Team:LZU-CHINA/Description

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Description

Show you how we deal with pancreatic cancer...
Inspiration Introduction Ras-raf pathway Composability ALKBH5 HIF-1 Exosome Logic Gate

Inspiration

Why do we want to treat pancreatic cancer?

Pancreatic cancer is a growing international problem and is a digestive tract malignancy that is difficult to diagnose and treat. Pancreatic cancer is a growing international problem and is a digestive tract malignancy.This has led the United Nations (UN) and the World Health Organization (WHO) to declare pancreatic cancer one of the greatest threats to global public health. The early symptoms of pancreatic cancer are not obvious, so most of them are found to be advanced, resulting in high mortality. The mortality rate of pancreatic cancer in China has risen from the 15th in the 1960s to the 5th now. Surgery is still the only way to achieve long-term survival.

Why is pancreatic cancer mortality high and difficult to cure?

The pancreas has both internal and external secretion functions, its volume is very small, its anatomical position is deep, and it is adjacent to the organs such as the stomach and the duodenum and common bile duct. Therefore, the clinical symptoms of pancreatic cancer appear later and are not easy to be removed. Because pancreatic cancer lacks specific clinical manifestations and early diagnostic markers at an early stage, most patients are diagnosed with advanced disease. In addition, the distal metastasis and local invasiveness of the tumor greatly shortens the survival time of the patient. The problem of metastasis and infiltration of pancreatic cancer is an important cause of high mortality in pancreatic cancer. Adenocarcinoma cells "reform" fibroblasts to remodel the matrix and interact with other less aggressive cancer cells to enhance tumors. The invasive ability of the tissue to form a microenvironment that protects both cancer cells and their transfer. Therefore, pancreatic cancer is extremely difficult to cure. Regardless of surgery or not, 80% of pancreatic cancer eventually died of tumor metastasis, leading to a stalemate in the study of pancreatic cancer diagnosis and treatment.

How do we choose this goal?

The RAS-RAF pathway can transduce extracellular stimuli into the nucleus, regulate cell growth, differentiation, proliferation, apoptosis, etc., so we try to achieve our goal by regulating the RAS-RAF system. m6A demethylase ALKBH5 is underexpressed in pancreatic cancer, and decreased expression of ALKBH5 promotes proliferation and invasion of pancreatic cancer cells. The decrease of ALKBH5 expression promotes proliferation and invasion and metastasis of pancreatic cancer cells. Therefore, we can regulate the metastasis of pancreatic cancer by regulating the content of ALKBH5. problem. In addition, because we used exosomes last year and the effects of exosomes are very good, we still use exosomes to introduce them into cancer cells this year.

What is the current research progress of pancreatic cancer treatment?

Although the current pancreatic cancer surgery has continuous improvement technology, a wide range of application atmospheres, and an endless stream of developed chemotherapy drugs, people’s resistance to drugs has made the treatment effect worse. Although there are many innovative therapies at present, the results are poor and the side effects are large. Therefore, there is still a need for a new treatment to solve this problem. Although many proposals have been made to solve this problem and a lot of efforts have been made, there is no clear solution.

What do we want to do?

A more in-depth study of the pathogenesis of pancreatic cancer and the search for new specific and effective pancreatic cancer standards have far-reaching significance for improving the early diagnosis and efficacy of patients and prognosis. So we try to solve this problem with our strength and knowledge.

How do we achieve our goals?

We extracted TIL cells from the patient's cancer cells and injected the engineered TIL cells back into the patient. TIL cells can target tumor masses where cancer cells localize and function, and exosomes automatically encapsulate mRNA and send it to cancer cells. This allows us to take different responses depending on the current state of the cell. If the cells are healthy, then the system will not start, so as to avoid harming normal cells to the greatest extent; if only a few cells are cancerous, the system will increase the amount of ALKBH5 through the ras-raf pathway receptor to attenuate the expression of the wnt pathway. Thereby inhibiting proliferation and invasion and metastasis; if a large number of cells have undergone cancer, the system will initiate an apoptosis program through the hypoxia-inducible factor system, releasing a large amount of casp3 to promote apoptosis of cancer cells.

Why is the effective application of synthetic biology?

We have designed a system that can respond to pancreatic cancer by determining the different states of the cell to produce different responses. We designed three parts, an anoxic induction system, an exosomes, and a RAS-RAF signaling pathway. The system we designed can play a large role in the early treatment of pancreatic cancer and is also desirable in the treatment of other cancers.

Introduction

In our project, we designed a system which can display different reactions by determining the current state of the cells. We used the regulatory line of protein-protein interactions to achieve this goal.
We stabilized the gene into engineered TIL cells, which transcribe the needed mRNA and deliver it to the target cells through the exosomes. We ligated N-TEVP on RAS and linked C-TEVP on RAF. The gene for TVMVP will be ligated after the HIF-1 hypoxia-inducible promoter.
For distantly metastatic cancer cells, the RAS-RAF-MAPK pathway is activated to produce active TEVP. For invasively growing cancer cells, the cells consume oxygen and form an anoxic microenvironment to induce promoter initiation and transcribe the mRNA of TVMVP.
We inserted a TEVP target site between N-ALKBH5 and C-ALKBH5. After activation of RAS-RAF, active TEVP is produced, and the target sequence between ALKBH5 is cleaved by TEVP to synthesize an active protein. Reversible expression of ALKBH5 low expression promotes pancreatic cancer proliferation, invasion and metastasis.
We linked the target site of TEVP between C-casp3 and N-casp3, and the TVMVP target site was ligated between N-casp3 and the efferent signal peptide. Cleavage by TEVP produces active casp3 (but it is still linked to the signal peptide, which cannot remain in the cell); when it is affected by the hypoxic microenvironment of the tumor, it causes a large amount of TVMVP to be expressed. The casp3 is cleaved to the efferent signal peptide, and the active casp3 is left in the target cell to exert a cell killing effect.

RAS-RAF pathway

The RAS-RAF pathway is an important link in the MAPK signaling pathway. The MAPK signal transduction pathway is the junction or the last common pathway for a variety of membrane receptor-mediated growth signal transmembrane transport, mediating growth, development, division, differentiation, apoptosis and intercellular interaction processes. Mainly have the following components：
1.GRB2: The junction of different signal transduction molecules in the GRB2 Grb2 (linker protein) signal transduction pathway, which links the upstream signal transduction molecule and the downstream signal transduction, mediates the protein interaction through the protein interaction domain.
2. SOS: SOS is a positive regulator of low molecular weight G protein and contains a motif structure that can be recognized and bound by SH3 of Grb2. In the Ras pathway, SOS is activated after binding to Grb2, acting on the low molecular weight G protein switch Ras, which promotes the release of GDP binding to GTP by Ras protein.
3. RAS: The RAS protein is a small GTP-binding protein with a GTPase domain that binds GTP to an inactive state and binds GTP to an activated state, so the RAS protein has a molecular switch function.
4. Mapk system MAPK system includes MAPKKK/Raf, MAPKK, MAPK/ERK.
Ras combines with Raf and activates Raf. Raf binds to and phosphorylates the Ser/Thr of MAPKK to activate it. MAPKK phosphorylates the Ser/Thr of MAPK to activate it. The system activates the entire signaling pathway by sequentially catalyzing the phosphorylation of lower levels of protein kinase.
ERK is a member of the MAPK family, whose signaling pathway is at the heart of a signaling network involved in regulating cell growth, development, and division. RAS-RAF signaling pathway activates ERK. Phosphorylation-activated ERK1/2 is translocated from cytoplasm to nucleus. Internally, it mediates the transcriptional activation of Elk-1, ATF, Ap-1, c-fos and c-Jun, and participates in cell proliferation and differentiation, cell morphology maintenance, cytoskeletal construction, apoptosis and cell carcinogenesis. Biological reactions.

Composability

Composability is a more stringent criterion than either orthogonality or modularity. Parts that are composable are modular units that have matching inputs and outputs and are designed so that any two parts can be connected to each other and yield predictable behavior. Standardization of parts and their interfaces is one way to develop such “plug-and-play” capability. Even electronic circuits, often considered easy to engineer compared to biological ones, are not necessarily composable, and combining parts can fail if input and output impedances are not designed appropriately; a similar concept in biological circuits has been termed retroactivity. Thus, synthetic biologists must carefully engineer natural parts to gain composability.
Composability can also be applied to the multicellular level, thinking of individual cells as composable units that can be programmed to interact with one another in predictable ways. Synthetic multicellular systems hold promise to aid in tissue regeneration and to program structured living materials capable of responding to the environment.
In the future, composability is likely to accelerate synthetic biology, especially when circuits at different levels are integrated to achieve much more powerful systems than currently feasible. With the long-standing limits of synthetic biology being crossed by means of composable toolkits for sequential, protein-based, and multicellular systems, the future is bright for the engineering of new capabilities in biomedicine and for gaining deeper biological understanding.

ALKBH5

ALKBH5 is an important demethylase that demethylates mRNA in the nucleus, has an alanine-rich region at the N-terminus and a unique coiled-coil structure. After knocking down ALKBH5 in the cell line, the level of m6A modification on mRNA was significantly increased. m6A demethylase ALKBH5 is under-expressed in pancreatic cancer, and decreased expression of ALKBH5 promotes pancreatic cancer cell proliferation and invasion. Metastasis of ALKBH5 expression promotes pancreatic cancer cell proliferation and invasion and metastasis. The wild-type TP53 protein promotes transcription of the ALKBH5 gene, whereas the TP53 mutant protein loses the function of promoting ALKBH5 transcription. In pancreatic cancer tissues with mutated TP53, TP53 promotes the attenuation of ALKBHS transcription, and the expression of ALKBH5 is decreased in cancer tissues, which may be an important regulatory mechanism upstream of ALKBH5. In pancreatic cancer, ALKBH5 is under-represented, the inhibition of the β-catenin/wnt/TCF4 pathway is weakened, and the pathway is activated to achieve its function.

HIF-1

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that acts as a major regulator of oxygen homeostasis and is ubiquitous in human and mammalian cells. It is also expressed under normoxia (21% O2), but synthetic. HIF-1 protein is rapidly degraded by the intracellular oxygen-dependent ubiquitin protease degradation pathway, and HIF-1 can be stably expressed only under hypoxic conditions. When hypoxia is localized, HIF-1 is overexpressed and promotes the progression of pancreatic cancer in a variety of ways. First, HIF-1 enhances the invasiveness of pancreatic cancer by regulating Fascin and LASP-1 expression of actin-binding protein, which is highly expressed in pancreatic cancer. Secondly, CX3CR1 forms a positive feedback loop, which mediates tumor cells to nerve tissue. Infiltration, while maintaining the expression of two proteins in pancreatic cancer, promotes the metabolic rearrangement of pancreatic cancer; again, HIF-1 also regulates the expression of related molecules that affect the proliferation of pancreatic cancer.
Fig. The 3D Structure of HIF-1

Exosome

Exosome are nanoparticles that can mediate intracellular communication by carrying RNA, DNA, and proteins. They can derive from membrane of donor cells (TIL cells) and can mix with recipient cells (cancer cells). There are three genes boost the production of exosomes。
Exosome booster:
 STEAP3,boost exosomes biogenesis.
 hSDC4,support budding of endosomal membranes.
 nadB,Enhance TCA cycle.
They combine with each other to boost exosomal transfer efficiency .

Logic Gate

AND: We use the target sequences of tevp and tvmvp to link YFP and CFP, and the YFP in the middle while two CFPs attached to each other on both sides. We implement the pathway function through this part. When FRET is used, the energy of the CFP is transferred to the YFP, and the YFP emits would be light. Only when the two cleavage enzymes are present at the same time, CFP should be separated from YFP, and when FRET is used again, the energy transfer of CFP to YFP is interrupted, and the CFP which is bascally not luminescent emits light and it's luminous intensity is large. If only one enzyme is present, CFP has general luminescence.
OR: We use the target sequence and tandem sequences of TEVP and TVMVP to link CFP and YFP,in order to achieving the function or pathway function. When FRET is used, the energy of the CFP is transferred to the YFP, and the YFP emits light. As long as we add any kind of cleavage enzyme (TEVP or TVMVP), we can cut off the energy transfer from CFP to YFP, and the CFP light that is bascally not luminescent.
NOR: The link we used was to link the C-terminus and the N-terminus of PPVp; P2A is a small peptide that can be self-cleaved, and a ppvp target sequence is added to YFP and CFP. This constitutes the nor path. When neither tevp nor tvmvp is present, active ppvp is produced, and then the target sequence is cleaved, and the energy transfer from CFP to YFP is cut off, and the CFP which is basically not luminescent emits light.
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