Project Description

Each year, more than half a million women are diagnosed with cervical cancer and the disease results in over 300 000 deaths worldwide [1]. Approximately 570 000 cases of cervical cancer and 311 000 deaths from the disease occurred in 2018 [2]. According to statistics, it accounts for 80% of malignant tumors in the female reproductive system and shows a trend of increasing occurrence in younger patients[3]. Patients with cervical cancer experience persistent vaginal discharge, and the metastasis of these cancer cells can induce multi-organ disease and infertility. There are various histologic subtypes of cervical cancer; the most common histopathologic subtype is squamous cell carcinoma (SCC), followed by adenomatous carcinoma (AC)[4]. Surgery is currently the mainstay treatment for cervical cancer, but patients with advanced or recurrent cervical cancer are often resistant to traditional radiotherapy, and effective treatments are still scarce [5-6]. Therefore, research on biomarkers and therapeutic targets with predictive value for cervical cancer is particularly important for improving patient survival and prognosis.

The Limitation of Current Cervical Cancer Detection Methods

Despite technological advances in diagnostic cervical cancer testing techniques, the accurate diagnosis and stage determination of cervical cancer still relies heavily on the morphological assessment of cervical epithelial cells by experienced pathologists. For example, cervical cancer epithelium has an abnormal nuclear composition compared to normal cells, with coarse chromatin, prominent nucleoli, irregular nuclear contour, and a high nuclear-to-plasmic ratio. However, this clinical case diagnostic assessment is usually subjective, which is an important reason behind the underestimation of the true incidence of cervical cancer [7]. In addition, it has been previously reported that some adjunctive imaging techniques for cervical cancer diagnosis, such as FEC-PET/CT, FDG-PET/ CT, and Diffusion-Weighted MRI, are characterized by low sensitivity in cervical cancer, which creates the risk of potential missed diagnoses [8]. Therefore, the development of a new, highly accurate, and efficient screening technique is essential for the detection of cervical cancer in clinical practice.

Therefore, our team is committed to design a new method based on new biomarkers that can diagnose cervical cancer with specificity, accuracy and ease, which also has the potential to be used in cancer treatment in the future.

Non-coding RNA in Cervical Cancer Detection

Non-coding RNAs (ncRNAs) are ribonucleic acid (RNA) molecules that are not translated into protein products. The ncRNAs can be divided into two categories according to length: one category is is long non-coding RNAs (lncRNAs) (>200 bp), and the other one is small ncRNAs (< 200 bp), including microRNA [9].

Long non-coding RNA (lncRNA) is functionally defined as a class of RNA transcripts with a length of 200 nt that lacks protein-coding potential. Their numbers reach into the tens of thousands, many of which are expressed only in differentiated tissues or specific types of cancer [10]. In recent years, an increasing number of studies have found that the lncRNA Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) may be involved in the occurrence and development of cervical cancer [11]. For instance, research has shown that the expression levels of MALAT1 are significantly elevated in patients’ tissues, and MALAT1 is also overexpressed in several cervical cancer cells [12] (Fig 1). Additionally, the survival curves of cervical intraepithelial neoplasia and cervical cancer indicate that higher expression of MALAT1 is correlated with lower patient survival rates [13].

Figure 1 Expression of MALAT1 in cervical cancer cells. Ect1/E5E7 is normal cervical cell line, while Hela, CaSki, SiHa and C33A are cervical cancer cell lines.

MicroRNAs (miRNAs) are small, non-protein coding RNAs of 18–25 nucleotides involved in post-transcriptional gene silencing or translational repression by binding to 3′ untranslated region (UTR) of its target messenger RNAs (mRNAs) [14]. A Research has also discovered that miR-203-3p, miR-22-3p, and miR-145 are significantly downregulated in cervical cancer [15]. Both miRNA and LncRNA play fundamental role in the cervical cancer progression. Thus they can be act as biomarkers in the cancer detection.

LncRNAs Act as Sponges to Compete miRNAs in Cervical Cancer

lncRNAs play an important role in a variety of physiological processes, including epigenetic regulation of gene expression, RNA decay, microRNA regulation, RNA splicing, protein folding[14].

Recently, several studies indicated that lncRNAs could act as sponges to compete miRNAs. miRNA Sponges contain complementary binding sites to a miRNA of interest, which inhibit miRNA activity [15] (Fig 2). Currently, Reports have shown that the lncRNA MALAT1 can act as a "sponge" for miR-202-3p, thereby regulating the invasion and epithelial-mesenchymal transition of cervical cancer cells [16]. Additionally, it has been reported that MALAT1 can also act as a "sponge" for miR-429, thereby regulating the migration, apoptosis, and other metabolic activities of cervical cancer cells [17-18]. This mechanism gives rise to our idea of fusing a sponge 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.

Figure 2 Molecular mechanisms of lncRNA-miRNA axes.

MiRNAs regulate gene expression by binding to 3’ UTR elements in mRNA and degrading them. 2.lncRNAs have been thought to interact with miRNAs as “sponges” or competing endogenous RNAs (ceRNA), attenuating the repression of mRNAs by miRNA.

Visualization of non-coding RNA

The imaging system has been developed for decades. Although naturally occurring fluorescent proteins exist, fluorescent RNAs have yet to be discovered in nature. Thus, in early RNA research, scientists developed a series of tools that visually detect RNA, based on fluorescent proteins[19].

In 2019, professor Yang Ge and Zhu Linyong collaborated to develop a new fluorescent RNA, Pepper-HBC [20]. This newly designed fluorophore molecule, HBC, can be modified to obtain a series of HBC derivatives, which can then be combined with the RNA aptamer Pepper, to emit different hues of fluorescence (from cyan fluorescence at 485 nanometers, green fluorescence at 530 nanometers, orange fluorescence at 599 nanometers, to red fluorescence at 620 nanometers). The new rational design of HBC targets and binds with RNA aptamers more easily, contains weaker background fluorescence, and has no cytotoxicity. At the same time, Pepper itself is a monomer, without G-quadruplexes, and with a strong binding force for HBC that is capable of reaching nanomolar levels. In addition, Pepper fluorescence is consistently bright and stable, regardless of whether it is being utilized in vitro or in live cells. This feature allows Pepper to facilitate research on RNA with different functions and different levels of abundance without affecting the RNA’s spatial distribution and dynamic changes.

We use the “Pepper ” to visualize LncRNA MALAT1 in order to reflect miRNA expression in vivo. The pepper-MALAT1 plasmid will emit green fluorescence when HBC is added. In the healthy people, there were fewer miR-22/miR-145. So fewer miRNA could bind to MALAT1.More free MALAT1 will emit fluorescence through reporter “Pepper assay”. 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. This result a weak fluorescence in the patients cells of cervical cancer. In summary, the miRNA expression could be detected through our “sensor”-pepper-MALAT1 fluorescence (Fig 3).

Figure 3 cervical cancer detection “sensor” model

In summary, our YiYe-China team aims to develop a “sensor” for cervical cancer screening, based upon the fluorescent variations of MALAT1-miRNA complexes in cervical cancer cells that can quantitatively reflect the disease’s progress in real-ime, thus providing a powerful support for the early diagnosis of cervical cancer. This novel, efficient, and inexpensive detection method will pave the way for accurate diagnosis and clinical staging of cervical cancer, as well as revolutionizing a new intellectual approach to the subject.

Reference

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