Article introduction
Prostate cancer (PCa), the second most common cancer in males, seriously affects the reproductive health and quality of life of patients. Because prostate cancer is regulated by a variety of genes, and there is currently no in-depth study on related mechanisms, the treatment effect is often not satisfactory. With the development of high-throughput technology, it is possible to study the genes and mechanisms of action of PCa-related molecular markers. At present, it has been reported that many star LncRNAs such as SChLAP1, HOTAIR, PCA3, PCAT1, NEAT1 and CTBP1-AS play an important role in PCa. So the question is, these LncRNAs are mostly located on the autosomes, but PCa can be used as a male-specific disease. Should the main genes regulating its function be located on the Y chromosome?
With this problem, the author of this article uses the LncRNA on the Y chromosome as an entry point to rapidly target a highly differentially expressed LncRNA in PCa by high-throughput screening technology, and confirmed it through a large number of clinical samples. It is indeed highly expressed in PCa patients. Finally, it was confirmed by molecular research that this process was regulated by miRNA-let-7, the target genes CDK6 and FN1, and the transcription factor foxa1. 
Article content
1. LncRNA-TTTY15 is highly expressed in prostate cancer tissues
This time, the research team conducted deep bioacoustic mining on the LncRNA part of the whole transcriptome sequencing (the cloud sequence organism can provide this service) data, and found that LncRNA-TTTY15 located on the Y chromosome is highly expressed in prostate cancer tissues. The results of the experiment were published again in the journal European Urology. Through LncRNA quantitative PCR , the authors in the sequencing sample (65:65), increased clinical samples (35:35) for large sample low-throughput validation, mining sequencing data from other ethnic prostate cancer patients in the TCGA database, and through larger clinical samples Transcriptome sequencing (145: 145) further confirmed the high expression of TTTY15 in prostate cancer tissues. At the same time, in situ hybridization experiments at the tissue level also confirmed its specific expression in cancer tissues.
2. TTTY15 promotes prostate cancer cell proliferation and migration
The authors selected a total of six cell lines, and found that TTTY15 is highly expressed in cancer cells by LncRNA quantitative PCR. Subsequently, the authors selected several highly expressed cell lines, and inhibited and overexpressed the TTTY15 gene, respectively, and found that the proliferation rate and number of cells were significantly affected. However, after overexpressing TTTY15 in an androgen-related cell line, the LncRNA was found to be insensitive to androgen, suggesting that its upstream factor may not be an androgen receptor.
3. CRISPR/Cas9 inhibits the promotion of LncRNA TTTY15 on prostate cancer cells
The authors of this article knocked out LncRNA TTTY15 by two CRISPR/Cas9 strategies, the first knocking out the entire TTTY15 fragment; the second is terminating TTTY15 transcription by knocking in the transcription terminator. Regardless of the method of knockout, both proliferation and migration were inhibited at the cellular level, and complementation experiments showed that inhibition could be restored. At the same time, after the authors transplanted knockdown cells into mice, the mice in the transplant group were smaller than the control group. Overall, the authors have fully demonstrated that TTTY15 promotes prostate cancer from both cell and animal dimensions.
4. LncRNA TTTY15 plays a sponge mechanism by adsorbing miRNA
First, the authors confirmed by cell localization experiments that LncRNA TTTY15 is mainly distributed in the cytoplasm, which indicates that the LncRNA may be related to the sponge mechanism. Next, the possible binding of miRNAs was predicted by a joint analysis of miRDB and miRanda software. The dual luciferase reporter gene experiments showed that there were 5 miRNAs bound to it, and the expression of miRNA let-7 was negatively correlated with LncRNA by miRNA quantitative PCR . Next, the authors verified 22 target genes that have been reported to bind to let-7 in LncRNA knockdown and overexpressing cell lines by mRNA quantitative PCR , and screened two target genes co-expressed with LncRNA. They are CDK6 and FN1. At the RNA, protein level and biosignal GSEA predictions, the expression level of the target gene was confirmed to be inversely proportional to let-7.
5. FOXA1 is the upstream transcription factor of LncRNA TTTY15
By prediction, the authors predicted many hypersensitive and epigenetic molecular marker binding sites on the LncRNA TTTY15 promoter. The TCGA database showed that only the expression levels of FOXA1 and FOXB13 were positively correlated with LncRNA. After knocking down the two transcription factors, the authors found that only FOXA1 inhibited the expression of LncRNA, and FOXA1 was also highly expressed in prostate cancer tissues. In the end, the authors confirmed that the two are directly related by ChIP sequencing (the cloud sequence organism can provide this service) .
to sum up
It is worth mentioning that Yunzo Bio has been very fortunate to participate in the transcriptome data mining of 130 cancer and paracancerous tissues of prostate cancer patients in this article, which provides the basic work for the discovery of LncRNA TTTY15.
Review the idea of ​​this article: Firstly, through the whole transcriptome sequencing data, we screened a LncRNA TTTY15 with the highest expression in prostate cancer tissue and located on the Y chromosome, and then combined with biosignal prediction, quantitative PCR, dual luciferase reporter gene and In experiments such as ChIP sequencing, the authors confirmed that LncRNA TTTY15 adsorbs let-7 through sponge action and promotes the expression of target genes CDK6 and FN1, thereby promoting the development of prostate cancer.
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Whole transcriptome sequencing
Circular RNA sequencing
LncRNA sequencing
ChIP sequencing
RNA pull down
RIP sequencing
Circular RNA quantitative PCR
LncRNA quantitative PCR
miRNA quantitative PCR
Quantitative PCR
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