Inhibition of invasion and transferring of prostatic cancer by utilization of targeting polar molecule Par3

A prostate cancer, 1.par3 technology, applied in the field of biomedicine, can solve the problems that the specific mechanism of prostate cancer metastasis is not yet clear, the upstream signal and signal transduction regulation mechanism are unknown, etc.

Inactive Publication Date: 2016-01-20
RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the molecular mechanism of the Hippo-YAP pathway itself has been basically clarified, the upstream signal that activates the pathway and the regulatory mechanism of signal transduction are still unclear. Not only that, but also the specific mechanism for the pathway to regulate prost

Method used

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  • Inhibition of invasion and transferring of prostatic cancer by utilization of targeting polar molecule Par3
  • Inhibition of invasion and transferring of prostatic cancer by utilization of targeting polar molecule Par3
  • Inhibition of invasion and transferring of prostatic cancer by utilization of targeting polar molecule Par3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] A subcloned cell line PC3-shPar3 stably knocking down Par3 and a control PC3-con were constructed and identified in the prostate cancer cell line PC3 (the human prostate cancer cell line PC3 is used here as an example, but not limited thereto).

[0043] Prostate cancer cell line PC3 was purchased from the Cell Bank of the Chinese Academy of Sciences, and Par3 interference plasmids (pshPar3#1, pshPar3#2) and control plasmids were purchased from Origen Company (Cat. The designed shRNA against Par3 (i.e. shPar3#1 and shPar3#2), also contains a control backbone plasmid). Will 1x10 6 PC3 cells were seeded in 6-well plates, and 24 hours later, the Par3 interference plasmid and the control plasmid were transfected with lipofectarmine2000 transfection reagent (purchased from LifeTechnology Company). 72 hours after transfection, the cells were transferred to a culture dish with a diameter of 10 cm by a conventional adherent cell subculture method and expanded to culture until t...

Embodiment 2

[0046] In vitro transwell experiments (using PC3-shPar3 and control PC3-con cells as examples, but not limited thereto).

[0047] BD company's transwell cell culture plate (the size of the mesh at the bottom of the upper chamber with a pore size of 8 microns) was used to conduct tumor cell migration (without matrigel coating) and invasion (with matrigel coating) experiments. Adherently cultured PC3-con and PC3-shPar3 cells were serum-starved for 24 hours, digested and prepared a serum-free cell suspension at a density of 5x10 5 / ml. 100 microliters of cell suspension was added to the upper chamber, 700 microliters of medium containing 10% FBS was added to the lower chamber, and the cell culture plate was routinely cultured in a cell culture incubator. For the migration experiment, after 4 hours of conventional culture, the upper chamber was taken out, and the residual cell suspension inside the chamber and on the screen was sucked off using a micropipette. The upper chamber ...

Embodiment 3

[0049] Scratch test (using PC3-shPar3 and control PC3-con cells as an example, but not limited thereto).

[0050] PC3-con and PC3-shPar3 cells that were conventionally adhered to the wall were digested with trypsin and seeded in a 6-well plate at a seeding density of 70%-80%. Routine culture in the cell incubator for 24 hours after inoculation to ensure that the cells completely cover the bottom of the culture plate. Use a 1ml micropipette tip perpendicular to the orifice plate and use a scale to assist in scratching, try to ensure that the width of each scratch is consistent. After scratching, the cell culture medium was sucked off, and the plate was washed twice with PBS to wash away the cell debris produced by the scratching. Add DMEM containing 2% serum for routine culture in a cell culture incubator. Three time points of 0 hour, 24 hours and 48 hours after the scratch were selected, and microscopic photography was performed under bright field using a phase contrast micr...

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Abstract

The invention relates to inhibition of invasion and transferring of prostatic cancer by utilization of a targeting polar molecule Par3. A prostate cancer cell subcloning cell line of the stable knock-down Par3 is constructed, a corresponding nude mouse prostate in-situ transplanting tumor model is established, and the effects of the stable knock-down Par3 on inhibition of invasion and transferring of the prostatic cancer are verified in vitro and in vivo. After the stable knock-down Par3 is discovered, through damage of formation of a Par3-aPKC-KIBRA ternary complex, phosphorylation of an important member MST1/2 and Lats1 in a Hippo passage is enhanced, therefore the Hippo passage is activated, a protooncogene YAP at the downstream is phosphorylated, thus the cytoplasm ofthe YAP protein is retarded, nuclear import of the non-phosphorylated YAP protein is reduced, and finally interaction of YAP and TEAD transcription factors in cell nuclei is weakened, and transcription of a series of invasion/ transferring promoting genes is inhibited. The fact that the stable knock-down Par3 has functions of inhibiting invasion and transferring of prostatic cancer cells is verified in vitro, in vivo, on phenotypes and in molecular mechanism.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a polar molecule partitioningdefective3homolog (Par3) expressed by eukaryotic cells. Using specific shRNA to stably target and knock down Par3 in prostate cancer cells can effectively inhibit the invasion and metastasis of prostate cancer, suggesting that Par3 as a potential drug target can be applied to the invasion and metastasis of prostate cancer in targeted gene therapy. Background technique [0002] Prostate cancer is a common malignant tumor in elderly men, and its incidence rate ranks second among male tumors worldwide. In my country, the incidence of prostate cancer increases significantly after the age of 60 and reaches a peak at the age of 75-79 [1]. Data in recent years have shown that the 5-year survival rate of patients with localized prostate cancer is almost 100%, while the 5-year survival rate of patients with distant metastasis is only 29% [2], revealing...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P35/04A61P35/00
Inventor 高维强方煜翔周培杰
Owner RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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