Knocking out the vitamin D receptor enhances malignancy and decreases responsiveness to vitamin D3 hydroxyderivatives in human melanoma cells

Active forms of vitamin D3, including $1,25(OH)_{2}D3$, 20(OH)D3 and $1,20(OH)_{2}D3$, inhibited cell proliferation, migration rate and the ability to form colonies and spheroids in the wild-type melanoma cell line, while cells with the vitamin D receptor (VDR) silenced showed an increased but not complete resistance to their action. Furthermore, silencing of the VDR in melanoma cells enhanced their proliferation as well as spheroid and colony formation and increased their migration rate. Previous clinicopathological studies have shown an inverse correlation between VDR expression, melanoma progression and poor outcome of the disease. Thus, the expression of VDR is not only necessary for the inhibition of melanoma growth by active forms of vitamin D, but the VDR can also function as a melanoma tumor suppressor gene. Vitamin D3 is not only involved in calcium and phosphate metabolism in humans, but it can also affect proliferation and differentiation of normal and cancer cells, including melanoma. The mechanism of the anti-cancer action of vitamin D3 is not fully understood. The nuclear vitamin D receptor (VDR) is crucial for the phenotypic effects of vitamin D hydroxyderivatives. VDR expression shows an inverse correlation with melanoma progression and poor outcome of the disease. In this study we knocked out the VDR in a human melanoma cell line using CRISPR methodology. This enhanced the proliferation of melanoma cells grown in monolayer culture, spheroids or colonies and their migration. Activated forms of vitamin D, including classical $1,25(OH)_{2}D3$, 20(OH)D3 and $1,20(OH)_{2}D3$, inhibited cell proliferation, migration rate and the ability to form colonies and spheroids in the wild-type melanoma cell line, while VDR KO cells showed a degree of resistance to their action. These results indicate that expression of VDR is important for the inhibition of melanoma growth induced by activated forms of vitamin D. In conclusion, based on our previous clinicopathological analyses and the current study, we suggest that the VDR can function as a melanoma tumor suppressor gene

The Novel Gene <i>CRNDE</i> Encodes a Nuclear Peptide (CRNDEP) Which Is Overexpressed in Highly Proliferating Tissues

<div><p><i>CRNDE</i>, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the <i>CRNDE</i> gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two <i>CRNDE </i>transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of <i>CRNDE</i>. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our <i>in silico</i> results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the <i>CRNDE</i> gene.</p></div

Molecular studies on CRNDEP.

<p>A) Simultaneous overexpression of the 6xHis-CRNDEP-EGFP and DsRed Monomer-6xHis-CRNDEP fusion proteins in HeLa cells, visualized under a fluorescence microscope. The former protein glows green and the latter glows red in these conditions. Yellow glow is caused by a co-localization of these two fusion proteins. Nuclei were stained blue with DAPI. The same shot with only the green (B) or the red (C) channel shown. D) Western blot-based verification of the size of the 6xHis-CRNDEP-EGFP fusion protein. M—Spectra Multicolor Low Range Protein Ladder (Thermo-Fisher Scientific), 1—the EGFP reporter protein (26.9 kDa), 2—6xHis-CRNDEP-EGFP (39.2 kDa). E) Western blot-based verification of the specificity of our custom-made polyclonal anti-CRNDEP antibody. M—Spectra Multicolor Low Range Protein Ladder; 1—DsRed Monomer-6xHis-CRNDEP (340 aas, 38.5 kDa); 2—purified 14 kDa protein containing the 6xHis tag, 1.4 μg (a negative control of the antibody's specificity, non-commercial); 3—6xHis-CRNDEP-EGFP (346 aas, 39.2 kDa); 4—empty; 5—EGFP (239 aas, 26.9 kDa, a negative control); 6—DsRed Monomer (232 aas, 26.2 kDa, a negative control). A loading control (the PVDF membrane used in this experiment, stained with Ponceau S) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127475#pone.0127475.s013" target="_blank">S13 Fig</a>. F–G) Detection of the overexpressed 2xFLAG-CRNDEP protein (~14 kDa) in a total protein lysate from 0.25 million HeLa cells with either the anti-FLAG (F) or anti-CRNDEP (G) antibody. H) Immunoprecipitation of 2xFLAG-CRNDEP using the anti-CRNDEP antibody (2) and control IgG (1) (both from a rabbit). A total protein lysate before immunoprecipitation was loaded for comparison (3). After precipitation, the 2xFLAG-CRNDEP protein was detected on the PVDF membrane using the anti-FLAG antibody. The correct bands in Fig F–H are encircled.</p

Evaluation of shRNA-mediated knockdown of <i>CRNDE</i>.

<p>The effects of <i>CRNDE</i> gene silencing were evaluated at either mRNA (A) or protein level (B-C). The strongest decrease in the amount of the CRNDEP-coding transcript (by ~65%) was observed for the SH1 silencing construct (A). The effects of this knockdown were detectable at the protein level as well (B, C), leading to a statistically significant decline in the amount of CRNDEP (red signal) in the cells transfected with the silencing construct (green signal). As expected, such a correlation did not occur in the cells transfected with the construct encoding a control (scrambled) shRNA molecule (SH SCR). The transfected cells are marked with white arrows.</p

Primary and secondary antibodies used in the present study.

<p>¹) Western Blot.</p><p>²) Immunofluorescence.</p><p>³) IHC (Immunohistochemistry).</p><p>⁴) Two of three anti-CRNDEP antibodies were not purified (Western blots were performed using rabbit sera with relatively low concentration of specific antibodies). IHC—immunohistochemistry; HRP—horseradish peroxidase; N/A—not applicable.</p><p>Primary and secondary antibodies used in the present study.</p

The endogenous CRNDEP peptide expression in different human tissues evaluated by immunohistochemical stainings.

<p>A) Epithelial ovarian cancer (serous carcinoma) with heterogeneous nuclear expression; B) The same section of ovarian carcinoma incubated with a blocking peptide; C) Normal proliferative phase endometrium with strong nuclear expression within the glandular epithelium and heterogeneous staining in stromal cells; D) Atrophic endometrium with negative staining in the nuclei; Normal tonsil (E-F) with heterogeneous (weak to moderate) nuclear expression in the germinal center (E) and strong nuclear expression in the parabasal layer of the squamous epithelium (F); G) Normal intestine: strong nuclear expression in intestinal crypts; H) Seminiferous tubules of an atrophic human testis: strong nuclear expression in spermatocytes.</p

The most important differences between the cloning experiments performed herein.

<p>¹) PCR products shown in this table were first cloned into the pGEM-T Easy vector and then subcloned into one of two target expression vectors listed above. This approach facilitated the cleavage of DNA inserts with restriction enzymes.</p><p>²) The pCR3-FL2-CRNDEP plasmid was created by subcloning the CRNDEP insert from the pEGFP-N1_CRNDEP plasmid (without PCR reactions); N/A—not applicable.</p><p>The most important differences between the cloning experiments performed herein.</p

Sequences of primers used for PCR, sequencing and Real-Time PCR, followed by sequences of a TaqMan probe, shRNAs, and CRNDEP epitopes used in the present study.

<p>¹) Restriction enzyme recognition sites inserted in primer overhangs were <u>underlined</u>.</p><p>²) shRNA-coding sequences: sense regions were marked with a <u>wavy</u> line, whereas antisense regions were indicated with a <u>double wavy</u> line; sticky ends, specific to BamHI/HindIII digestion, were <u>double-underlined</u>.</p><p>Sequences of primers used for PCR, sequencing and Real-Time PCR, followed by sequences of a TaqMan probe, shRNAs, and CRNDEP epitopes used in the present study.</p