Indian Journal of Cancer Home 

ORIGINAL ARTICLE
[Download PDF]
Year : 2015  |  Volume : 52  |  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

Correspondence Address:
L Yi
Department of General Surgery, Suzhou Kowloon Hospital Shanghai Jiao Tong University, School of Medicine, Suzhou, Jiangsu 215021
P.R. China

Abstract

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.



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;52:186-189


How to cite this URL:
Yi L, Yuan Y. MicroRNA-618 modulates cell growth via targeting PI3K/Akt pathway in human thyroid carcinomas. Indian J Cancer [serial online] 2015 [cited 2020 Jul 7 ];52:186-189
Available from: http://www.indianjcancer.com/text.asp?2015/52/7/186/186577


Full Text

 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. [1] Although WDTC represents the majority of TCs, PDTC and anaplastic thyroid carcinoma account for most TC-related death. [2],[3],[4] 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. [5],[6],[7],[8] However, about 10% of the PDTC patients present with recurrences and distant metastases, which is associated with increasing mortality. [9] Meanwhile, the prognosis for these patients is very poor as they have no therapeutic options. [10],[11] 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. [12]

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. [13],[14] 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. [15] However, the potential implication of miRNAs in the tumorigenesis of TC remains largely unknown. [16]

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, [17] male breast cancer, [18] and Barrett's esophageal cancer. [19] However, to our knowledge, there are only two functional works that have been done to explicate miR-618 connection to human malignancies: Lymphomagenesis [20] and anaplastic TC. [21] 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.

Western blotting

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.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay

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.

Statistical analysis

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, [21] 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 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}

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}

 Discussion



The downregulation of miR-618 has been observed in TC cell lines. [21] 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. [17],[18],[19],[20] 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. [21] 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. [21] 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. [22],[23],[24] 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

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Kondo T, Ezzat S, Asa SL. Pathogenetic mechanisms in thyroid follicular-cell neoplasia. Nat Rev Cancer 2006;6:292-306.
2Krook KA, Fedewa SA, Chen AY. Prognostic indicators in well-differentiated thyroid carcinoma when controlling for stage and treatment. Laryngoscope 2015;125:1021-7.
3Lu ZZ, Zhang Y, Wei SF, Li DS, Zhu QH, Sun SJ, et al. Outcome of papillary thyroid microcarcinoma: Study of 1,990 cases. Mol Clin Oncol 2015;3:672-6.
4Nguyen QT, Lee EJ, Huang MG, Park YI, Khullar A, Plodkowski RA. Diagnosis and treatment of patients with thyroid cancer. Am Health Drug Benefits 2015;8:30-40.
5Li DD, Zhang YF, Xu HX, Zhang XP. The role of BRAF in the pathogenesis of thyroid carcinoma. Front Biosci (Landmark Ed) 2015;20:1068-78.
6Yeganeh MZ, Sheikholeslami S, Hedayati M. RET proto oncogene mutation detection and medullary thyroid carcinoma prevention. Asian Pac J Cancer Prev 2015;16:2107-17.
7Links TP, Verbeek HH, Hofstra RM, Plukker JT. Endocrine tumours: Progressive metastatic medullary thyroid carcinoma: First- and second-line strategies. Eur J Endocrinol 2015;172:R241-51.
8Russ G, Leboulleux S, Leenhardt L, Hegedüs L. Thyroid incidentalomas: Epidemiology, risk stratification with ultrasound and workup. Eur Thyroid J 2014;3:154-63.
9Lang BH, Wong KP, Wan KY, Lo CY. Significance of metastatic lymph node ratio on stimulated thyroglobulin levels in papillary thyroid carcinoma after prophylactic unilateral central neck dissection. Ann Surg Oncol 2012;19:1257-63.
10Schlumberger M, Sherman SI. Clinical trials for progressive differentiated thyroid cancer: Patient selection, study design, and recent advances. Thyroid 2009;19:1393-400.
11Kojic KL, Kojic SL, Wiseman SM. Differentiated thyroid cancers: A comprehensive review of novel targeted therapies. Expert Rev Anticancer Ther 2012;12:345-57.
12Petrulea MS, Plantinga TS, Smit JW, Georgescu CE, Netea-Maier RT. PI3K/Akt/mTOR: A promising therapeutic target for non-medullary thyroid carcinoma. Cancer Treat Rev 2015;41:707-13.
13Calin GA, Croce CM. MicroRNA-cancer connection: The beginning of a new tale. Cancer Res 2006;66:7390-4.
14Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004;116:281-97.
15Di Leva G, Croce CM. Roles of small RNAs in tumor formation. Trends Mol Med 2010;16:257-67.
16Forte S, La Rosa C, Pecce V, Rosignolo F, Memeo L. The role of microRNAs in thyroid carcinomas. Anticancer Res 2015;35:2037-47.
17Abdalla MA, Haj-Ahmad Y. Promising candidate urinary microRNA biomarkers for the early detection of hepatocellular carcinoma among high-risk hepatitis C virus Egyptian patients. J Cancer 2012;3:19-31.
18Fassan M, Baffa R, Palazzo JP, Lloyd J, Crosariol M, Liu CG, et al. MicroRNA expression profiling of male breast cancer. Breast Cancer Res 2009;11:R58.
19Fassan M, Volinia S, Palatini J, Pizzi M, Baffa R, De Bernard M, et al. MicroRNA expression profiling in human Barrett's carcinogenesis. Int J Cancer 2011;129:1661-70.
20Fu A, Hoffman AE, Liu R, Jacobs DI, Zheng T, Zhu Y. Targetome profiling and functional genetics implicate miR-618 in lymphomagenesis. Epigenetics 2014;9:730-7.
21Cheng Q, Zhang X, Xu X, Lu X. MiR-618 inhibits anaplastic thyroid cancer by repressing XIAP in one ATC cell line. Ann Endocrinol (Paris) 2014;75:187-93.
22Xu W, Yang Z, Lu N. A new role for the PI3K/Akt signaling pathway in the epithelial-mesenchymal transition. Cell Adh Migr 2015;9:317-24.
23Wei L, Zhang B, Cao W, Xing H, Yu X, Zhu D, et al. Inhibition of CXCL12/CXCR4 suppresses pulmonary arterial smooth muscle cell proliferation and cell cycle progression via PI3K/Akt pathway under hypoxia. J Recept Signal Transduct Res 2015;35:329-39.
24Song R, Tian K, Wang W, Wang L. P53 suppresses cell proliferation, metastasis, and angiogenesis of osteosarcoma through inhibition of the PI3K/AKT/mTOR pathway. Int J Surg 2015;20:80-7.