مقالات پذیرفته شده در پنجمین کنگره بین المللی زیست پزشکی
Design efficient RNAi sequences to postpone the metastasis by down-regulating ZEB1 in pancreatic cancer
Design efficient RNAi sequences to postpone the metastasis by down-regulating ZEB1 in pancreatic cancer
Mahbod Djamshidi,1,*Ghazal Mohammadbeigi,2
1. Department of Biology, Faculty of Science, University of Guilan 2. Department of Biology, Faculty of Science, University of Guilan
Introduction: The pancreas is a gland that produces chemical substances for food digestion (the exocrine component) and hormones (the endocrine component) that can give the creatures a helping hand to control the rate of sugar in blood [15]. Cancer is known as a heterogeneous disease, which is regulated by complex mechanisms that could promote both tumor initiation and progression [1]. In pancreas cancer, normal cells undergo stopping work and grow out of control to form a mass named tumor [15]. Cancer is a leading cause of death universally. Pancreatic cancer has given rise to 4.2% deaths in males and 4.9% in females in 2018 [1].
1.2. Metastasis
Most of the cancer deaths are caused by “metastasis” which means the spread of primary cancer to distant sites [2]. In other words, metastasis is the circulating of cancer cells via the vascular system and its establishment in a new location causing a secondary tumor. For many types of cancers, metastasis is also called stage IV (4) cancer or advanced cancer [2,3,15]. Clinically, it is diagnosed when observing cancer cells under a microscope having features like primary cancer cells but not resembled to the cells of the tissue they have found [2].
Cancer can spread to different sites. For example, in our case “pancreatic cancer”, tends to spread to the liver, lung, and peritoneum [2].
Treat metastatic cancer is hard and depends on cancer’s origin (where it started), the much of the spread, health, age, and some other things. Often the goal of treating metastatic cancer is to control it by stopping or slowing its growth.[3]
Main types of treatment for metastasis are:
• Systemic therapy: treatment affects the entire body including chemotherapy, hormone therapy, targeted therapy and immunotherapy.
• Local therapy: treatment for the area with cancer including surgery, radiation therapy, and some other treatments.
Metastasis has some steps which are demonstrated bellow in order [2,4]:
1. Invasion: growing into, or invading nearby normal tissues
2. Intravasation: moving through the walls of nearby lymph nodes or blood vessels
3. Survival: traveling through the lymphatic system and bloodstream to other parts of the body and survival within blood
4. Arrest: stopping in small blood vessels at a distant location
5. Extravasation: invading via blood vessel walls (hematogenous spread), and moving into the surrounding tissue like lung, liver, bone etc. (2way: first, moving through the blood vessel and second, cell gathering in capillaries till erupting the vessel)
6. Microenvironment: growing in target tissue until a tiny tumor form by resisting the target tissue ECM
7. Metastatic Colonization (MC): causing new blood vessels to grow, which creates a blood supply that allows the metastatic tumor to continue growing
Initiation of metastasis depends on invasion. A method that could suppress invasion can be a way to postpone metastasis. This may happen by suppressing effective signals and factors in invasion so it’s important to know about invasions incidents.
1.3. Metastasis and Invasion
Invasion is the mechanism by which affected cells penetrate into neighboring tissues [5]. As can be discovered in the following figure, metastasis and invasion are different mechanisms. Invasion refers to the ability of a tumor to expand into surrounding tissues while metastasis refers to the ability to penetrate into new tissue or distal organ at a different location [5]. The main process of invasion is penetration but metastasis, includes invasion, intravasation, and extravasation so invasion is a part of metastasis [5].
During the invasion in cancer progression, a variety of tumor cells show alterations in their plasticity by morphological and phenotypical conversions, including the epithelial to mesenchymal transition (EMT) so it is obvious that EMT initiates metastatic cascade [1] [6,12,].
1.4. EMT
EMT is a biologic process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype, which involves enhanced migratory capacity, remodeling of the cytoskeleton, the disruption of cell-cell adhesion and cellular polarity, invasiveness, elevated resistance to apoptosis and greatly increased production of ECM components [7,12-16]. EMT is a normal cellular process that regulates embryogenesis, tissue regeneration, organ fibrosis, and wound healing [1] [7-11,13,]. Metastatic tumor cells with a mesenchymal phenotype are believed to undergo a reverse transition (MET) that facilitates metastatic colonization at the site of metastasis to gain the pathology of their corresponding primary tumors by disseminated tumor cells [10,12]. As mentioned, the induction of EMT in epithelial cancer cells promotes migration, invasion, and dissemination [10].
There are three types of EMT [7,10]:
• Type I: EMT during implantation, embryogenesis and organ development
• Type II: EMT associated with tissue regeneration and organ fibrosis
• Type III: EMT associated with cancer progression and metastasis
The activation of tumor EMT typically occurs during signal swaps between tumor cells and adjacent stromal cells [8]. One hallmark of EMT is the downregulation or even loss of epithelial (E-)cadherin, which is an essential component of adherence junctions [6]. The subsequent upregulation of mesenchymal markers such as vimentin and neuronal (N-)cadherin is one of the hallmarks for mesenchymal cells [6]. This shift from E- to N-cadherin expression, termed cadherin-switch, leads to enhanced motility of EMT-transformed cells [6].
Initiate an EMT and orchestrating it triggered by complex regulatory networks including some transcription factors (EMT-TFs), such as the Snail, Twist, and ZEB families and some other things like expression of specific cell-surface proteins, reorganization and expression of cytoskeletal proteins, production of ECM-degrading enzymes, and changes in the expression of specific microRNAs (such as the miR-200 family, miR-205 and miR-9) [1][7,10,13,]. The complex regulatory networks facilitate EMT by downregulating epithelial genes (such as E-cadherin) and upregulating mesenchymal genes. [1]
1.5. EMT regulation
As EMT-TFs can down-regulate the metastasis-suppressor protein (E-cadherin) and up-regulate mesenchymal phenotype, it’s expected that consumption and down-regulating of these TFs may postpone metastasis and inhibit the metastatic dissemination. All of these factors and their relations are available in the figure 1.
1) Snail: The Snail family includes Snail1, Snail2 (Slug), and Snail3 (Smuc) [8,9]. These factors regulate epithelial and mesenchymal markers [8,9]. Snail and Slug, as two master regulators of the epithelial-mesenchymal transition, mainly mediate E-cadherin repression and are overexpressed in cancer cells during EMT [9].
2) Twist: Twist factor induces EMT by influencing other EMT-ATFs and contains two proteins (Twist1 and Twist2) [8,9]. These factors are absent in normal epithelium but are induced in many human carcinomas [8]. Usually, Twist is identified as Twist1 and Twist1 is thought more significant in cancer metastasis than Twist2 [9]. Twist1 represses E-cadherin by inducing Snail1 or Snail2 and then binding to its promoter so it decreases E-cadherins transcription indirectly [8,14]. Twist1 and Twist2 are upregulated at the invasive front of carcinomas in cancer and stromal cells [8].
3) ZEB: The zinc finger enhanced binding (ZEB1(also known as TCF8 and δEF1) [10,11] and ZEB2(also known as SIP1) [11,14]) transcription factors inhibit the miR-200 family at the transcriptional level, whereas these miRNAs themselves post-transcriptionally repress the EMT-inducers ZEB1/2 [6,14]. ZEB induces EMT by inhibiting various epithelial genes [8,10,14] and can initiate stem cell properties by upregulation of Klf4, Sox2 and Bmi1 which are normally repressed by the miR-200 family [6]. ZEB1 can repress E-cadherin expression and reciprocally repress the expression of the miR200s so the EMT process would be resulted and tumorgenicity would be enhanced [11,14].
4) miRNAs: MiRNAs play an important role in controlling tumor growth and progression. Some miRNAs function as oncogenes and tumor suppressors [8]. The miR-200 family including miR-200a, miR-200b, miR-200c, miR-429 and miR-141 leads to epithelial differentiation [8,14]. The mechanism of miR-200s suppress ZEB1 so they would be used as a cancer treatment [11].
These microRNAs work in concert to repress EMT by targeting ZEB1 and ZEB2, which are direct repressors of E-cadherin. ZEB1 and ZEB2 also transcriptionally repress miR-200c in a double-negative feedback loop, facilitating the maintenance of a mesenchymal state [11,14]. Interestingly, the knockdown of ZEB1 in mesenchymal-like pancreatic cancer cell lines reversed the EMT phenotype [10].
Snail, Twist, and ZEB1 can enhance invasion and promote the degradation of E-cadherin [8,14]. Zeb1 can directly bind to specific DNA sequences named E-boxes (the gene encoding E-cadherin) and drive EMT and caner progression [10]. Expression of ZEB1 correlates with loss of E-cadherin (downregulation) [9,14]. The expression of both ZEB1 and ZEB2 is regulated by Snail1 in certain contexts, and Snail2 activates ZEB1 by directly binding to its promoter [14]. The aberrant expression of ZEB1 is thought to be connected with tumorigenesis and poor prognosis in various tumors, especially in breast cancer [11].
Other transcription factors also induce EMT and tumor invasiveness. The homeobox factor goosecoid induces EMT by activating mesenchymal genes and repressing epithelial markers [8]. The growth factor TGF-β induces goosecoid in breast epithelial cells, and goosecoid is overexpressed in ductal breast carcinomas and atypical ductal hyperplasia [8]. It is directly activated by hypoxia via hypoxia-inducible factor 1 alpha (HIF1α) [14]. HIF1α can also induce epigenetic regulation of EMT by transcriptionally targeting HDAC3, which cooperates with the EMT-associated TF Snail1 to mediate gene repression of epithelial-specific promoters [14]. TGF-β is an important suppressor of epithelial cells [15]. It stimulates cells to lose epithelial markers, such as E-cadherin, and also to gain mesenchymal markers, such as vimentin. TGF-β is related to cell proliferation, and when this growth factor is mutated, it contributes to the uncontrolled proliferation of cancer cells [15].
2. Aim
This article will focus on the EMT transcription factor “ZEB1” and its correlation with stemness-repressor miRNAs to design a siRNA to inhibit this EMT-TF and postpone the metastasis of cancer.
Methods: 1. NCBI [16] used to identify special sequences and their homology in comparison with other resembled ones.
2. Gene-Runner exploited to predict Hairpin loops, Dimers, Bulge loops, and match sites in a given sequence’s binding site.
3. RNA Central [17] utilized to confirm the NCBI data by using 3-dimensional graphs and certified information
4. IDT Oligo Analyzer [18] used to recheck Gene-runner’s data’s accuracy
5. RNA Fold [19] utilized to be aware of the binding location’s both specificity and entropy that culminated in binding affinity.
6. Zincdb, Bindingdb and Drugdb [24-26] used as resources of official and existed ligands.
Results: 3.1. It was assumed that Zinc Finger E-box Binding homeobox 1 [Homo sapiens] has 44 significant transcripts, which are all protein-coding mRNAs. ZEB1 transcript variant 1 that contains 6192bp, not only is known as the longest transcript variant of the ZEB1 gene (Location: 10p11 .22, Exon count; 20) but also covers all other variants of the gene transcripts at least on 91% in transcript variant 25 and the most on 100% in transcript variant 7. [16] Moreover, all these data rechecked by RNA central and proved with same results. [17]
3.2. Ten significant RNAis, which are available in chart 3.1 are designed by Gene Runner computer-based engine concerning highly-effective siRNA guideline. [20] Firstly, these sequences are analyzed by the oligo analyze tool in the application. How calculations illustrate the condition; all sequences checked by their attributes and potential in making secondary structures such as Hairpin loops, Dimers, Bulge loops, and match sites. Furthermore, the sequences were rechecked and revised with a more powerful engine, which is known as the IDT analyzer tool. [18] A few novel characteristics were found out in the new results and the estimated Tm was a bit different (this difference is based on diverse Tm methods that are used by these analyzers) from the former.
3.3. In addition, the specificity of designed RNAis blasted by the NCBI BLAST tool separately. Results depict that three substantial RNAis have non-specific binding to other mRNAs based on Nucleotide Collection (nt) database. Therefore, all these three sequences, which are specified in the figure by red color, were eliminated and the study continued with the other distinguished RNAis.
3.4. Besides, these remained RNAis checked for binding and entropy potential according to data is achieved from RNAfold Webserver diversely. [19] The results are based on base-pair capability and second structure occurrence capability.
3.5. Finally, a common 5’- TCAAGAG-3’ hairpin was added in the middle of the sequence to prepare the most effective mature RNAi structures, which are predicted in this study for injecting into the cell for more in vivo/in vitro experiments. The ultimate results, which are rosed from our filters that were elaborated before in the article, are available in chart 3.2.
In the current study, we focused on optimizing novel RNAis based on any accessible feature to increase the efficiency of deactivating all ZEB1 transcripts, which over-expressed in an astonishing number of pancreatic cancers patients. Unfortunately, many of the cases with signs of the over-expression of ZEB1 and consequently, the lack of E-cadherin as one of the main characters in cell-to-cell connection. This circumstance, will promote the metastasis process and lead patients to die.
Conclusion: Whereas ZEB1 plays a critical role in metastatic cell specially in the preparation process of EMT, it would be both beneficial and practical to cut the cascade as early as feasible or in crucial points; therefore, ZEB1 was chosen because of its unique characteristics. According to bindingdb [24], drug bank [25] and Zincdb [26], there is no official and specific ligand to inhibit ZEB1 as main vertex in the network of many of the effective elements in EMT process such as Twist, Snail, Slug, miR-200, miR-205 and SOX family. In other words, if there is any specific ligand to down-regulate the over-expressed ZEB1 in pre-EMT condition. Here, we suggested a number of RNAi sequences to inhibit the over-expressed ZEB1 in pancreatic tumor cell expeditiously as our first step in the full study in this case.
In this research, we designed several specific RNAis with an optimized format by using the strongest engines in each part. We also used different well-known protocols to optimize many parameters such as Tm, length, starting nucleotide, GC content, selected region, and thermodynamic properties. All of these, besides to target’s entropy and binding probability, support our results fidelity.
In the next step, we will check to be aware of the exact rate of the RNAis’ efficiency. We hope that our outcome will be used as the first official, a specific and effective nucleotide-based ligand to postpone EMT and subsequently, metastasis in cancer patients besides to other discovered drugs. Of course, new approaches in the drug delivery field can increase the effect of our RNAis.
What is your perspective about this notion? “We are going to use accurate nucleotide treatments to reduce side effects and many problems that common drugs have.”
If you are interested in this field of study, you can check these useful references for further information. [21]-[23]