Unlock instant, AI-driven research and patent intelligence for your innovation.

Application of single-stranded guide RNA in preparation of targeted drugs for treating cervical cancer and like

A technology for cervical cancer and cervical cancer cells, applied in the field of targeted drugs, can solve the problems of tumor invasion and poor prognosis, and achieve the effect of inhibiting proliferation and migration

Active Publication Date: 2021-08-03
TIANJIN MEDICAL UNIV
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to the physiological role, the EphA7 gene is also related to the occurrence of tumors, but its gene expression is tissue-specific, such as in hepatocellular carcinoma, glioblastoma multiforme, pancreatic adenocarcinoma, gallbladder carcinoma, squamous cell carcinoma of the tongue and High expression in non-small cell lung cancer, which is related to tumor invasion and poor prognosis

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Application of single-stranded guide RNA in preparation of targeted drugs for treating cervical cancer and like
  • Application of single-stranded guide RNA in preparation of targeted drugs for treating cervical cancer and like
  • Application of single-stranded guide RNA in preparation of targeted drugs for treating cervical cancer and like

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0071] 1. Reduce the level of gene methylation and activate gene transcription and expression:

[0072] (1) Design and synthesis of sgRNA

[0073] 1. Design of sgRNA

[0074] According to the design principles of sgRNA, a total of 6 sgRNAs that can activate gene expression were designed in the PAX1 / EphA7 promoter region, and the sequences are as follows,

[0075]

[0076] 2. Synthesis and purification of sgRNA

[0077] Based on the synthesized sgRNA sequence, the U6-sgRNA backbone with a U6 promoter was used to express the recombinant plasmid, and the complete sgRNA was synthesized by two-step PCR ( figure 1 A), purified after synthesis, and verified the quality of sgRNA by agarose gel ( figure 1 B), stored at -20°C.

[0078] (2) Construction of cell lines

[0079] Cervical cancer cell lines Caski and Siha cells in logarithmic growth phase were inoculated in 6-well plates one day in advance, and transfected when the density reached 75-80%.

[0080] 2 μg of recombinant p...

Embodiment approach

[0126] (3.1) Cell proliferation curve

[0127] 1×10^5 cervical cancer Siha cells were inoculated on a 24-well plate, and the experimental group (dCas9-Tet1(Addgene#84475)) and the control group were evenly spread in 15 wells (3 replicate wells in each group, a total of 5 days). The sgRNA combination targeting PAX1 and the sgRNA combination targeting EphA7 were co-transfected in equal proportions with dCas9-Tet1 (Addgene #84475) into the cervical cancer Siha cells of the experimental group, and digestion and counting began after 24 hours, and each group digested every day The number of cells in the 3 wells was counted every 24 hours.

[0128] (3.2) Migration experiment

[0129] Siha cells in the logarithmic growth phase were seeded in 6-well plates one day in advance, and when the density reached 70%, the sgRNA combination targeting PAX1 and the sgRNA combination targeting EphA7 were mixed with dCas9-Tet1 (Addgene# 84475) were co-transfected into the Siha cell line, scratched...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the technical field of gene engineering, and discloses an application of a single-stranded guide RNA in preparation of targeted drugs for treating cervical cancer and the like. An sgRNA gene sequence for activating and regulating the transcription expression level of a PAX1 / EphA7 gene can be used for effectively reducing the methylation level of the PAX1 / EphA7 gene. The sgRNA gene sequence for inhibiting the transcription expression level of the PAX1 / EphA7 gene can be used for effectively increasing the methylation level of the PAX1 / EphA7 gene. According to the invention, sgRNA and a connection effector (activating / inhibiting protein; the dCas9 recombinant plasmids (such as methylated / demethylated enzyme and the like) act on regulation regions of the PAX1 and EphA7 genes in a targeting manner, so that the methylation level is reduced or increased, and the transcriptional expression of the PAX1 and EphA7 genes is endogenously regulated.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the use of a single-stranded guide RNA (sgRNA) in preparing targeted drugs for treating cervical cancer and the like. Background technique [0002] Current studies have confirmed that the occurrence and development of cancer are closely related to the regulation of epigenetics. Among them, DNA methylation (DNA methylation) is one of the most important modification methods of epigenetic regulation. Methylation modification in the promoter region of some tumor suppressor genes can reduce its transcriptional activity, silence gene expression, and lead to Abnormal proliferation of cells to produce malignant tumors. Therefore, abnormal DNA methylation is not only expected to become a biomarker for identifying and diagnosing diseases, but also targets DNA methylation for treatment, resets methylation imbalance, regulates oncogene transcription to regulate gene ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N5/10A61K31/7105A61P35/00C12Q1/6886
CPCC12N15/1135C12N15/85A61K31/7105A61P35/00C12Q1/6886C12N2310/10C12Q2600/154
Inventor 王蓉张文帆
Owner TIANJIN MEDICAL UNIV