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

Olasz EB, Seline LN, Schock AM, Duncan NE, Lopez A, Lazar J, Flister MJ, Lu Y, Liu P, Sokumbi O, Harwood CA, Proby CM, Neuburg M, Lazarova Z - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Immunohistochemical analysis of LZTS1 protein levels in cells transfected with miR-135b mimic.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. Bar = 1000um.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4418692&req=5

pone.0125412.g005: Immunohistochemical analysis of LZTS1 protein levels in cells transfected with miR-135b mimic.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. Bar = 1000um.

Mentions: To directly test whether miR-135b regulates LZTS1 expression in cSCC in vitro, the PM1, MET1, and MET4 cell lines were transfected with synthetic mirVana miR-135b inhibitor, mirVana miR-135b mimic, or mirVana negative control and tested in functional assays. At 48 hours post-transfection LZTS1 expression was assessed by qRT-PCR, and quantitative immunofluorescent staining of the cSCC cell lines. Compared with the negative control, the miR-135b inhibitor significantly increased LZTS1 mRNA expression in PM1 (2.1 ± 0.7-fold; p < 0.05), MET1 (4.0 ± 1.2-fold; p< 0.05), and to a lesser extent in MET4 (1.5 ± 0.5-fold) (Fig 3B). Similar to changes in LZTS1 mRNA transcript expression, LZTS1 staining intensity was significantly increased in PM1 (3.34 ± 2.0-fold; p<0.001) and MET1 (1.7 ± 0.7-fold; p<0.001) cells treated with the miR-135b inhibitor, whereas LZTS1 staining intensity in MET4 did not change (Fig 4A and 4B). In contrast, all cSCC cell lines transfected with miR-135b mimics (to increase endogenous miR-135b expression) demonstrated further downregulation of LZTS1 (PM1–1.00 ± 0.11-fold; MET1–1.60 ± 0.15-fold; MET4–1.77 ± 0.07-fold) within 24 hours post-transfection when compared to the negative control as shown in Fig 5A and 5B. Collectively, these data demonstrate that LZTS1 expression is regulated by miR-135b in cSCC cells in vitro.


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

Olasz EB, Seline LN, Schock AM, Duncan NE, Lopez A, Lazar J, Flister MJ, Lu Y, Liu P, Sokumbi O, Harwood CA, Proby CM, Neuburg M, Lazarova Z - PLoS ONE (2015)

Immunohistochemical analysis of LZTS1 protein levels in cells transfected with miR-135b mimic.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. Bar = 1000um.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418692&req=5

pone.0125412.g005: Immunohistochemical analysis of LZTS1 protein levels in cells transfected with miR-135b mimic.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. Bar = 1000um.
Mentions: To directly test whether miR-135b regulates LZTS1 expression in cSCC in vitro, the PM1, MET1, and MET4 cell lines were transfected with synthetic mirVana miR-135b inhibitor, mirVana miR-135b mimic, or mirVana negative control and tested in functional assays. At 48 hours post-transfection LZTS1 expression was assessed by qRT-PCR, and quantitative immunofluorescent staining of the cSCC cell lines. Compared with the negative control, the miR-135b inhibitor significantly increased LZTS1 mRNA expression in PM1 (2.1 ± 0.7-fold; p < 0.05), MET1 (4.0 ± 1.2-fold; p< 0.05), and to a lesser extent in MET4 (1.5 ± 0.5-fold) (Fig 3B). Similar to changes in LZTS1 mRNA transcript expression, LZTS1 staining intensity was significantly increased in PM1 (3.34 ± 2.0-fold; p<0.001) and MET1 (1.7 ± 0.7-fold; p<0.001) cells treated with the miR-135b inhibitor, whereas LZTS1 staining intensity in MET4 did not change (Fig 4A and 4B). In contrast, all cSCC cell lines transfected with miR-135b mimics (to increase endogenous miR-135b expression) demonstrated further downregulation of LZTS1 (PM1–1.00 ± 0.11-fold; MET1–1.60 ± 0.15-fold; MET4–1.77 ± 0.07-fold) within 24 hours post-transfection when compared to the negative control as shown in Fig 5A and 5B. Collectively, these data demonstrate that LZTS1 expression is regulated by miR-135b in cSCC cells in vitro.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus