Abstract
Background: miR-96 is reported to inhibit reversion cysteine-rich Kazal motif (RECK), which is associated with tumor invasion, in solid cancer types (e.g. breast cancer, non-small cell lung cancer, esophageal cancer). The purpose of this study is to clarify whether miR-96 is similarly associated with tumor invasion in colorectal cancer. Materials and Methods: We performed western blotting to investigate the expression of RECK when miR-96 mimics or inhibitors were transferred into HCT-116 colorectal cancer cells. The RECK mRNA level was assessed by a reverse transcription polymerase chain reaction. An invasion assay was used to evaluate tumor invasion. Results: The expression of RECK was inhibited by the transfection of miR-96 mimics. RECK mRNA level was reduced by miR-96 mimics and increased by miR-96 inhibitor. In the invasion assay, miR-96 mimics were shown to promote tumor invasion. Conclusion: miR-96 may be associated with tumor invasion through inhibition of RECK expression in colorectal cancer.
Colorectal cancer (CRC) is the third most common form of cancer worldwide (1). Chemotherapy and operative techniques for CRC have improved; however, one-third of patients with CRC who receive surgical treatment die within 5 years of surgery; thus, the prognosis of CRC remains poor (2, 3). The prognosis of metastatic colorectal cancer is worse. Thus, elucidating the mechanisms of metastasis and tumor invasion is important for improving the prognosis of this disease (4).
MicroRNAs (miRs), which are small non-coding RNAs, unite with the 3’-untranslated region on mRNA to regulate the expression of proteins via degradation of mRNA or by inhibiting the translation of proteins (5). The expression of miR in tumors is different from that in normal tissue; the miR level in carcinoma is reported to be associated with tumor invasion, proliferation, metastasis and resistance to anticancer agents (6-9). However, there are many unanswered questions regarding the effect of changes in the levels of miRs in cancer tissue.
More than 3,000 miRs have been reported. In particular, miR-96 is up-regulated more highly in breast (10), non-small cell lung (11), esophageal (12), hepatocellular (13), endometrial (14) and colorectal cancer (15). The up-regulation of miR-96 is associated with a poor prognosis in CRC (15); thus, miR-96 is referred to as an ‘onco-miR’ in CRC (15, 16).
miR-96 activates matrix metalloproteinases (MMP), which regulate the remodeling of the extra cellular matrix (ECM) via the expression of reversion cysteine-rich Kazal motif (RECK), to induce cell invasion in breast (10), non-small cell lung (11), and esophageal (12) cancer. However, the functions of miRs differ in each of these cancer types and it is unclear whether miR-96 is associated with tumor invasion in CRC via RECK.
The aim of this study was to clarify whether miR-96 regulates tumor invasion in CRC via RECK.
Materials and Methods
Cell line. A colon cancer cell line, HCT-116, was used. HCT-116 was obtained from the American Type Culture Collection (Rockville, MD, USA) and were incubated in Dulbecco's modified Eagle's medium (DMEM; Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS; Nichirei, Tokyo, Japan), 100 IU/ml penicillin (Wako), 100 mg/ml streptomycin (Wako) and 0.5 mM sodium pyruvate (Wako) at 37°C with 5% CO2.
Transfection of miR mimic and inhibitor. HCT-116 cells were transfected with mirVana® miRNA mimic (MC12537), mirVana miRNA inhibitor (MH12537), mirVana® miRNA Inhibitor negative control (NC) (4464076) or mirVana miRNA Mimic NC (4464058) according to the manufacturer's instructions (Applied Biosystems). A total of 1×106 cells were then incubated in lysis medium containing 9 μl of lipofectamine RNAiMAX® (Life Technologies, Carlsbad, CA, USA), 150 μl of OptiMEM® (Life Technologies), and 30 pmol miR mimic or 30 pmol miR inhibitor, in a 6-well dish for 5 min in room air. Then 1.7 ml of DMEM containing 2% FBS was added and the cells were incubated for 48 h.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was extracted from transfected and parentaI HCT-116 cells using an miReasy Mini kit (Life Technologies, Carlsbad, CA, USA), and RNA was transcribed for cDNA using ReverTra Ace® qPCR RT Master Mix (Toyobo, Osaka, Japan), according to the manufacturer's instructions.
We verified the quantification of total RNA using a NanoDrop ND-1000 spectrometer (NanoDrop, Wilmington, DE, USA), and the optical density (OD) ratio at 260/280 nm was utilized for quality control and to determine the concentration of total RNA. A Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used for reverse transcription with a TaqMan® Gene Expression Assay (Thermo Fisher Scientific, Waltham, MA, USA) for RECK (Hs01019185; Applied Biosystems) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Hs02758911; Applied Biosystems). GAPDH was used as an endogenous control. The cycle threshold (Ct) values were used to calculate the relative expression ratios between the control and treated cells. The PCR cycle conditions were follows: 30 s at 95°C, followed by 40 cycles at 10 s at 95°C, 60 s at 60°C and 30 s at 70°C. The relative quantification of the gene expression was performed using the 2−ΔΔCt method. Each sample was examined in triplicate.
Western blotting. The protein expression was analyzed by western blotting. The transfected cells were washed with phosphate-buffered saline (PBS) (Wako), then centrifuged for 20 s at 13,000 × g. The cells were dissolved by 400 μl PRO-PREPTM (iNtRON, Seongnam-shi, Korea) according to the manufacturer's instructions. The lysis solution was centrifuged at 4°C for 5 min at 13000 × g and the supernatant was retrieved. We then determined the concentration of protein using an ND-1000 spectrometer™ (NanoDrop). We adjusted the protein level of each sample, and performed electrophoresis using NuPAGE® Tris-Acetate SDS Running Buffer (Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer's protocol. The samples were then transferred to polyvinylidene difluoride membranes (Trans-Blot Turbo Transfer Pack®; Bio-Rad, Hercules, CA, USA). After blocking the membranes with skim milk, they were incubated with primary antibody for RECK (ab88249; Abcam, Cambridge, MA, USA) and β-actin (#4863; Cell Signaling Technology, Danvers, MA, USA) using SNAP i.d. (Merck, Darmstadt, Germany). We used an enhanced chemiluminescence system (ImageQuant LAS 4000 mini; General Electric, Fairfield, CA, USA) to detect the bands.
Cell invasion assay. A total of 5×104 cells which had been transfected miR mimics or miR inhibitor, were plated in the top chamber of a Transwell with a Matrigel-coated membrane with pores of 8-mm in diameter (Millipore, Darmstadt, Germany). DMEM (1.7 ml) was added. The lower chamber contained 750 μl of DMEM (Wako) containing 10% FBS (Nichirei), 100 IU/ml penicillin (Wako), 100 mg/ml streptomycin (Wako) and 0.5 mM sodium pyruvate (Wako). After 48 h incubation, we removed the cells on the upper membrane, and counted the number of cells in five random fields (×200) on each membrane. The cells were incubated for 48 h. The mean number of cells was calculated after the lower membranes were fixed and stained using Diff-Quik (Sysmex, Kobe, Japan), according to manufacturer's instructions.
Statistical analysis. JMP 11 software (SAS Institute, Cary, NC, USA) was used to analyze the data. Differences between groups were analyzed using the Mann–Whitney test and Student's t-test. p-Values of less than 0.05 were considered to indicate statistical significance.
Results
miR-96 reduced RECK mRNA and inhibited the expression of RECK. The RT-PCR revealed that miR-96 mimic significantly reduced the mRNA level of RECK in comparison to NC mimic. In contrast, miR-96 inhibitor increased the RECK mRNA level in comparison to NC inhibitor (Figure 1).
Western blotting confirmed that miR-96 mimic reduced the expression of RECK to less than that of the control and miR-96 inhibitor increased RECK expression in comparison to the control (Figure 2).
miR-96 up-regulated tumor cell invasion. miR-96 mimic increased tumor cell invasion in comparison to NC mimic, while miR-96 inhibitor reduced tumor cell invasion in comparison to NC inhibitor (Figure 3).
Discussion
The present study revealed that miR-96 mimic increased tumor cell invasion by inhibiting the expression of RECK in the HCT-116 CRC cell line.
RECK is a 110 kDa glycoprotein of the cell membrane anchor type. miR-96 has been reported to inhibit RECK expression in different types of cancer (10-12). The remodeling of the ECM is important for the invasion of cancer cells and RECK controls MMPs that are involved in the remodeling of the ECM (17, 18). MMP2, MMP9 and membrane type 1 (MT1)-MMP induce a decrease in adhesion between the ECM and cells, which leads to tumor cell invasion (17, 18). RECK inhibits the change of proMMP2 to intermediate MMP2 and from intermediate MMP2 to activated MMP2, and reduces proMMP9 secretion. In this way RECK reduces MMP levels leading to a decrease in the degradation of the ECM, thereby inhibiting tumor cell invasion (Figure 4) (17, 18). RECK can be considered to be a tumor-suppressor gene. In previous reports, the prognosis of patients with CRC with reduced expression of RECK was significantly poor (17, 18).
It is reported that miR-96 is also associated with the inhibition of expression of forkhead box protein O1 and 3a, which are transcription activity factors associated with survival and tumor growth (19, 20).
In the future, drugs targeting miR-96 may be applied in the treatment of CRC. However, the factors associated with the progression of cancer, including RECK, are controlled by more than one miR. mir-21 (21) and miR-221 (22) also control the expression of RECK. The further elucidation of the mechanisms underlying the effects of miRs will be necessary before treatments targeting miRs can be used clinically.
Conclusion
miR-96 may be associated with tumor invasion through inhibition of RECK expression in CRC cells.
Acknowledgements
The Authors thank Brian Quinn who provided medical writing services on behalf of JMC, Ltd. and the staff of the Central Laboratory of Osaka City University Medical School for their technical support.
Footnotes
Conflicts of Interest
The Authors declare that they have no competing interests in regard to this study.
- Received January 23, 2018.
- Revision received February 15, 2018.
- Accepted February 26, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved