Non-coding RNA, splicing regulators and RNA promoters: an insight into the molecular basis of colorectal cancer
Next generation sequencing provides insight into the molecular basis of different diseases. Cancer remains as one of the most investigated diseases so far, due its high incidence and mortality rates. In this study, researchers were able to identify a small protein that dysregulated RNA splicing, leading to a substantial cancer cell growth and proliferation and, thus, worsening a patient’s prognosis.
Colorectal cancer (CRC) is still considered a significant cause of death among cancer-related mortality rates . The guidelines promoted by different medical associations keep pushing and promoting effective screening programs, which have increased the early diagnosis of CRC and vastly improved their chances of recovery.
However, the 5-year survival rate amongst late-stage CRC patients remains low, particularly due the lack of effective therapeutic choices. Understanding the molecular basis of the disease will shed some light on potential therapeutic targets, diminishing the odds of death and increasing their life quality.
Tumorigenesis is a long and notoriously difficult process to understand. Thankfully, advancement in the next generation sequencing data services have allowed researchers to unravel this herculean task. Many oncogenic genes are simultaneously responsible for altering transcription factors (TF) and their signaling pathways.
The importance of non-coding RNA, such as long-noncoding RNAs (lncRNAs), have increased drastically, as previous studies have demonstrated a possible role in many diseases, such as different types of cancer . Thus, figuring out where lncRNAs are involved in the tumorigenesis of CRC and its interaction with other proteins and their TF will prove useful.
In this study, researchers demonstrated the lncRNA LOC90024 is responsible for encoding a small protein, but with enormous implications. Said protein will then interact with other transcription factors, unleashing the chain of events that culminate in cancer growth.
Tumor tissue of CRC patients, noncancerous tissue of patients and parental cells lines were used. These samples were subjected to ribosome-bound RNAs purification and RNA sequencing, to identify if the LOC90024 lncRNA could, or not, bind to any ribosome.
Sequencing matching techniques and other techniques, such as immunochemistry assay, were used as well to determine the expression of certain proteins.
The findings demonstrated that one of the encoding products of LOC90024 directly participated in the tumorigenesis, cell proliferation, colony formation and more. To reach this conclusion, several ideas were postulated, based on the current study.
1.LncRNA LOC90024 is capable of encoding a small protein
The purification of ribosome-bound RNAs, together with the RNA-seq process, showed that LOC90024 was indeed capable of binding to a ribosome and, thus, it can be translated into a protein/peptide. Further research confirmed that this lncRNA does have an Open Reading Frame (ORF), composed of 392 nucleotides, capable of encoding a 130-amino acid small protein. Said protein was named “Splicing Regulatory Small Protein” (SRSP).
2.Upregulation and expression levels of LOC90024 and SRSP determine the prognosis in CRC patients
Upregulation of this lncRNA was confirmed in numerous cancerous samples, such as highly metastatic colorectal, ovarian, nasopharyngeal and breast cancer cells. Subsequently, researchers matched the CRC tissue samples with nontumor tissue samples. Immunochemistry assays were further used to confirm the findings.
Researchers found that both LOC90024 and SRSP were upregulated in cancerous tissue samples. Not just that, but their expression levels were significantly higher in these types of cells than in their non tumoral matchings.
3. It’s not LOC90024 itself that promotes cancer growth, but the work of SRSP
The previous findings confirm that either the lnc-RNA LOC90024, or its encoding protein, play a certain role in the cancerogenesis of CRC. However, they don’t demonstrate which one is more directly responsible. To help distinguish between the both, LOC90024 knockout (KO) and knockdown (KD) models were employed.
In both, cancer cell growth, proliferation, metastasis and vascular invasion were all inhibited. Afterwards, alterations of these KO models were restored, which led to a reexpression of SRSP and an increase in cancer cell growth and more. However, 5′UTR‐ORFmut‐Flag (MUT) vector construct did not reexpress SRSP and there was no significant change in the cancer cells, compared to the other models.
Moreover, RNA-seq confirmed that SRSP overexpression leads to a much higher transcriptome mutational rate than LOC90024 overexpression itself. Thus, these findings seem to indicate that it’s SRSP that promotes CRC growth.
4. SRSP protein interaction induces cancerogenesis
Mass spectrometry and Gene ontology were used to identify which proteins or peptides SRSP interacts with to unleash its carcinogenic implications. The results suggested this newly discovered protein mainly interacts with RNA splicing processing. Thus, the researchers’ aim became to find out which splicing regulators SRSP interacts with.
Out of all the possible candidates, one particularly stood out. SRSF3 had previously been linked to progression of several human cancers . Interactomics analysis helped determine the protein interactions SRSP has and GO confirmed that the most significant interaction was with SRSF3.
KD SRSF3 models were used to further confirm that this splicing regulator is involved in the signaling pathway of CRC, as cell growth, colony formation, migration and invasion were all inhibited, compared to non-KD models.
5. SRSP helps bind SRSF3 to a transcription factor
SRSP primarily interacts with splicing regulators, specifically an important role in pre-mRNA splicing by interacting with the aforementioned splicing regulator. RNA-seq helped found that SRSF3 promotes the inclusion of exon 3 to the TF Sp4. This transcription factor binds to the GC promoter region of a variety of genes, it has also been previously associated with other diseases, such as bipolar disorder, and other types of cancer .
After the exon inclusion, the long Sp4 isoform (L-Sp4) is formed. Essentially, what SRSP does is enhance the binding properties of SRSF3, promoting the splicing process and overexpression of this L-Sp4 and inhibiting the short isoform (S-Sp4).
L-Sp4 was confirmed to have oncogenic effects, as demonstrated by Sp4 KD models, where the long isoform was inhibited, while the S-Sp4 continued to be expressed. These animal models had inferior levels of cell growth, colony formation, cell migration and invasions.
The lnc-RNA LOC90024 is, in itself, not an oncogenic promoter. However, it’s capable of encoding a small protein (SRSP). This protein interacts with splicing regulators, thus, altering and disregulating their splicing capabilities, as it happens with SRSF3. Instead, SRSF3 further binds to certain transcription factors, such as Sp4. This leads to an overexpression of the carcinogenic L-Sp4 isoform.
Novogene’s exceeding and trustworthy capabilities allow to provide professional and comprehensive sequencing technology. Non-coding RNA is gaining attention, as more and more researchers gradually uncover its possible roles in many pathologies, from neurodegenerative disorders to cancer.
Long non-coding RNA sequencing service (lncRNA-seq) is a comprehensive next-generation method to detect the transcripts with a length of over 200nt, which do not encode protein and perform as regulatory elements in multiple biological processes.
However, the progressive library preparation enables information enrichment and gene expression profiling for both coding and non-coding transcripts from a high-sensitive transcriptomic perspective. Bioinformatic analysis does not reveals the quantification and functional enrichment of the target transcripts, but also indicates the strand orientation and regulatory relation of lncRNA targeted mRNA.
RNA-seq, and other NGS technologies, will continue providing the biomedical community more tools to search for potential therapeutic targets.
1.Brody H. Colorectal cancer. Nature. 2015 May 14;521(7551):S1. doi: 10.1038/521S1a. PMID: 25970450. [Nature]
2.Lim KG, Lee CS, Chin DHJ, et al. Clinical characteristics and predictors of 5-year survival among colorectal cancer patients in a tertiary hospital in Malaysia. J Gastrointest Oncol. 2020;11(2):250-259. doi:10.21037/jgo.2020.02.04 [J Gastrointest Oncol.]
3.John S. Mattick, Igor V. Makunin, Non-coding RNA, Human Molecular Genetics, Volume 15, Issue suppl_1, 15 April 2006, Pages R17–R29 [Oxford Academic]
4.Wang FW, Cao CH, Han K, Zhao YX, Cai MY, Xiang ZC, Zhang JX, Chen JW, Zhong LP, Huang Y, Zhou SF, Jin XH, Guan XY, Xu RH, Xie D. APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome production. J Clin Invest. 2019 Feb 1;129(2):727-743. doi: 10.1172/JCI122478. Epub 2019 Jan 14. PMID: 30511962; PMCID: PMC6355227. [J Clin Invest]
5.Kumar D, Das M, Sauceda C, Ellies LG, Kuo K, Parwal P, Kaur M, Jih L, Bandyopadhyay GK, Burton D, Loomba R, Osborn O, Webster NJ. Degradation of splicing factor SRSF3 contributes to progressive liver disease. J Clin Invest. 2019 Aug 8;129(10):4477-4491. doi: 10.1172/JCI127374. PMID: 31393851; PMCID: PMC6763247. [J Clin Invest]
6.Xiao W, Adhikari S, Dahal U, Chen YS, Hao YJ, Sun BF, Sun HY, Li A, Ping XL, Lai WY, Wang X, Ma HL, Huang CM, Yang Y, Huang N, Jiang GB, Wang HL, Zhou Q, Wang XJ, Zhao YL, Yang YG. Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing. Mol Cell. 2016 Feb 18;61(4):507-519. doi: 10.1016/j.molcel.2016.01.012. Epub 2016 Feb 11. Erratum in: Mol Cell. 2016 Mar 17;61(6):925. PMID: 26876937. [Mol Cell]
7.Zhou X, Tang W, Greenwood TA, Guo S, He L, Geyer MA, Kelsoe JR. Transcription factor SP4 is a susceptibility gene for bipolar disorder. PLoS One. 2009;4(4):e5196. doi: 10.1371/journal.pone.0005196. Epub 2009 Apr 9. PMID: 19401786; PMCID: PMC2674320. [PLoS One]
8.Hedrick E, Cheng Y, Jin UH, Kim K, Safe S. Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are non-oncogene addiction genes in cancer cells. Oncotarget. 2016 Apr 19;7(16):22245-56. doi: 10.18632/oncotarget.7925. PMID: 26967243; PMCID: PMC5008359. [Oncotarget]