SiRNA interfering with HSPA13 expression and application thereof

A gene expression and molecular technology, applied to siRNA that interferes with HSPA13 expression and its application field, can solve the problem of not studying the EMT process of HSPA13 epithelial cells, and achieve the effect of reducing recurrence and avoiding risks.

Active Publication Date: 2019-01-15
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous research reports have confirmed that HSPA13 gene is highly expressed in liver cancer, squamous head and neck cancer

Method used

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  • SiRNA interfering with HSPA13 expression and application thereof
  • SiRNA interfering with HSPA13 expression and application thereof
  • SiRNA interfering with HSPA13 expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0047] Example 1

[0048] Establish an in vitro EMT cell model to detect the expression level of HSPA13

[0049] Establish an in vitro EMT cell model: When the confluence of ARPE19 cells is about 60-70%, they are treated in serum-free medium overnight, and then ARPE19 cells are treated with 10ng / ml TGFβ1 to induce EMT in ARPE19 cells. The induced RPE cell morphology changed from epithelial to fibrous morphology, and at the same time lost tight junctions, and cell migration was also accelerated.

[0050] Q-PCR, Western blot and immunofluorescence to detect the expression level of HSPA13

[0051] Q-PCR, Western blot and immunofluorescence were used to detect the expression level of HSPA13 before and after ARPE19 cells were induced.

[0052] (1) Q-PCR detection

[0053] The normal control and TGFβ1 induced cells were collected with 1ml Trizol in a 1.5ml EP tube for total RNA extraction. After reverse transcription and real-time quantitative PCR analysis, a semi-quantitative method is used...

Example Embodiment

[0094] Example 2

[0095] Detection of HSPA13 and EMT related gene expression after plasmid transfection and TGFβ1 induction

[0096] 1. Cell preparation: Inoculate the cells in a 6cm Petri dish, and when the cell growth confluence reaches 60-70%, starve the cells overnight with serum-free medium.

[0097] 2. The siRNA molecule has the sequence shown in SEQ ID NO. 17, namely GCCUGACGUCUUCCACGUC. The long hairpin DNA molecule corresponding to the siRNA molecule was annealed in vitro and then ligated with the empty plasmid pSuper digested with BglII and Hind III to obtain the plasmid HSPA13siRNA. The sequence was verified by a small amount of plasmid extraction and sequencing.

[0098] 3. Plasmid transfection: use Invitrogen's Lipofectamine 3000 transfection reagent for plasmid transfection. Operate the prepared empty plasmid pSuper and plasmid HSPA13siRNA according to the transfection reagent instructions.

[0099] 4. Treat with TGFβ1 after 48h of transfection.

[0100] 5. After 48 hour...

Example Embodiment

[0103] Example 3

[0104] Lentivirus infection and streaming screening

[0105] 1. Cell treatment: inoculate the cells in a 6-well plate and perform the corresponding cell treatment (as described in step 1 of Example 2);

[0106] 2. Infection: Use lentivirus of appropriate concentration and volume, add it to serum-free cell culture medium, make a label, and change to a complete medium containing 10% fetal bovine serum after four hours.

[0107] 3. Microscopic observation: After 36 hours of infection, observe the green fluorescence under a fluorescence microscope, that is, whether there is expression of green fluorescent protein.

[0108] 4. Flow cytometry: After the green fluorescence is observed, perform flow cytometry. The specific steps are roughly described as follows: After the cells to be screened are trypsinized and centrifuged, the cells are dispersed into individual cells using a 0.7um filter and collected into the flow Place them in a BD tube and place them on ice; prepare tw...

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Abstract

The invention relates to siRNA interfering with HSPA13 expression and application thereof, in particular to siRNA molecule interfering with HSPA13 expression and application of the siRNA molecule as therapeutic drug for preparing proliferative vitreoretinopathy. The siRNA molecule is adopted to interfere with the expression of HSPA13, realizes the occurrence and development of effective treatmentof PVR from the gene molecule level, and can avoid the risks caused by other treatment methods.

Description

technical field [0001] The present invention relates to the research of proliferative vitreoretinopathy (PVR), especially relates to the siRNA that interferes with the expression of HSPA13 and its application, and the application of the siRNA that interferes with the expression of HSPA13 as a drug for the preparation of proliferative vitreoretinopathy. The siRNA interferes with the expression of HSPA13 To suppress or alleviate the symptoms of PVR and delay its progression. Background technique [0002] Proliferative vitreoretinopathy (PVR) is a response to wound healing in the retina, and it is also the most common cause of failure of rhegmatogenous retinal detachment surgery. PVR can also be seen as the growth and shrinkage of cell membranes in the vitreous cavity and on the surface of the retina. Further contraction and stretching of these membranes can re-open successfully treated retinal tears or create new holes, deform the macula, and form traction retinal detachment....

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/7105A61P9/10A61P27/02
CPCA61K31/7105A61P9/10A61P27/02C12N15/113C12N2310/141
Inventor 吕立夏徐国彤李梦雯王娟张介平
Owner TONGJI UNIV
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