|Year : 2015 | Volume
| Issue : 7 | Page : 186-189
MicroRNA-618 modulates cell growth via targeting PI3K/Akt pathway in human thyroid carcinomas
L Yi, Y Yuan
Department of General Surgery, Suzhou Kowloon Hospital Shanghai Jiao Tong University, School of Medicine, Suzhou, Jiangsu 215021, P.R. China
|Date of Web Publication||20-Jul-2016|
Department of General Surgery, Suzhou Kowloon Hospital Shanghai Jiao Tong University, School of Medicine, Suzhou, Jiangsu 215021
Source of Support: None, Conflict of Interest: None
OBJECTIVE: MicroRNAs (miRNAs) were popularly investigated in many cancers. The aim of this study was to evaluate the expression, role, and mechanism of microRNA-618 (miR-618) in human thyroid cancer (TC) cells. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction was carried out to examine the expression level of miR-618 in 20 TC tissues with 15 adjacent normal tissues. Synthesized mimics medicated miR-618 overexpression model was done in TC TPC-1 cell line. The effects of cell growth were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide method. In addition, PI staining followed by flow cytometry was performed to analyze cell cycle. Then, we performed Western blotting to analyze the impact of miR-618 overexpression on the classical PI3K/Akt signaling pathway. RESULTS: We confirmed previous findings that miR-618 was downregulated in TC. Functionally, we found that forced expression of miR-618 suppressed cell proliferation and led to G2/M arrest in TPC-1 cells. Mechanically, we showed that miR-618 overexpression induced a significant inhibition of PI3K/Akt signaling pathway in TPC-1 cells. Importantly, restoration of Akt reversed the growth inhibitory effects of miR-618. CONCLUSION: Taken together, our results described a growth-suppressive role of miR-618 in TC cells partially targeting the PI3K/Akt signaling pathway.
Keywords: MicroRNA-618, PI3K/Akt, proliferation, thyroid cancer
|How to cite this article:|
Yi L, Yuan Y. MicroRNA-618 modulates cell growth via targeting PI3K/Akt pathway in human thyroid carcinomas. Indian J Cancer 2015;52, Suppl S3:186-9
| » Introduction|| |
Thyroid cancer (TC), with the increasing incidence worldwide, ranks the most common type of endocrine malignancy, which is the follicular cell origin. Three categories of TC can be defined according to the specific histological features: Well-differentiated thyroid carcinomas (WDTCs), poorly differentiated thyroid carcinomas (PDTCs), and undifferentiated thyroid carcinomas.  Although WDTC represents the majority of TCs, PDTC and anaplastic thyroid carcinoma account for most TC-related death. ,, Recent advances have improved our understanding of the pathogenesis of TC. Moreover, this information can provide us powerful ancillary diagnostic tools and can also be used to identify novel therapeutic targets. ,,, However, about 10% of the PDTC patients present with recurrences and distant metastases, which is associated with increasing mortality.  Meanwhile, the prognosis for these patients is very poor as they have no therapeutic options. , The pathogenesis of TC is complex and involves multiple genetic events that lead to activation of oncogenic pathways, such as PI3K/Akt pathway. This pathway has emerged as an important player in the pathogenesis of TC. 
MicroRNAs (miRNAs) are a class of small noncoding RNAs that with 19-22 nucleotides in length. They are highly conserved and function as posttranscriptional modulators by base-pairing to the 3'- UTR of target mRNAs, in a sequence-specific manner, thus resulting in gene silencing and/or translation repression. , The involvement of miRNA in human cancers is well established: They represent a new level of gene regulation and a potential tool as diagnostic, prognostic, and therapy response biomarkers.  However, the potential implication of miRNAs in the tumorigenesis of TC remains largely unknown. 
Among the many dysregulated miRNAs in TC, microRNA-618 (miR-618) was poorly understood. Its deregulation has previously been linked to a number of malignancies, including hepatocellular carcinoma,  male breast cancer,  and Barrett's esophageal cancer.  However, to our knowledge, there are only two functional works that have been done to explicate miR-618 connection to human malignancies: Lymphomagenesis  and anaplastic TC.  In the latter report, Cheng et al. proved that miR-618 was downregulated in TC cell lines. However, the biological role of miR-618 in TC has not been studied. Hence, the aim of this study was to characterize the expression of miR-618 in TC tissues, to identify its function in TC cells in vitro, and to determine its utility in TC diagnosis.
| » Materials and Methods|| |
Human tissues and cell line
Twenty human thyroid carcinoma and 15 normal tissues were provided by Suzhou Kowloon Hospital affiliated with Shanghai Jiao Tong University School of Medicine. All human tissues were obtained from patients who provided informed consent.
Human TPC-1 cell line was purchased from the Cell Line Resource Center, Shanghai Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). TPC-1 cells were maintained in DMEM supplemented with 10% fetal bovine serum (Gibco, Life Technologies, USA) at 37°C, 5% CO 2 in a humidified incubator.
Proteins from the cells were extracted in radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China). The whole cell extracts were boiled with equal amounts of loading dye for 10 min at 100°C and separated by 10% polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. Membranes were blocked in phosphate-buffered saline with 0.1% Tween-20 containing 5% nonfat milk for 1 h and then incubated with primary and secondary antibodies. The primary antibodies p-PI3K, p-Akt, and GAPDH were purchased from Cell Signaling Technology.
Quantitative real-time polymerase chain reaction
Total RNA of tissues and TPC-1 cells was extracted with TRIzol Reagent (Invitrogen). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of miRNA expression was performed on ABI7900 (Applied Biosystems). All reactions were run in triplicate. U6B were used as endogenous controls for detection of miRNA. For data analysis, the 2−ΔΔCt method was used to calculate fold change.
MicroRNA mimics and transfection
MiR-618 mimics and negative control were purchased from GenePharma (Shanghai, China). Transient transfection was performed with Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol.
Vector construction and transfection
The coding sequence of Akt was cloned into pcDNA3.1 to construct the Akt constitutively active expression plasmid (myc-Akt-ca). Transfection was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.
TPC-1 cells (2000 cells/well) were plated in 96-well plates. Following transient transfection at 24 h, cells were cultured continually. At 24, 48, 72, and 96 h, 10 μL of 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) was added to each well. Cells were incubated at 37°C for 4 h; the medium removed, and precipitated formazan dissolved in 100 μL dimethyl sulfoxide. After shaking for 30 min, absorbance was detected at 490 nm on a microplate spectrophotometer.
Cell cycle assay
The transfected TPC-1 cells were harvested, fixed with cold 70% ethanol for 30 min at 4°C, and incubated in the dark with RNase (100 mg/ml) and propidium iodide (50 mg/ml) for 30 min at 37°C. A total of 10,000 nuclei were examined by flow cytometry.
Each experiment was repeated at least three times. All data were presented as mean ± standard deviation. The difference between means was analyzed with Student's t-test. All tests performed were two-sided. P <0.05 was considered to be statistically significant.
| » Results|| |
MicroRNA-618 is frequently decreased in human thyroid cancer tissues
Although it was previously reported a downregulation of miR-618 in TC cell lines,  more substantial works including its expression in clinical TC tissues are lacking. To verify its downregulation in TC tissues, we applied the qRT-PCR method to quantify its expression level. We found miR-618 was significantly decreased in TC tissues (tumor) comparing with adjacent normal thyroid tissues (normal) (P < 0.0001) [Figure 1]. These results provide novel evidence for miR-618 downregulation in human TC tissues.
|Figure 1: MicroRNA-618 was downregulated in human thyroid carcinomas tissues. Relative expression level of microRNA-618 was performed in normal tissues versus thyroid carcinomas. MicroRNA-618 mRNA expression by quantitative real-time polymerase chain reaction normalized to U6B (normal tissues: n = 15; thyroid carcinomas: n = 20) (**P < 0.01)|
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MicroRNA-618 suppresses cell growth and induces cell cycle arrest in thyroid cancer cells
To determine whether miR-618 regulates TC cell growth, we constructed miR-618 overexpression models in TPC-1 cells transfected with mimics carrying the miR-618 gene. As shown in [Figure 2]a, forced expression of miR-618 suppressed cell proliferation compared with negative control as well as the mock groups as demonstrated by the MTT assay. Cell cycle analysis by flow cytometry showed that miR-618 induced G2/M cell cycle arrest [Figure 2]b. Successful increased expression of miR-618 upon transfection in the TPC-1 cell line was validated by qRT-PCR [Figure 3]a. These results suggest that miR-618 can inhibit cell growth of TC cells.
|Figure 2: Effects of transfection with microRNA - 618 or nonspecific control on TPC-1 cell viability. (a) Effect of microRNA-618 on PTC-1 cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay. *P < 0.05. **P < 0.001. (b) Effect of microRNA-618 on PTC-1 cell cycle was measured by flow cytometry. G2/M arrest was increased by microRNA-618 overexpression. The results represent three independent experiments|
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PI3K/Akt pathway is involved in the microRNA-618-induced cell growth inhibition in thyroid cancer cells
Given that PI3K/Akt signaling is intensively investigated in TC tumorigenesis, we first examined the phospho-PI3K (p85) and phospho-Akt levels in the miR-618 overexpressed TC cells. As depicted in [Figure 3]b, a marked decrease in phospho-PI3K (p85) and phospho-Akt expression was found. We also tested whether restoration of phospho-Akt could reverse the growth inhibitory effects of miR-618. The Akt1-ca plasmid that expressing sustained activation of phospho-Akt was cotransfected into the miR-618-overexpressed TPC-1 cells. As shown in [Figure 3]c, cell growth rate was rescued markedly after Akt-ca plasmid was cotransfected with miR-618. These findings indicate that miR-618 inhibits PI3K/Akt signaling, and its growth-suppressive role was partially mediated by this pathway in TC cells.
|Figure 3: MicroRNA-618 inhibits TPC-1 cells viability partly through Akt. (a) TPC-1 cells were transfected with microRNA-618 mimics or negative controls and subjected to quantitative real-time polymerase chain reaction analysis. (b) The expression of p-PI3K and p-Akt was analyzed by Western blotting. GAPDH was used as an internal control. (c) Reverse experiment of co-transfected microRNA-618 mimics with myc-Akt-ca plasmid in TPC-1 cells by 3 - (4,5 - dimethylthiazol - 2 - yl) - 2,5 - diphenyl - tetrazoliumbromide assay. *P < 0.05. **P < 0.001|
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| » Discussion|| |
The downregulation of miR-618 has been observed in TC cell lines.  Our data further validated its downregulation in clinical TC tissues than in normal thyroid tissues. The forced expression of miR-618 was shown to suppress cell proliferation and induce cell cycle arrest. In addition, for the first time, we proved that miR-618 could inhibit the PI3K/Akt signaling, which is known to regulate TC initiation and progression in TC cells. Importantly, we report that its growth inhibitory effect was at least partially mediated by Akt activation. This is the first study to examine the biological function of miR-618 in TC cells and may be a diagnostic biomarker in TC.
A growing body of evidence strongly supports dysregulation of miR-618. ,,, Nevertheless, little is known of its roles in tumorigenesis and tumor progression. Cheng et al. first reported that miR-618 inhibits anaplastic TC by repressing X-linked inhibitor of apoptosis protein (XIAP) in one TC cell line.  In their paper, they showed that miR-618 could inhibit the growth, invasion, and migration of the TC 8305C cells. They used luciferase assay and found that XIAP was a target gene of miR-618.  Our functional study revealed miR-618 could inhibit cell growth, and this activity could be explained by the effect of miR-618 on the PI3K/Akt pathway. Accumulating evidence indicates that the PI3K/Akt pathway regulates epithelial-mesenchymal transition (EMT), cell cycle, angiogenesis, and apoptosis. ,, Thus, miR-618 overexpression is a new mechanism that activates PI3K/Akt pathway in human TC.
The current study shows that miR-618 expression leads to the inhibition of Akt phosphorylation. Moreover, we found that the growth inhibitory effects of miR-618 overexpression could be reversed by transducing Akt-ca plasmid. We thus gave some explanations to interpret its growth suppressive function in TC cells. However, whether miR-618 could impact on other biological processes including cell apoptosis, EMT, migration, and invasion? There are still many substantial works need to be done to explore it. In addition, we had not found the direct target of miR-618 in TC cells. If other targets besides XIAP existed, these above all questions need to be further answered.
This study identified miR-618 as a tumor suppressor miRNA in human TC by inhibiting PI3K/Akt signaling, consequently resulting in slower cell growth and cell cycle arrest. Our findings suggest that miR-618 may be a potential target for future prevention and treatment of human TC.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]