CN112294957A

CN112294957A - Application of human melanoma-related cyclic RNAcirROR 1 target point in preparation of melanoma treatment drug

Info

Publication number: CN112294957A
Application number: CN202011130236.9A
Authority: CN
Inventors: 周建大; 刘彦; 谢慧清; 周玥; 李佩雯; 吴先睿; 史可; 李哲琳
Original assignee: Third Xiangya Hospital of Central South University
Current assignee: Third Xiangya Hospital of Central South University
Priority date: 2020-10-21
Filing date: 2020-10-21
Publication date: 2021-02-02
Anticipated expiration: 2040-10-21
Also published as: CN112294957B

Abstract

The invention relates to the technical field of biological medicines, and discloses application of a circular RNA circROR1 target spot related to human melanoma in preparation of a medicine for treating melanoma. The invention shows that the circular RNA circROR1 and the binding protein PTBP1 related to the human melanoma have potential effects in melanoma drug treatment, melanoma prognosis evaluation and melanoma curative effect monitoring, the invention provides more choices for more strengthening and treating the melanoma clinically, and the drug and the related action mechanism of the invention can become a new strategy for improving the melanoma treatment.

Description

Application of human melanoma-related cyclic RNAcirROR 1 target point in preparation of melanoma treatment drug

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a human melanoma-related cyclic RNAcircROR1 target spot in preparation of a medicine for treating melanoma.

Background

The latest NCCN clinical practice guidelines state that: the melanoma metabolic disorder is one of the main reasons for chemotherapy resistance and radiotherapy resistance of melanoma cells, and the accurate treatment aiming at the melanoma cell metabolic disorder is a brand new direction for treating melanoma. With the rapid development of tumor molecular biology, genomic high-throughput sequencing and metabonomics, the treatment of melanoma is more and more deeply explored towards the direction of precise target molecule treatment. Melanoma cells differ from normal cells in that the ubiquity of hypoxic regions in melanoma cells and the mutation of the relevant genes under hypoxic conditions lead to melanoma cells turning on the potential for unlimited proliferation in the glycolytic metabolic mode, the Warburg effect. The process by which melanoma cells are metabolized by the Warburg effect is faster than normal cells and grows unrestrictedly in the surrounding tissues, mediating widespread invasion and distant metastasis of melanoma. Therefore, the search for molecular mechanisms of action that block glycolysis in skin melanoma has been reluctant to search for new targets for metabolic therapy.

Circular RNA (circRNA) is a kind of endogenous non-coding RNA different from linear RNA, and in eukaryotic cells, circRNA is formed in a nucleus, and the most common synthesis mode is to directly connect 3 'ends and 5' ends by covalent bonds through trans-splicing to form a closed circular structure, and the closed circular structure is mainly transported to cytoplasm after being generated to play a role. The CircRNA has no 3 'end polyA and 5' end cap structure, and is not easy to be degraded by exonuclease, so that the CircRNA can be stably and widely distributed in different tissue structures. The circRNA can be used as a sponge to adsorb miRNA by a CERNA mechanism, positively regulates the expression level of a downstream target gene, can adsorb protein in cytoplasm to form circRNPs to perform a function, can be combined with the protein to regulate the post-transcriptional modification process of other genes, can also be combined with RNA to interfere the splicing processing process of other genes, can be translated into polypeptide and the like, and plays a great role in the occurrence and development of malignant tumors. However, the research on circRNA is not sufficient at present, and the research on melanoma is worth further exploration. The common database of Cancer-specific-circrnadatatase (CSCD) shows that circROR1 is a tumor-specific high-expression circular RNA, and further searching the common database of TCGA and GEPIA shows that circROR1 is highly expressed in metastatic melanoma tissues through a biological analysis.

In conclusion, the inventor invents the application of the human melanoma related cyclic RNAcircROR1 target point in the preparation of drugs for treating melanoma.

Disclosure of Invention

Based on the problems, the invention provides the application of the human melanoma-related cyclic RNAcircROR1 target point in preparing drugs for treating melanoma, and the invention provides more choices for more intensive clinical treatment of melanoma.

In order to solve the technical problems, the invention provides the following technical scheme:

application of circular RNA circROR1 target spot related to human melanoma in preparation of drugs for treating melanoma.

Furthermore, the medicament achieves the aim of treating the melanoma by reducing the expression of the human melanoma-associated circular RNAcircROR1 target or silencing the human melanoma-associated circular RNA circROR1 target.

Further, the action mechanism of the medicine is as follows: the expression of a splicing silencing factor PTBP1 is regulated by preventing or preventing the formation of an RNA-protein complex by human melanoma-related cyclic RNAcircROR1, further preventing or preventing the formation of a splicing silencer by human melanoma-related cyclic RNAcircROR1 through the formation of the RNA-protein complex, further preventing or preventing the recruitment of hnRNPA1 to form a splicing isomer, further preventing the expression regulation of a downstream PKM gene and an ABCB gene, reducing the expression of the PKM2 protein and slowing down the rate-limiting process of the last step of glycolysis on the one hand, and reducing the expression of the ABCB5 protein, reducing the expression of an ATP transport protein involved in the glycolysis process on the other hand, slowing down the glucose metabolism process of melanoma in key nodes and processes, further preventing the glycolysis metabolism of melanoma, and finally preventing the proliferation and invasion and metastasis of the melanoma.

Compared with the prior art, the invention has the beneficial effects that: the invention shows that the human melanoma-related cyclic RNAcirROR 1 and the binding protein PTBP1 have potential effects in melanoma drug treatment, melanoma prognosis evaluation and melanoma curative effect monitoring, the invention provides more choices for more strengthening and treating melanoma clinically, and the drug and the related action mechanism of the invention can become a new strategy for improving melanoma treatment.

Drawings

FIG. 1 is a diagram illustrating the screening results of a non-coding group chip according to an embodiment of the present invention;

FIG. 2 is a graph showing the results of the change in PKM and ABCB after overexpression of circROR1 in melanoma cell lines according to an embodiment of the present invention;

FIG. 3 is a graph showing the results of sequencing analysis of the circROR1 binding to PTBP1 protein binding String;

FIG. 4 is a graph of the significant correlation between PTBP1 and patient prognosis poor in melanoma clinical samples according to an embodiment of the present invention;

FIG. 5 is a graph of the results of the interaction between circROR1 and PTBP1 according to an embodiment of the present invention;

FIG. 6 is a graph of the results of the interaction between circROR1 and PTBP1 according to an embodiment of the present invention;

FIG. 7 is a schematic diagram of the mechanism of action of the cyclic RNAcircROR1 and the binding protein PTBP1 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limitations of the present invention.

Example (b):

in order to search the reason for the high invasion and metastasis of melanoma, the inventor researches the non-coding group chip screening of a normal melanoma cell line HM, a low invasion metastatic cell line WM35 and a high invasion metastatic cell line WM451, identifies remarkably changed non-coding RNA, and finds that the circular RNAcircROR1 (hereinafter, the same is called as circROR1) related to human melanoma is remarkably and highly expressed in the high invasion metastatic melanoma cell line, and the result is shown in the attached drawing 1; after circROR1 in melanoma cells is over-expressed, the migration and invasion capacity of the melanoma cells are remarkably enhanced; by silencing circROR1, the migration and invasion capacity of melanoma cells are obviously weakened. The results all show that the circROR1 target point can be applied to the preparation of sensitive molecular markers or kits for evaluating the prognosis of melanoma or evaluating the recurrence of melanoma or predicting the metastasis of melanoma.

Previous GO functional enrichment prediction analysis shows that circRNA with up-regulated expression in the high-metastatic WM451 is enriched in glycolysis of a metabolic pathway, and occurrence of glycolysis and changes of key proteins PKM and ABCB families of glycolysis are detected after the circROR1 is over-expressed in a WM451 cell line, and the results are shown in figure 2, and show that after the circROR1 is over-expressed, the expressions of PKM2 and ABCB5 are obviously increased, and the expression trends of proteins and mRNAs of different subtypes of the PKM and ABCB families show different change trends and are even completely opposite. The experimental results show that circROR1 can be used as a target point to be applied to the preparation of medicines for treating melanoma.

Previous inventors showed by String sequencing analysis that the downstream effect binding protein of circROR1 was PTBP 1; later tests of the binding capacity of circROR1 and PTBP1 showed that the two have a binding probability of 71%, a differential potency of 98%, a free binding interaction energy of-29.5331 kcal/mol, and a red marker indicating the predicted binding sequence site, as shown in FIG. 3.

The inventor then carries out survival analysis on 461 clinical sample tissue data of skin melanoma, the total survival time of the melanoma with high expression of PTBP1 is obviously shortened (p is less than 0.05), and the result is shown in figure 4. The results of the spearman correlation analysis of PTBP1 and hnRNPA1 using GEPIA showed that PTBP1 and hnRNPA1 were significantly positively correlated in melanoma (P <0.05), and String protein interaction showed that PTBP1 could bind to proteins hnRNPA1, hnRNPk, hnRNPC, etc. The PTBP1 belongs to RNA binding protein, and can form a complex with heterogeneous nuclear RNA to participate in the formation of pre-mRNA in cell nucleus and the metabolism and transportation of mRNA, thereby influencing the expression level of mRNA. The results of the study herein suggest that PTBP1 may be involved in melanoma variable excision in concert with hnRNPA 1.

The experimental results show that circROR1, PKM2 and ABCB5 can influence the glycolysis and invasion and metastasis of melanoma. Referring to the attached figures 5 and 6, the inventor further indicates that circROR1 interacts with PTBP1 through Co-IP results, immunofluorescence double-color detection indicates that the interaction position of circROR1 and PTBP1 is mainly in the nucleus, and protein stability experiments indicate that the circROR1 can promote PTBP1 high expression; the functional recovery experiment results show that: under the action of cisplatin, compared with a group for knocking out circROR1+ over-expression PTBP1, the circrorR 1 in WM451 cells is knocked out, the cell proliferation and migration capacity is reduced, the apoptosis rate is increased, the tumor body growth speed is increased and the tumor body size is obviously reduced in an animal experiment, immunohistochemistry indicates that Ki-67 is reduced, and the apoptosis related index is obviously increased.

In summary, this example shows that circROR1 and its downstream target protein PTBP1 can be applied to the preparation of drugs for treating melanoma, and can exert effective effects to guide clinical decisions.

The example shows that circROR1 and binding protein PTBP1 have potential effects in treatment, prognosis evaluation and efficacy monitoring of melanoma, and also shows that circROR1 can regulate the expression of a splicing silencing factor PTBP1 through the form of RNA-protein complex formation, simultaneously recruit hnRNPA1 to form a splicing isomer, participate in the expression regulation of PKM gene and ABCB gene, on one hand, increase the expression of PKM2 protein and accelerate the final speed-limiting process of glycolysis, on the other hand, increase the expression of ABCB5 protein, increase the expression of ATP transport protein participating in the glycolysis process, and regulate the melanoma sugar metabolism process in key nodes and processes; promotes the glycolysis metabolism of the melanoma, and finally promotes the rapid proliferation, invasion and metastasis of the melanoma. Therefore, the PKM2 and the ABCB5 can also be used as targets to be applied to the preparation of medicines for treating melanoma. The mechanism of action is shown in figure 7.

The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the process of verifying the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all the equivalent structural changes made by applying the content of the specification of the invention should be covered by the scope of the invention.

Claims

1. Application of circular RNA circROR1 target spot related to human melanoma in preparation of drugs for treating melanoma.

2. The use according to claim 1, wherein the medicament achieves the treatment of melanoma by reducing expression of human melanoma associated circular RNA circROR1 target or silencing human melanoma associated circular RNA circROR1 target.

3. The use according to claim 1, wherein the mechanism of action of the drug is: the expression of a splicing silencing factor PTBP1 is regulated by preventing or preventing the circular RNA circROR1 related to the human melanoma from forming an RNA-protein complex, further preventing or preventing the circular RNA circROR1 related to the human melanoma from forming an RNA-protein complex, further preventing or preventing the recruitment of hnRNPA1 to form a splicing isomer, further preventing the expression regulation of a downstream PKM gene and an ABCB gene, reducing the expression of the PKM2 protein and slowing down the rate-limiting process of the last step of glycolysis on the one hand, and reducing the expression of the ABCB5 protein, reducing the expression of ATP transport proteins involved in the glycolysis process on the other hand, slowing down the glucose metabolism process of the melanoma in key nodes and processes, thereby stopping the glycolysis metabolism of the melanoma and finally stopping the proliferation and invasion and metastasis of the melanoma.