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Preparation and application of triple artificial miRNA in restraining of VEGFRs

A VEGFR2, artificial technology, applied in the field of biomedicine, can solve the poor prognosis of pancreatic cancer patients and other problems, achieve the effect of reducing growth, reducing cell migration and invasion ability, and promoting apoptosis

Active Publication Date: 2017-01-04
AFFILIATED HOSPITAL OF NANTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, studies have reported that EMT is involved in the metastasis of pancreatic cancer. It not only plays an important role in the invasion and metastasis of malignant tumors derived from epithelial cells, but also is closely related to the phenomenon of tumor resistance to apoptosis. The prognosis of pancreatic cancer patients with EMT is significantly poor.

Method used

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  • Preparation and application of triple artificial miRNA in restraining of VEGFRs
  • Preparation and application of triple artificial miRNA in restraining of VEGFRs
  • Preparation and application of triple artificial miRNA in restraining of VEGFRs

Examples

Experimental program
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Effect test

Embodiment 1

[0033] 1) Large-scale pancreatic cancer tissue chips (including 168 pancreatic cancers and 40 benign pancreatic diseases), clinical data include gender, age, tumor diameter, location, degree of differentiation, metastasis, 5-year overall survival rate, etc. The samples are used by the hospital The ethics committee agreed. Paraffin tissue microarray sections were repaired with high-temperature antigens, and the primary antibody was incubated overnight at 4°C. According to the number of positive cells and staining intensity, they were divided into high expression, low or no expression. It was confirmed by statistical analysis that the three subtypes of vascular endothelial growth factor receptor The expression of VEGFRs in cancer tissues is higher than that in benign pancreatic tissues. VEGFR1 and VEGFR3 are not only highly expressed in tumor cells, but also have different degrees of positive expression in tumor stroma; the combined high expression of VEGFRs in cancer and pancrea...

Embodiment 2

[0052] The artificial miRNA (artificial miRNA, amiRNA) targeting the three target genes VEGFR1, VEGFR2, VEGFR3 were constructed in pcDNA TM 6.2-GW / miR vector, such as image 3 As shown, it is used to express a specific artificial miRNA, which is engineered to have 100% homology with the target gene sequence and can cause cleavage of the target molecule. The vector is transfected into the cell, and the engineered Pre-miRNA formed by transcription from the strong CMV type II (Pol II) promoter contains the flanking sequence and loop sequence of mouse miR-155, in which the 5' and 3' The flanking sequence makes the transcribed Pre-miRNA similar in structure to miR-155, and the efficiency of cutting the target gene is improved by artificially optimizing the loop sequence. Cloning of miRNA by quick ligation protocol and transfection can immediately express pre-miRNA, and the expressed premiRNA is processed by endogenous cellular machinery (including Drosha enzyme) in the nucleus, t...

Embodiment 3

[0080] The proliferation experiment of embodiment 3 cell count (CCK8)

[0081] For two cell lines, SW1990 and PANC-1, the cell concentration was adjusted to 5×10 4 cells / mL, inoculated in 96-well culture plate, 100 μL per well, cultured in 37°C, 5% CO2 incubator for 24 hours. Drugs with different concentration gradients were added to the 96-well plate, 150 μL per well, and each group had three replicate wells. After 6h, 12h, 24h, 48h, and 72h, CCK8 (product of Sigma Company) was detected. Add 15 μL of CCK8 per well at 37°C, 5% CO 2 Incubate in the incubator for 3 h in the dark. The OD value at the same time point was measured by an enzyme label detector (Thermo MK3 type) at a wavelength of 492 nm, and the effect of cell proliferation was analyzed using the measured OD value.

[0082] The result is as Figure 6 As shown, compared with the control cells, the triple artificial miRNA targeting VEGFRs inhibited the proliferation of PANC-1 and SW1990; the inhibition rate of SW1...

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Abstract

The invention discloses a triple artificial miRNA carrier in restraining of VEGFRs and a construction method and application thereof. According to the triple artificial miRNA carrier in restraining of VEGFRs, artificial miRNAs are constructed in a pcDNATM6.2-GW / miR carrier and targeted to three target genes, namely VEGFR1, VEGFR2 and VEGFR3, the three artificial miRNAs are connected in series to be connected to a skeleton carrier of psliencer4.1 to form the triple amiRNA carrier, and the triple amiRNA carrier exerts the effect of silencing the three genes, namely VEGFR1, VEGFR2 and VEGFR3 at the same time. The triple amiRNA reduces proliferation of pancreatic cancer cells, accelerates apoptosis, and reduces the capacity of cell migration and invasion; in a nude mouse pancreatic cancer transplanting model, the triple amiRNA-VEGFRs obviously reduces the growth speed of a tumor, achieves a synergistic effect with chemotherapy drugs, and is related to restraining of epithelium-mesenchyme conversion; the pancreas shape and the insulin and blood glucose level of peripheral blood are not influenced. In this way, the triple artificial miRNA in restraining of VEGFRs can be well applied to preparation of medicine for treating a pancreatic cancer and other malignant tumors with high expression VEGFRs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to the preparation and application of a triple artificial miRNA inhibiting VEGFRs. Background technique [0002] Pancreatic cancer is one of the common malignant tumors of the digestive system, with high malignancy, low early diagnosis rate, rapid progression, and extremely poor prognosis. Its case fatality rate ranks fourth among males and third among females among malignant tumors. Although a variety of methods have made some progress in the diagnosis and treatment of pancreatic cancer in recent years, its prognosis is extremely poor, the rate of postoperative recurrence and metastasis is high, it is not sensitive to radiotherapy and chemotherapy, it is prone to multidrug resistance, and the 5-year survival rate is still low at 4%. Improving the therapeutic effect of pancreatic cancer is a difficult problem facing the medical community, so it is of great signific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66A61K31/7105A61K48/00
CPCA61K31/7105C12N15/63C12N15/66
Inventor 黄剑飞王志伟咸云曹学敏李洁莹张筱静
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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