Reagent for interfering with expression of hsa-mir-449a and application thereof

A technology of hsa-mir-449a and sh-mir-449a, which is applied in the field of genetic engineering, can solve the problems that have not been clearly reported and the regulatory mechanism of tumor stem cells is unclear, so as to delay the growth of tumors in vivo, inhibit self-renewal, and inhibit proliferation Effect

Active Publication Date: 2020-08-11
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the internal regulatory mechanism of tumor stem cells is still unclear, and whether inhibitors targeting tumor stem cell regulatory molecules can be used to assist tumor therapy has not been clearly reported.

Method used

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  • Reagent for interfering with expression of hsa-mir-449a and application thereof
  • Reagent for interfering with expression of hsa-mir-449a and application thereof
  • Reagent for interfering with expression of hsa-mir-449a and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Detection of the expression of Hsa-miR-449a in liver cancer stem cell-like cell populations

[0054] The human primary liver cancer cell line PLC / PRF / 5 was cultured into sphere-like cells by the method of sphere culture, which was recorded as PLC / PRF / 5sphere; Nanog-positive liver cancer stem cells were sorted by flow cytometry (remark for Nanog pos ) and Nanog-negative non-liver cancer stem cells (denoted as Nanog neg ). PLC / PRF / 5sphere were detected by RT-PCR; Nanog pos and Nanog neg The expression of Hsa-miR-449a in the cells and the sorafenib-resistant cell line PLC / PRF / 5DrugResistance constructed in our laboratory.

[0055] Experimental results such as figure 1 Shown: Hsa-miR-449a in Nanog-positive liver cancer stem cells (denoted as Nanog pos ) and high expression in sorafenib-resistant cell lines, suggesting that Hsa-miR-449a may be an oncogene and is related to the regulation of liver cancer stem cells.

Embodiment 2

[0056] Example 2 Construction method of reagents for interfering with Hsa-miR-449a expression

[0057] The synthesis of shRNA in the nucleus of transfected / transduced cells forms a hairpin structure that can recognize target mRNA and degrade it.

[0058] (1) Packaging of lentivirus Lv-sh-miR-449a and Lv-sh-NC

[0059] Spread 2 to 5×10 in a 10cm petri dish 6 For 293T cells, the culture medium contained 10% FBS in DMEM (v / v). When the cells reached 70% confluence, change to fresh 10% FBS in DMEM (v / v) culture medium, and used for transfection after 3-4 hours. dye.

[0060] Take 15-20μg of pLv-sh-miR-449a and pLv-sh-NC lentiviral expression plasmids, and add 50ul of 2.5M CaCl dropwise to the mixture 2 , and then add sterile water to a total volume of 500 μL, mix well and then add 500 μL 2×HBS buffer solution, shake and mix well after the addition, leave at room temperature for 5 minutes, add the liquid to the cultured HEK 293T and mix well, 6- After 12 hours, replace with fre...

Embodiment 3

[0064] Example 3 Nanog that interferes with Hsa-miR-449a pos Liver cancer stem cell construction

[0065] According to the method sorting Nanog of embodiment 1 pos Liver cancer stem cells were planted in a 6-well plate until the cells grew to 1×10 6 At the same time, lentivirus Lv-sh-miR-449a and Lv-sh-NC particles were added, and the interference efficiency of Hsa-miR-449a was detected by qRT-PCR after 72 hours. The result is as image 3 The indicated effect of Lv-sh-miR-449a on Nanog pos Liver cancer stem cells have high interference efficiency, and obtained Nanog after interference with Hsa-miR-449a pos Liver cancer stem cells.

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Abstract

The invention belongs to the field of genetic engineering, and relates to a reagent for interfering with expression of Hsa-miR-449a and application of the reagent and mainly relates to the applicationof the reagent in preparing medicine for inhibiting self-renewal of liver cancer stem cells. The nucleotide sequence of a cancer gene Hsa-miR-449a for promoting the self-renewal of the liver cancer stem cells is shown in SEQ ID NO:1. The invention discloses the expression of Hsa-miR-449a in the liver cancer stem cells for the first time, and the expression is crucial to maintaining the self-renewal of the liver cancer stem cells. The application of the Hsa-miR-449a gene in inhibiting the self-renewal of the liver cancer stem cells is to inhibit the self-renewal of the liver cancer stem cellsby interfering with the expression of Hsa-miR-449a in the liver cancer stem cells. The reagent for interfering with the expression of Hsa-mi-449a can be prepared into the medicine for inhibiting the self-renewal of the liver cancer stem cells, is used for targeted treatment of liver cancer and has an important significance in prolonging the survival time of a liver cancer patient.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a reagent for interfering with the expression of Hsa-miR-449a and its application, in particular to the application in the preparation of a drug for inhibiting the self-renewal of liver cancer stem cells. Background technique [0002] Liver cancer is one of the most common malignant tumors in the world. Its pathogenesis has not yet been clarified. The current treatment of liver cancer is not ideal. Postoperative recurrence and metastasis are still a major problem to be solved. Recent studies have made the concept of cancer stem cells (CSCs) deeply rooted in the hearts of the people, and more and more evidences show that CSCs are closely related to tumor growth, drug resistance, recurrence and metastasis. So far, the existence of tumor stem cells has been confirmed in more than 30 common tumors such as blood system tumors, glioma, liver cancer, breast cancer, and colorectal cancer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K48/00A61K31/713A61P35/00C12N15/867
CPCA61K31/713C12N15/1135C12N15/86C12N2310/141C12N2740/15043
Inventor 张茜真杨智徐艳敏赵文旭陈军齐亚男沈俊杰钱程
Owner CHONGQING PRECISION BIOTECH CO LTD
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