Difference between revisions of "Team:NJU-China/Proof"

Anti-KRAS siRNA (siRNA for KRAS gene silencing)

Introduction

This part is an artificially designed RNA strand. It serves as an element of the Team NJU-CHINA RNAi module which can be used for down-regulation of KRAS expression in lung adenocarcinoma cells. We designed specific KRAS siRNA with algorithm based on a software developed by SYSU-Software team. This tool can find the best siRNA sequence on target gene KRAS to ascertain the maximum gene-specificity and silencing efficacy and also designs the pair of oligonucleotides needed to generate short hairpin RNAs (shRNAs) in the plasmid. Then we synthesize the shRNA sequence from a DNA synthesis company (Genscript).

Usage and Biology

We packaged KRAS siRNA into exosomes by transfecting HEK293 cells with a plasmid expressing KRAS siRNA and then collected siRNA-encapsulated exosomes. When modified exosomes being intravenously injected, they will specifically recognize integrin receptors and fuse with lung adenocarcinoma cells under the direction of the iRGD peptide. Once inside cells, KRAS siRNA will bind to KRAS mRNA through base-pairing and digest the mRNA, resulting in sharp decrease of K-ras in lung cancer cells. As a consequence, K-ras protein’s reduction and disturbed function will both result in the inhabitation of the proliferation of cancer cells, which ultimately have some therapeutic effects on lung cancer (non-small cell lung cancer in this case).

Characterization

Interference efficiency of anti-KRAS siRNA plasmid

To ensure the interference efficiency of anti-KRAS siRNA plasmid, we transfected it into human lung adenocarcinoma cell line A549 and then extracted protein from these cells to perform western blot. Significant down-regulation of K-ras can be observed in A549 cells treated with anti-KRAS siRNA, demonstrating that anti-KRAS siRNA has the gene silencing effect on lung cancer cells.

A coding sequence of iRGD peptide and position it outside the membrane.

Usage and Biology

iRGD is a tumor-penetrating peptide that can increase vascular and tissue permeability. Importantly, this effect did not require the drugs to be chemically conjugated to the peptide. To enhance the accuracy of drug delivery system and improve targeting index of drugs, iRGD peptide was displayed on the surface of the exosome containing our previously designed siRNA, allowing us to target recipient cells in vivo efficiently. Lamp-2b is a protein found specifically abundant in exosomal membranes. So we connect iRGD with Lamp2b by a glycine-linker, and promote the expression using cmv promoter. We engineered our chassis, human embryonic kidney 293 (HEK293) cells, to express iRGD-Lamp2b fusion protein. Therefore, the iRGD exosomes (iRGD-Exos) are endowed with site-specific recognition ability and were purified from cell culture supernatants and loaded with Dox by electroporation.

Characterization

The iRGD-Lamp2b expressing vector was thoroughly described in Tian’s article (Yanhua Tian, et al. Biomaterials, 2013). He showed that exosomes, endogenous nano-sized membrane vesicles secreted by most cell types, could deliver chemotherapeutics such as doxorubicin (Dox) to tumor tissue in BALB/c nude mice. To reduce immunogenicity and toxicity, mouse immature dendritic cells (imDCs) were used for exosome production. Tumor targeting was facilitated by engineering the imDCs to express a well-characterized exosomal membrane protein (Lamp2b) fused to αν integrin-specific iRGD peptide (CRGDKGPDC). Purified exosomes from imDCs were loaded with Dox via electroporation, with an encapsulation efficiency of up to 20%. iRGD exosomes showed highly efficient targeting and Dox delivery to αν integrin-positive breast cancer cells in vitro as demonstrated by confocal imaging and flow cytometry. Intravenously injected targeted exosomes delivered Dox specifically to tumor tissues, leading to inhibition of tumor growth without overt toxicity. The results suggested that exosomes modified by targeting ligands could be used therapeutically for the delivery of Dox to tumors, thus having great potential value for clinical applications in our project.

1.1 KRAS siRNA interference efficiency verification in vitro

To ensure the interference efficiency of anti-KRAS siRNA, we transfected the plasmid loaded with this siRNA into human lung adenocarcinoma cell line A549 and extracted protein to perform western blot. Significant down-regulation of K-ras can be observed in A549 cells treating with anti-KRAS siRNA plasmid, compared with the control group, demonstrating that anti-KRAS siRNA has a gene silencing effect on lung cancer cells.

1.2 TEM imaging of exosomes carrying KRAS siRNA and expressing iRGD peptide on their membrane

After co-transfection of the two plasmids mentioned above, we performed a transmission electron microscopy (TEM) to characterize the iRGD-KRAS-siRNA-exosomes. The TEM image showed that the exosomes presented normal morphological characteristics after outside modification and siRNA loading, with a diameter of approximately 200 nanometer and a double-layer membrane.

1.3 Nanoparticle tracking analysis to ascertain the relationship between protein concentration and exosomes quantity

We then asked NUDT_CHINA to help us perform nanoparticle tracking analysis (NTA) for a further evaluation of the quantity and size of secreted exosomes. The use of Nanosight enabled quantification and size determination of the extracellular vesicles, as nanoparticles can be automatically tracked and sized based on Brownian motion and the diffusion coefficient. The size of exosomes attained ranged around 270nm. Basing the particle size and relative intensity, we also created a 3D plot for a visual explanation. Under measurement condition listed, the exosomes secreted by HEK293 cells were assayed for 2.95 E8 particles each milliliter. Then the relationship between particle number and protein was determined that exosomes in 1 ng protein were equivalent to 6277.95 particles, according to the dilution multiple (24) and protein concentration (1127.756 ng/ul) we have tested. All the data collected helped us decide the transfection dosage of siRNA and dosing of treatment prepared for animal experiment.

1.4 The iRGD-KRAS-siRNA-exosomes suppressed KRAS expression in A549 cells in vitro

We next evaluate the effect of iRGD-KRAS-siRNA-exosomes on KRAS expression in vitro. The KRAS expression level was assayed in A549 cells after co-cultured with exosomal KRAS siRNA. Non-loaded iRGD-exosomes were used as control to ascertain that any RNAi response observed did not derive from the exosomes per se. The western electrophoresis and knockdown data obtained from qPCR analysis of KRAS gene expression indicated that KRAS protein and mRNA levels both dramatically decreased in the cells incubating with iRGD-KRAS-siRNA-exosomes compared with cells treating with nude exosomes or without any treatment. This result suggests that iRGD-KRAS-siRNA-exosomes can deliver siRNA into target cells and finally reduce the KRAS expression.

1.5 The iRGD-KRAS-siRNA-exosomes efficiently arrests cancer cell proliferation in vitro

KRAS over-expression has been demonstrated to promote the growth of lung adenocarcinoma cells and be involved in migration and invasion of lung cancer. For further verification, we examined the role of iRGD-KRAS-siRNA-exosomes in cell proliferation. An EDU assay using Cell-Light™ EdUTP Apollo®567 TUNEL Cell Detection Kit, was carried out after siRNA-iRGD-exosome incubation and as a control, nude exosomes were also treated to A549 cells. The result indicated that KRAS knockdown had an anti-proliferation effect on lung tumor cells while nude exosomes were not capable of inhibiting the growth of tumor cells.

2.1 Establishment of none-small cell lung cancer mouse model with 40 mice to perform validation experiment in vivo

The iRGD-KRAS-siRNA-exosomes can be released into A549 cells and suppress the expression of KRAS in vitro. To examine the consequence of KRAS knockdown by anti-KRAS siRNA in vivo, we built a non-small cell lung cancer mouse model for in vivo experiment. Forty mice were subcutaneously injected A549-LUC cells to realize tumor implantation. Then tumor volume were measured through bioluminescent imaging several times after injection. The areas that emit fluorescence in the mice bodies represent labeled tumors (tumors developed from the implanted A549-LUC cells), which helps us to monitor the tumor growth, location and metastasis.

2.2 Mice were divided to receive different treatment, testing the function of iRGD-KRAS-siRNA-exosomes

Mice were randomly assigned to 2 groups (n=20 per group) and treated differently. One group received PBS injections, the other group is treated with iRGD-KRAS-siRNA-exosomes via tail-vein injections. The administrations were given five times for 2 successive weeks since contamination of HEK293 cells resulted in exosomes shortage and the treatment of mice were delayed.

2.3 The measurement of tissues harvested indicates the function of KRAS siRNA on the tumor treatment

Subsequently, mice were sacrificed for tumor harvest after in vivo imaging. All the tumors were measured for length, volumes and weight. Small pieces from every tumor was cut independently and fixed by paraformaldehyde to prepare for histopathological examination.

2.4 Pathological section to observe tumor cells

After measurement of tumors, HE staining was conducted to verify the function of iRGD-exosomal siRNA. HE staining enabled better visualization of tissue structure and cell morphology, which can be used for morphological observation of normal and diseased tissue. The detection result showed that tumor cell necrosis rate in experimental group was much higher than control group. In other words, iRGD exosomes KRAS siRNA can effectively induce tumor cell necrosis and suppress cell proliferation

Figure 11. Pathological section of tumor tissues from model mice treated with PBS or iRGD-KRAS-siRNA-exosomes (exosome).

2.5 iRGD-KRAS-siRNA-exosomes reduce the KRAS expression in tumor cells in vivo

Then, total protein and RNA were extracted from the rest tumor tissues to evaluate the expression level of KRAS in vivo. Results showed both KRAS protein and mRNA level were reduced in tumor cells of mice injected with KRAS-siRNA-iRGD-exosomes compared with mice treated with PBS. Though the down-regulation is not that evident, it still demonstrated that iRGD-KRAS-siRNA-exosomes can efficiently be delivered into tumor cells and regulate target gene expression, taking the short time of treatment into account.

Figure 12. Quantitative RT-PCR analysis of KRAS mRNA level in tumor cells after treatment of PBS and KRAS-siRNA-iRGD-exosomes (exosome). (***: p < 0.001)

Endotoxin is a type of natural pyrogen that was found in outer cell membrane of Gram-negative bacteria and can make impact on over 30 biological activities. To ensure the safety of our drug system, avoid toxicities, thus prove that our achievement is of great value for clinical application, a detecting assay was carried out using an endotoxin test kit. The result was negative, demonstrating that our drug system satisfies the safety requirement.

KRAS mutation was identified in NSCLC more than 20 years ago, but its clinical importance in cancer therapy just began to be appreciated. Our project aimed to develop a drug system that employed modified exosomes with iRGD peptide on its surface to deliver KRAS-siRNA into lung cancer cells specifically, thus target KRAS gene and down-regulate K-ras protein expression to treat lung cancer cases. Our results had demonstrated that iRGD-KRAS-siRNA-exosomes can be delivered into tumor cells and efficiently down-regulate KRAS expression both in vitro and in vivo. In silencing validation, we transfected KRAS siRNA into A549 cells using Lipo 2000 and performed western blot to test the function of our siRNA. Subsequently, we collected iRGD-modified exosomes loaded with KRAS siRNA, evaluating its effect on lung cancer cells (A549) by examining KRAS expression after transfection. The result confirmed that iRGD-KRAS-siRNA-exosomes could effectively down-regulate KRAS transcription level and reduce its protein expression. Later, the EDU assay further ascertained the biological role of KRAS siRNA in cell proliferation suppression in vitro.

To perform in vivo validation, none-small cell cancer mouse model was established after implanting tumor in 40 mice by subcutaneous injection with A549-LUC cells. After a short-time treatment, tumors harvested from killed mice were measured, then protein and RNA extracted from these tissues were examined, results supporting that KRAS siRNA can efficiently suppress KRAS expression, inhibit cell proliferation and thus have its potential to be taken as a cancer treatment. Besides, endotoxin detecting validated that our drug system satisfies the safety requirement and won’t impact on biological activities. To be more meaningful, we purified the batch of iRGD-KRAS-siRNA-exosomes for liquid fill, completing a full process including experiment design, compounds synthesis, effect test and drug production.