MicroRNA-135b Regulates Leucine Zipper Tumor Suppressor 1 in Cutaneous Squamous Cell Carcinoma

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin malignancy and it presents a therapeutic challenge in organ transplant recipient patients. Despite the need, there are only a few targeted drug treatment options. Recent studies have revealed a pivotal role played by microRNAs (miRNAs) in multiple cancers, but only a few studies tested their function in cSCC. Here, we analyzed differential expression of 88 cancer related miRNAs in 43 study participants with cSCC; 32 immunocompetent, 11 OTR patients, and 15 non-lesional skin samples by microarray analysis. Of the examined miRNAs, miR-135b was the most upregulated (13.3-fold, 21.5-fold; p=0.0001) in both patient groups. Similarly, the miR-135b expression was also upregulated in three cSCC cell lines when evaluated by quantitative real-time PCR. In functional studies, inhibition of miR-135b by specific anti-miR oligonucleotides resulted in upregulation of its target gene LZTS1 mRNA and protein levels and led to decreased cell motility and invasion of both primary and metastatic cSCC cell lines. In contrast, miR-135b overexpression by synthetic miR-135b mimic induced further down-regulation of LZTS1 mRNA in vitro and increased cancer cell motility and invasiveness. Immunohistochemical evaluation of 67 cSCC tumor tissues demonstrated that miR-135b expression inversely correlated with LZTS1 staining intensity and the tumor grade. These results indicate that miR-135b functions as an oncogene in cSCC and provide new understanding into its pathological role in cSCC progression and invasiveness

miR-135b overexpression increases cell migration and invasion in MET1, PM1 and MET4 cSCC cell lines.

<p>A, B) miR-135b mimic significantly increases cSCC cell migration in the wound scratch assay within 24 hours post-transfection. C, D) miR-135b mimic significantly increases cSCC cell invasion in the Matrigel transwell invasion assay in PM1 cell line. * indicate p values less than 0.01. A) <i>Bar = 100um</i>; B) <i>Bar = 200um</i>.</p

Immunohistochemical analysis of LZTS1 protein levels in cells transfected with miR-135b mimic.

<p>A) All cSCC cell lines transfected with miR-135b mimic showed decreased LZTS1staining intensity 48 hours post-transfection when compared to negative control. B) Mean fluorescent intensity per cell was quantified using NIH image J program in cell transfected with miR-135b mimic and negative control oligonucleotiides. * indicates p values less than 0.001. <i>Bar = 1000um</i>.</p

Statistical analysis of differentially expressed miRNAs in cSCC tissues from organ transplant recipients.

<p>Statistical analysis of differentially expressed miRNAs in cSCC tissues from organ transplant recipients.</p

Immunohistochemical analysis of LZTS1 protein levels in normal human skin and cSCC tissues.

<p>A, C) LZTS1 staining is localized in the normal epidermis, but is largely absent in the tumor tissue. Arrows point towards the tumor nests. The absence of staining with control rabbit IgG antibody confirms the specificity of LZTS1 antibody. B, D) Evaluation of LZTS1 staining intensity shows significant reduction of LZTS1 staining in the tumor tissue when compared to normal non-lesional epidermis. * indicate p values less than 0.001. <i>Bar = 100um</i>.</p

Statistical analysis of differentially expressed miRNAs in cSCC tissues from immunocompetent patients.

<p>Statistical analysis of differentially expressed miRNAs in cSCC tissues from immunocompetent patients.</p

Differentially expressed miRNAs in human cSCC tissue.

<p>A) PCR array analysis of miRNA expression in cSCC tumors from immunocompetent patients (n = 32). B) PCR array analysis miRNA expression in cSCC tumors from organ transplant recipients (n = 11).</p

Functional assays of miR-135b inhibition and overexpression.

<p>A) Baseline mRNA miR-135b expression in 3 different OTR-derived cSCC cell lines was established by qRT-PCR. B) Effect of miR-135b inhibition on LZTS1 mRNA expression in cSCC lines by qRT-PCR within 48 hours post-transfection. C) Effect of miR-135b overexpression on LZTS1 mRNA expression in cSCC lines by qRT-PCR within 48 hours post-transfection. * indicate p values less than 0.005.</p