CN106139164B

CN106139164B - Application of miR-5001 in preparation of medicine for treating leukemia

Info

Publication number: CN106139164B
Application number: CN201610635414.0A
Authority: CN
Inventors: 崔健; 姜薇
Original assignee: Beijing Belife Bio-Medical Technology Ltd
Current assignee: Shandong Meisen artificial lawn Co.,Ltd.
Priority date: 2016-08-04
Filing date: 2016-08-04
Publication date: 2020-02-21
Anticipated expiration: 2036-08-04
Also published as: CN106139164A

Abstract

The invention relates to application of miR-5001 in preparation of a medicament for treating leukemia, and a medicinal composition taking miR-5001 as an active ingredient, wherein the medicinal composition can inhibit the development process of leukemia through the inhibition effect of miR-5001 on the transfer capacity of leukemia cells, and the inhibition efficiency is up to 60%.

Description

Application of miR-5001 in preparation of medicine for treating leukemia

Technical Field

The invention relates to the technical field of biology, in particular to application of microRNA in preparation of a medicament for treating leukemia.

Background

Leukemia is a malignant clonal disease of hematopoietic stem cells. Clonal leukemia cells proliferate and accumulate in bone marrow and other hematopoietic tissues in large quantities due to mechanisms such as uncontrolled proliferation, differentiation disorder, and apoptosis inhibition, infiltrate other tissues and organs, while normal hematopoiesis is inhibited. Different degrees of anemia, bleeding, infectious fever, and swelling of the liver, spleen, lymph nodes and bone pain are seen clinically. The incidence of leukemia in various regions of our country is reported to be the sixth among various tumors. Clinically, surgery or radiotherapy is effective for the limited leukemia, but is difficult to cure for the metastatic leukemia, and there is still no effective drug for the treatment of the metastatic leukemia. miRNA is endogenous non-coding small molecular RNA for regulating gene expression, regulates gene expression at the level after transcription, and participates in physiological processes such as cell cycle, apoptosis, development, differentiation, metabolism and the like. The misexpression of miRNA in cells can cause the occurrence of various diseases including cancer, and the latest research shows that some miRNA are abnormally expressed in leukemia, but no consensus is achieved on which miRNA is related to the occurrence and development of leukemia.

Therefore, it is necessary to find miRNA related to the occurrence and development of leukemia, so as to provide an effective means for clinically treating metastatic leukemia.

Disclosure of Invention

The invention aims to provide a pharmaceutical composition containing miRNA for inhibiting leukemia development, which provides possibility for clinically treating metastatic leukemia.

The inventor discovers that the expression of miR-5001 in leukemia cells Daudi and Jurkat is remarkably reduced in the process of researching the transfer mechanism of leukemia, and simultaneously discovers that miR-5001 can inhibit the proliferation and migration of cancer cells and promote the apoptosis, and miR-5001 can inhibit the transfer of lymphoma cells in a nude mouse. The expression level of miR-5001 in leukemia cells is high and low, and the miR-5001 can be used as a molecular marker for cancer cell metastasis.

One aspect of the invention provides an application of miR-5001 in preparation of a medicament for treating leukemia, wherein a nucleotide sequence of the miR-5001 is shown in SEQ ID No. 1.

Alternatively, the use comprises the manufacture of a medicament for inhibiting migration, invasion and proliferation of leukemia cells.

Optionally, the application comprises compounding the miR-5001 and a carrier and then compounding with an anti-tumor drug and/or a pharmaceutically acceptable auxiliary material to prepare the pharmaceutical composition.

One aspect of the invention provides application of miR-5001 in preparation of a medicine for inhibiting migration capacity of leukemia cell highly-metastatic cell lines Daudi and/or Jurkat.

The invention also provides a pharmaceutical composition, which takes miR-5001 as an active ingredient, wherein the nucleotide sequence of the miR-5001 is shown as SEQIDNo.1.

Optionally, the pharmaceutical composition contains miR-5001 combined with a carrier and/or pharmaceutically acceptable auxiliary materials.

In the pharmaceutical compositions provided herein, the vector may be of the kind commonly used in the art for expression of mirnas in host cells, for example, the vector may be a liposome, chitosan, or lentiviral expression vector.

In one embodiment of the invention, the expression vector is a lentiviral expression vector, preferably, the lentiviral expression vector may be a pwxl, pmd2.g or psPAX2 lentiviral expression vector.

In a preferred embodiment of the present invention, the pharmaceutical composition further optionally comprises one or more other tumor drugs effective against leukemia, optionally, the pharmaceutical composition comprises a platinum-based anti-tumor drug, and further comprises other tumor drugs conventionally used in combination chemotherapy based on platinum-based anti-tumor drugs, which are well known to those skilled in the art.

Optionally, the anti-tumor drug is mitomycin, cisplatin or carboplatin.

When the miR-5001 is used in combination with a platinum anti-tumor medicament, the dosage of the platinum anti-tumor medicament can be reduced to 20-80% of that in a conventional chemotherapy scheme.

In the present invention, the administration route of the pharmaceutical composition is intravenous, intra-arterial infusion or local injection, etc., and tumor targeted administration can also be performed by using a targeted administration technology known to those skilled in the art.

The pharmaceutical composition provided by the invention can inhibit the development process of leukemia through the inhibition effect of miR-5001 on the transfer capacity of leukemia cells, and the inhibition efficiency is up to 50%. See fig. 3.

Drawings

FIG. 1 shows the results of miR-5001 experiments on cell proliferation inhibition and colony formation.

Wherein (A) miR-5001 is used for transfecting Daudi, and Q-PCR is used for detecting the expression quantity of miR-5001; (B) detecting cell proliferation after miR-5001 transfects Daudi; (C) the cell clonality is tested.

FIG. 2 is a bar graph of miR-5001 capable of inhibiting the progression of leukemia cell cycle.

FIG. 3 is a graph of the results of miR-5001 inhibiting leukemia cell migration and invasion.

Wherein, (A) the miR-5001 transfuses Daudi cells and then detects the cell migration and invasion and the number of the cell migration and invasion in a visual field, and (B) the miR-5001 transfuses Jurkat cells and then detects the cell migration and invasion and the number of the cell migration and invasion in the visual field.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments.

In the present invention, the term "miR-5001" refers to a microRNA comprising the sequence shown in SEQ ID No.1 or a sequence homologous thereto. Various sources of miR-5001 are known in the art, e.g., human, murine, rabbit, etc., and these homologous sequences are encompassed by the term miR-5001 of the present invention. The term of the invention also comprises derivative RNA of the naturally-occurring miR-5001 sequence which is subjected to substitution, deletion or addition of one or more nucleotides, or is biologically modified and still has the biological activity of miR-5001.

In the invention, the miR-5001mimics which are artificially synthesized and have miR-5001 biological activity and can be obtained by purchasing a commercial product in the market also belong to the protection scope of the invention.

In addition, miR-5001 described in the invention can also be in the form of a precursor, and the miR-5001 precursor refers to a precursor which can be processed into miR-5001 in the cells or in vivo of a subject to be administered. Methods for obtaining naturally occurring miR-5001 precursors are well known to those skilled in the art.

As is well known to those skilled in the art, the initial transcription product of miR-5001 undergoes a series of processing to form mature miR-5001. The miR-5001 precursor has corresponding biological functions only after being processed into mature miR-5001.

The composition provided by the invention can be used for treating leukemia. The composition contains an effective amount of miR-5001 disclosed by the invention.

In the present invention, the pharmaceutically acceptable excipients include various excipients, diluents and adjuvants. The adjuvant is not an essential active ingredient per se and is not excessively toxic after use. Such adjuvants include, but are not limited to: physiological saline, buffer, glucose, water, glycerol, ethanol, etc.

In one embodiment of the invention, the composition is in a form suitable for: direct naked microRNA injection, liposome-encapsulated RNA direct injection, plasmid DNA carried by reproduction-defective bacteria or target DNA carried by replication-defective adenovirus, etc.

The effective dosage of the miR-5001 can be correspondingly adjusted according to the administration mode, the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art by a combination of factors. Such factors include, but are not limited to: pharmacokinetic parameters of miR-5001, health of the patient being treated, body weight, route of administration, and the like.

In the present invention, the administration route of the pharmaceutical composition is intravenous, intra-arterial infusion, local injection, etc., and may be performed by using an administration technique well known to those skilled in the art.

The pharmaceutical composition of the invention can be used in combination with other therapeutic means for the treatment of leukemia.

Example 1

This example demonstrates the inhibitory effect of miR-5001 on leukemia cell proliferation and colony formation

A lymphoma cell line Daudi is transfected by miR-5001mimics (miR-5001 mimics) of miR-5001 through a transfection reagent lipofectamine 2000, and the expression quantity of the miR-5001mimics is detected through Q-PCR (Q-polymerase chain reaction) as shown in a figure 1A, and the result shows that the miR-5001 expression quantity in cells is remarkably increased after the miR-5001mimics transfects DAUDI, and the fact that the mimics can simulate the expression of miR-5001 is proved. After 24, 36, 48, 72 and 96h of DAUDI cells transfected by miR-5001mimics, cell proliferation is detected by a CCK-8 kit in vitro, and miR-5001 can obviously inhibit the proliferation of leukemia cells as shown in figure 1B.

The cell population dependence and proliferation capacity were examined by soft agar culture clonogenic experiments. The cells in the logarithmic growth phase of the monolayer culture were digested with 0.25% trypsin and blown into single cells, and the cells were suspended in a 1640 culture medium of 10% fetal bovine serum for use. And (3) diluting the cell suspension by multiple times, inoculating the cell suspension into a soft agar culture plate, standing and culturing for 10-14 days, and calculating the clone formation rate, wherein the result shows that miR-5001 can remarkably inhibit the clone formation capability of DAUDI as shown in figure 1C.

Example 2

This example demonstrates the effect of miR-5001 on inhibiting the cell cycle progression of leukemic cells

After miR-5001mimics are transfected into Daudi, after the Daudi is cultured for 24 hours, cells are harvested and PI staining is carried out, and the proportion of a cell cycle is detected by using flow cytometry, wherein G1/G0 is a cell resting phase, S is a DNA synthesis phase, G2/M is a mitosis phase, and the lower the proportion of G2/M is, the slower the cell proliferation is indicated.

The results are shown in FIG. 2: miR-5001mimics transfected Daudi can obviously reduce the proportion of G2/M phase cells, and proves that miR-5001 can obviously inhibit cell cycle process of Daudi, thereby inhibiting cell division.

Example 3

This example demonstrates the inhibitory effect of miR-5001 on leukemia cell migration and invasion.

miR-5001mimics transfects Daudi and Jurkat cells, the cells are cultured in a cell culture box for 24 hours, and then the cells are transferred to a transwell culture dish and observed for 48 hours. The migration and invasion capacities of the cells are verified by using a transwell invasion experiment, and as shown in FIG. 3, the result shows that miR-5001mimics transfect Daudi and Jurkat, so that the migration and invasion capacities of two leukemia cells are obviously reduced.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

The application of miR-5001 in preparing medicines for inhibiting leukemia cell proliferation and clone formation, cell cycle process, cell migration and invasion is disclosed, wherein the nucleotide sequence of miR-5001 is shown in SEQ ID No.1, and the leukemia cell is a cell line Daudi.
2. The application of claim 1, which comprises compounding miR-5001 and a carrier and then compounding with an anti-tumor drug and/or a pharmaceutically acceptable adjuvant to prepare a pharmaceutical composition.
The application of miR-5001 in preparing the medicine for inhibiting the migration capability of leukemia cell highly-metastatic cell lines Daudi and/or Jurkat is characterized in that the nucleotide sequence of miR-5001 is shown in SEQ ID No. 1.