Project Design

Cervical cancer

Cervical cancer is a widespread disease that caused over 300,000 deaths worldwide in 2020. Women 35 to 44 years old are most likely to get it. More than 20% of new cases are in women over age 65, however, especially those who have not been getting regular screening [1]. The cause of cervical cancer is due to cells change in women’s cervix, which connects the uterus and vagina. This cancer can affect the deeper tissues of their cervix and may spread to other parts of their body, often lungs, liver, bladder, vagina, and rectum. HPV types 16 and 18 are responsible for 70% of cervical cancer cases [2]. Cervical smear tests are the most common diagnostic tools, reducing the incidence and mortality rate by 60-90%. Other methods like vaginal colposcopy, cone biopsy, and imaging tests are also available, but they are expensive and have limitations [3]. Therefore, our team plan to create a more efficient early diagnose system about cervical cancer.

Potential biomarker

Based on previous literature, we found that LncRNA MALAT1 is significantly upregulated in patient tissues and highly expressed in cervical cancer cells [4][5]. Conversely, miR-203-3p, miR-22-3p, and miR-145 are significantly downregulated in cervical cancer [6][7][8]. By constructing a recombinant plasmid, we were able to use the MALAT1-miRNA complex as an early diagnostic indicator of cervical cancer.

RNA visualization

Recently, Chinese scientists found peppers are monomeric, bright, and stable fluorescent proteins that can visualize RNA species in live cells with minimal disturbance. [9] Using Peppers as RNA aptamers makes early screening of cancer more accurate, especially cervical cancer(Figure1). Pepper is a fluorogenic RNA aptamer tag that binds to a variety of (4-((2-hydroxyethyl)(methyl)amino)-benzylidene)-cyanophenylacetonitrile (HBC) derivatives with tight affinity and activates their florescence. LncRNA MALAT1 is highly expressed in cervical cancer, which is more than twice as much as in normal people. With the utilization of Pepper, it could be clearly observed under a microscope, making it a promising technique for detecting cervical cancer.

Figure 1 HBC bind with Pepper model

HBC is a GFP fluorophore-like synthetic dye. It contains a structurally rigid electron acceptor and a strong electron donor. HBC is nonfluorescent in solution, but strongly fluoresces upon constraining intramolecular motion. Pepper aptamers bound with HBC and exhibit bright fluorescence.

Part design

Figure 2 Experiment Protocol Design

In the first part, we constructed a recombinant plasmid by amplifying the part sequence of LncRNA MALAT1 using PCR technology and connecting it to the pepper plasmid. Secondly, Hela cells were transfected with the plasmid. In the disease group, we transfected the pepper plasmid with MALAT1. In the diagnosis group, we transfected the pepper plasmid with both MALAT1 and miRNA mimic. Finally, we added HBC fluorescent dye and incubated it for approximately two hours. (Figure 2)

Figure 3 Our Research Model

Using confocal microscopy fluorescence observation, the pepper plasmid containing the part sequence of lncRNA MALAT1 in the disease group will emit strong green fluorescence. Conversely, in the diagnosis group, the pepper plasmid with both MALAT1 and miRNA mimic will exhibit weak green fluorescence. Due to the normal expression of miRNA in normal cells, the cells will emit fluorescence when the HBC pepper dye binds to the adapter. However, due to the high expression of miR-22/miR-145 in cells in cervical cancer patients, miR-22/miR-145 will bind with MALAT1, hence suppressing fluorescence when we add dyes, resulting in low fluorescence in the cells that have cervical cancer.(Figure 3).

In summary, our mission is to develop a new method to diagnose cervical cancer . We will synthesis miR-22/miR-145 and clone LncRNA MALAT1 sequence into the pepper plasmids. And we will test the potential role of MALAT1 as sponge to find the abnormal expression of cervical cancer. In the future, we will promote our idea to the public like reagent kits and more patients could benefit from our work.

Reference

[1] Cohen, P. A., Jhingran, A., Oaknin, A., & Denny, L. (2019). Cervical cancer.

[2] Muñoz, N., Castellsagué, X., de González, A. B., & Gissmann, L. (2006). HPV in the etiology of human cancer. Vaccine, 24, S1-S10.

[3] Wilkinson, C., Jones, J. M., & McBride, J. (1990). Anxiety caused by abnormal result of cervical smear test: a controlled trial. BMJ: British Medical Journal, 300(6722), 440.

[4] Castellsagué, X. (2008). Natural history and epidemiology of HPV infection and cervical cancer. Gynecologic oncology, 110(3), S4-S7.

[5] Wilkinson, C., Jones, J. M., & McBride, J. (1990). Anxiety caused by abnormal result of cervical smear test: a controlled trial. BMJ: British Medical Journal, 300(6722), 440.

[6] Jiang, Y., Li, Y., Fang, S., Jiang, B., Qin, C., Xie, P., ... & Li, G. (2014). The role of MALAT1 correlates with HPV in cervical cancer. Oncology letters, 7(6), 2135-2141.

[7] Yang, L., Bai, H. S., Deng, Y., & Fan, L. (2015). High MALAT1 expression predicts a poor prognosis of cervical cancer and promotes cancer cell growth and invasion. European Review for Medical & Pharmacological Sciences, 19(17).

[8] Zierau, O., Helle, J., Schadyew, S., Morgenroth, Y., Bentler, M., Hennig, A., ... & Kretzschmar, G. (2018). Role of miR‐203 in estrogen receptor‐mediated signaling in the rat uterus and endometrial carcinoma. Journal of cellular biochemistry, 119(7), 5359-5372.

[9] Lv, K. T., Liu, Z., Feng, J., Zhao, W., Hao, T., Ding, W. Y., ... & Gao, L. J. (2018). MiR-22-3p regulates cell proliferation and inhibits cell apoptosis through targeting the eIF4EBP3 gene in human cervical squamous carcinoma cells. International journal of medical sciences, 15(2), 142.

[10] Lu, H., He, Y., Lin, L., Qi, Z., Ma, L., Li, L., & Su, Y. (2016). Long non-coding RNA MALAT1 modulates radiosensitivity of HR-HPV+ cervical cancer via sponging miR-145. Tumor Biology, 37, 1683-1691.

[11] Rees HC, Gogacz W, Li NS, Koirala D, Piccirilli JA. Structural Basis for Fluorescence Activation by Pepper RNA. ACS Chem Biol. 2022 Jul 15;17(7):1866-1875. doi: 10.1021/acschembio.2c00290. Epub 2022 Jun 27. PMID: 35759696; PMCID: PMC9969808.