways that involve endogenous pro-and antiapoptotic Bcl-2 family proteins [35].The results of the Annexin V assays provided evidence that the depletion of Nanog promoted apoptosis of CCSCs, which is in accordance with the results of inhibition of Nanog and aggravation of apoptosis in non-small cell lung cancer [36].Moreover, the results of JC-1 assays suggested that a loss in mitochondrial transmembrane potential (Δψm) was observed in the Nanog siRNA group compared with that in the mock and negative siRNA control groups.In addition, we analyzed the mRNA levels and protein expression of several apoptosis-related genes since previous studies have shown that resistance to apoptosis is one of the leading causes of tumorigenesis [7,14,37].
Caspase-3 is a downstream molecule that is activated by upstream molecules such as caspase-8 or caspase-9, leading to cell apoptosis.Pro-apoptotic Bax promotes the release of pro-apoptotic molecules by forming oligomers in the mitochondrial outer membrane, thus promoting cell apoptosis.Anti-apoptotic Bcl-2 blocks mitochondrial apoptosis by blocking the release and oligomerization of Bax [38].Thus, Caspase-3 and the Bcl-2 family proteins play an important role in the regulation of apoptosis [39].Nanog silencing activated cleaved caspase-3 expression, promoted the expression of pro-apoptotic BAX gene, and inhibited the expression of anti-apoptotic Bcl-2 gene.These results indicate that Nanog may play a vital role in the induction of apoptosis in CCSCs via the caspase-3 cascade.
It is well known that the invasive ability of CSCs is crucial for cancer metastasis [40].Further, high expression levels of matrix metalloproteinases (MMPs), which are identi ed as potential major regulators of invasion, are associated with tumor progression and metastasis in diverse human cancers [38].The tissue inhibitors of metalloproteinases (TIMPs), which are inhibitors of MMPs, have been shown to impede tumor progression [41].In our study, Transwell invasion assay and analyses of mRNA and protein expressions of MMP-2, MMP-9, and TIMP-1, in line with the results of the regulation of MMP-2, 9, and TIMP-1, promote the invasion and metastasis of renal cell carcinoma and MMP-9 (TIMP-1 is its inhibitor) in the degradation of extracellular matrix.This enhances metastasis in breast cancer; thus revealing that Nanog silencing signi cantly suppressed the invasive potential of CCSCs [42,43].Thus, Nanog may play a crucial role in the invasive potential of CSCCs [44].Consistent with our in vitro results, in vivo results showed that Nanog siRNA signi cantly suppressed xenograft tumor growth and prolonged mice survival.

Conclusions
In conclusion, our results demonstrated that silencing of Nanog expression suppressed the proliferation, invasion, and tumorigenesis, as well as induced apoptosis of CCSCs in vitro and in vivo, all of which are crucial for cancer development.Furthermore, our ndings revealed for the rst time the key role of Nanog in tumor growth in CRC.Thus, Nanog-targeted siRNA may provide a possible novel strategy for cancer stem cell-based targeted therapies in CRC.The exact mechanism by which Nanog silencing exerts its antitumor ability may be worth exploring in future investigations.