BackgroundWith the increasing discovery of long noncoding RNAs (lncRNAs), the application of functional techniques that could have very specific, efficient, and robust effects and readouts is necessary. Here, we have applied and analyzed three gene knockout (KO) strategies to ablate the CCAT1 gene in different colorectal adenocarcinoma cell lines. We refer to these strategies as “CRISPR excision”, “CRISPR HDR”, and “CRISPR du-HITI”.ResultsIn order to obstruct the transcription of lncRNA or to alter its structure, in these strategies either a significant segment of the gene is removed, or a transcription termination signal is inserted in the target gene. We use RT-qPCR, RNA-seq, MTT, and colony formation assay to confirm the functional effects of CCAT1 gene ablation in knockout colorectal adenocarcinoma cell lines. We applied three different CRISPR/Cas9 mediated knockout strategies to abolish the transcription of CCAT1 lncRNA. CCAT1 knockout cells displayed dysregulation of genes involved in several biological processes, and a significant reduction for anchorage-independent growth. The du-HITI strategy introduced in this study removes a gene segment and inserts a reporter and a transcription termination signal in each of the two target alleles. The preparation of donor vector for this strategy is much easier than that in “CRISPR HDR”, and the selection of cells in this strategy is also much more practical than that in “CRISPR excision”. In addition, use of this technique in the first attempt of transfection, generates single cell knockouts for both alleles.ConclusionsThe strategies applied and introduced in this study can be used for the generation of CCAT1 knockout cell lines and in principle can be applied to the deletion of other lncRNAs for the study of their function.Electronic supplementary materialThe online version of this article (10.1186/s12575-018-0086-5) contains supplementary material, which is available to authorized users.

BackgroundLong noncoding RNAs (lncRNAs) are involved in different pathogenesis pathways including cancer pathogenesis. The adenoma-carcinoma pathway in colorectal cancer may involve the aberrant and variable gene expression of regulatory RNAs. This study was conducted to analyse the expression and prognosis prediction ability of two natural antisense transcripts, protein kinase C theta antisense RNA 1 (PRKCQ-AS1), and special AT-rich sequence binding protein 1 antisense RNA 1 (SATB1-AS1) in colorectal low-grade adenoma, advanced adenoma, and adenocarcinomas.MethodsIn this study, from two RNA-seq analyses of CCAT1-ko cells and colorectal carcinoma biopsies having diminished and increased levels of CCAT1 transcription, respectively, we nominated two antisense lncRNAs of PRKCQ-AS1 and SATB1-AS1. Samples from colorectal low-grade adenomas, advanced adenomas, adenocarcinomas, and adjacent tissue were subjected to RT-qPCR to determine the expression of PRKCQ-AS1, SATB1-AS1 along with colon cancer-associated transcript 1 (CCAT1) and cMYC. In addition, we used different bioinformatics analyses and webservers (including GEPIA 2, TCGA, and CancerMine) to elucidate the prognosis prediction value, the expression correlation of sense–antisense pair of genes, and the expression profile of these antisense transcripts at the presence or absence of mutations in the driver genes, or the corresponding sense genes.ResultsPRKCQ-AS1 showed a wide range of expression levels in colorectal adenoma, advanced adenoma, and adenocarcinoma. Upregulation of PRKCQ-AS1 was related to a significant decrease in survival of colorectal cancer (CRC) patients. The expression levels of PRKCQ-AS1 and PRKCQ were strong and significantly concordant in normal and cancerous colorectal tissues. While SATB1-AS1 showed a wide range of expression in colorectal adenoma, advanced adenoma, and adenocarcinoma as well, its expression was not related to a decrease in survival of CRC patients. The expression levels of SATB1-AS1 and SATB1 (the sense gene) were not strong in normal colorectal tissues. In addition, where SATB1 gene was mutated, the expression of SATB1-AS1 was significantly downregulated.ConclusionsWe found the expression of PRKCQ-AS1 and SATB1-AS1 at a given stage of CRC very variable, and not all biopsy samples showed the increased expression of these antisense transcripts. PRKCQ-AS1 in contrast to SATB1-AS1 showed a significant prognostic value. Since a significantly concordant expression was observed for SATB1-AS1 and SATB1 in only cancerous, and for PRKCQ-AS1 and PRKCQ in both normal and cancerous colorectal tissues, it can be concluded that common mechanisms may regulate the expression of these sense and antisense genes.

Background: The methylation of the CpG islands of the LINE-1 promoter is a tight control mechanism on the function of mobile elements. However, simultaneous quantification of promoter methylation and transcription of LINE-1 has not been performed in progressive stages of colorectal cancer. In addition, the insertion of mobile elements in the genome of advanced adenoma stage, a precancerous stage before colorectal carcinoma has not been emphasized. In this study, we quantify promoter methylation and transcripts of LINE-1 in three stages of colorectal non-advanced adenoma, advanced adenoma, and adenocarcinoma. In addition, we analyze the insertion of LINE-1, Alu, and SVA elements in the genome of patient tumors with colorectal advanced adenomas. Methods: LINE-1 hypomethylation status was evaluated by absolute quantitative analysis of methylated alleles (AQAMA) assay. To quantify the level of transcripts for LINE-1, quantitative RT-PCR was performed. To find mobile element insertions, the advanced adenoma tissue samples were subjected to whole genome sequencing and MELT analysis. Results: We found that the LINE-1 promoter methylation in advanced adenoma and adenocarcinoma was significantly lower than that in non-advanced adenomas. Accordingly, the copy number of LINE-1 transcripts in advanced adenoma was significantly higher than that in non-advanced adenomas, and in adenocarcinomas was significantly higher than that in the advanced adenomas. Whole-genome sequencing analysis of colorectal advanced adenomas revealed that at this stage polymorphic insertions of LINE-1, Alu, and SVA comprise approximately 16%, 51%, and 74% of total insertions, respectively. Conclusions: Our correlative analysis showing a decreased methylation of LINE-1 promoter accompanied by the higher level of LINE-1 transcription, and polymorphic genomic insertions in advanced adenoma, suggests that the early and advanced polyp stages may host very important pathogenic processes concluding to cancer.

This study was designed to evaluate the in vitro chondrogenic differentiation potential of equine adipose tissue-derived mesenchymal stem cells (AT-MSCs) in response to different culture conditions. Fat tissue cells after collection were cultured in optimized conditions until passage 3 (P3). P3 cells were cultured as micropellets under different chondrogenic conditions for 21 days in 5 groups: basic medium as a control, basic chondrogenic medium (BCM), BCM supplemented with transforming growth factor beta 3 (TGF-β 3 ; 10 ng/mL), BCM supplemented with bone morphogenetic protein 6 (BMP-6; 10 ng/mL), and BCM supplemented with both TGF-β 3 and BMP-6. The growth rate of pellets was measured and differentiation progression was assessed by specific stainings and gene expression profiling. Results showed that the largest pellets of cells belonged to the group containing both growth factors. All treated cells showed different levels of chondrogenic differentiation compared to the control group (P < 0.05). The differentiation score in groups containing TGF-β 3 and both growth factors was significantly greater than that in other groups. Moreover, the expression of aggrecan, as a cartilage-specific gene, was confirmed in all groups expect the control group. Taken together, these results indicate that the addition of both TGF-β 3 and BMP-6 to BCM potentiates the chondrogenic differentiation of equine AT-MSCs.

Background: LINE-1, Alu, and SVA elements are non-LTR retrotransposons that create approximately one-third of the human genome. The loss of tight control mechanisms on the function of mobile elements has been implicated in many human diseases. The methylation of the CpG islands of the LINE-1 promoter is one of these mechanisms. In this study, we determined the promoter methylation and the expression of LINE-1 in three stages of colorectal non-advanced adenoma, advanced adenoma, and adenocarcinoma. In addition, we analyzed the insertion of LINE-1, Alu, and SVA elements in the genome of colorectal advanced adenomas.Results: We found that the LINE-1 hypomethylation index in advanced adenoma and adenocarcinoma were significantly higher than that in non-advanced adenomas. The copy number of LINE-1 transcripts in advanced adenoma was significantly higher than that in non-advanced adenomas, and in adenocarcinomas was significantly higher than that in the advanced adenomas. Analysis of the genome of colorectal advanced adenomas revealed that at this stage de novo insertions of LINE-1, Alu, and SVA were approximately 16%, 51%, and 74%, respectively.Conclusions: Our findings showing a decreased methylation of LINE-1 promoter accompanied by the higher level of LINE-1 transcription, and de novo genomic insertions in advanced (high-grade) adenoma, a precancerous stage before colorectal carcinoma, suggests that the early and advanced polyp stages may host very important pathogenic processes concluding to cancer.