MicroRNA181a Is Overexpressed in T-Cell Leukemia/Lymphoma and Related to Chemoresistance

MicroRNAs (miRs) play an important role in tumorogenesis and chemoresistance in lymphoid malignancies. Comparing with reactive hyperplasia, miR181a was overexpressed in 130 patients with T-cell leukemia/lymphoma, including acute T-cell lymphoblastic leukemia (n = 32), T-cell lymphoblastic lymphoma (n = 16), peripheral T-cell lymphoma, not otherwise specified (n = 45), anaplastic large cell lymphoma (n = 15), and angioimmunoblastic T-cell lymphoma (n = 22). Irrespective to histological subtypes, miR181a overexpression was associated with increased AKT phosphorylation. In vitro, ectopic expression of miR181a in HEK-293T cells significantly enhanced cell proliferation, activated AKT, and conferred cell resistance to doxorubicin. Meanwhile, miR181a expression was upregulated in Jurkat cells, along with AKT activation, during exposure to chemotherapeutic agents regularly applied to T-cell leukemia/lymphoma treatment, such as doxorubicin, cyclophosphamide, cytarabine, and cisplatin. Isogenic doxorubicin-resistant Jurkat and H9 cells were subsequently developed, which also presented with miR181a overexpression and cross-resistance to cyclophosphamide and cisplatin. Meanwhile, specific inhibition of miR181a enhanced Jurkat and H9 cell sensitivity to chemotherapeutic agents, further indicating that miR181a was involved in acquired chemoresistance. Collectively, miR181a functioned as a biomarker of T-cell leukemia/lymphoma through modulation of AKT pathway. Related to tumor cell chemoresistance, miR181a could be a potential therapeutic target in treating T-cell malignancies.


Introduction
Malignancies derived from the T-cell lineages encompass a heterogeneous group of neoplasm. The World Health Organization (WHO) classification recognizes distinctive subtypes of immature T-cell malignancies, like acute T-cell lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL), as well as mature T-cell malignancies, mainly including peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma (ALCL), and angioimmunoblastic T-cell lymphoma (AITL) [1]. Varied from clinicopathological features and biological behavior, they are generally more aggressive than their B-cell counterpart, characterized by resistance to conventional chemotherapy and poor prognosis of the patients [2]. Therefore, biomarkers related to tumor progression and chemoresistance remain to be investigated and may become potential targets for future therapy in T-cell leukemia/lymphoma. MicroRNAs (miRs), a class of 19-to 23-nucleotide noncoding RNA molecules, regulate gene expression by targeting mRNA at the 3 untranslated region (UTR) [3]. Growing evidences suggested that miRs are critical regulators in tumorigenesis and drug resistance [4,5]. MiR181 is essential for lymphocyte differentiation and maturation in thymus [6].

BioMed Research International
More recently, it has been reported that miR181 overexpression promotes cell proliferation and activates PI3K/AKT signaling transduction pathway [7,8]. Activated in lymphoid malignancies [9], AKT plays a pivotal role in tumor progression and resistance to chemotherapeutic agents [10,11]. Here we assessed miR181a expression, as well as its relation to AKT activation and chemoresistance in T-cell leukemia/lymphoma.

Patients.
One hundred and thirty patients diagnosed with T-ALL or T-cell lymphoma were enrolled in this study, including 32 T-ALL, 16 T-LBL, 45 PTCL-NOS, 15 ALCL, and 22 AITL. Histologic diagnoses were established according to the WHO classification [1]. PTCL cases (PTCL-NOS, ALCL, and AITL) were treated with CHOP-based chemotherapy. T-LBL and T-ALL cases were treated with HyperCVAD-A/B regimens as previously reported [12,13]. Response rates were assessed according to the criteria as reported [12,13]. The clinicopathological data of the patients was listed in Table 1. Thirty-four age-and sex-matched cases with reactive hyperplasia were referred to as controls. The study was approved by the Institutional Review Board with informed consent obtained in accordance with the Declaration of Helsinki.

Cell Lines and Reagents
. T-leukemia/lymphoma cell lines Jurkat, H9, and embryonic kidney cell line HEK-293T were available from American Type Culture Collection (Manassas, VA, USA). Doxorubicin-resistant Jurkat and H9 cells were established by exposure to gradually increasing concentrations of doxorubicin in vitro, as described by Huang et al. [14].

Cell Proliferation Assay.
Cell proliferation was measured by MTT and EdU incorporation assay. Cells were seeded in 96-well plates and incubated with the indicated concentrations of reagents at 37 ∘ C. After 72 h incubation, 0.1 mg of MTT was added to each well and the absorbance was measured at 490 nm by spectrophotometry. EdU assay was conducted using Cell-Light EdU imaging kit (RiboBio, Guangzhou, China) according to the manufacturer's instruction.

MiR181a Detection.
Total RNA was extracted from 20 m thick paraffin ( = 100) or frozen sections ( = 64) using RecoverAll total nucleic acid isolation kit or Trizol agent following the manufacturer's protocol. MiR181a expression was analyzed by real-time quantitative RT-PCR using miRNA reverse transcription kit, hsa-miR 181a assay, and 7500HT Fast Real-time PCR system (Applied Biosystems, CA, USA). RNU24 was used as endogenous control and Jurkat cells for calibration. A relative quantification was calculated using the ΔΔ CT method [15].

Statistical Analysis.
Differences of miR181a expression among groups were assessed using Mann-Whitney test. The association between miR181a and p-AKT expression in human tumor samples was analyzed by Fisher's exact test. In vitro experimental results were expressed as mean ± S.D. of data obtained from three separate experiments and determined using -test to compare variance. < 0.05 was considered statistically significant.

MiR181a Was Overexpressed in T-Cell Leukemia/Lymphoma and Related to AKT Activation.
Compared with reactive hyperplasia, miR181a was overexpressed in T-cell leukemia/ lymphoma ( < 0.0001, Figure 1(a)). No significant difference was observed among T-ALL and subtypes of T-cell lymphoma ( = 0.5153, Figure 1(b)).
The median value of relative miR181a expression in Tcell leukemia/lymphoma was 2136. The patients with miR181a expression level over and equal to the median value were regarded as high miR181a expression, whereas those below the median value were included in the low miR181a expression. Patients with high miR181a expression had significantly lower overall response rate (ORR) than those with low miR181a expression (Table 1). P-AKT expression was detected by immunohistochemistry in primary tumor sections of 12 T-cell lymphoma patients (6 cases from high miR181a expression group and 6 cases from low miR181a expression group, Figure 2(a)). High miR181a expression was associated with increased positivity of p-AKT ( = 0.0152, Figure 2(b)).

MiR181a Overexpression Corresponded to Chemoresis
Acquired drug resistance is an important obstacle that impairs the success of cancer treatment. Isogenic doxorubicin-resistant sublines were developed as previously reported [14,17] Figure 4(f)), consistent with AKT activation (Figures 4(g) and 4(h)). Increased miR181a expression was linked to AKT phosphorylation, not only in PTEN-negative Jurkat cells [18] but also in PTEN-positive H9 cells (Figures  4(g) and 4(h)). Therefore, regulation of AKT phosphorylation could be independent of PTEN expression in T-cell leukemia/lymphoma. Further drug-sensitivity test showed that these resistant sublines with miR181a overexpression also had cross-resistance to other chemotherapeutic agents (Table 2). Therefore, exposure to chemotherapeutic agents could induce miR181a expression and AKT activation, which is closely related to acquired chemoresistance.

MiR181a Inhibition Enhanced T-Leukemia/Lymphoma
Cell Sensitivity to Chemotherapeutic Agents. Specific inhibition of miR181a in Jurkat and H9 cells as well as their resistant sublines, using an antagomir, significantly increased cell sensitivity to doxorubicin (Figures 5(a) and 5(b)) and decreased AKT phosphorylation (Figures 5(c) and 5(d)). Similar to doxorubicin, decreased miR181a expression was also related to reduced IC50 of cisplatin and cyclophosphamide in doxorubicin-resistant Jurkat and H9 cells (Table 3).

Discussion
In addition to genetic abnormalities, epigenetic aberrations, particularly those of miRs, participate in human carcinogenesis [5,19]. MiR181a is critically involved in hematological malignancies. In acute myelogenous leukemia, increased miR181a expression was significantly associated with a higher complete remission rate of the patients [20]. However, as recently reported in lymphoid malignancies like acute lymphoblastic leukemia and multiple myeloma, miR181 overexpression was related to advanced stage and tumor progression [7,21]. In accordance with malignant T cells, miR181a is upregulated in normal T-cell counterpart and miR181a/bdeficient mice show severe defects in T-cell development [6,8]. Therefore, miR181 may have different roles in hematological malignancies. Our study showed that miR181a, independent of histological subtypes, was overexpressed and these patients were less responding to treatment, referring to miR181a as a common biomarker of chemoresistance in T-cell leukemia/lymphoma. Chemoresistance, determining therapeutic effect and clinical outcome of the patients, is one of the control factors in cancer treatment, including T-cell leukemia/lymphoma [22]. Here miR181a was closely related to chemoresistance   in T-leukemia/lymphoma. This was consistent with previous reports in B-cell lymphoma that high expression of miR181 could lead to decreasing proapoptotic protein Bim and increasing resistance to chemotherapy [23]. AKT is a key tuning point in tumor cell growth and chemosensitivity [24,25]. MiR181 is a central regulator of PI3K pathway, since miR181a/b-deficient mice showed severe defects in lymphoid development and T-cell homeostasis associated with impaired PI3K/AKT cascade [8]. As mechanism of action, miR181 targets PTPN, DUSP5, and DUSP6, resulting in PI3K/AKT activation and tumorigenesis in murine T-cell leukemia [7,8]. Our results showed that ectopic expression of miR181a leads to AKT phosphorylation, enhancing cell proliferation and inducing cell resistance to chemotherapy in T-cell leukemia/lymphoma. This correlation of miR181a overexpression with AKT activation was observed not only in cell lines but also in primary tumor samples of patients with Tleukemia/lymphoma. Apart from primary chemoresistance, acquired chemoresistance is also an important factor of treatment failure. MiR181a expression of Jurkat cells was significantly upregulated after exposure to chemotherapeutic agents and linked to increased AKT phosphorylation. Meanwhile,  isogenic doxorubicin-resistant cell lines were developed, which were resistant to doxorubicin and had cross-resistance to other chemotherapeutic drugs. The relative resistance to chemotherapeutic agents was along with increased miR181a expression and subsequent AKT activation, further confirming that miR181a induced AKT activation and contributed to chemoresistance in T-cell leukemia/lymphoma.

Conclusions
MiR181a was involved in T-cell leukemia/lymphoma through modulation of AKT pathway. Functioned as a critical regulator of chemosensitivity, miR181a could thus be a promising therapeutic target in treating T-cell malignancies resistant to chemotherapy.