MALAT1-KTN1-EGFR regulatory axis promotes the development of cutaneous squamous cell carcinoma

Long noncoding RNAs (LncRNAs), including MALAT1, are critical regulators of tumor development. However, the roles and molecular mechanisms of LncRNAs in cutaneous squamous cell carcinoma (cSCC) remain underexplored. In this study, functional studies using in vitro cellular and in vivo xenograft models confirmed the pro-carcinogenic roles of MALAT1 in cSCC. Further, MALAT1 was identified to regulate epidermal growth factor receptor (EGFR) protein expression but did not affect EGFR mRNA expression. Transcriptomic sequencing identified kinectin 1 (KTN1) as the key mediator for MALAT1 regulation of EGFR. Mechanistic study revealed that MALAT1 interacts with c-MYC to form a complex and directly binds to the promoter region of KTN1 gene and enhances its transactivation to positively regulate EGFR protein expression. Our findings, therefore, establish a novel c-MYC-assisted MALAT1-KTN1-EGFR axis, which contributes to cSCC development and may serve as novel target for therapeutic intervention.

of the patients were >50 years of age. Fresh samples obtained during surgery were immediately frozen in liquid nitrogen for subsequent total RNA extraction, protein extraction, and paraffin embedding. Tumors were classified according to the SCC Broders Pathological Classification [1]: grade I (well differentiated) with 75-100% differentiated cells, grade II (moderately differentiated) with 50-75% differentiated cells, and grades III and IV (poorly differentiated) with 0-50% differentiated cells. Of the 113 cases, 61 were well differentiated (54.0%), 36 were moderately differentiated (31.9%), and 16 were poorly differentiated (14.2%).
DNA constructs. The full-length MALAT1 sequence was analysed using the Vienna RNA secondary structure server [2] and separated into 5 fragments based on the mountain plot of whereas enhanced green fluorescent protein (EGFP) was cloned into the XbaI site of pcDNA3.1(+) for in vitro transcription as described in a previous study [3].
In vitro transcription. For generating sense transcripts of the deletion mutant fragments of MALAT1, in vitro transcription was performed using a MAXIscript T7/T3 kit (Ambion) after linearisation of the plasmids.
All the generated RNA-seq datasets can be found in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (Accession codes: GSE100399, GSM2680311 and GSM2680312).
Biotinylated MALAT1 fragments and LacZ RNAs were synthesized using a MAXIscript T7/T3 in vitro transcription kit (Ambion) and Biotin RNA labelling Mix (Roche).
ChIP-qPCR analysis. ChIP experiments were performed as recommended by the EZ ChIP TM Chromatin Immunoprecipitation kit manual (Cat. no. 17-371, Merck Millipore) using 5 μg antibodies against c-MYC (CST) or isotype IgG (Merck Millipore) used as a negative control.
Relative enrich of c-MYC at KTN1 locus was quantified by qRT-PCR as previously described [3,5]. Primers used are listed in Supporting Information Table 4.
Electrophoretic mobility shift assay (EMSA). EMSA experiments were carried out using forward strand 5Cy5-labelled-dsDNA (Life Technologies) as previously described [3]. 100 nM dsDNA probe containing the E-box motif at KTN1 promoter was tested for binding with purified c-MYC protein (Cat. no. ab169901, Abcam). Supershift assay was performed by incubating c-MYC antibody with the above E-box motif-containing probe and c-MYC protein and detecting the formation of supershift bind. The bands were detected using the ODYSSEY infrared imaging system (LI-COR Biosciences).
Chromatin isolation by RNA purification (ChIRP) assay. Anti-sense DNA tiling probes for the selective retrieval of MALAT1 were designed using an online probe designer at http://singlemoleculefish.com/ and synthesised by Life Technologies Inc. with BiotinTEG at the 3-prime end. The ChIRP assay was performed according to the kit instructions of Magna ChIRP™ RNA Interactome Kits (Merck Millipore) and also with reference to previous studies [3,6]. Specifically, retrieved MALAT1 RNA and DNA regions expected to bind MALAT1 were quantified by qRT-PCR as previously described [3,6].
Xenograft mouse model. Briefly, 1 × 10 7 cells were subcutaneously implanted into the left and right flanks of female athymic nude mice (4-5 weeks old). At 8 days after implantation, NC ASO or MALAT1 ASO was injected into the left or right tumor, respectively; and the injection was repeated every other day. Oligos were prepared by pre-incubating 3 nM ASO per mouse with Lipofectamine 2000 (Life Technologies) for 15 min; injections were made using a final volume of 50 μl in serum free DMEM (Life Technologies). The tumor diameters were measured and recorded every day to generate a tumor growth curve. After tumor growth assessment, the tumors were excised and snap-frozen for RNA and protein extraction or paraffin-embedded for IHC staining.
Immunoblotting and immunohistochemistry assays. Total cell extracts were prepared and assayed by western blot as previously described [3,7]. The following primary antibodies and   Supplementary Figure