CN114645050B - miRNA and application thereof in preparation of medicines for treating breast cancer - Google Patents

Abstract

The present invention relates to molecular biology, cell biology and pharmacology, and specifically relates to a kind of miRNA and its application in preparing drugs for treating breast cancer. The miRNA is named ach-miR-276a-3, and its sequence is shown in SEQ ID NO. 1. It can significantly inhibit the proliferation of breast cancer cells and the growth of tumors, providing a new way for the treatment of breast cancer.

Description

A kind of miRNA and its application in preparing drugs for treating breast cancer

Technical field

The present invention relates to molecular biology, cell biology and pharmacology. Specifically relates to a kind of miRNA and its application in preparing drugs for treating breast cancer.

Background technique

Aspongopus chinensis (Dallas, 1851) is an insect in the family Dinidoridae of the order Hemiptera. It is commonly known as black pocket bugs, black melon bugs, fart board bugs, dung beetles, spanking bugs, and fart bugs. It is distributed in Guizhou. , Anhui, Fujian, Guangdong, Jiangsu, Jiangxi, Sichuan, Shanghai, Yunnan, Tibet, Taiwan, Vietnam, Laos, Indonesia, Japan, Myanmar, the Malay Peninsula and India. In the 2015 edition of the Chinese Pharmacopoeia, Nine-flavored Cockroach is listed in the categories of medicinal materials and decoction pieces. It has the effects of regulating qi and relieving pain, warming the middle and supporting yang. The traditional Chinese medicine Nine-flavored insect is the dried form of Nine-flavored insect. It was first recorded in the "Compendium of Materia Medica" as "produced in the Chishui River in Yongningwei, Guizhou... It flies out after the awakening of stings and is no longer useful. Odor: salty, warm, It is non-toxic and mainly treats qi stagnation in the diaphragm and kidneys, and strengthens yang." The compound prescriptions of Jiuxiang Chong are clinically used to treat epigastric pain, endometriosis and other diseases, and are also used to treat cancer pain. They all have good curative effects, but there are few studies on its mechanism of action.

MicroRNAs (miRNAs) are non-coding, short sequence small RNAs composed of 15-28 nucleotides. Approximately 28,000 miRNAs have been discovered so far, and studies have shown that these miRNAs can regulate the expression of approximately 60% of human genes and subsequently participate in a series of human physiological activities. Therefore, studying the distribution, expression and regulatory effects of miRNAs in tissues is important for Understanding the occurrence, development and treatment of human diseases (such as cancer) plays an important role. MiRNAs mainly bind to the 3’UTR region of target mRNA, destroy the stability of the mRNA or prevent its translation, and inhibit the expression of its target genes. Research shows that miRNAs regulate almost all growth and development, physiological and pathological processes in organisms. miRNAs play an important role in tumor progression. They can affect most tumor-related pathways, such as cell proliferation, cell migration, autophagy, and apoptosis pathways, by regulating the expression of target genes, thereby regulating tumor growth, migration, and apoptosis. Apoptosis and immune escape, etc., it may be a promising natural anti-tumor nucleic acid drug.

In view of the important role of miRNAs, discovering more miRNAs related to human diseases is a technical problem that those skilled in the art continue to solve.

Contents of the invention

In order to solve the above technical problems, the present invention provides miRNA and its application in preparing drugs for treating breast cancer.

A first aspect of the present invention provides a miRNA, the sequence of which is shown in SEQ ID NO.1.

A second aspect of the present invention provides the use of the miRNA in preparing drugs for treating breast cancer.

The third aspect of the present invention provides the coding gene of the miRNA.

A fourth aspect of the present invention provides a lentiviral vector containing the encoding gene.

A fifth aspect of the present invention provides the use of the lentiviral vector in the preparation of drugs for treating breast cancer.

A sixth aspect of the present invention provides a drug for treating breast cancer, the drug comprising the miRNA or the lentiviral vector, and a pharmaceutically acceptable carrier.

Further, the pharmaceutically acceptable carrier is selected from one or more of chitosan, cholesterol, liposomes and nanoparticles.

Furthermore, the medicine is an oral preparation or injection.

A seventh aspect of the present invention provides a method for non-therapeutic inhibition of breast cancer cell proliferation in vitro, comprising the following steps:

The miRNA was transfected into breast cancer cells cultured in vitro.

Compared with the prior art, the present invention has the following beneficial effects:

For the first time, the present invention has screened a highly expressed miRNA in the worm, named ach-miR-276a-3p. The experimental results of the present invention prove that ach-miR-276a-3p can significantly inhibit breast cancer MCF both in vivo and in vitro. -7 cells and inhibit the growth of breast cancer tumors in tumor-bearing mice, providing a new approach for the treatment of breast cancer.

Description of drawings

Figure 1 shows the expression of miRNA in the hemolymph of the worm.

Figure 2 shows the effect of ach-miR-276a-3p on the proliferation of breast cancer cells MCF-7 (A) and MDA-MB-453 (B) in vitro.

Figure 3 is a diagram of the tumor body of the MCF-7 human breast cancer nude mouse model.

Figure 4 is a statistical diagram of tumor volume in the MCF-7 human breast cancer nude mouse model.

Figure 5 is a statistical graph of tumor weight in the MCF-7 human breast cancer nude mouse model.

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but should not be understood as limitations of the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following examples can all be obtained from commercial sources, unless otherwise specified.

Example 1: Acquisition of ach-miR-276a-3p sequence

(1) Collection and pretreatment of nine-scented insects: Wash the nine-scented insects with clean water and store them in a -80°C refrigerator;

(2) Extraction of hemolymph from the worm: First, take out the frozen worm and place it in a 4°C refrigerator to thaw. After thawing, remove the cephalothorax, take the abdomen and physically squeeze it, and place the squeezed contents in a centrifuge tube. , centrifuge at 12,000×g, 4°C for 10 min. After centrifugation, divide into three layers. Aspirate the dark red liquid in the middle layer and place it in a new centrifuge tube. Centrifuge at 10,000×g, 4°C for 5 min. , take the dark red liquid to obtain the hemolymph of the Nine-flavored Insect.

(3) The extracted hemolymph was sent to BGI Group for sequencing. Through analysis and screening, the miRNA sequence highly expressed in the worm was obtained: UAGGAACUUCAUACCGUGCUCU, as shown in SEQ ID NO.1, with a length of 22 nucleotides. , named ach-miR-276a-3p (Figure 1).

Example 2: Application of ach-miR-276a-3p in inhibiting breast cancer cell proliferation in vivo and in vitro

(1) In vitro experiments

First, human breast cancer MCF-7 and MDA-MB-453 cell lines were selected, and NC mimic (negative control) at a concentration of 50 nM and ach-miR-276a-3p mimic (synthesized by Suzhou Jima Gene Co., Ltd. ) were transfected into two breast cancer cells respectively, and the absorbance value at 450 nm was measured at 24 h, 48 h, 72 h and 96 h using the CCK-8 method and the full-wavelength microplate reader Multiskan GO to calculate the cell viability. The results are shown in Figures 2 and 3. Compared with the NC-treated group, ach-miR-276a-3p significantly inhibited the proliferation of two human breast cancer cell lines, MCF-7 and MDA-MB-453.

(2) Animal experiments

The human breast cancer MCF-7 cell line was transfected with NC mimic (negative control) and ach-miR-276a-3pmimic at a concentration of 50 nM respectively. After stable expression in the cells, the cells were injected into nude mice to construct MCF-7 human breast cancer nude mouse model. After 8 days, the mice were sacrificed by cervical dislocation. The tumors were dissected, weighed and measured. The results are shown in Figures 4 and 5: Compared with the NC treatment group, ach-miR-276a-3p can significantly inhibit the growth of tumors.

Although the preferred embodiments of the present invention have been described, those skilled in the art will be able to make additional changes and modifications to these embodiments once the basic inventive concepts are apparent. Therefore, it is intended that the appended claims be construed to include the preferred embodiments and all changes and modifications that fall within the scope of the invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the invention. In this way, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and equivalent technologies, the present invention is also intended to include these modifications and variations.

sequence list

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<120> A kind of miRNA and its application in preparing drugs for treating breast cancer

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<212> RNA

<213> Artificial sequence

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uaggaacuuc auaccgugcu cu 22

Claims (6)

1. The application of a miRNA in the preparation of a drug for inhibiting breast cancer cell proliferation, characterized in that the sequence of the miRNA is shown in SEQ ID NO. 1. 2. The use of a lentiviral vector in the preparation of drugs for the treatment of breast cancer, characterized in that the lentiviral vector contains a gene encoding the miRNA described in claim 1. 3. A drug for treating breast cancer, characterized in that the drug contains the miRNA described in claim 1 or the lentiviral vector described in claim 2, and a pharmaceutically acceptable carrier. 4. The medicine according to claim 3, characterized in that the pharmaceutically acceptable carrier is selected from one or more of chitosan, cholesterol, liposomes and nanoparticles. 5. The medicine according to claim 4, characterized in that the medicine is an injection. 6. A method for non-therapeutic inhibition of breast cancer cell proliferation in vitro, characterized by comprising the following steps: The miRNA described in claim 1 is transfected into breast cancer cells cultured in vitro.