阅读说明：本技术 Opa1异形体蛋白在调控肝癌细胞增殖能力中的用途 (Application of Opa1 allotrope protein in regulating and controlling proliferation capacity of liver cancer cells ) 是由 刘兴国 杨亮 唐海特 于 2019-02-01 设计创作，主要内容包括：本发明提出了含有Exon4b的Opa1异形体蛋白在调控肝癌细胞增殖能力中的用途。发明人通过实验惊喜地发现,敲低肝癌细胞中含有Exon4b的Opa1异形体蛋白的表达水平,肝癌细胞的增殖能力下降；之后若回补含有Exon4b的Opa1异形体蛋白,肝癌细胞的增殖能力回复。因此,通过调节含有Exon4b的Opa1异形体蛋白的表达水平,可以特异性调控肝癌细胞的增殖能力,或可为肝癌的治疗提供一条有效的途径。(The invention provides application of Opa1 allotrope protein containing Exon4b in regulating the proliferation capacity of liver cancer cells. The inventor surprisingly finds out through experiments that the expression level of Opa1 allotrope protein containing Exon4b in liver cancer cells is reduced, and the proliferation capacity of the liver cancer cells is reduced; then, if the Opa1 isoform protein containing Exon4b is supplemented, the proliferation capacity of the hepatoma cells is recovered. Therefore, by regulating the expression level of Opa1 isoform protein containing Exon4b, the proliferation capacity of liver cancer cells can be specifically regulated, or an effective approach can be provided for treating liver cancer.)

1. Use of Opa1 isoform protein containing Exon4b in regulating the proliferation capacity of hepatoma cells.

2. A kit for regulating the proliferative capacity of a hepatoma cell, comprising: an agent for modulating the expression level of an Opa1 isoform protein containing Exon4 b.

3. The kit of claim 2, wherein the Opa1 isoform protein containing Exon4b comprises at least one protein selected from Opal iso3, 5, 6, 8;

optionally, the kit is used for inhibiting the proliferative capacity of liver cancer cells, and the reagent is used for reducing the expression level of Opa1 isoform protein containing Exon4 b;

optionally, the agent is an siRNA or shRNA that knockdown an isoform protein containing Exon4 b;

optionally, the agent is an siRNA having the sequence as set forth in SEQ ID NO: 1;

optionally, the kit is used for enhancing the proliferation capacity of liver cancer cells, and the reagent is used for increasing the expression level of Opa1 isoform protein containing Exon4 b;

optionally, the reagent is an expression vector for over-expressing Opa1 isoform protein containing Exon4 b.

4. Use of an agent for modulating the expression level of Opa1 isoform protein containing Exon4b in the preparation of a kit for modulating the proliferative capacity of a hepatoma cell.

5. The use according to claim 4, wherein the agent is used for reducing the expression level of Opa1 isoform protein containing Exon4b, and the kit is used for inhibiting the proliferative capacity of hepatoma cells;

optionally, the agent is an siRNA or shRNA that knockdown an isoform protein containing Exon4 b;

optionally, the agent is an siRNA having the sequence as set forth in SEQ ID NO: 1;

optionally, the reagent is used for increasing the expression level of Opa1 isoform protein of Exon4b, and the kit is used for enhancing the proliferation capacity of liver cancer cells;

optionally, the reagent is an expression vector for over-expressing Opa1 isoform protein containing Exon4 b.

6. A method for regulating the proliferation ability of a liver cancer cell, which comprises increasing or decreasing the expression level of Opa1 isoform protein containing Exon4b of the liver cancer cell during culture so as to control the proliferation ability of the liver cancer cell;

optionally, the liver cancer cell is a liver cancer cell line;

optionally, the liver cancer cell line is SMMC-7721, Bel-7402, Bel-7404, Bel-7405, MHCC97, HepG2, Hep3B, Hep1, Huh-7, PLC/PRF/5, H22, HHCC, HCCLM3, HB611, QGY-7701, QGY-7703;

optionally, the liver cancer cell is a primary cell;

optionally, the expression level of Opa1 isoform protein containing Exon4b in the liver cancer cell is increased, and the proliferation capacity of the liver cancer cell is enhanced;

optionally, the expression level of Opa1 isoform protein containing Exon4b in the hepatoma cells is reduced, and the proliferative capacity of the hepatoma cells is decreased.

7. A pharmaceutical composition comprising an agent for reducing the expression level of Opa1 isoform protein containing Exon4b, said pharmaceutical composition for inhibiting the proliferation of hepatoma cells;

optionally, the agent is an siRNA or shRNA that knockdown an isoform protein containing Exon4 b;

optionally, the agent is an siRNA having the sequence as set forth in SEQ ID NO: 1.

8. Use of an inhibitor of function of Opa1 isoform protein containing Exon4b in the manufacture of a medicament for inhibiting proliferation of hepatoma cells;

optionally, the functional inhibitor comprises at least one selected from the group consisting of an agent that specifically knockdown the Opa1 isoform protein containing Exon4b, an inhibitor of the Opa1 isoform protein containing Exon4 b;

optionally, the agent that specifically knockdown the Opa1 isoform protein containing Exon4b is an siRNA having the amino acid sequence of SEQ ID NO: 1.

9. A method of screening for a drug for inhibiting proliferation of hepatoma cells, comprising:

contacting the liver cancer model with a candidate drug;

comparing the expression level of Opa1 isoform protein containing Exon4b in the liver cancer model before and after the contact, and judging whether the candidate drug is the target drug.

10. The method of claim 9, wherein a decreased level of expression of the Opa1 isoform protein containing Exon4b in the liver cancer model after exposure as compared to before exposure is indicative that the drug candidate is a drug of interest.

Technical Field

The present invention relates to the field of bioengineering. Specifically, the invention relates to application of Opa1 isoform protein in regulation of proliferation capacity of liver cancer cells, and more specifically relates to application of Opa1 isoform protein containing Exon4b in regulation of proliferation capacity of liver cancer cells, a kit, application of a reagent in preparation of the kit, a method for regulating proliferation capacity of liver cancer cells, a pharmaceutical composition, application of a functional inhibitor of Opa1 isoform protein containing Exon4b in preparation of a medicament, and a method for screening the medicament.

Background

Liver cancer is one of the five most common malignant tumors in the world, and the death rate of the liver cancer is second to that of stomach cancer and esophageal cancer. The liver cancer patients are not treated, the survival rate of 5 years is less than 10 percent, but the survival rate of 5 years after radical excision of 'small liver cancer' can reach 70 percent. Moreover, liver cancer does not have obvious symptoms in the early stage, and is often ignored by people, and the clinical symptoms are in the middle and late stages, so that the operation chance is lost, and the early treatment of liver cancer becomes particularly important. However, current early treatments for liver cancer lack specificity and specificity.

Therefore, if a specific method for treating liver cancer can be found, the method is of great significance for the early treatment of liver cancer.

Disclosure of Invention

The present application is based on the discovery and recognition by the inventors of the following facts and problems:

the human OPA1 gene is located on chromosome 3 (Christiane Alexander et al, 2000) and consists of 28 exons, the product OPA1 (see FIGS. 1-2 of Christiane Alexander et al, 2000) being a nuclear-encoded mitochondrial dynamics-related protein that is a component of the mitochondrial network structure and has similar dynamics-related GTPase activity. In human cells, the OPA1 gene is transcribed and then subjected to complex alternative splicing to form 8 transcripts (Delettre et al, 2001), which are then translated into 8 OPA1 isoform (iso) proteins (OPA 1iso-1, 2, 3, 4, 5, 6, 7, 8). These 8 proteins differ mainly by alternative splicing of exons 4, 4b, 5b of their mRNA segments (see FIGS. 1-32 in Ste 'phane Duvezin-Caubet al, 2007) (Ste' phane Duvezin-Caubet al, 2007).

The inventor surprisingly finds out through experiments that the expression level of Opa1 allotrope protein containing Exon4b in liver cancer cells is reduced, and the proliferation capacity of the liver cancer cells is reduced; then, if the Opa1 isoform protein containing Exon4b is supplemented, the proliferation capacity of the hepatoma cells is recovered. Therefore, by regulating the expression level of Opa1 isoform protein containing Exon4b, the proliferation capacity of liver cancer cells can be specifically regulated, or an effective approach can be provided for treating liver cancer.

To this end, in a first aspect of the invention, the invention proposes the use of the Opa1 isoform protein containing Exon4b for modulating the proliferative capacity of a liver cancer cell. As described above, the OPA1 gene was transcribed and then subjected to complex alternative splicing to form 8 transcripts (Delettre et al, 2001), which were then translated into 8 OPA1 isoform proteins (OPA 1iso-1, 2, 3, 4, 5, 6, 7, 8). These 8 proteins differ mainly by alternative splicing of exons 4, 4b, 5b of their mRNA segments. The "Opa 1 isoform protein containing Exon4 b" in the present invention refers to an isoform protein formed by translation of mRNA containing Exon4 b. In some embodiments, the Opa1 isoform protein containing Exon4b comprises at least one selected from Opal iso3, Opal iso 5, Opal iso 6, Opal iso 8. As described above, the inventors surprisingly found in the experiment that the proliferation ability of the liver cancer cell is decreased by knocking down the expression level of Opa1 isoform protein containing Exon4b in the liver cancer cell; then, if the Opa1 isoform protein containing Exon4b is supplemented, the proliferation capacity of the hepatoma cells is recovered. Therefore, by regulating the expression level of Opa1 isoform protein containing Exon4b, the proliferation capacity of hepatoma cells can be specifically regulated. It should be noted that the use can be used for non-therapeutic purposes, such as scientific research, for example, by regulating the expression level of Opa1 isoform protein containing Exon4b, thereby regulating the proliferation capacity of liver cancer cells, and further obtaining a corresponding liver cancer cell model for subsequent experimental study of liver cancer.

In a second aspect of the invention, a kit is provided. According to an embodiment of the present invention, the kit is used for regulating the proliferation capacity of the liver cancer cells, and comprises: an agent for modulating the expression level of an Opa1 isoform protein containing Exon4 b. As described above, the expression level of Opa1 isoform protein containing Exon4b in liver cancer cells is knocked down, and the proliferation capacity of the liver cancer cells is reduced; then, if the Opa1 isoform protein containing Exon4b is supplemented, the proliferation capacity of the hepatoma cells is recovered. Thus, the reagent is used to regulate the expression level of Opa1 isoform protein containing Exon4b, and thus, the proliferation ability of liver cancer cells can be specifically regulated using the kit comprising the reagent according to an embodiment of the present invention.

According to an embodiment of the present invention, the kit may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the Opa1 isoform protein containing Exon4b comprises at least one selected from Opal iso3, 5, 6, 8. The inventor finds that the proliferation capacity of the liver cancer cells is remarkably restored by supplementing the Opa1 isoform protein containing Exon4b, such as at least one of Opal iso3, Opal iso 5, Opal iso 6 and Opal iso 8, after the knock-down, so that the proliferation capacity of the liver cancer cells can be effectively regulated by using the kit according to the embodiment of the invention.

The kind of the reagent is not particularly limited as long as the reagent can specifically regulate the expression amount of Opa1 isoform protein containing Exon4b, and those skilled in the art will understand that the expression amount may refer to either an absolute expression amount or a relative expression amount, and that the expression amount of Opa1 isoform protein containing Exon4b in the control sample may be used as a reference to relatively represent the expression amount of Opa1 isoform protein containing Exon4b in the sample to be tested, or the expression amount of Opa1 isoform protein containing Exon4b in the sample to be tested may be used as a reference to the expression amount of housekeeping gene.

According to an embodiment of the present invention, the kit is used for inhibiting the proliferative capacity of liver cancer cells, and the reagent is used for reducing the expression level of Opa1 isoform protein containing Exon4 b. In some embodiments, the agent is an siRNA or shRNA that knockdown an isoform protein containing Exon4 b. In some embodiments, the agent is an siRNA having the sequence as set forth in SEQ ID NO: 1 or other siRNA nucleotide sequences capable of knocking down Opa1 isoforms containing Exon4 b. As described above, the expression level of Opa1 isoform protein containing Exon4b in liver cancer cells was knocked down, and the proliferation ability of liver cancer cells was decreased. Thus, the reagent is used for reducing the expression level of Opa1 isoform protein containing Exon4b, and the kit according to the embodiment of the invention can specifically inhibit the proliferation capacity of liver cancer cells.

AAGTCATAGGAGCTTCTGACCTA(SEQ ID NO：1)。

According to an embodiment of the present invention, the kit is used for enhancing the proliferation ability of liver cancer cells, and the reagent is used for increasing the expression level of Opa1 isoform protein with Exon4 b. In some embodiments, the agent is an expression vector for over-expressing Opa1 isoform protein containing Exon4 b. As described above, the expression level of Opa1 isoform protein containing Exon4b in liver cancer cells is knocked down, and the proliferation capacity of the liver cancer cells is reduced; then, if the Opa1 heteromorphic protein containing Exon4b is supplemented, the proliferation capacity of the liver cancer cells is recovered. Therefore, the reagent is used for increasing the expression level of Opa1 isoform protein containing Exon4b, and the kit according to the embodiment of the invention can be used for specifically enhancing the proliferation capacity of liver cancer cells.

In a third aspect of the invention, the invention proposes the use of an agent for modulating the expression level of Opa1 isoform protein containing Exon4b in the preparation of a kit for modulating the proliferative capacity of a liver cancer cell. As described above, the expression level of Opa1 isoform protein containing Exon4b in liver cancer cells is knocked down, and the proliferation capacity of the liver cancer cells is reduced; then, if the Opa1 isoform protein containing Exon4b is supplemented, the proliferation capacity of the hepatoma cells is recovered. Therefore, the kit prepared by using the reagent can regulate the expression level of Opa1 isoform protein containing Exon4b, and further regulate the proliferation capacity of the liver cancer cells.

According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

according to an embodiment of the present invention, the reagent is used for reducing the expression level of Opa1 isoform protein containing Exon4b, and the kit is used for inhibiting the proliferation ability of hepatoma cells. In some embodiments, the agent is an siRNA or shRNA that knockdown an isoform protein containing Exon4 b. In some embodiments, the agent is an siRNA having the sequence as set forth in SEQ ID NO: 1 or other siRNA nucleotide sequences capable of knocking down Opa1 isoforms containing Exon4 b. As described above, the expression level of Opa1 isoform protein containing Exon4b in liver cancer cells was knocked down, and the proliferation ability of liver cancer cells was decreased. Therefore, the reagent is used for reducing the expression level of Opa1 isoform protein containing Exon4b, and a kit prepared by using the reagent can specifically inhibit the proliferation capacity of liver cancer cells.

According to an embodiment of the present invention, the reagent is used for increasing the expression level of Opa1 isoform protein of Exon4b, and the kit is used for enhancing the proliferation ability of liver cancer cells. In some embodiments, the agent is an expression vector for over-expressing Opa1 isoform protein containing Exon4 b. As described above, the expression level of Opa1 isoform protein containing Exon4b in liver cancer cells is knocked down, and the proliferation capacity of the liver cancer cells is reduced; then, if the Opa1 heteromorphic protein containing Exon4b is supplemented, the proliferation capacity of the liver cancer cells is recovered. Therefore, the reagent is used for increasing the expression level of Opa1 isoform protein containing Exon4b, and a kit prepared by using the reagent can specifically enhance the proliferation capacity of liver cancer cells.

In a fourth aspect of the present invention, a method of modulating the proliferative capacity of a hepatoma cell is presented. According to the embodiment of the invention, the expression level of Opa1 isoform protein containing Exon4b of liver cancer cells in the culture process is increased or decreased so as to control the proliferation capacity of the liver cancer cells. It should be noted that the method can be used for non-therapeutic purposes, such as scientific research, for example, by regulating the expression level of Opa1 isoform protein containing Exon4b, so as to regulate the proliferation capacity of liver cancer cells, and further obtain a corresponding liver cancer cell model for subsequent experimental research on liver cancer.

According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:

according to an embodiment of the present invention, the liver cancer cell is a liver cancer cell line. In some embodiments, the liver cancer cell lines are SMMC-7721, Bel-7402, Bel-7404, Bel-7405, MHCC97, HepG2, Hep3B, Hep1, Huh-7, PLC/PRF/5, H22, HHCC, HCCLM3, HB611, QGY-7701, QGY-7703 and the like. In other embodiments, the hepatoma cells are primary cells.

According to the embodiment of the invention, the expression level of Opa1 isoform protein containing Exon4b in the liver cancer cell is increased, and the proliferation capacity of the liver cancer cell is enhanced. According to the embodiment of the invention, the expression level of Opa1 isoform protein containing Exon4b in the liver cancer cell is reduced, and the proliferation capacity of the liver cancer cell is reduced. As described above, the expression level of Opa1 isoform protein containing Exon4b in liver cancer cells is knocked down, and the proliferation capacity of liver cancer cells is reduced; then, if the Opa1 isoform protein containing Exon4b is supplemented, the proliferation capacity of the hepatoma cells is recovered. Therefore, the proliferation capacity of the liver cancer cells can be specifically regulated by regulating the expression level of Opa1 isoform protein containing Exon4 b.

In a fifth aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the present invention, the pharmaceutical composition comprises an agent for decreasing the expression level of Opa1 isoform protein containing Exon4b, and the pharmaceutical composition is for inhibiting proliferation of liver cancer cells. As described above, the expression level of Opa1 isoform protein containing Exon4b in liver cancer cells was knocked down, and the proliferation ability of liver cancer cells was decreased. Thus, the pharmaceutical composition comprising the agent for decreasing the expression level of Opa1 isoform protein containing Exon4b can effectively inhibit the proliferation ability of liver cancer cells, or can provide an effective approach for the treatment of liver cancer.

According to an embodiment of the present invention, the above pharmaceutical composition may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the agent is siRNA or shRNA that knockdown a isoform protein containing Exon4 b. In some embodiments, the agent is an siRNA having the sequence as set forth in SEQ ID NO: 1 or other siRNA nucleotide sequences capable of knocking down Opa1 isoforms containing Exon4 b.

In a sixth aspect of the invention, the invention proposes the use of a functional inhibitor of the Opa1 isoform protein containing Exon4b in the manufacture of a medicament for inhibiting the proliferation of hepatoma cells. As described above, the expression level of Opa1 isoform protein containing Exon4b in liver cancer cells was knocked down, and the proliferation ability of liver cancer cells was decreased. Therefore, the agent for reducing the expression level of Opa1 isoform protein containing Exon4b, namely the functional inhibitor of Opa1 isoform protein containing Exon4b, can effectively reduce the proliferation capacity of liver cancer cells, or can provide an effective way for treating liver cancer.

According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

according to an embodiment of the invention, the functional inhibitor comprises at least one selected from the group consisting of an agent that specifically knockdown the Opa1 isoform protein containing Exon4b, an inhibitor of the Opa1 isoform protein containing Exon4 b. It is noted that an inhibitor of the Opa1 isoform protein containing Exon4b refers to an inhibitor that competitively inhibits the activity of the Opa1 isoform protein containing Exon4b or acts as a complementary protease active site. In some embodiments, the agent that specifically knockdown the Opa1 isoform protein containing Exon4b is an siRNA having the amino acid sequence of SEQ ID NO: 1 or other siRNA nucleotide sequences capable of knocking down Opa1 isoforms containing Exon4 b.

In a seventh aspect of the present invention, the present invention provides a method of screening a drug for inhibiting proliferation of liver cancer cells. According to an embodiment of the invention, the method comprises: contacting the liver cancer model with a candidate drug; comparing the expression level of Opa1 isoform protein of Exon4b in the liver cancer model before and after the contact to determine whether the candidate drug is the target drug. As described above, the expression level of Opa1 isoform protein containing Exon4b in liver cancer cells is knocked down, and the proliferation capacity of the liver cancer cells is reduced; then, if the Opa1 isoform protein containing Exon4b is supplemented, the proliferation capacity of the hepatoma cells is recovered. Furthermore, by comparing the expression levels of Opa1 isoform protein containing Exon4b in the liver cancer model before and after exposure, it can be effectively determined whether the drug candidate is a target drug.

According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:

according to embodiments of the invention, a decreased expression level of the Opa1 isoform protein containing Exon4b in the liver cancer model after exposure as compared to before exposure is indicative that the drug candidate is a drug of interest. In other words, if the expression level of Opa1 isoform protein containing Exon4b is decreased in the liver cancer model after exposure as compared to before exposure, the drug candidate is a drug of interest and may effectively inhibit proliferation of liver cancer cells.

Drawings

FIG. 1 shows a proliferative capacity profile of SK-Hep1 cells according to one embodiment of the invention; and

FIG. 2 shows a graph of Exon4b repopulation according to one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

In addition, iso or isoform refers to an allotrope; flag refers to the tag.