Anti-tumor effect of miR-1291 in colon cancer cells

Background: Cancer stem cells (CSCs) are drug-tolerant and cause distant metastasis and recurrence in various cancers, including colorectal cancer (CRC). Thus, CSC-targeted therapy may be an effective curative approach in CRC. MiR-1291 has an anti-tumor effect in carcinoma of kidney, esophagus, pancreas, and prostate. However, there is no report about the effect of miR-1291 on CRC. Methods: In this study, we took CSC marker DCLK-1 as a target gene, and screened promising miRNAs that may suppress DCLK-1 by using TargetScan Human. We performed luciferase reporter assay, quantitative real-time PCR analysis, and Western blot analysis to verify the interaction between DCLK-1 and miR-1291 in CRC cells. We also conrmed the function of miR-1291 on cancer stemness by identifying the expression of Bmi1 and CD133 in CRC cells by quantitative real-time PCR analysis, Western blot analysis, and ow cytometric analysis, as well as performing sphere formation assay. We also explored the effect of miR-1291 on cell proliferation, invasion, and wound-healing, colony formation, and cell cycle regulation. Results: We found a 7-base seed sequence of miR-1291 that matches the 3’ UTR sequence of DCLK-1 using TargetScan Human. A luciferase reporter assay showed that miR-1291 directly bound the 3’ UTR sequence of DCLK-1 and suppressed its expression at both the mRNA and protein levels. In addition, miR-1291 suppressed CSC markers Bmi1 and CD133 as well as sphere formation ability in CRC cells. Moreover, miR-1291 signicantly suppressed the proliferation, invasion, wound-healing, and colony formation capability of colon cancer cell lines. MiR-1291 caused altered expression of the cell cycle-regulatory proteins representatively, CDK inhibitors p21 WAF1/CIP1 and p27 KIP1 . Conclusions: Taken together, these ndings indicate that miR-1291 has an anti-tumor effect by modulating multiple functions, including, cancer stemness, cell cycle, and invasiveness. Our data suggest that miR-1291


Background
Colorectal cancer (CRC) is the third most widespread cancer and the second most deadly cancer in the world. Approximately 1.8 million new cases of CRC and 881,000 related deaths were estimated in 2018 [1,2]. In the past few decades, improved treatment options have become available, including surgery, radiotherapy, chemotherapy, and molecular-targeted therapy for advanced CRC [3][4][5][6]. However, the 5-year survival rate of CRC is < 65% due to cancer relapse [2].
Cancer stem cells (CSCs) possess characteristics associated with stem cells and they are hypothesized to exist as a top class of hierarchy within solid tumors or hematological cancers [7,8]. Such cells are also believed to have distinct capability for self-renewal, unlimited proliferation, reduced capacity to undergo apoptosis, and multi-potential differentiation [9,10].
Conventional anticancer drugs and radiotherapy may reduce tumor bulk, but CSCs can still survive, as they confer resistance to these therapies and cause distant metastases and recurrence in various cancers [11][12][13]. Thus, the development of CSC-targeted therapy may be an effective approach for overcoming the shortage of current therapies and completely cure CRC patients [14][15][16].
MicroRNAs (miRNAs) are short (18-25 nucleotides) internally originated non-coding RNAs that mainly bind to the 3'-untranslated region (3' UTR) of target mRNAs, contributing to mRNA cleavage or translational suppression [26,27]. MiRNAs play an important role in many biological progresses, including tumor growth, apoptosis, invasion, and survival [28], which are closely related to oncogenesis and tumor progression. Recent studies have shown that the pathological mechanisms underlying CRC depend on a variety of signaling pathways, including Wnt/β-catenin, EGFR, TGF-β, TP53, and epithelial-tomesenchymal transition, and miRNAs play a pivotal role in regulating these pathways [29][30][31]. For example, miR-4689 has an anti-tumor effect on mutant KRAS CRC by inhibiting the EGFR pathway [32]. In addition, miR-34a can inhibit cell proliferation and increase the expression of p21 WAF1/CIP1 in HCT116 and RKO colon cancer cells [33]. Through in silico analysis and in vitro selection, we focused on miR-1291 as a possible upstream modulator for DCLK-1. In recent years, miR-1291 has been demonstrated to have anti-tumor effects in carcinoma of the kidney, esophagus, pancreas, and prostate [34][35][36][37]. However, to the best of our knowledge, miR-1291 in CRC has not been reported. Therefore, we investigated the antitumor effect of miR-1291 in CRC cells in an effort to improve our understanding of the potential mechanisms of miR-1291 in CRC.

Cell lines and cell culture
Human CRC cell lines DLD-1, HT29, HCT116, human pancreatic adenocarcinoma cell line Panc-1, and human non-tumor cell line HEK293 were purchased from the American Type Culture Collection (Rockville, MD, USA). These cell lines were authenticated by morphological inspection, short tandem repeat pro ling, and mycoplasma testing. DLD-1, HT29, and Panc-1 cells were cultured in RPMI 1640 medium, and HCT116 cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured in the humidi ed incubator at 37°C and 5% CO 2 .

Transduction of the degron reporter
The degron sequence of ornithine decarboxylase (ODC) is recognized directly by proteasomes, which leads to the immediate destruction of the involved protein. The retroviral expression vector pQCXIN-ZsGreen-cODC, containing green uorescence ZsGreen-labeled degron ODC (Gdeg), was kindly provided by Dr. Frank Pajonk (UCLA's Jonsson Comprehensive Cancer Center, CA, USA). The vector was transfected into Platinum retroviral packaging cells, and the retrovirus collected from the supernatant was used to infect pancreatic cancer Panc-1 cells. Stable transfectants were selected with G418 solution (Roche, Germany). ZsGreen + cells were sorted by the ow cytometry (Cell Sorter SH800, SONY, Japan).
Luciferase reporter assay Cells were seeded in 96-well plates at a density of 10,000 cells per well and co-transfected with 50 ng of the pmirGLO plasmid vector and 50 nM of either miR-NC or miR-1291. After 24 hours of transfection, cells were assayed for both re y and renilla luciferase using the Dual-Luciferase Reporter Assay System (Promega).

RNA isolation
Total RNA, including miRNA, was isolated from cell lines using the miRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Total RNA concentration and purity were measured using a NanoDrop one spectrophotometer (Thermo Fisher Scienti c).  Table S1.

MiRNA expression
TaqMan miRNA analysis (Applied Biosystems) was used to measure miRNA expression. The reverse transcription reaction was performed with the TaqMan MicroRNA RT Kit (Applied Biosystems) according to the manufacturer's protocol. Quantitative real-time PCR was performed using the 7900 HT Sequence Detection System (Applied Biosystems). Ampli cation data were normalized to endogenous RNU6B expression. The relative expression level was quanti ed by the 2 -ΔΔCt method.

Proliferation assay
Cells were seeded in 96-well plates at a density of 4000-8000 per well and were transfected with miR-NC or miR-1291 at a nal concentration of 30 nM the second day after seeding. Twenty-four, 48, and 72 hours after transfection,10 µl of Cell Counting Kit-8 (DOJINDO Molecular technologies, Inc., Kumamoto, Japan) was added to each well, and the 96-well plates were shaded for 2 hours. After that, the absorbance was detected by Multiskan Go (Thermo Fisher Scienti c) to determine cell number.

Matrigel invasion assay
Cells were seeded in BD BioCoat Matrigel Invasion Chambers (BD Biosciences, San Jose, CA, USA) at a density of 50,000-100,000 cells per chamber. The cells were transfected with the miRNAs at a nal concentration of 50 nM. After 48-72 h of transfection, invaded cells were stained with hematoxylin.
Wound healing assay Cells were seeded in ibidi culture 2-well inserts (ibidi, Gräfel ng, Germany) in 24-well plates at a con uent density. The inserts were removed after 24 hours to create wounds. The miRNAs were transfected at a nal concentration of 30 nM. The areas of the wounds were measured at 0-48 hours using ImageJ software.
Colony formation assay Cells were transfected with miR-NC or miR-1291 at a nal concentration of 30 nM for 8 hours and then seeded in 6-well plates at a density of 500 cells per well. After 10 days, the cells were stained by crystal violet and counted.
Cell cycle assay Cells were starved in serum-free medium for 48 hours. Twenty-four hours before the end of starvation, miR-NC or miR-1291 was transfected at a nal concentration of 30 nM ( Sphere formation assay Single cells were seeded 24 hours after transfection of miR-negative control or miR-1291 in 96-Well Clear Ultra Low Attachment Microplates (Corning Inc., USA) at the density of 1000 cells per well. And the cells were cultured in DMEM/F-12 serum-free medium (Invitrogen, USA) supplemented with 20 ng/ml epithelial growth factor, 10 ng/ml basic broblast growth factor-2 (PeproTech, USA), and 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured in the humidi ed incubator at 37°C and 5% CO 2 . The number of spheres ≥40 µm was counted 4 days after seeding.
Flow cytometric analysis for CD133 marker expression Suspensions of HCT116 single cells were stained with antibodies against human CD133 (APCconjugated, No. 130-113-106, Miltenyi Biotec, Bergisch Gladbach, Germany). Dead cells were excluded by utilizing forward and side scatter. One million cells were incubated with antibodies on ice for 20 minutes in the dark, centrifuged, and washed twice with PBS containing 2% FBS. Spectral Analyzer (SA3800, Sony Biotechnology, Inc.) was used for ow cytometric analysis.

Statistical analysis
Data are presented as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 (San Diego, CA, USA) and Microsoft Excel. The statistical differences between the miR-NC and miR-1291 groups were analyzed by student's t-test (two-tailed). Comparisons among more than two groups were performed by one-way analysis of variance (ANOVA). P<0.05 was considered signi cant.

Screening of candidate miRNAs
Firstly, we identi ed 1749 miRNAs that target to DCLK-1 in TargetScan Human. Using Ingenuity Pathway Analysis' microRNA Target Filter, we screened candidate miRNAs whose target genes correlate with Notch Signaling, Wnt/β-catenin Signaling or Wnt/Ca 2+ Signaling pathway. Eventually thirty candidate miRNAs were selected for cell viability assessment (Fig. 1a). For the assessment, we made a CSC model by transducing ornithine decarboxylase (ODC)-degron to pancreatic cancer Panc-1 cells [38] to test the effects of these miRNAs on stem (degron (+)) cells and non-stem (degron (-)) cells. MiR-34a, which reached phase I clinical trial as a therapy for human solid tumor [39], was used as a therapeutic control in this experiment. Among these 30 miRNAs, miR-1291 (the 16 th miRNA) obviously suppressed both the stem (degron (+)) and non-stem (degron (-)) Panc-1 cells viability compared to either negative control miR (NC) or even positive control miR-34a (Fig. 1b). This result is consistent with the report that miR-1291 presented anti-tumor function in pancreatic cancer [36]. However, there is no previous report about miR-1291's effect in CRC, therefore, in this study we tried to clarify the function of miR-1291 in CRC.
MiR-1291 directly targeted DCLK-1 Using miRNA target prediction algorithm TargetScan Human, we identi ed a target site in the 3' UTR of DCLK-1 mRNA that is complementary to the seed sequence of miR-1291 (Fig. 2a). MiR-1291 signi cantly suppressed the expression of DCLK-1 at both the mRNA and protein levels (Fig. 2b, c). Using plasmid containing the 3' UTR sequence of DCLK-1, miR-1291 signi cantly inhibited luciferase activity in the luciferase reporter assay in DLD-1, HT29, and HCT116 cells, indicating a direct interaction between the DCLK-1 3' UTR and miR-1291 (Fig. 2d). These ndings indicate that CSC marker DCLK-1 is a direct target of miR-1291.

MiR-1291 suppressed CRC stem-like properties
We assessed stemness of CRC cell after treatment of miR-1291. We demonstrated that other CSC markers in addition to DCLK-1, including Bmi1 and CD133, were signi cantly down-regulated by miR-1291 overexpression at mRNA level (Fig. 3a, b). MiR-1291 treatment also down-regulated the protein level of Bmi1 by Western blot analysis (Fig. 3c). Moreover, the ratio of CD133 positive cells decreased with treatment of miR-1291 by ow cytometric analysis (Fig. 3d). Furthermore, miR-1291 treatment signi cantly decreased the ability of sphere formation in HCT116 cells (Fig. 3e). These ndings suggest that miR-1291 may be involved in the regulation of stem cell properties through direct inhibition of DCLK-1.

Expression of miR-1291 in CRC cells
We evaluated the expression of miR-1291 in nine CRC cell lines and non-tumor human HEK293 cells by qRT-PCR. The expression of miR-1291 was apparently lower in ve of the nine CRC cell lines compared to HEK293 cells (Fig. S2).

MiR-1291 inhibited cell proliferation
To con rm the anti-tumor effect of miR-1291 in CRC cell growth, we detected the absorbance of cells transfected with miR-NC or miR-1291 as determined by Cell Counting Kit-8. The miR-1291 transfection group presented a signi cant low absorbance compared to the miR-NC transfection group in DLD-1, HT29, and HCT116 cells after 48 and 72 hours of transfection (Fig. 4b). miR-1291 signi cantly suppressed the proliferation ability of the three cell lines.
MiR-1291 inhibited cell invasion miR-NC or miR-1291 was transfected into CRC cells to evaluate the effect on invasion ability. The cells invading through Matrigel were stained with hematoxylin 48 hours for DLD-1 or 72 hours for HT29 and HCT116 after transfection and then counted. The invasion ability of miR-1291-transfected cells was signi cantly inhibited compared to miR-NC-transfected cells in the three cell lines tested (Fig. 4c).

MiR-1291 inhibited cell migration ability
We evaluated the effect of miR-1291 on the wound-healing ability of CRC cells. The wound area of the cells was measured at the same location every 24 hours after miR transfection. The migration ability of DLD-1, HT29, and HCT116 cells was signi cantly suppressed in the miR-1291 group compared to the miR-NC group either at 24 hours or 48 hours or both (Fig. 5a).

MiR-1291 inhibited colony formation ability
To con rm the function of miR-1291 in cell colony formation ability, we observed and evaluated the colony formation of the CRC cells which were transfected with miR-NC or miR-1291. The colonies were stained with crystal violet 10 days after transfection, and we counted the number of the colonies. MiR-1291 signi cantly inhibited the colony formation potential compared to miR-NC in DLD-1, HT29, and HCT116 cells (Fig. 5b).

Effect of miR-1291 on cell cycle regulation
To determine the mechanism underlying the growth inhibitory effect of miR-1291, we performed cell cycle analysis in DLD-1 and HT29. Twelve hours refed with FBS, cells in G1 phase were signi cantly increased in miR-1291 treated cultures of DLD-1 cells (60.81% vs 67.89%, Fig. 6a). On the other hand, cell population in each cell cycle phase was not apparently changed in HT29 cells throughout the time points examined (Fig. 6a, Fig. S3).

Altered expression of cell cycle components when treated with miR-1291
Cells were examined for the change in cell cycle regulatory protein expression after transfection of miR-1291. In DLD-1 cells, the expression of CDK inhibitors p21 WAF1/CIP1 and p27 KIP1 were up-regulated and CDK4 and CDC25A level decreased at 48 hours after transfection with treatment of miR-1291 and these changes maintained till 72 hours (Fig. 6b). In HT29 cells, on the other hand, the expression of p21 WAF1/CIP1 and p27 KIP1 protein slightly increased at 48 hours but returned back to the basal levels of negative control cultures at 72 hours. Instead, Cyclin E1, CDK4, and CDK6 subsequently decreased at 72 hours with treatment of miR-1291 (Fig. 6c).
Discussion DCLK-1 is over-expressed in subsets of cancers and has an oncogenic function [40,41]. In the Caki-2 renal cancer cell line, knocking down DCLK-1 contributes to inhibition of cell proliferation, invasion, and wound-healing [42]. In 2013, DCLK-1 was reported as a marker distinguishing tumor stem cells from intestinal normal stem cells [21]. Accumulating evidence supports the involvement of DCLK-1 in the stemness of CRC [43,44]. In epigenetic regulation of DCLK-1, miR-137 suppresses the ability of cell growth by inhibiting the expression of DCLK-1 in the SW480 CRC cell line [45]. In the present study, we considered a CSC marker DCLK-1 as the target molecule, and extracted 30 miRNAs that possibly inhibit DCLK-1 and correlate with cancer stemness signal pathways including Notch Signaling, Wnt/β-catenin Signaling or Wnt/Ca 2+ Signaling pathway [43,46,47]. Among the candidate miRNAs, miR-1291 presented the notable effect of suppressing cell viability in the ODC-degron transduced Panc-1 cells which is previously reported as cancer stem-like cells [38]. Although Zs-green + cells were more resistant to miR-1291 treatment compared with Zs-green − non-stem cells, its growth inhibitory effect was even stronger than that given by positive control miR-34a.
Studies showed that miR-1291 had anti-tumor effects in multiple cancers including renal cancer, esophagus cancer, pancreatic cancer, and prostate cancer [34][35][36][37]. As the target for miR-1291, several molecules have been identi ed. These include SLC2A1/GLUT1 in A498 and 786-O renal cancer cells [34], MUC1 in human esophagus cancer EC9706 and EC-1 cells [35], the forkhead box protein A2-anterior gradient 2 (FOXA2-AGR2) pathway in PANC-1 pancreatic cancer cells [36]. Furthermore, miR-1291 has been shown to inhibit cell growth and tumorigenesis in prostate cancer by binding to MED1 [37]. However, to the best of our knowledge, there is no previous report of miR-1291 in CRC which is one of the widespread cancers in the world.
We veri ed that miR-1291 directly bound to the 3' UTR of the DCLK-1 mRNA sequence, leading to decreased expression of DCLK-1 at both the mRNA and protein levels. In addition to DCLK-1, miR-1291 lowered Bmi1 and CD133 expression, which are also representative CSC markers in CRC. Moreover, we con rmed that miR-1291 inhibited sphere formation ability of the CRC cells. These results support the notion that miR-1291 suppresses the cancer stemness through direct targeting DCLK-1, suggesting that miR-1291 may be a novel CSC-targeted therapeutic strategy for CRC.
We also found that the replacement therapy using mimic-miR-1291 played various anti-tumor roles in CRC cells, suppressing the cell proliferation, invasion, cell mobility, and colony-forming abilities. These ndings suggest that miR-1291 could serve as an inhibitor in CRC tumors.
One of the major effects given by miR-1291 was drastic change in the cell cycle components. Time course study after serum starvation for DLD-1 showed the delay in G1-S transition at 12 hours.
Concordantly, Western blot analysis showed increase in CDK inhibitors p21 WAF1/CIP1 and p27 KIP1 which bind to and block the G1-S accelerators Cyclin D1-CDK4/6 complex and Cyclin E1-CDK2 complex, leading to restraint from the G1 to S phase [48][49][50]. We also found that CDK4 and CDC25A were decreased. Since CDC25A is a crucial mediator that positively regulates the Cyclin D1-CDK4/6 complex and Cyclin E1-CDK2 complex [51], downregulation of CDC25A could be another possible explanation for G1 phase arrest and these schemes are simply summarized in Fig. S4. Although we could not nd signi cant dysregulation of cell cycle in the serum-starved HT29 cells, marked reduction of G1-S facilitating modulators Cyclin E1, CDK4, and CDK6 was observed at the later time point 72 hours. In addition, CDC25B that positively acts in the G2-M transition [52,53] was also down-regulated at 72 hours.
Collectively these results suggest that miR-1291 could cause dysregulation of cell cycle control even though its effectual action point and timing may differ by cell types.
In terms of the association of cell cycle regulators with DCLK-1, Chandrakesan et al. reported that DCLK-1-positive cells had higher expression of p21 WAF1/CIP1 and p27 KIP1 and maintained quiescence in normal small intestine [54]. However, the current study demonstrated that miR-1291 inhibited DCLK-1 and upregulated p21 WAF1/CIP1 and p27 KIP1 , which caused G1 phase arrest. These ndings indicate an opposite role of DCLK-1 in the cell cycle between normal and cancer cells. Further studies are required to de ne how DCLK-1 is associated with the cell cycle in CRC.

Conclusions
In conclusion, miR-1291 presented a strong anti-tumor effect on CRC cells and played an important role in both delaying the cell cycle and suppressing cancer stemness. To the best of our knowledge, this is the rst study that demonstrates the function of miR-1291 in CRC. Considering the anti-tumor effect of miR-1291 in the broad range of cancer type [34][35][36][37], this microRNA may be one of the candidates for the next generation nucleic acid medicine using the practical DDS systems [30,31,55,56].   Screening of candidate miRNAs. (a) There were 1749 miRNAs target to DCLK-1 in TargetScan Human.
Among the 1749 miRNAs, 30 miRNAs whose target genes related to Notch Signaling, Wnt/β-catenin Signaling or Wnt/Ca2+ Signaling pathway were extracted for cell viability experiment. (b) Ornithine decarboxylase (ODC)-degron transduced pancreatic cancer Panc-1 cells were used as a CSC model to test the effects of these miRNAs on stem (degron (+)) cells and non-stem (degron (-)) cells. Cell viability was evaluated by Cell Counting Kit-8 at 72 hours after transfection. Cell viability by each treatment was normalized to that of control cells without transfection. MiR-34a, a putative Anti-OncomiR was used as a positive control in this experiment. At 72 hours after transfection, among these 30 miRNAs, miR-1291 (the 16th miRNA) signi cantly inhibited the cell viability in both the stem (degron (+)) and non-stem (degron (-)) cell groups compared to either miR-NC group or positive control miR-34a.  The sphere formation ability was signi cantly suppressed in miR-1291 transfected HCT116 cells compared to the negative control miR (NC). The number of spheres ≥40 µm was counted 4 days after seeding. Representative images are shown on the left. In a, b, and e, the experiments were performed more than three times, and data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were performed more than three times. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5
The effects of miR-1291 on cell migration and colony-forming ability in colorectal cancer cells. (a) Wound healing assay in DLD-1, HT29, and HCT116 cells treated with negative control miR (NC) or miR-1291. The wound area was measured at the indicated times by ImageJ software. (b) The colony-forming ability was suppressed in DLD-1 and HT29 cells, but not in HCT116, 10 days after transfection with miR-1291. All experiments were performed more than three times. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.