Improvement

Our improved experiment is based upon the 2023 YiYe-China project [1]. The project aims to develop a “sensor” for cervical cancer screening. Cervical cancer is a major cancer worldwide, causing more than 660,000 cases and 350,000 deaths per year [2]. In cervical cancer, long non-coding RNA (lncRNA) MALAT1 is overexpressed as an oncogene [3]. MicroRNA 22 (miR-22) and microRNA-145 (miR-145) are two short RNA molecules that can bind with MALAT1 through complementary base pairing. As MALAT1 is overexpressed, miR-22 and miR-145 underexpressed [4, 5]. LncRNAs could bind to specific miRNA and act as sponges to compete miRNAs [4]. This mechanism gives rise to our idea about fusing a sponge RNA based on the sequences of lncRNA MALAT1 with binding sites complementary to the sequence of miRNA to a plasmid that has reporter gene, pepper for instance, which will monitor the expression of miRNA in the cells.

As miR-22 and miR-145 are underexpressed in cervical cancer cells, the 2023 YiYe-China team decided to use them as biomarkers. In the plasmid they constructed, the 2023 YiYe-China team fused a pepper sequence downstream the MALAT1 gene. Upon transcription, the MALAT1 lncRNA will be connected to a pepper structure. Afterward, they will transfect excess amounts of HBC530 molecules into the cells. HBC530 molecules can bind to pepper structures to show green fluorescence. In healthy cells, higher miR-22 and miR-145 concentrations will lead to more rapid degradation of MALAT1 and pepper structures. Subsequently, less HBC530 molecules will be able to bind to pepper structures, showing lower fluorescence. In cervical cancer cells, lower miR-22 and miR-145 concentrations will lead to slower degradation of MALAT1 and pepper structures. Subsequently, more HBC530 molecules will be able to bind to pepper structures, showing higher fluorescence. The experimental outline is shown in Figure 1.

To achieve this, the 2023 YiYe-China team designed the parts BBa_K4789004 and BBa_K4789005. BBa_K4789004 consists of the miR-22 target sequence from MALAT1 connected with a pepper sequence, as shown in Figure 2. Upon transcription, it will produce an mRNA containing the miR-22 binding site connected with a pepper structure.

BBa_K4789005 consists of the miR-145 sequence from MALAT1 connected with a pepper sequence, as shown in Figure 3. Upon transcription, it will produce an mRNA containing the miR-145 binding site connected with a pepper structure.

From 2023 YiYe-China’s results, we found that the miR-22 sensor worked more accurately and efficiently than miR-145. Therefore, we decided to design our improved experiment based on the miR-22 sensor.

This year, we redesigned the miR-22-sponge-pepper part by adding three additional miR-22 binding sites with 3-nt spacers for bulged sites based on the sequence of hsa-miR-22 according to a previous study [6]. Therefore, the new sensor will be more sensitive to the miR-22 concentration in cells. The part is called Improved miR-22-sponge-pepper (BBa_K5082017) and its sequence is shown in Figure 4.

The plasmid map for the plasmid constructed by the 2023 YiYe-China team is shown in Figure 5.

To amplify the miR-22 concentration difference between healthy and cervical cancer cells, we used Hela cell lines and transfected different concentrations of the pre-miR-22 plasmids (BBa_K4789002).

In a 24-well plate, we separated the Hela cells into two groups. For one group, we added 1 μg of pre-miR-22 plasmid into each well, while no pre-miR-22 plasmid was added for the other group. Afterward, we transfected 2 μg of the improved miR-22-sponge-pepper, into all cells in the 24-well plate, and cultured the cells for 48 hours. The cells are shown in Figure 6.

After 48 hours of cell culture, we added 2 μg of HBC530 fluorescent dye into each well. After 2 more hours of incubation, we observed the cells under a fluorescent microscope and measured the fluorescence value using SpectraMax i3. The qualitative observation results are shown in Figure 7, and the quantitative observation results are shown in Figure 8.

As shown in Figure 7 and 8, for both the original and improved sensor, the fluorescence value was lower in cells with additional miR-22, proving that both sensors could detect miR-22 concentration. Moreover, the difference between fluorescence values was larger in the Improved miR-22-sponge-pepper as compared to the miR-22-sponge-pepper. The improved one boost the affinity of a decoy target for its cognate microRNA.

In our project, we constructed two miR-22 sponge plasmid, miR-22-sponge-pepper and Improved miR-22-sponge-pepper, as the monitor to detect the expression of miR-22 in cells. Subsequently, we got two standard curves by transfected miR-22 sponge plasmid and Improved miR-22-sponge-pepper with different concentration of pre-miR-22 to Hela cells. Based on the values in cell treated with different concentration of pre-miR-22, the standard curve of the relationship between fluorescence and pre-miR-22 amount were made by EXCEL. We suggested the slope of the standard curve of Improved miR-22-sponge-pepper is better than miR-22-sponge-pepper,suggesting the Improved miR-22-sponge-pepper as a monitor.

In summary, by adding three more miR-22 binding sites to part BBa_K4789004 created by the 2023 YiYe-China team, we created part Improved miR-22-sponge-pepper, detecting miR-22 with a higher accuracy and sensitivity.

[1] YiYe-China. “Home | YiYe-China - IGEM 2023.” iGEM.wiki, 2023, 2023.igem.wiki/yiye-china/. Accessed 7 Aug. 2024.

[2] ---. “Cervical Cancer.” World Health Organization, 2019, www.who.int/health-topics/cervical-cancer#tab=tab_1. Accessed 8 Aug. 2024.

[3] JIANG, YAN, et al. “The Role of MALAT1 Correlates with HPV in Cervical Cancer.” Oncology Letters, vol. 7, no. 6, 24 Mar. 2014, pp. 2135–2141, https://doi.org/10.3892/ol.2014.1996. Accessed 9 Aug. 2024.

[4] Kazem Nejati, et al. “MicroRNA-22 in Female Malignancies: Focusing on Breast, Cervical, and Ovarian Cancers.” Pathology - Research and Practice, vol. 223, 1 July 2021, pp. 153452–153452, https://doi.org/10.1016/j.prp.2021.153452. Accessed 9 Aug. 2024.

[5] Sawant, Dwitiya, and Brenda Lilly. “MicroRNA-145 Targets in Cancer and the Cardiovascular System: Evidence for Common Signaling Pathways.” Vascular Biology, 23 Oct. 2020, https://doi.org/10.1530/vb-20-0012. Accessed 9 Aug. 2024.

[6] Ebert, Margaret S et al. “MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells.” Nature methods vol. 4,9 (2007): 721-6. doi:10.1038/nmeth1079. Accessed 9 Aug. 2024.