RNAi-mediated downregulation of CDKL1 inhibits growth and colony-formation ability, promotes apoptosis of human melanoma cells

https://doi.org/10.1016/j.jdermsci.2015.03.020Get rights and content

Highlights

  • Human melanoma cell lines A375 and MV3 expressed CDKL1 mRNA.

  • RNAi-mediated CDKL1 knockdown inhibited cell growth and colony formation.

  • CDKL1 knockdown also promoted cell apoptosis, and arrested cells in G1 phase by upregulation of p21 and downregulation of CDK2.

  • CDKL1 may be a valuable target for anti-melanoma therapeutic strategies.

Abstract

Background

Cyclin-dependent kinase-like 1 (CDKL1) is a member of cell division control protein 2 (CDC-2)-related serine threonine protein kinase family, and is reported to be overexpressed in malignant tumors such as breast cancer and gastric cancer.

Objective

This study aimed to evaluate the whether CDKL1 can serve as a potential molecular target for melanoma gene therapy.

Methods

CDKL1 expression in two melanoma cell lines, A375 and MV3 was measured by real-time PCR. To investigate the role of CDKL1 in cell growth of melanoma, we constructed CDKL1-siRNA expressing lentivirus and infected A375 and MV3 cells. The effects of RNAi-mediated CDKL1 downregulation on A375 and MV3 cell proliferation and colony-formation ability were detected by methylthiazoletetrazolium (MTT) assay and colony-formation assay. The effects of CDKL1 downregulation on A375 and MV3 cell cycle and apoptosis were analyzed by FACS analysis.

Results

Human melanoma cell lines A375 and MV3 expressed CDKL1 mRNA. Knockdown of CDKL1 in A375 and MV3 by CDKL1-siRNA lentivirus infection significantly inhibited cell growth and colony formation ability, promoted cell apoptosis, and arrested cells in G1 phase.

Conclusion

CDKL1 is associated with melanoma cell growth, colony formation, apoptosis, and cell cycle regulation. It may be considered as a valuable target for anti-melanoma therapeutic strategies.

Introduction

Malignant melanoma is a highly aggressive skin tumor with increasing incidence and poor prognosis [1]. Many attempts have been made to treat the disease. BRAF V600E inhibitors (e.g. vemurafenib, dabrafenib), the first target therapy, have improved both progression-free survival and overall survival, compared with chemotherapy in patients with BRAF V600E-mutated metastatic melanoma [2], [3], [4], [5], [6], [7]. However, clinical evidence of tumor resistance developed 5–7 months later in a subset of patients [8], [9], due to MAPK reactivation, driven by secondary mutations in NRAS and MEK1 genes [10].

Malignant tumors are characterized by inappropriate cell proliferation, which arises when checkpoint mechanisms that limit proliferation of cells with damaged DNA are degraded, permitting expansion of clones with genetic instability [11]. Disturbed regulation of the cell cycle is a hallmark of cancer [12]. Melanomagenesis is associated with not only defects in nucleotide excision repair of solar radiation-induced DNA damage, but also cell cycle checkpoints that arrest growth after DNA damage [13]. Defective checkpoints may represent potentially selective anti-melanoma therapeutic targets.

The cyclin-dependent protein kinase (CDK) protein family has been demonstrated to be an important regulator of cell division at the G1/S and G2/M checkpoints [14]. The CDK family regulates a wide range of cellular functions such as cell cycle progression, differentiation, and apoptosis [15]. CDKs form active complexes with a specific cyclin, thus controlling the expression of downstream genes involved in the cell cycle [14]. Cyclin-dependent kinase 1 (CDK1), also known as cell division control protein 2 (CDC2), is a key molecule among them. Cyclin-dependent kinase-like 1 (CDKL1) is a member of CDK1-related serine–threonine protein kinase family [16]. It was cloned on the basis of its similarity to the CDK1 kinase domain [17], and named after the amino acid sequence corresponding to the PSTAIRE motif of CDK1/CDC2 [18], [19]. CDKL kinase family is not known to interact with cyclins, but considered as a separate branch of CDK family that is similar to the MAP kinases group of signal transducing enzymes [23]. CDKL1 contains the conserved MAP kinase dual phosphorylation motif Thr-Xaa-Tyr (Thr-Asp-Tyr), and may therefore contribute to signal transduction [24]. Recent studies reported that CDKL1 was overexpressed in human breast cancer and gastric cancer, and loss of CDKL1 function in these cancer cell lines resulted in inhibition of cell proliferation and increase of apoptosis [20], [21]. However, up to now, the expression of CDKL1 in malignant melanoma and its function in melanoma cell regulation are not known.

In this study, we investigated the expression of CDKL1 in human melanoma cell lines A375 and MV3. Furthermore, we constructed CDKL1-siRNA expressing lentivirus, and evaluated the effects of RNAi-mediated CDKL1 downregulation on A375 and MV3 cell proliferation, colony-formation ability, cell cycle and apoptosis.

Section snippets

Cell culture

Human melanoma cell lines, A375 and MV3 were obtained from Shanghai institutes for biological science (SIBS) cell bank. A375 cells were maintained in DMEM and MV3 cells were maintained in RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) plus 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) in a 5% CO2 incubator at 37 °C.

Lentiviral CDKL-siRNA vector construction and packaging

Small interfering RNA (siRNA) target sequence (CTACTGTGATACCAAGAAA) for CDKL1 gene (NM_004196) was designed and a non-silencing siRNA sequence (TTCTCCGAACGTGTCACGT) was

Knockdown efficiency of CDKL1 by siRNA lentivirus system in human melanoma cells

To investigate the role of CDKL1in melanoma, siRNA targeting CDKL1 or non-silencing sequences were cloned into pGCSIL-GFP plasmid vector, respectively. Then, CDKL1 siRNA lentivirus or non-silencing siRNA lentivirus (negative control) expressing GFP were generated and infected into two human melanoma cell lines, A375 and MV3 cells. As shown in Fig. 1a, three days after infection, more than 80% of GFP expressing cells were observed under the fluorescence microscope, suggesting an infection

Discussion

Our study showed that CDKL1 is expressed in melanoma cell lines A375 and MV3. We then employed lentivirus-mediated knockdown to specifically inhibit CDKL1 gene expression and demonstrated that loss of CDKL1 function significantly inhibited cell proliferation and colony formation, and promoted apoptosis in A375 and MV3 cells. These results suggested that CDKL1 might be associated with tumor progression in malignant melanoma. It may become a probable target of anti-melanoma therapy. Besides, our

Acknowledgements

This work was funded by National Natural Science Foundation of China (No. 81102070).

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