阅读说明：本技术 miR-2052在制备抗肝癌药物中的应用 (Application of miR-2052in preparation of anti-liver cancer drugs ) 是由 张学武 *** 廖智斌 刘富荣 张必翔 陈孝平 于 2019-10-16 设计创作，主要内容包括：本发明提供了一种miR-2052的新用途-在制备抗肝癌药物中的应用,所述miR-2052的序列如SEQ ID NO.1所示；本发明还提供了一种抗肝癌药物,所述抗肝癌药物包括miR-2052 mimic、miR-2052真核表达载体、miR-2052病毒表达载体、以及所述miR-2052病毒表达载体包装形成的病毒中的至少一种。本发明通过大量的实验数据证明miR-2052能够明显抑制肝癌细胞的增殖、侵袭与转移,因此miR-2052可用于制备防治肝癌的药物。(The invention provides a new application of miR-2052in preparation of anti-liver cancer drugs, wherein the sequence of miR-2052 is shown in SEQ ID NO. 1; the invention also provides an anti-liver cancer drug, which comprises at least one of miR-2052 mici, miR-2052 eukaryotic expression vectors, miR-2052 virus expression vectors and viruses formed by packaging the miR-2052 virus expression vectors. A large amount of experimental data prove that miR-2052 can obviously inhibit proliferation, invasion and transfer of liver cancer cells, so that miR-2052 can be used for preparing medicines for preventing and treating liver cancer.)

The application of miR-2052in preparing anti-liver cancer drugs, wherein the sequence of miR-2052 is shown in SEQ ID NO. 1.

2. The use of claim 1, wherein the anti-liver cancer agent comprises an agent that inhibits proliferation of liver cancer cells.

3. The use of claim 1, wherein the anti-liver cancer agent comprises an agent that inhibits invasion and metastasis of liver cancer cells.

4. The anti-liver cancer drug is characterized by comprising at least one of miR-2052mimic, miR-2052 eukaryotic expression vectors, miR-2052 virus expression vectors and viruses formed by packaging the miR-2052 virus expression vectors.

5. The anti-liver cancer drug according to claim 4, wherein the sense chain sequence of miR-2052mimic is shown in SEQ ID NO. 2; the sequence of the antisense strand is shown in SEQ ID NO. 3.

6. The anti-liver cancer drug according to claim 4, wherein the sequence of the expression region of the eukaryotic expression vector of miR-2052 is shown as SEQ ID No. 4.

7. The anti-liver cancer drug of claim 4, wherein the miR-2052 viral expression vector comprises a miR-2052 lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, and a herpes viral vector.

8. The anti-liver cancer drug of claim 7, wherein the sequence of the expression region of the miR-2052 lentiviral vector is shown in SEQ ID No. 4.

9. The anti-liver cancer drug according to claim 4, wherein the anti-liver cancer drug is in the form of granules, sustained release agents, microinjection agents, transfection agents or surfactants; the anti-liver cancer medicine also comprises conventional auxiliary materials required by preparing different dosage forms.

Technical Field

The invention relates to the technical field of molecular biology, in particular to application of miR-2052in preparation of an anti-liver cancer drug.

Background

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. The pathogenesis of the virus is extremely complex and can have various reasons, including chronic hepatitis B virus or hepatitis C virus infection, aflatoxin B1 contact, alcoholic and non-alcoholic fatty diseases and the like. Despite recent research findings of many genes and pathways that affect liver cancer development, treatment and patient prognosis remain unsatisfactory.

Micro rna (mirna) is widely present in the body, highly conserved, and involved in a variety of pathophysiological processes in the body. miRNA is endogenous non-coding RNA consisting of 19-25 nucleotides and is involved in the control of the stability and translation of messenger RNA. It was originally discovered by Ambros et al in 1993. The MiRNA gene is a non-coding gene, the transcription product of which is MiRNA. Aberrant regulation of miRNAs is involved in the development and progression of a variety of diseases, including a variety of cancers, cardiovascular diseases, and degenerative diseases, among others. It plays a key role in normal physiological changes such as embryogenesis, cell differentiation regulation, proliferation and apoptosis in vivo. Therefore, the search for new miRNA related to liver cancer occurrence is of great significance for diagnosis and treatment of liver cancer.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a new application of miR-2052in preparation of anti-liver cancer drugs.

The invention is realized by the following steps:

firstly, the expression level of miR-2052in 42 pairs of liver cancer tissues and tissues beside the cancer is detected, and the result is shown in figure 1, and the expression level of miR-2052 is obviously reduced in the liver cancer tissues compared with the tissues beside the cancer.

Secondly, CCK8 experiment is adopted to detect the influence of miR-2052 on the proliferation of human liver cancer cells, and as shown in FIGS. 2A-B, the proliferation rate of human liver cancer cells HLF and 97H transfected with miR-2052 mimics (miR-2052mimic) is obviously lower than that of human liver cancer cells transfected with NC-mimic; as can be seen from FIGS. 2C-D, the proliferation rates of the human liver cancer cells HLF and 97H transfected with the miR-2052inhibitor (miR-2052inhibitor) are significantly lower than those of the human liver cancer cells transfected with the NC-inhibitor, which also shows that the miR-2052 can inhibit the proliferation rate of the human liver cancer cells. In conclusion, the CCK8 detection result shows that miR-2052 inhibits the proliferation of HCC cells.

Thirdly, the invention adopts a trans-well experiment to detect the influence of miR-2052 on the invasion and metastasis abilities of HCC cells, and as a result, trans-well analysis shows that miR-2052 mimics (miR-2052 micic) reduce the invasion (figure 3A) and metastasis (figure 3B) of human hepatoma cells HLF and 97H cells, and miR-2052 inhibitors (inhibitors) increase the invasion (figure 3C) and metastasis (figure 3D) of HLF and 97H cells.

Fourthly, the invention adopts a xenograft tumor model to research the action of miR-2052in a nude mouse, and the result shows that miR-2052 can inhibit the proliferation of liver cancer cells in the nude mouse.

In conclusion, miR-2052 can inhibit proliferation, invasion and metastasis of liver cancer cells.

Therefore, the miR-2052 can be applied as an anti-liver cancer drug. In specific application, the miR-2052mimic or a virus formed by packaging a miR-2052 virus expression vector can be prepared into anti-liver cancer drugs to be prepared into different dosage forms, and the expression vector (the expression region sequence is shown as SEQ ID NO.4, and comprises a eukaryotic expression vector and a virus expression vector) of the miR-2052 can be constructed for gene therapy.

The anti-liver cancer medicament is in the dosage form of granules, sustained release agents, microinjection agents, transfection agents or surfactants; the anti-liver cancer medicine also comprises conventional auxiliary materials required by preparing different dosage forms.

The invention has the beneficial effects that:

a large amount of experimental data prove that miR-2052 can obviously inhibit proliferation, invasion and transfer of liver cancer cells, so that miR-2052 can be used for preparing a medicament for preventing and treating liver cancer.

Drawings

Fig. 1 is an expression analysis detection result of miR-2052in a liver cancer tissue and a para-cancer tissue provided in embodiment 1 of the present invention, where N represents the para-cancer tissue and C represents the liver cancer tissue in an abscissa of the graph;

FIG. 2 is a result of CCK8 experiment provided in example 3 of the present invention to detect the effect of miR-2052 on human liver cancer cell proliferation; wherein panel A is the results of mimic and NC-mimic transfection of HLF cells; panel B shows the results of mimic and NC-mimic transfection of 97H cells; panel C shows the results of the transfection of HLF cells with inhibitor and NC-inhibitor; panel D shows the results of 97H cells transfected with inhibitor and NC-inhibitor; in the figure, the micic represents miR-2052 micic, the NC-micic represents a micic negative control, the inhibitor represents miR-2052inhibitor, and the NC-inhibitor represents an inhibitor negative control;

FIG. 3 is a result of detecting the effect of miR-2052 on the invasion and migration ability of HCC cells by using a trans-well experiment, which is provided in embodiment 4 of the present invention; wherein, FIG. 3A shows the results of invasion experiments of mimic and NC-mimic transfected HLF cells and 97H cells; FIG. 3B shows migration experiment results of mimic and NC-mimic transfected HLF cells and 97H cells; FIG. 3C shows the results of invasion experiments in which the inhibitor and NC-inhibitor transfected HLF cells and 97H cells; FIG. 3D shows the results of migration experiments in which inhibitor and NC-inhibitor were transfected into HLF cells and 97H cells;

FIG. 4 shows the results of tumor tissue changes in the xenograft mouse model provided in example 5 of the present invention; wherein, the A picture is a picture of tumor tissues; panel B is the volume of tumor tissue; panel C is the weight of tumor tissue;

FIG. 5 shows the expression result of miR-2052in immunohistochemical detection of exfoliated tumor tissue provided in example 5 of the present invention;

FIG. 6 shows the results of Ki67 expression in the immunohistochemical assay of detached tumor tissues provided in example 5 of the present invention;

FIG. 7 is a map of a GV369 lentiviral vector used in the construction of the lentiviral vector of example 2 of the present invention;

FIG. 8 shows the result of viral supernatant infection of the Pri-miRNA-2052 recombinant viral vector; wherein the picture (A) is a green fluorescence field; (B) the figure is a bright field view.

Detailed Description