|Year : 2015 | Volume
| Issue : 7 | Page : 182-185
MicroRNA-185 is a novel tumor suppressor by negatively modulating the Wnt/β-catenin pathway in human colorectal cancer
W Dong-xu, L Jia, Z Su-juan
Department of Gastroenterology and Hepatology, PLA 254 Hospital, Tianjin, PR China
|Date of Web Publication||20-Jul-2016|
Department of Gastroenterology and Hepatology, PLA 254 Hospital, Tianjin
Source of Support: None, Conflict of Interest: None
OBJECTIVE: The deregulation of microRNA-185 (miR-185) has been showed to be associated with many cancers and act as a tumor suppressor in many types of human malignancies. We hence tried to find out its role in human colorectal cancer (CRC). MATERIALS AND METHODS: miR-185 expression was investigated by real-time quantitative polymerase chain reaction. We carried out transfections to overexpress or knockdown of miR-185 by mimics or inhibitor, respectively. Functional study like cell counting kit-8 assay was performed to evaluate the proliferation. For addressing the impact of miR-185 on Wnt/β-catenin signaling, we further applied luciferase reporter assay and Western blotting for specific proteins in this pathway. RESULTS: miR-185 was decreased in CRC cell lines when compared with corresponding control cell line. We also proved that its overexpression in LoVo cells could remarkably suppress cell proliferation whereas knocked it down in SW480 cells has the opposite effect in vitro. Mechanically, we demonstrated that miR-185 could suppress the Wnt/β-catenin signaling and modulate the transcription and translation level of downstream molecules of this pathway, including MYC and CCND1. CONCLUSION: Taken together, these results suggested that miR-185 exerts its tumor suppressor activities probably through a negative modulation of the Wnt/β-catenin pathway.
Keywords: Colorectal cancer, microRNA-185, tumor suppressor, Wnt/β-catenin
|How to cite this article:|
Dong-xu W, Jia L, Su-juan Z. MicroRNA-185 is a novel tumor suppressor by negatively modulating the Wnt/β-catenin pathway in human colorectal cancer. Indian J Cancer 2015;52, Suppl S3:182-5
|How to cite this URL:|
Dong-xu W, Jia L, Su-juan Z. MicroRNA-185 is a novel tumor suppressor by negatively modulating the Wnt/β-catenin pathway in human colorectal cancer. Indian J Cancer [serial online] 2015 [cited 2019 Aug 21];52, Suppl S3:182-5. Available from: http://www.indianjcancer.com/text.asp?2015/52/7/182/186576
Dong-xu W, Jia L and Su-juan Z contributed equally to this work
| » Introduction|| |
Colorectal cancer (CRC) is now one of the most common malignancies and ranks as the third leading cause of cancer-related death in the Western countries.  In China, CRC is the fifth most common type of malignancy and the fourth most frequent cause of cancer-related death, with an increasing incidence in recent years. , Outcomes for patients with CRC remain poor, with an average survival of <30 months.  The development and progression of CRC is a multistep oncogenic process in which both genetic and epigenetic alterations accumulate in a temporal and spatial manner sequentially.  Although much effort was dedicated to explore the molecular mechanisms of CRC during the last two decades, the precise molecular pathogenesis of CRC remains unclear. Therefore, it is clinically urgent to understand the CRC development, which may facilitate to provide new targets for CRC diagnosis and prognosis.
MicroRNA (miRNA) is a group of endogenous small noncoding RNA that interferes the expression of target messenger RNA (mRNA) in a posttranscriptional manner. miRNAs are of highly conservation among species, indicating its vital roles in basic biological processes. Actually, through the previous intensive researches, it has been proved that miRNAs have important regulatory functions in many aspects of life sciences, including cell differentiation, proliferation, apoptosis, and the most attractive events, cell transformation, and tumorigenesis. ,, Till now, the number of verified human miRNAs is still expanding along with the continual cloning and identification studies. Recently, the key roles of miRNAs in human cancers have been well characterized. They can act as either oncogenes or tumor suppressors, through different mechanisms that finally contributes to tumor formation and progression. ,,, In the CRC research field, both overexpression and silencing or switching off of specific miRNAs, have been described in the carcinogenesis of CRC. Moreover, its aberrant expression in circulating blood is considered as a new and promising early diagnostic option. ,
miR-185 stands out as an important molecule among numerous miRNAs. Investigations in breast cancer, , liver cancer  and gastric cancer  all revealed that it functioned as a tumor suppressor. However, the biological functions as well as the underlying mechanisms of miR-185 in CRC have not been fully elucidated. Therefore, in this study, we set to explore its roles in CRC cells.
| » Materials and Methods|| |
Human normal intestinal epithelial cell CCD841 and CRC-derived cells SW480, SW620, RKO, LoVo, HT29 were cultured in RPMI1640, L15, or DMEM which was supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified incubator containing 5% CO 2 , as recommended by the American Type Culture Collection (Rockville, MD, USA).
Reagents and transfections
The hsa-miR-185 mimic, miR-185 inhibitor, mimic negative control (NC), and inhibitor NC sequences were purchased by the GenePharma Company (Shanghai, China). Colon cancer cells were transfected with miR-185 NC mimic, mimic, NC inhibitor, and inhibitor, using the lipofectamine2000 transfection reagent according to the manufacturer's instructions.
Luciferase reporter assay
Cells were plated at a subconfluent density and co-transfected with 0.05 μg of the reporter plasmid, miRNA mimics or inhibitor, and 0.02 μg of Renilla luciferase pRL-TK as an internal control for transfection efficiency. Cell lysates were prepared 24 h after transfection, and the reporter activity was measured using the Promega Dual-luciferase reporter assay system.
Cell counting kit-8 cell viability assay
Cell counting kit-8 (CCK-8) assay was performed to test cell viability in this study. Briefly, the LoVo and SW480 cells transfected with miR-185 mimics or inhibitors and compared controls. 24 h later, cells were trypsinized and seeded into 96-well plates at a density of 1.5 × 10 3 cells in 200 μl of full medium each well. After this, the plate was incubated at 37°C with 5% CO 2 . CCK-8 assay was performed using CCK-8 KIT as per the manufacturer's instructions for the nest continuous 4 days. Absorbance at 490 nm (OD490) was detected by using a microplate reader.
Quantitative analysis of microRNAs and real-time polymerase chain reaction of messenger RNAs
miRNA and total RNA was extracted from cultured cells using Trizol reagent (TaKaRa, Dalian, China) according to the manufacturer's instructions. miRNA expression levels were quantitated using an SYBR PrimeScript miRNA RT-PCR kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. For miRNA analysis, the relative amount of miRNA was normalized to U6 using comparative threshold cycle method. For quantitative analysis of mRNA expression, 2 μg of total RNA was used to synthesize complementary DNA (cDNA) using PrimeScript RT reagent kit (TaKaRa), and the corresponding cDNA was used for reverse transcriptional PCR. The relative amount of gene transcripts was normalized to GAPDH.
Cells were lysed using radioimmunoprecipitation assay buffer. Protein concentration was determined with the bicinchoninic acid methods. Equal amounts of total proteins were separated in 9% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked for 1 h at room temperature with 5% milk in PBST containing 0.05% Tween20, incubated overnight with primary antibody (MYC, CYCLIND1, 1:1000, Santa Cruz; GAPDH, 1:3000, Santa Cruz), washed and incubated with secondary antibody, and visualized by chemiluminescence.
All data presented in this study have been repeated for at least three times from three independent experiments and are expressed as the mean ± standard error of mean. The Student's t-test was performed to determine the significance of each respective group for each experimental test condition and **P < 0.01 or *P < 0.05 indicated a significant difference.
| » Results|| |
Altered expression of microRNA-185 in colorectal cancer cell lines
Although it has been reported that miR-185 is dysregulated in various human cancers, ,,, the precise expression level of miR-185 in CRC cells remains unclear. To gain insights into its role in CRC tumorigenesis, we determined its expression in five CRC cell lines (SW480, SW620, RKO, LoVo, HT29) and compared with normal colon epithelial cell line CCD-841 by quantitative RT-PCR. As shown in [Figure 1], altered and different expression of miR-185 were observed in these cell lines, in which SW480 had the increased while LoVo and HT29 had the decreased expression, significantly. Therefore, we chose SW480 and LoVo cell lines for the following functional assays.
|Figure 1: MicroRNA-185 expression is downregulated in colorectal cancer cell lines. microRNA-185 expression was detected in human normal intestinal epithelial cell CCD841 and colorectal cancer-derived cells SW480, SW620, RKO, LoVo, HT29. By quantitative real-time polymerase chain reaction. U6B was used as internal control|
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MicroRNA-185 regulated colorectal cancer cell proliferation in vitro
To understand the function of miR-185 in CRC cells, we first introduced miR-185 mimics into LoVo cells, which containing a low endogenous expression of miR-185 by transfection. Transfection of the mimics successfully increased miR-185 in the cells, compared with NC transfected cells. CCK-8 cell viability assay was carried out in these cells, and the results come out a significant suppression of cell growth by miR-185 overexpression [Figure 2]a. To the contrast, we inhibited the endogenous expression of miR-185 in SW480 cells, which expressing a relative high level of miR-185. This could lead to elevated proliferation compared to the NC group [Figure 2]b. These results supported a tumor suppressor role of miR-185 in CRC cells, via suppressing cell proliferation in vitro.
|Figure 2: MicroRNA-185 inhibits cell proliferation of colorectal cancer cells. (a) Left panel: LoVo cells were transfected with microRNA-185 mimics duplexes or control duplexes. In 48 h, the cells were harvest for testing the overexpression effect. Right panel: LoVo cells were transfected with microRNA-185 mimics duplexes or control duplexes. In 24 h, cells were trypsinized and seeded into four 96-well plates at a density of 1.5 × 103 cells for cell counting kit-8 assay. (b) Left panel: SW480 cells were transfected with microRNA-185 inhibitor oligoes or control oligoes. In 48 h, the cells were harvest for testing the knockdown effect. Right panel: SW480 cells were transfected with microRNA-185 inhibitor oligoes or control oligoes. In 24 h, cells were trypsinized and seeded into four 96-well plates at a density of 1.5 × 103 cells for cell counting kit-8 assay|
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MicroRNA-185 negatively regulate the Wnt/β-catenin signaling pathway
To investigate the molecular mechanisms through which miR-185 exerts its effects, we started to screen the signaling pathway it might regulate. Wnt/β-catenin signaling is a key pathway that aberrantly activated in CRC.  We first measured the TOPFlash luciferase reporter activity after miR-185 mimics or inhibitor introduction. As illustrated in [Figure 3]a, the normalized TOP activity was decreased after miR-185 overexpression, while elevated after its inhibition. The results showed a negative modulation of this pathway by miR-185. In particular, the downstream transcription and protein expression of MYC, CCND1 were significantly impaired, which were consistent with the TOP reporter analysis [Figure 3]b. Our findings of decreased T-cell factor/lymphoid enhancer factor/β-catenin activity together with reduction levels of MYC and CCND1 suggest that miR-185 functions as a suppressor for the Wnt/β-catenin signaling pathway in CRC.
|Figure 3: MicroRNA-185 inhibits Wnt/ƒÀ -catenin signaling in colorectal cancer cells. (a) TOPFlash reporter activity in LoVo and SW480 cells. Cells were co-transfected with TOPFlash as well as either negative control or microRNA-185 mimics/inhibitor. TOPFlash transcription was determined by the TOPFlash luciferase activity. Reporter activities were normalized to internal control Renilla. (b) Reverse transcriptional polymerase chain reaction and immunoblotting to examine expression for two Wnt/ƒÀ -catenin signaling pathway target proteins, MYC and CYCLIND1 in LoVo and SW480 cells|
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| » Discussion|| |
miR-185 has been shown to function as a tumor suppressor in many types of cancers. ,,, In this study, we also proved that it was downregulated and could inhibit cell proliferation of human CRC cells, partially through negative modulation of the Wnt/β-catenin signaling pathway. Our findings not only enriched the knowledge of miR-185 in CRC but also strongly suggested that miR-185 could be used as a potential therapeutic target and even a potential therapeutic agent for CRC.
The functional analysis only showed the growth inhibitory activity of miR-185. We did not measure the apopsis, migration, invasion and angiogenesis of CRC cells after overexpression or inhibition of miR-185. Hence, to clearly recognize the biological functions of miR-185, we need to carry out a lot more experiments to give more details. When characterizing the proliferation inhibition of miR-185 in CRC cells, we used two respective cell lines: SW480 cells for miR-185 inhibition and LoVo for miR-185 overexpression. Interestingly, we obtained an accordant conclusion that miR-185 suppressed cell proliferation. This phenomenon importantly supported the significant growth inhibitory effects governed by miR-185 in CRC cells. Nevertheless, an in vivo tumorigenesis experiment is still required to discuss the tumor-suppressing role of miR-185 in CRC cells.
The mechanistic studies further showed that miR-185-mediated tumor suppressor could be through modulating the Wnt/β-catenin signaling. Previous studies have demonstrated that adenomatous polyposis coli (APC) mutation and Wnt/β-catenin signaling activation, and chronic inflammation in colon, are the two major causes for CRC formation. ,, The studies from us have shown that forced expression of exogenous miR-185 causes reduced TOPFlash reporter activity while knockdown of endogenous miR-185 causes elevated TOPFlash reporter activity. More importantly, the downstream targets of β-catenin including MYC and CCND1 changed synergistically. In fact, Li et al. have reported that miR-185-3p regulates nasopharyngeal carcinoma radio resistance by targeting WNT2B in vitro,  which is another evidence to connect miR-185 with Wnt/β-catenin signaling.
Taken together, our results revealed a decreased expression of miR-185 in CRC cell lines, and its inhibitory effect on CRC cell proliferation via suppressing of Wnt/β-catenin pathway in vitro. The detail mechanisms responsible for this signaling regulation are still unclear and must be clarified. Our data suggest that miR-185 may be an important tumor suppressor in CRC cells.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]