ncRNA is closely relating to disease, so how can we study its function?

ncRNA is closely relating to disease, so how can we study its function?

The sequencing results of the human genome show that there are more than 20,000 genes responsible for encoding proteins, accounting for less than 2% of the total genome length. Many protozoa also have more than 20000 protein coding genes, but unlike humans, the proportion of coding genes in these creatures in the total gene length is much higher. Therefore, interesting questions came out: Does the 98% non-coding RNA (ncRNA) contribute to the complexity of the human body? How does ncRNA participate in life activities?

Figure 1. Proportion of ncRNAs in different species

At first, people had limited knowledge of ncRNA, and believed that ncRNAs are junk genes, which might be just a "shelter" to avoid external adverse environments, such as rays, viruses, and other damage to human genes. With the deepening of research, scientists gradually discovered the importance of ncRNAs. ncRNAs do not encode proteins, but they are not non-transcribed. In fact, these ncRNAs are also continuously transcribed to participate in the protein synthesis process. Among them, the most familiar one is tRNA that transport amino acids, rRNA and snoRNA that participate in ribosome synthesis. At present, lncRNA, microRNA and circRNA are the three main types highly related to cancer, which play important roles in cancer cell proliferation, migration, cell death resistance, etc. In clinical cancer screening, ncRNAs have also become an important hallmark, providing great help for early screening of cancer. ^[1-4]

Figure 2. ncRNAs have become hallmarks of cancer

microRNA is referred to as miRNA, which is complementary to mRNA through the principle of base pairing, leading to mRNA cleavage and degradation. Although the principle of miRNA is single, it plays a regulatory role in all aspects of cancer development. If miRNA can combine with the mRNA of tumor suppressor genes, miRNA can promote tumor survival and proliferation. On the contrary, if it is bound to the mRNA of oncogenes, miRNA can inhibit the expansion of tumor cells.

The functions of lncRNA are more diverse: ① lncRNA directly binds with mRNA to inhibit its translation, or combines with protein to form a transcription regulatory complex, which acts as a transcription regulatory factor to regulate the transcription level of downstream genes; ② It acts as a miRNA sponge, specifically binds to some miRNA, and continuously interferes with the function of miRNA; ③ As the backbone of the intracellular complex, it enables the stable combination of proteins and RNAs.

circRNA is lncRNA in essence. This long chain non coding RNA will form a single chain closed loop after being transcribed. Its mechanism of action is similar to that of IncRNA, which can regulate the transcription of oncogenes and tumor suppressor genes, and can also act as a molecular sponge to competitively inhibit miRNA. It has been suggested that it can act as a molecular scaffold to provide a binding site for complexes.

Functional study methods and knockout strategies of ncRNAs

To study the function of ncRNA, it is necessary to edit its genes. RNAi technology is a convenient and commonly used means in scientific research. However, due to its low efficiency and off-target possibility, better regulation methods are required. Compared with RNAi technology, CRISPR/Cas9 system has the advantages of low off-target effect, more thorough knockout, and the ability to interfere with RNA in the nucleus that is difficult to interfere with RNAi. CRISPR/Cas9 is more suitable for ncRNA intervention and gene knockout.^5,6 The following are CRISPR/Cas9 knockout strategies for miRNA, lncRNA, and circRNA:

The miRNA fragment is small, so it is relatively simple to knockout. The target sequence can be designed on the mature miRNA, at the stem ring. Or design a pair of gRNAs to directly knockout the entire miRNA sequence, but it should be noted that the knockout region cannot overlap with other coding genes.

LncRNA is relatively long, and it is difficult to have a great impact on the function by knockout some bases, so it is recommended to knockout as long fragment as possible. Generally, large fragment gene knockout strategy is used, which is to design 2-4 gRNAs from upstream to downstream.

However, circRNAs often overlap with the exons of coding genes, so the knockout is more difficult and complex - it is necessary to avoid destroying the exons while knockout circRNA. It is recommended to knockout the circRNA loop forming element located in the intron, which can destroy the function of circRNA without affecting the coding gene (Figure 3).

Figure 3. circHIPK3 knockout strategy - Alu looping element knockout ^[8]

Ubigene has developed CRISPR-U™ system, which is 10 times more efficient in editing. And Ubigene has mastered three knockout strategies: small fragment knockout, indel frameshift mutation, and large fragment knockout. More than 5000 successful cases of gene editing in 200+ cell lines. Easily achieve the knockout of miRNA, lncRNA, and circRNA. Express knockout cell line service as fast as four weeks, deliver high-quality positive clones. To learn more>>

After understanding the roles of ncRNA in tumor and the strategies of ncRNA knockout, let's go over some cases of CRISPR/Cas9 mediated ncRNA research.

Study of drug resistance mechanism of tumor by CRISPR/Cas9 knockout miR-137

       In the article microRNA-137 promotes apoptosis in ovarian cancer cells via the regulation of XIAP⁷ published by the British Journal of Cancer, Jiang’s team from the State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University clarified that miR-137 plays an important role in cisplatin induced apoptosis of ovarian cancer cells.^[7]

At present, it can be confirmed that cisplatin can achieve the therapeutic effect by promoting tumor cells to apoptosis, but its mechanism is still unclear. To uncover its mechanism, Jiang's team screened eight miRNAs that can regulate apoptosis regulator XIAP (the most effective apoptosis regulator in mammalian cells) through the dual luciferase reporter gene. Among them, miR-137 has high significance, and the high expression of this miR-137 also plays an important role in inhibiting other tumors. Therefore, the team chose miR-137 for further study. By overexpressing miR-137, ovarian cancer cell A2780 downregulates apoptosis regulator XIAP, making ovarian cancer cells more prone to apoptosis when induced by cisplatin.

Figure 4. miR-137 promotes apoptosis of ovarian cancer cells

In order to further confirm that miR-137 plays the role of tumor suppressor gene in the apoptosis of ovarian cancer cells, Jiang's team designed three types of sgRNAs to target the upstream, downstream and precise regions of mature miR-137, and knockout the miR-137 in A2780 cell line using CRISPR/Cas9 system. It was found that after miR-137 was deleted, XIAP was subsequently up-regulated, and the apoptosis rate was reduced.

Figure 5. After knockout miR-137, XIAP is subsequently up-regulated

Study of the mechanism of tumor cell proliferation and colony forming ability by CRISPR/Cas9 knockout SNHG1

Juan's team from Center for Computational and Systems Biology of National Taiwan University found that the high expression of SNHG1 was highly correlated with the poor prognosis of neuroblastoma (NB). So Juan's team designed two gRNAs and knockout the endogenous SNHG1 of SK-N-BE(2)C cell line (a NB cell line) using CRISPR/Cas9 technology. In the article LncRNA SNHG1 regulates neuroblastoma cell fate via interactions with HDAC1/2⁸, Juan's team suggested that the knockout of SNHG1 inhibited the proliferation of NB cells. Moreover, the analysis of cell line transcriptome suggests that SNHG1 is closely related to a variety of biological processes, such as cell migration, growth and apoptosis resistance. Moreover, SNHG1 can affect the expression of genes in the core regulatory circuit (CRC), which includes PHOX2B, HAND2, GATA3, ISL1, TBX1 and MYCN.

Figure 6. SNHG1 affects the core regulation loop

Juan’s team further used ChIP-seq and ATAC-seq to study the possible interaction between SNHG1 and HDAC1/2, and then further confirmed the interaction through pull-down and RIP.

Figure 7. Results of pull-down and RIP

The above Juan’s team analyzed the action mechanism of SNHG1 in NB, and suggested that blocking the interaction between SNHG1 and HDAC1/2 would be essential to inhibit the proliferation and colony formation of NB cells, providing a potential therapeutic strategy for the treatment of NB.

Study the signal pathway of hepatocellular carcinoma development by CRISPR/Cas9 technology

Compared with normal liver tissues, circSOD2 is highly expressed in tumor tissues of HCC patients. Inhibition of circSOD2 can significantly inhibit the proliferation of liver cancer cells and slow down the tumor formation in vivo. The Tu’s team of Wenzhou Medical University proposed a new signal pathway in the CircSOD2 induced epigenetic alteration drives hepatocellular carcinoma progression through activating JAK2/STAT3 signaling pathway published in the Journal of Experimental & Clinical Cancer Research, which provides new ideas for the prevention and drug intervention of liver cancer.

Tu’s team analyzed the RNA interactome database, used Ago2 pull-down and biotin pull-down techniques, and observed the expression level of miR-502-5p after silencing and overexpressing CircSOD2. They found that CircSOD2 acts as a "miRNA sponge" in liver tumor cells, specifically binding to miR-502-5p, effectively inhibiting the expression of miR-502-5p.

Figure 8. CircSOD2 inhibits the expression of miR-502-5p

Subsequently, Tu’s team made use of the above similar experimental principle, and found that after adding the analog of miR-502-5p, the expression of DNMT3a, a protein highly expressed in various liver cancer cell lines, was significantly down regulated.

Figure 9. Down-regulation of DNMT3a by miR-502-5p

In order to further study the molecular mechanism of DNMT3a in HCC, the team constructed DNMT3a-KO HEPG2 and HUH7 cell lines by CRISPR/Cas9 technology. The WB results show that DNMT3a may regulate the proliferation of HCC cells through SOCS3/JAK2/STAT3 signal pathway. According to the clue that DNMT3a is a DNA methyltransferase, the team further verified the reliability of the signal pathway by using the dual luciferase reporter gene experiment and CHIP-qPCR experiment. The final conclusion is that DNMT3a upregulation promotes SOCS3 promoter methylation and inhibits SOCS3 expression, thereby further activating JAK2/STAT3 signal pathway.

Figure 10. DNMT3a inhibits SOCS3 expression, thereby activates JAK2/STAT3 signaling pathway

Summary

Many ncRNAs have been proved to play important roles in promoting tumor growth, migration and escaping cell death. Meanwhile, some other ncRNAs play the role of tumor suppressor, prevent cells from entering the division cycle, activate programmed death, maintain genomic stability, and directly or indirectly inhibit tumor occurrence.

With the help of CRISPR/Cas9 gene editing technology, the above cases have achieved ideal results in the functional research of ncRNAs. Ubigene has collected 200+ cell lines gene editing parameters, and developed CRISPR-U^TM system with 10X efficiency, which can provide services for lncRNA, miRNA, and circRNA knockout in various cell lines. High-quality homozygous KO clones deliver as fast as 4 weeks!

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Reference:

Winkle, M., El-Daly, S. M., Fabbri, M. & Calin, G. A. Noncoding RNA therapeutics - challenges and potential solutions. Nat Rev Drug Discov 20, 629-651, doi:10.1038/s41573-021-00219-z (2021).

Renganathan, A. & Felley-Bosco, E. Long Noncoding RNAs in Cancer and Therapeutic Potential. Adv Exp Med Biol 1008, 199-222, doi:10.1007/978-981-10-5203-3_7 (2017).

Pichler, M. & Calin, G. A. MicroRNAs in cancer: from developmental genes in worms to their clinical application in patients. British Journal of Cancer 113, 569-573, doi:10.1038/bjc.2015.253 (2015).

Kristensen, L. S., Hansen, T. B., Venø, M. T. & Kjems, J. Circular RNAs in cancer: opportunities and challenges in the field. Oncogene 37, 555-565, doi:10.1038/onc.2017.361 (2018).

Yang, J. et al. CRISPR/Cas9-mediated noncoding RNA editing in human cancers. RNA Biol 15, 35-43, doi:10.1080/15476286.2017.1391443 (2018).

Dimitri, A., Herbst, F. & Fraietta, J. A. Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing. Molecular Cancer 21, 78, doi:10.1186/s12943-022-01559-z (2022).

Hsu, C. L. et al. LncRNA SNHG1 regulates neuroblastoma cell fate via interactions with HDAC1/2. Cell Death Dis 13, 809, doi:10.1038/s41419-022-05256-z (2022).

Li, X. et al. microRNA-137 promotes apoptosis in ovarian cancer cells via the regulation of XIAP. Br J Cancer 116, 66-76, doi:10.1038/bjc.2016.379 (2017).

Zhao, Z. et al. CircSOD2 induced epigenetic alteration drives hepatocellular carcinoma progression through activating JAK2/STAT3 signaling pathway. J Exp Clin Cancer Res 39, 259, doi:10.1186/s13046-020-01769-7 (2020).