CN111748625A - Therapeutic application of miR-515-3p as esophageal cancer molecular marker - Google Patents

Therapeutic application of miR-515-3p as esophageal cancer molecular marker Download PDF

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CN111748625A
CN111748625A CN202010594259.9A CN202010594259A CN111748625A CN 111748625 A CN111748625 A CN 111748625A CN 202010594259 A CN202010594259 A CN 202010594259A CN 111748625 A CN111748625 A CN 111748625A
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李斌
胡会芳
何庆瑜
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Jinan University
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Abstract

The invention belongs to the field of esophageal cancer treatment, and particularly relates to treatment application of miR-515-3p as an esophageal cancer molecular marker. The invention discovers for the first time that miR-515-3p and oligonucleotide synthesized according to miR-515-3p sequence can effectively inhibit the metastasis of esophageal cancer cells, and miR-515-3p can be used as an esophageal cancer treatment or prognosis marker, and has important significance for the treatment of esophageal cancer.

Description

Therapeutic application of miR-515-3p as esophageal cancer molecular marker
Technical Field
The invention belongs to the field of esophageal cancer treatment, and particularly relates to treatment application of miR-515-3p as an esophageal cancer molecular marker.
Background
Esophageal cancer is a common high-grade digestive tract malignant tumor in the world which cannot be effectively controlled. The five-year survival rate is between 15 and 25 percent. In recent years, cancer treatment has been greatly advanced, but the metastasis characteristics of the cancer are easy to relapse, and radical treatment is difficult. Epithelial-to-mesenchymal transition (EMT) is a biological process by which epidermal cells acquire mesenchymal characteristics and is an important feature of cancer cell metastasis. The high lethality rate of most cancers and the important reason for poor prognosis are epithelial-mesenchymal transition of tumor cells. The EMT process involves numerous proteins such as fibrinectin, N-cadherin, E-cadherin, and the like. microRNAs have also been shown to play an important role in the development of EMT in cells.
Cancer cell metastasis is a dynamic, continuous biological process that involves detachment from the primary tumor, entry into blood vessels, formation of metastases, and the like. There are studies that show that miRNAs are closely related to cancer cell metastasis or can be used as a means for treating cancer cell metastasis. miRNAs can eventually cleave the target mRNA by complete complementary binding to the target gene or by incomplete complementary binding to the target gene, thereby repressing translation without affecting mRNA stability. miRNA is a regulatory molecule in the process of gene expression and protein translation, and plays a pivotal role in regulation and control in the process of occurrence and development of tumors. 10% -30% of the human gene transcriptome is the direct target of mirnas. It can be used as the therapeutic means of tumor or more effectively. However, no mirnas have been found in the prior art that are effective in treating or inhibiting esophageal cancer.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide application of miR-515-3p as an esophageal cancer treatment or prognosis marker and application of the miR-515-3p in preparation of a medicine for inhibiting esophageal cancer cell metastasis. The invention also aims to provide application of the miR-515-3p oligonucleotide in preparation of a medicament for inhibiting esophageal cancer cell metastasis.
The purpose of the invention is realized by the following technical scheme:
application of miR-515-3p in preparation of a kit for treatment or prognosis of esophageal cancer.
Preferably, miR-515-3p is used as a marker for esophageal cancer treatment or prognosis in the kit.
Application of miR-515-3p in preparation of medicines for treating esophageal cancer.
Preferably, the miR-515-3p is used for inhibiting the metastasis of esophageal cancer cells.
More preferably, the miR-515-3p is used for inhibiting invasion of esophageal cancer cells.
Preferably, the miR-515-3p is used for inhibiting the EMT process.
More preferably, miR-515-3p is used for down-regulating the expression of fibronectin and N-cadherin and up-regulating the expression of ZO-1 and E-cadherin.
Application of miR-515-3p oligonucleotide in preparation of medicine for treating esophageal cancer.
Preferably, the miR-515-3p oligonucleotide is used for inhibiting the metastasis of esophageal cancer cells.
More preferably, the miR-515-3p oligonucleotide is used for inhibiting migration and invasion of esophageal cancer cells.
Compared with the prior art, the invention has the following advantages and effects:
the invention discovers for the first time that miR-515-3p and oligonucleotide synthesized according to miR-515-3p sequence can effectively inhibit the metastasis of esophageal cancer cells, and miR-515-3p can be used as an esophageal cancer treatment or prognosis marker, and has important significance for the treatment of esophageal cancer.
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FIG. 1 shows the staining analysis of normal esophageal tissue and esophageal cancer tissue, the survival analysis of the corresponding patients, and the expression of miR-515-3p in esophageal cancer tissue in example 1.
FIG. 2 shows the effect of miR-515-3p on the metastatic invasion of esophageal cancer cells in example 2.
FIG. 3 shows the ability of miR-515-3p to inhibit esophageal cancer cell metastasis in nude mice in example 3.
FIG. 4 shows the effect of miR-515-3p oligonucleotide on the inhibition of esophageal cancer cell metastasis in example 4.
FIG. 5 shows the ability of miR-515-3p oligonucleotide to inhibit esophageal cancer cell metastasis in nude mice in example 5.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
Example 1 expression of miR-515-3p in esophageal cancer tissue
Based on 70 normal esophageal tissues and 90 esophageal cancer tissues, staining analysis, survival analysis of corresponding patients and detection of miR-515-3p expression in esophageal cancer tissues are respectively carried out, and the results are shown in FIG. 1. In FIG. 1a, the expression level of miR-515-3p in 45.7% (32/100) normal tissue is significantly higher than that in cancer tissue, while only 22.0% (22/100) cancer tissue has higher expression of miR-515-3 p. And negative and weak are classified into low expression, modete and strong are classified into high expression, and miR-515-3p low expression indicates worse survival rate according to the graph 1 b. In addition, 20 normal tissues, 8 primary tumor tissues (T1&2), 9 primary tumor tissues (T3&4) and 9 metastatic tumor tissues were screened to investigate the relationship of miR-515-3p to tumor stage. FIG. 1c shows that the expression level of miR-515-3p in normal tissues is significantly higher than that of primary tumors (T1&2, T3&4) and metastatic tumors (metastic tumors).
Example 2 inhibitory Effect of miR-515-3p on invasion of esophageal cancer cells
A cell line KYSE150-Luc-LM3 with high transfer capacity is obtained by screening in a nude mouse, and a cell line KYSE410-Luc-I6 with high invasion capacity is established by using a Transwell technology, wherein the specific method is shown in Li, B.et.Significance of PI3K/AKT signaling pathway in metastasis of esophageal cancer cell a target for anti-metastasizing. Subsequently, two cell lines described above were constructed in which esophageal cancer stably overexpressed and knocked-down miR-515-3p and miR-CON (control group), and the results are shown in FIGS. 2 a-c. In FIGS. 2a and 2b, miR-515-3p overexpression significantly inhibits invasion of esophageal cancer. Moreover, miR-515-3p can down-regulate the expression of Fibronectin and N-cadherin and up-regulate the expression of ZO-1 and E-cadherin (FIG. 2 c).
In addition, KYSE150-Luc and EC109 cell lines for stable esophageal Cancer overexpression and miR-CON knock-down were constructed based on similar methods, wherein the screening of KYSE150-Luc is described in Xu, W.W.et al Cancer cell-segmented IGF2 infected tissues and bone marrow-derived vascular Cancer cells to promoter progression.8,14399(2017), and EC109 can be directly purchased from Chinese Academy of Sciences (Chinese Academy of Sciences, Shanghai). FIG. 2d shows that an esophageal cancer cell line with miR-515-3p knocked down is successfully constructed, and invasion experiments show that miR-515-3p knocked down can remarkably promote invasion capacity of esophageal cancer cells (FIG. 2e) and promote an EMT process (FIG. 2 f).
Example 3 in vivo experiments on the action of miR-515-3p
20 female nude mice (balb/c-nu/nu) aged 6 weeks were selected and divided into 4 groups: 5 in the overexpression group (miR-515-3p, miR-CON) and the knock-down group (ZIP-miR-515-3p, ZIP-miR-CON).
Constructing a tumor metastasis model:
(1) each nude mouse needs to be injected with 106(ii) esophageal cancer cells, resuspended in PBS;
(2) the resuspended cells were then taken out by a 25G needle microinjector and injected into the tail vein of nude mice.
(3) After 5 to 6 weeks, D-Luciferin (purchased from GOLDBIO) was intraperitoneally injected, and live fluorescence photographs were taken to observe the metastasis of cancer cells, and the results are shown in FIG. 3. FIG. 3a shows that miR-515-3p is overexpressed to inhibit metastasis of esophageal cancer cells in vivo. FIG. 3b shows that the knock-down of miR-515-3p can promote the in vivo metastasis of esophageal cancer cells, indicating that miR-515-3p can be used as a marker for the treatment and prognosis of esophageal cancer.
Example 4 inhibitory Effect of miR-515-3p oligonucleotide on metastasis of esophageal cancer cells
The esophageal cancer cell KYSE150-Luc-LM3 is used for transfecting miR-515-3p and miR-CON oligonucleotides, and the influence of the miR-515-3p oligonucleotides on the migration invasion capacity of the esophageal cancer cell is detected through a migration invasion experiment, and the result is shown in figure 4. The results in FIG. 4 show that the miR-515-3p over-expression oligonucleotide can inhibit invasion and migration of esophageal cancer cells KYSE150-Luc-LM 3.
Example 5 in vivo experiments on the Effect of miR-515-3p oligonucleotides
10 female nude mice (balb/c-nu/nu) with the age of 6 weeks, 5 control groups and 5 experimental groups are selected to construct a tumor metastasis model.
(1) Each nude mouse needs to be injected with 106The esophageal cancer cells KYSE150-Luc-LM3 are resuspended in PBS;
(2) the resuspended cells were then taken out by a 25G needle microinjector and injected into the tail vein of nude mice.
(3) The miR-515-3p and miR-CON oligonucleotides were injected into the experimental group and the control group from the tail vein, respectively, approximately two weeks after the cells were injected.
(3) After 9 weeks, D-Luciferin (purchased from GOLDBIO) was intraperitoneally injected, and live fluorescence photographs were taken to observe metastasis of cancer cells, and the results are shown in FIG. 5.
FIG. 5 shows the results of miR-515-3p oligonucleotide in vivo. FIG. 5a shows the effect of miR-515-3p and miR-CON oligonucleotides on metastasis in nude mice, and the right side quantification result shows that the esophageal cancer metastasis ability is obviously inhibited by the miR-515-3p oligonucleotides. The HE staining results in FIG. 5b also show that the 515-3p oligonucleotide inhibits the metastasis of esophageal cancer cells.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

  1. Application of miR-515-3p in preparation of a kit for treatment or prognosis of esophageal cancer.
  2. 2. The use according to claim 1, wherein miR-515-3p is used in the kit as a marker for the treatment or prognosis of esophageal cancer.
  3. Application of miR-515-3p in preparation of medicines for treating esophageal cancer.
  4. 4. The use of claim 3, wherein miR-515-3p is used for inhibiting metastasis of esophageal cancer cells.
  5. 5. The use of claim 4, wherein miR-515-3p is used for inhibiting invasion of esophageal cancer cells.
  6. 6. The use according to claim 3, wherein miR-515-3p is used for inhibiting the EMT process.
  7. 7. The use of claim 6, wherein miR-515-3p is used to down-regulate the expression of fibronectin and N-cadherin and up-regulate the expression of ZO-1 and E-cadherin.
  8. Application of miR-515-3p oligonucleotide in preparation of medicines for treating esophageal cancer.
  9. 9. The use of claim 8, wherein the miR-515-3p oligonucleotide is used to inhibit metastasis of esophageal cancer cells.
  10. 10. The use of claim 9, wherein the miR-515-3p oligonucleotide is used to inhibit the migration and invasion of esophageal cancer cells.
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