siRNA‐induced CD44 knockdown suppresses the proliferation and invasion of colorectal cancer stem cells through inhibiting epithelial–mesenchymal transition

Abstract CD44 has shown prognostic values and promising therapeutic potential in multiple human cancers; however, the effects of CD44 silencing on biological behaviors of cancer stem cells (CSCs) have not been fully understood in colorectal cancer. To examine the contribution of siRNA‐induced knockdown of CD44 to the biological features of colorectal CSCs, colorectal CSCs HCT116‐CSCs were generated, and CD44 was knocked down in HCT116‐CSCs using siRNA. The proliferation, migration and invasion of HCT116‐CSCs were measured, and apoptosis and cell‐cycle analyses were performed. The sensitivity of HCT116‐CSCs to oxaliplatin was tested, and xenograft tumor growth assay was performed to examine the role of CD44 in HCT116‐CSCs tumorigenesis in vivo. In addition, the expression of epithelial–mesenchymal transition (EMT) markers E‐cadherin, N‐cadherin and vimentin was quantified. siRNA‐induced knockdown of CD44 was found to inhibit the proliferation, migration and invasion, induce apoptosis, promote cell‐cycle arrest at the G1/G0 phase and increase the sensitivity of HCT116‐CSCs to oxaliplatin in HCT116‐CSCs, and knockdown of CD44 suppressed in vivo tumorigenesis and intrapulmonary metastasis of HCT116‐CSCs. Moreover, silencing CD44 resulted in EMT inhibition. Our findings demonstrate that siRNA‐induced CD44 knockdown suppresses the proliferation, invasion and in vivo tumorigenesis and metastasis of colorectal CSCs by inhibiting EMT.

Previous studies have shown that CSCs play important roles in cancer initiation, growth, migration, invasion, metastasis and recurrence, and contribute to resistance to chemotherapy, radiotherapy and targeted therapy. [5][6][7][8][9] Thus, elimination of CSCs may reverse the resistance to chemotherapy, radiotherapy and targeted therapy, improve the prognosis and yield long-lasting responses in cancer patients. [10][11][12] Colorectal CSCs have been therefore considered as a promising therapeutic target for colorectal cancer. [12][13][14] Identification of CSCs is a prerequisite to the therapeutic use of these specific cells. 15 Currently, the biomarkers for identification of CSCs mainly include CD molecules (CD133, CD166, CD44, CD24 and CD138), ATP-binding cassette (ABC) transporters (ABCG2 and ABCB5), EpCAM, ALDH1 and CXCR4, Lgr5, ALDH1, Msi-1, DCAMLK1 and EphB receptors, in which CD molecules are the most common markers for identifying CSCs. [16][17][18] CD44, one of the most common CSC surface marker, is widely accepted as a key regulator of cancer stemness. 19,20 In addition, CD44 has shown prognostic values and promising therapeutic potential in multiple human cancers. [21][22][23][24] However, the effects of CD44 silencing on biological behaviors of CSCs remain to be investigated in colorectal cancer. This study was therefore designed with aims to examine the contribution of siRNA-induced knockdown of CD44 to the biological features of colorectal CSCs.

| Animals
Four-week-old male athymic BALB/c nude mice were purchased from Nanjing Experimental Animal Center of the Chinese Academy of Sciences (Nanjing, China). All animals were maintained in a specific pathogen-free facility and given free access to water and food.

| Cell culture and HCT116-CSCs preparation
Human colorectal cancer HCT116 cell line was purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and cultured in McCoy's 5A medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; GIBCO), 100-IU/ml penicillin (GIBCO) and 100μg/ml streptomycin (GIBCO). HCT116-CSCs were enriched from HCT116 cells with the continuous cell microsphere culture and incubated in complete DMEM/F12 medium containing B27 (10 ng/ml), epidermal growth factor (EGF; 20 ng/ml), basic fibroblast growth factor (bFGF; 10 ng/ml) and leukemia inhibitory factor (LIF; 10 ng/ml). Briefly, log-phase HCT116 cells were harvested and digested with pancreatin containing 0.25% EDTA and terminated with serum-containing medium. Following centrifugation, the supernatant was discarded, and the sediment was washed twice with PBS, and re-suspended in complete stem cell culture. The number of cells was counted. Cells were then seeded onto ultra-low adhesive petri dishes at a density of 1 × 10 4 cells/ml and incubated at 37°C in a humidified atmosphere containing 5% CO 2 . Semiquantitative medium changes were done once every 2-3 days, and cell passage was completed once the microsphere formation was observed to become larger and the structures to become loose. The culture medium containing microspheres in the petri dishes was collected during passaging, centrifuged at a low speed (500-700 r/min), and the supernatant was discarded. The microspheres were digested with a small amount of 0.25% trypsin-EDTA (100-200 μl) according to the amount of cells, and the centrifuge tube was flicked. The microspheres were observed to be digested into a single-cell suspension under an inverted microscope, and PBS was used to resuspend cells with 20 times of the amount of trypsin digestion, centrifuged at 1000 r/min for 5 min and washed twice with PBS. The number of cells was counted. Cells were then incubated in completely stem cell culture at a density of <10 4 cells/ml. This method was used to subculture microsphere cells for at least 10 passages to enrich CSCs from HCT116 cells, which were named as HCT116-CSCs.

| Cell transfection
HCT116-CSCs were incubated in completely stem cell culture without double antibodies. Cells were seeded onto 6-well plates at a density of 1 × 10 5 cells/well and incubated for 6 h. HCT116-CSCs were

| MTT assay
The cell viability was measured using MTT assay every 24 h with the Cell Proliferation Reagent Kit I (Roche Applied Science) according to the manufacturer's protocol, and the half-maximal inhibitory concentration (IC 50 ) was calculated. Briefly, HCT116-CSCs were seeded onto 96-well plates (Corning, Inc.) at a density of 3000 cells/ well and transfected with siRNAs. Cells were then seeded onto 96-  Following incubation for 48 h, cells were exposed to MTT solutions (0.5 mg/ml; Sigma-Aldrich) for further 4 h, and then, the medium was substituted with 150μl dimethyl sulfoxide (DMSO; Sigma-Aldrich) and vortexed for 10 min. The absorbance of each well was measured at 490 nm. In addition, the cell viability was evaluated at 0, 24, 48, 72 and 96 h using 0.5-mg/ml MTT solution without oxaliplatin treatment. Each assay was repeated at least in triplicate.

| Colony formation assay and migration and invasion assays
For the colony formation assay, a total of 600 transfected cells were seeded onto 6-well plates (Corning, Inc.) and maintained in DMEM-F12 medium supplemented with 10% FBS for 2 weeks, displacing the medium every 3-4 days. After incubation for 14 days, cells were fixed with methanol and stained with 0.1% crystal violet The assays were independently repeated in triplicate.

| qPCR assay
Total RNA was extracted from cells or tissues with the TRIzol reagent (Invitrogen) following the manufacturer's instructions. Total RNA (1 μg) was reversely transcribed into cDNA using the PrimeScript RT reagent kit (TaKaRa), and the CD44, E-cadherin, N-cadherin and vimentin mRNA expression was quantified with the SYBR Premix Ex Taq (TaKaRa) using the designed primers (Table 1) on an ABI 7500 real-time PCR system (Applied Biosystems), while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. The relative gene expression was estimated using the 2 −ΔΔCT method. All assays were performed in triplicate.

| Xenograft tumor growth assay
To examine the impact of CD44 knockdown on in vivo tumorigenesis and metastasis of HCT116-CSCs, HCT-CSCs were stably transfected with shRNA-CD44 and sh-NC (Dharmacon, Inc.), and digested with pancreatin, seeded onto petri dishes and incubated with 2-to 10μg/ ml puromycin (Sigma-Aldrich). Medium containing fresh puromycin was changed once every 3-4 days, and cells growing to approximately

| Data management
All measurement data were described as mean ±standard error (SE).
Data were tested for statistical significance with the Student's t test, one-way analysis of variance (ANOVA) and the Mann-Whitney

| Ethics approval
The study protocol was reviewed and approved by the Ethics Review

| Knockdown efficiency of CD44 expression in HCT116-CSCs
To investigate the knockdown efficiency of CD44 expression in HCT116-CSCs, qPCR assay was performed to detect CD44 expression in HCT116-CSCs transfected with different CD44 siRNAs 48 h posttransfection. A greater knockdown efficiency was seen by CD44-siRNA 1# and 2# than by CD44-siRNA 3# ( Figure 1A), and CD44-siRNA 1# and 2# were therefore selected for the subsequent experiments.

| CD44 silencing inhibited HCT116-CSCs proliferation, migration and invasion
To evaluate the functional role of CD44 in HCT116-CSCs, we first examined the effect of CD44 silencing on cell proliferation. MTT assays revealed that cell proliferation was significantly inhibited in HCT116-CSCs following CD44-siRNA transfection ( Figure 1B).
Colony formation assays then showed that knockdown of CD44 expression suppressed the colony formation of HCT116-CSCs, which reflected the self-renewal and differentiation abilities of the CSCs ( Figure 1C). Furthermore, Transwell migration and invasion assays revealed that siRNA-induced knockdown of CD44 expression inhibited the migration and invasion of HCT116-CSCs as compared to si-NC ( Figure 1D). These data demonstrate that CD44 knockdown inhibits the migratory phenotype of HCT116-CSCs.

| Knockdown of CD44 induces apoptosis and promotes cell-cycle arrest at the G1/G0 phase of HCT116-CSCs
To further detect the effects of CD44 knockdown on apoptosis and cell cycle of HCT116-CSCs, flow cytometric analysis was performed. Flow

| Knockdown of CD44 promotes the sensitivity of HCT116-CSCs to oxaliplatin
We then examine the effect of CD44 knockdown on the sensi-

As described above, Transwell migration and invasion assays
showed that the metastatic ability of HCT116-CSCs was significantly weakened by CD44 silencing ( Figure 1D). Next, we exam-

| Knockdown of CD44 inhibits HCT116-CSCs tumorigenesis in vivo
Since CD44 knockdown was found to suppress HCT116-CSCs migration and invasion in vitro, xenograft tumor growth assay was performed to examine the effects of CD44 knockdown on in vivo tumorigenicity and metastasis in nude mice. qPCR and Western blotting assays showed lower CD44 expression in HCT116-CSCs transfected with shRNA-CD44 than in those transfected with sh-NC at both transcriptional and translational levels, confirming a high knockdown efficiency ( Figure 5A,B). All mice were found to develop xenograft tumors at the injection site 42 days postinjection ( Figure 5C), and the mean weight of the xenograft tumors derived from shRNA-CD44-transfected HCT116-CSCs was significantly lower than from sh-NC-transfected HCT116-CSCs ( Figure 5D). During the 42-day study period, the mean volumes of the xenograft tumors derived from shRNA-CD44-transfected HCT116-CSCs were all significantly smaller than from sh-NC-transfected HCT116-CSCs ( Figure 5E).
Next, we observed intrapulmonary metastatic nodules in mice following subcutaneous injection of HCT116-CSCs. Except one natural death in the sh-NC group during the study period, intrapulmonary metastatic nodules were seen in other four mice, while one of the five mice presented intrapulmonary metastatic nodules in the shRNA-CD44 group ( Figure 5G,H,H O ). Collectively, these findings demonstrate that CD44 knockdown suppresses in vivo tumorigenesis and metastasis of HCT116-CSCs through inhibiting EMT.

| DISCUSS ION
CD44, a cell adhesion molecule, has shown an important role in tumor progression and metastasis. 26 Previous studies have shown the roles of CD44 as prognostic factors and therapeutic targets in human cancers. 20 Roosta and colleagues 27 identified the clinical association of CD44 with the stage of breast cancer and systematic reviews and meta-analyses revealed that CD44 expression is a prognostic factor for pharyngolaryngeal cancer, 28 non-small-cell lung cancer, 29 renal cell carcinoma 30 and colorectal cancer. 31 In  Figure 5H at a magnification of 400×. **p < 0.01; ***p < 0.001 tumor cells by maintenance of low stemness state. 41 In addition, targeting colorectal CSCs is proposed to become a promising approach for the future cure of colorectal cancer. 42  Epithelial-mesenchymal transition, which is characterized by In summary, the results of the present study demonstrate that siRNA-induced CD44 knockdown suppresses the proliferation, migration and invasion of colorectal CSCs by inhibiting EMT. Our findings confirm that targeting colorectal CSCs is a promising therapy for colorectal cancer, and CD44 may be a novel therapeutic target for the treatment of colorectal cancer.

ACK N OWLED G EM ENTS
This study was supported by the grants from Natural Science Research Project of Anhui Educational Committee (grant no.

KJ2019A0295, KJ2021A0792) and the Research and Innovation
Team of Bengbu Medical College (grant no. BYKC201908).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.