The Long Noncoding RNA HOTAIR Promotes Colorectal Cancer Progression by Sponging miR-197.

The long noncoding RNA HOX transcript antisense RNA (HOTAIR) has been found to be overexpressed in many human malignancies and involved in tumor progression and metastasis. Although the downstream target through which HOTAIR modulates tumor metastasis is not well known, evidence suggests that microRNA-197 (miR-197) might be involved in this event. In the present study, the significance of HOTAIR and miR-197 in the progression of colorectal cancer was detected in vitro and in vivo. We found that HOTAIR expression was significantly increased in colorectal cancer cells and tissues. In contrast, the expression of miR-197 was obviously decreased. We further demonstrated that HOTAIR knockdown promoted apoptosis and inhibited cell proliferation, migration, and invasion in vitro and in vivo. Moreover, HOTAIR modulated the progression of colorectal cancer by competitively binding miR-197. Taken together, our study has identified a novel pathway through which HOTAIR exerts its oncogenic role and provided a molecular basis for potential applications of HOTAIR in the prognosis and treatment of colorectal cancer.

INTRODUCTION

Colorectal cancer is the third leading cause of cancer-related deaths worldwide, and its incidence is on the rise1. Although it is well known that multiple carcinogens and varying genetic backgrounds are involved in the tumorigenesis and progression of colorectal cancer, the detailed interactions and regulatory mechanisms are still obscure. Therefore, a deeper understanding of the molecular and genetic mechanisms that induce the initiation and progression of colorectal cancer is crucial.

Circulating, long noncoding RNAs (lncRNAs) are newly recognized diagnostic and prognostic biomarkers for malignant tumors2,3. lncRNAs regulate gene expression at the level of chromatin modification, transcription, and posttranscriptional processing4. HOTAIR [homeobox (Hox) transcript antisense intergenic RNA] is one of the most well-studied lncRNAs which was first identified by Rinn et al. in 20075. Recently, HOTAIR has been identified as an oncogenic molecule in different cancers including lung cancer6, hepatocellular carcinoma7, and cervical cancer8. Meanwhile, HOTAIR knockdown inhibited the proliferation, migration, and invasion and induced apoptosis of tumor cells9,10. A recent study revealed that HOTAIR knockdown inhibited proliferation and improved radiosensitivity in colorectal cancer11. However, the precise molecular mechanism is not well explored.

MicroRNAs (miRNAs) are a conserved family of small noncoding RNA molecules that posttranscriptionally regulate gene expression12. It was estimated that about 30% of genes can be regulated by miRNAs13. miR-197 is a recently identified cancer-related miRNA. It was reported to be downregulated in many cancers, such as thyroid cancer14, gastric cancer15, and breast cancer16. A recent study determined that miR-197 regulated 5-fluorouracil (5-FU) resistance via thymidylate synthase in colorectal cancer17. However, the exact expression of miR-197 and the molecular mechanism underlying its role in colorectal cancer require further exploration.

In this study, we explored the impacts of HOTAIR knockdown in colorectal cancer tissues, cell lines, and mouse models. The underlying mechanisms, especially the interaction between HOTAIR and miR-197, were further examined.

MATERIALS AND METHODS

Tissue Samples

Colorectal tumor tissues and matched adjacent nontumor tissues were obtained from 30 patients with colorectal cancer (diagnosed from January 2016 to March 2017 at the Department of Gastrointestinal Surgery in the Affiliated Hospital of Jining Medical University, Jining, P.R. China). None of the patients had received any preoperative therapy. The informed consent forms were signed by all participants. This study was approved by the Ethics Committee of the Affiliated Hospital of Jining Medical University.

Cell Culture

HCT116, SW480, LOVO, HT29, and FHC cells (ATCC, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) complemented with 10% fetal bovine serum (FBS; v/v; Life Technologies, Grand Island, NY, USA). All cells were cultured at 37°C in a 5% CO2 incubator.

Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from cells or tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Equal amounts of RNA were reverse transcribed to cDNA with SuperScript Reverse Transcriptase Kit (Thermo Fisher Scientific, Waltham, MA, USA). Then the total cDNA was amplified and analyzed by SYBR Green PCR Master Mix (Thermo Fisher Scientific) in a Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The following primers were used: HOTAIR, 5′-GGTAGAAAAAGCAACCACGAAGC-3′ (sense) and 5′-ACATAAACCT CTGTCTGTG AGTGCC-3′ (anti-sense); miR-197, 5′-AGTTGTTCACCACCTTCTCCAC-3′ (sense) and 5′-TATCGTTGTACTCCAGTCCAAGTC-3′ (anti-sense). The original Ct values were adjusted to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

HOTAIR Knockdown

HCT116 cells were transfected with four HOTAIR small interfering RNAs (siRNAs) (50 nM) using DharmaFECT reagent (Life Technologies) according to the manufacturer’s instructions. The following sequences were used: #1, 5′-GAACGGGAGUACAGAGAGAUU-3′; #2, 5′-CCACAUGAACGCCCAGAGA UU-3′; #3, 5′-UAACAAGACCAGAGAG CUGUU-3′; #4, 5′-GAGGAA AAGGGAAAAUCUAUU-3′; si-green fluorescent protein (siGFP), 5′-CUACAACAGCCACAACGUCdTd-3′.

Cell Viability Analysis

HCT116 cells were cultured on a 96-well plate and transfected with siGFP or siHOTAIR for various times. Cell viability was then measured by the cell counting kit-8 (CCK-8; Beyotime Biotechnology, Shanghai, P.R. China) according to the manufacturer’s instructions.

Flow Cytometry

HCT116 cells were transfected with siGFP or siHOTAIR for 24 h. After washing with ice-cold phosphate-buffered saline (PBS), the cells were resuspended in annexin V binding buffer and incubated with fluorescein isothiocyanate (FITC)-conjugated annexin V antibody (Cell Signaling Technology, Danvers, MA, USA) and propidium iodide for 15 min at room temperature. The cells were then analyzed with a Beckman Counter.

Western Blot

Total protein samples from cells and tissues were prepared with a standard protocol. Equal amounts of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat milk, combined with primary antibody [Ki-67, proliferating cell nuclear antigen (PCNA), cleaved caspase 3, cleaved caspase 9, matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), and β-actin; all purchased from Abcam, Cambridge, MA, USA] and secondary antibody, and then subjected to immunodetection procedure.

Cell Migration Assay

HCT116 cells transfected with siHOTAIR or scrambled RNA were cultured in a 24-well chamber. The confluent cell monolayer was stroked with a pipette tip. Cells were washed to remove detached and damaged cells. The cell migrations were monitored microscopically, and the migration distance was measured from five preset positions for each treatment condition by the ImageJ software [National Institutes of Health (NIH), Bethesda, MD, USA].

Transwell Invasion Assay

Cell invasive capacity was detected using a 24-well plate with Transwell chamber (8-μm pore size; Corning, New York, NY, USA) inserts precoated with Matrigel (BD Biosciences, San Jose, CA, USA). Briefly, HCT116 cells were seeded into the upper chamber in serum-free medium. Then complete medium (containing 10% FBS) was added into the lower chamber. After 24 h of incubation, cells in the upper surface of the membranes were removed with cotton buds. The invaded cells on the underside of the chamber were stained with hematoxylin. The stained cells were photographed and counted in 10 random fields.

Animal Work and Experimental Protocols

Athymic BALB/c mice (nu/nu, 20–25 g body weight) were purchased from the Animal Center of Jining Medical University (Jining, P.R. China). Mice were housed under controlled conditions (25 ± 2°C, 70% humidity and 12-h light–dark periods) and fed on regular sterile chow diet and water ad libitum. The mice were randomly divided into two groups (n = 6). One group was subcutaneously inoculated in the back with 5 × 106 HCT116 cells which had been transfected with siHOTAIR, and another group was treated with scrambled HCT116 cells. Tumor volumes were calculated at 8–35 days after injection. All the animal experiments were performed according to relevant national and international guidelines and were approved by the Animal Experimental Ethical Committee.

Immunohistochemistry

Tumor sections were prepared essentially as previously described18. The sections were incubated with PCNA and MMP-9 antibody (Cell Signaling Technology) overnight at 4°C, followed by incubation with fluorophore-conjugated secondary antibody (Invitrogen) for 1 h. Sections were visualized with a fluorescence microscope and quantified by ImageJ software.

Bioinformatics Data Set

Prediction of the interaction between HOTAIR and miR-197 was performed using DIANA Tools (http://diana.imis.athena-innovation.gr/DianaTools).

Luciferase Reporter Assay

The 3′-untranslated region (3′-UTR) of HOTAIR mRNA containing miR-197 binding sites was PCR amplified and inserted downstream of a Renilla luciferase reporter gene in the pGL3 vector. A mutant construct containing mutations within the binding sites was generated using the TaKaRa MutanBEST Kit (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. The luciferase activities were measured with a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Renilla luciferase intensity was normalized to firefly luciferase intensity.

Statistical Analysis

All results were presented as mean ± standard deviation (SD). The statistical significance of the studies was analyzed using one-way ANOVA. The difference was considered statistically significant at a value of p < 0.05.

RESULTS

HOTAIR Is Highly Expressed in Colorectal Cancer Tissues and Cell Lines

To understand the biological significance of lncRNA HOTAIR in colorectal cancer, the mRNA levels of HOTAIR were examined in colorectal cancer tissues and corresponding noncancerous tissues from 30 patients. The results showed that the expressions of HOTAIR in colorectal cancer tissues were significantly higher than in normal tissues (Fig. 1A). The expressions of HOTAIR were subsequently measured in FHC cells and four colorectal cancer cell lines (HCT116, HT29, SW480, and LOVO). As shown in Figure 1B, the mRNA expression levels of HOTAIR were significantly elevated in colorectal cancer cells compared with FHC cells. These results indicate that HOTAIR has a high expression in colorectal cancer cells.

Figure 1

Homeobox (Hox) transcript antisense intergenic RNA (HOTAIR) is highly expressed in colorectal cancer (CRC) tissues and cell lines. (A) The mRNA levels of HOTAIR in 30 CRC tissues and adjacent normal tissues were assayed by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). (B) The mRNA levels of HOTAIR in FHC, HCT116, HT29, SW480, and LOVO cells were assayed by qRT-PCR. All the experiments were repeated at least three times. *p < 0.05 versus normal tissues or FHC cells.

HOTAIR Knockdown Suppresses Proliferation and Metastasis of Colorectal Cancer Cells

Based on the above observations, we next investigated the role of HOTAIR in colorectal cancer cell proliferation. Four siRNAs were designed and transfected into HCT116 cells. As shown in Figure 2A, all the siRNAs significantly decreased the expression of HOTAIR, and siHOTAIR#1 displayed the highest efficiency. Therefore, siHOTAIR#1 was used for further studies. As shown in Figure 2B, siRNA-mediated knockdown of HOTAIR significantly decreased cell proliferation at 48–96 h posttransfection. In addition, HOTAIR knockdown markedly increased the percentage of annexin V+ apoptotic cells (Fig. 2C and D). Moreover, the expressions of proliferation-related proteins Ki-67 and PCNA were evidently decreased when compared with the control group (Fig. 2E–G). By contrast, the protein levels of cleaved caspase 3 and cleaved caspase 9, which are closely associated with cell apoptosis, were significantly upregulated (Fig. 2H–J). Furthermore, the wound healing assay and Matrigel Transwell assay showed that the cell migration levels and invasion capacities were obviously decreased after HOTAIR knockdown (Fig. 2K–N). Since VEGF and MMP-9 play an important role in tumor progression by promoting migration and invasion19,20, the effect of HOTAIR knockdown on the expression levels of these proteins was determined in HCT116 cells. As anticipated, the expression levels of VEGF and MMP-9 were significantly decreased after the cells were transfected with siHOTAIR (Fig. 3O–Q). Taken together, these findings indicate that HOTAIR knockdown may inhibit proliferation and metastasis of colorectal cancer cells.

Figure 2

HOTAIR knockdown suppresses proliferation and metastasis of CRC cells. (A) HCT116 cells were transfected with four different small interfering RNAs (siRNAs) or siRNA green fluorescent protein (siGFP) for 24 h, and the mRNA levels of HOTAIR were measured by qRT-PCR. (B) HCT116 cells were transfected with HOTAIR siRNA or scramble for 24, 48, 72, and 96 h, and cell viability was assayed by cell counting kit-8 (CCK-8). (C) Cell apoptosis was analyzed by annexin V flow cytometry. (D) Apoptotic cell quantification for three independent experiments. (E) Protein levels of Ki-67 and proliferating cell nuclear antigen (PCNA) were assayed by Western blot. (F, G) Quantification of (E). (H) Protein levels of cleaved caspase 3 and cleaved caspase 9 were assayed by Western blot. (I, J) Quantification of (H). (K) Cell migration assay. (L) Cell migration distances were measured by ImageJ software. (M) Matrigel invasion assay. (N) Quantification of (M). (O) The protein levels of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) were assayed by Western blot. (P, Q) Quantification of (O). All the experiments were repeated at least three times. β-Actin was used as loading control. *p < 0.05 versus scramble.

Figure 3

HOTAIR knockdown inhibits tumor progression in vivo. (A) Mice were injected subcutaneously with HOTAIR overexpression or scramble HCT116 cells, and the tumor volumes were measured at 8–35 days. (B) Tumor weight at 35 days after injection. (C) The protein levels of PCNA and MMP-9 in tumor tissues were detected by immunohistochemistry. (D) Semiquantification of immunohistochemistry (the percentage of brown color area over the whole field area from stained sections). n = 6. *p < 0.05 versus scramble.

HOTAIR Knockdown Inhibits Tumor Progression In Vivo

We next assessed the role of HOTAIR on tumor progression in a xenograft mouse model of colorectal cancer. As shown in Figure 3A, the tumor volume in the HOTAIR knockdown mouse model was markedly smaller than in the scrambled model at 20–35 days after injection. The tumor weight was still decreased at 35 days after HOTAIR knockdown (Fig. 3B). Furthermore, immunohistochemistry staining showed that the expression level of PCNA and VEGF in HOTAIR knockdown mouse model was significantly decreased (Fig. 3C), indicating that HOTAIR knockdown may modulate colorectal cancer cell proliferation and metastasis through the downregulation of PCNA and VEGF in a mouse colorectal cancer model.

miR-197 Suppresses HOTAIR Function in Colorectal Cancer Cells

To determine the relationship between HOTAIR and miR-197, bioinformatics analysis was performed. The result showed that miR-197 and HOTAIR have a targeted correlation (Fig. 4A). Then qRT-PCR showed that the expression levels of miR-197 in colorectal cancer tissues and cells were significantly decreased compared to the normal control tissue or cells (Fig. 4B and C). In addition, HOTAIR knockdown markedly promoted the expression of miR-197 (Fig. 4D). Cells transfected with the miR-197 mimic significantly decreased the expression of HOTAIR, whereas the miR-197 inhibitor increased it (Fig. 4E). To further determine the bonding effect between HOTAIR and miR-197, a luciferase reporter containing exact or mutant miR-197 binding sites was established and transfected into HCT116 cells. The results showed that the miR-197 mimic significantly decreased the luciferase activity of the wild-type HOTAIR reporter plasmid, and the miR-197 inhibitor notably enhanced it. However, the effect of the miR-197 mimic or inhibitor was abolished on mutant HOTAIR reporter plasmid (Fig. 4F). Furthermore, the proliferation, migration, and invasion of HCT116 cells were significantly inhibited by the miR-197 mimic, but increased by the miR-197 inhibitor (Fig. 4G–I). Taken together, these results demonstrate that HOTAIR is targeted by miR-197.

Figure 4

miR-197 suppresses HOTAIR function in CRC cells. (A) Bioinformatics analysis of HOTAIR and miR-197. (B) The mRNA levels of miR-197 in 30 CRC tissues and adjacent normal tissues were assayed by qRT-PCR. (C) The mRNA levels of miR-197 in FHC, HCT116, HT29, SW480, and LOVO cells were assayed by qRT-PCR. (D) HCT116 cells were transfected with siGFP or siHOTAIR for 24 h, and the mRNA levels of miR-197 were assayed by qRT-PCR. (E) HCT116 cells were transfected with miR-197 mimic or miR-197 inhibitor for 24 h, and the mRNA levels of HOTAIR were assayed by qRT-PCR. (F) HCT116 cells were transfected with wild-type or mutant HOTAIR plasmid and cotransfected with miR-197 mimic or miR-197 inhibitor for 24 h. Cell lysates were assayed for luciferase activity. HCT116 cells were transfected with miR-197 mimic or miR-197 inhibitor for 24 h, and cell proliferation (G), migration (H), and invasion (I) were measured as before. All the experiments were repeated at least three times. *p < 0.05 versus scramble or mimic control.

DISCUSSION

In this study, we have investigated the role of HOTAIR in colorectal cancer and made several novel observations. (1) HOTAIR is highly expressed in colorectal cancer. (2) Knockdown of HOTAIR mitigates the progression of colorectal cancer. (3) miR-197 has a low expression in colorectal cancer cells. (4) miR-197 can target and downregulate the expression of HOTAIR.

Accumulating evidence suggests that lncRNAs play diverse roles in human carcinomas21,22. Since then, many lncRNAs have been linked to tumorigenesis, either as oncogenes or tumor suppressors. While the underlying mechanism of many of these lncRNAs remains to be elucidated, it is clear that lncRNAs contribute to the dysregulation of gene expression in colorectal cancer, which then results in cancer initiation, development, and progression23. The lncRNA HOTAIR used in this study has been demonstrated to make a critical effect on most biological processes in cancer and would be a potential new target in tumor treatment24. Up to now, HOTAIR had been found overexpressed in many human malignancies and acted as a negative prognostic predictor6,10,25. In our study, we found that HOTAIR was overexpressed in colorectal cancer tissues and cells lines. In addition, HOTAIR knockdown notably decreased cellular proliferation, migration, and invasion and accelerated cell apoptosis. These findings thoroughly illuminated the biological significance of HOTAIR in colorectal cancer.

The expression levels of certain miRNAs are upregulated or downregulated and function either as oncogenes or tumor suppressor genes by regulating target genes in malignant tumors26–28. The altered expression of miR-197 has been found to be associated with many types of cancer. Fused in sarcoma 1 (FUS1), located on human chromosome 3p21.3, has been shown to be a novel tumor suppressor gene in different tumors29. In non-small cell lung cancer, overexpression of miR-197 repressed FUS1 expression by targeting its 3′-UTR30. Fiori et al. described that miR-197 decreased apoptosis through inhibiting the expression of tumor protein 53 (p53), one of the most altered tumor suppressors31. In addition, miR-197 induces epithelial–mesenchymal transition in pancreatic cancer cells by targeting p120 catenin δ132. In contrast, miR-197 inhibits cell proliferation by targeting growth factor receptor bound protein 2-associated protein 2 (GAB2) in glioblastoma33. Furthermore, miR-197 mediates the response of colorectal cancer cells to 5-FU by regulating thymidylate synthetase (TYMS) expression17. In this study, we confirmed that miR-197 was significantly downregulated in colorectal cancer cell lines and tissues. Aberrant expression of miR-197 was closely related to the progression of colorectal cancer.

The importance of lncRNAs in human disease might be correlated with their ability to impact cellular functions through different mechanisms9,34. Recently, HOTAIR has been reported to bind different miRNAs in several cancer cells. For example, HOTAIR competitively bound miR-331-3p to regulate human epidermal growth factor receptor 2 (HER2) expression in gastric cancer25. HOTAIR modulates c-KIT expression through sponging miR-193a in acute myeloid leukemia35. Similarly, Liu et al. demonstrated that overexpression of long intergenic noncoding RNA LINC00312 downregulated miRNA-197-3p to inhibit the invasion and migration of thyroid cancer cells36. Using an online software, we identified HOTAIR as a possible target of miR-197. Furthermore, the potential negative correlation of miR-197 and HOTAIR expression via their interactions was verified in vitro by gain-of-function, loss-of-function, and mutation studies. Therefore, our study has discovered a crucial upstream event through which HOTAIR exerts tumorigenic function, although the precise mechanism needs further investigation.

In summary, we have identified that HOTAIR is upregulated in human colorectal cancer tissues and serves as a negative prognostic factor. Silencing HOTAIR inhibits colorectal cancer cell proliferation and invasion and induces apoptosis. Mechanistically, HOTAIR functions as an oncogene in colorectal cancer, at least partly, through sponging miR-143. Understanding the precise molecular mechanism is vital for exploring new potential strategies for early diagnosis and therapy. Our experimental data also suggested that targeting miR-197-HOTAIR may represent a novel therapeutic application in colorectal cancer.