CN115990257A - Application of MARCH8 gene in preparing medicine for treating pancreatic cancer - Google Patents

Abstract

The invention discloses application of MARCH8 gene in preparing a medicine for treating pancreatic cancer, belonging to the technical field of tumor treatment. The invention utilizes CRISPR-based transcription activation technology to improve the expression level of MARCH8 protein in high-metastatic AsPC-1 cells, and results show that the protein can obviously inhibit proliferation and migration of tumor cells after the protein expression is up-regulated. Meanwhile, the invention also proves that the down regulation of the protein expression not only promotes the growth of tumor, but also can promote the lung metastasis of tumor cells through a chick embryo allantoic membrane (CAM) model. It can be seen that the MARCH8 gene can be used as a new target for pancreatic cancer treatment.

Description

Application of MARCH8 gene in preparing medicine for treating pancreatic cancer

Technical Field

The invention belongs to the technical field of tumor treatment, and particularly relates to application of MARCH8 gene in preparation of a medicine for treating pancreatic cancer.

Background

Pancreatic cancer belongs to a highly lethal tumor, and the poor clinical prognosis is closely related to factors such as early diagnosis difficulty, rapid metastasis, high treatment resistance and the like. Although surgery is the current treatment of choice, most patients enter advanced stages of the disease when diagnosed, losing the opportunity for surgical treatment; the drug treatment of advanced pancreatic cancer is still based on the combined chemotherapy of cytotoxic drugs, and the overall curative effect is poor. For many years, a great deal of research on development, metastasis and treatment resistance of pancreatic cancer has been carried out internationally, and in view of key roles of K-RAS tumor gene mutation and related signal paths in pancreatic cancer, attempts to develop molecular targeted drugs for tumor cell K-RAS mutation are research hotspots, but clinical success has not been achieved so far; in addition, in recent years, due to the discovery of a close association between pancreatic tumor stroma and tumor malignant phenotype, treatment strategies by targeting pancreatic tumor stroma have also been tried internationally, but the clinical efficacy is limited. These facts highlight the difficulty and difficulty of clinical pancreatic cancer treatment.

Ubiquitin system (ubiquitin system) is an intracellular protein post-translational modification system, which relies on the synergistic action of ubiquitin activating enzyme (E1), ubiquitin binding enzyme (E2) and ubiquitin ligase (E3) to jointly catalyze covalent binding between ubiquitin and substrate, and finally leads to ubiquitination of substrate protein. Depending on the ubiquitin signaling differences, the ubiquitinated proteins can be sorted and degraded by proteasomes, lysosomes or autophagosomes (autophagosomes), or can be involved directly in the regulation of downstream related signaling pathways by non-degradation means. Ubiquitin ligases can be divided into HECT and RING families based on the difference in functional protein domains. MARCH ubiquitin ligases are a family of ubiquitin ligases containing a RING-CH domain, comprising a total of 11 members of the MARCH8 protein. Whereas most members contain a typical transmembrane domain, MARCH ubiquitin ligases are classified as membrane-associated ubiquitin ligases.

Disclosure of Invention

One of the purposes of the invention is to provide the application of MARCH8 in preparing a medicine for treating pancreatic cancer.

Preferably, the pancreatic cancer is pancreatic ductal adenocarcinoma.

More preferably, the medicament comprises an effective amount of a MARCH8 expression promoter and pharmaceutically acceptable excipients.

It is a second object of the present invention to provide the use of MARCH8 gene as a drug target for screening, alleviating or/and treating pancreatic cancer drugs.

Preferably, the pancreatic cancer is pancreatic ductal adenocarcinoma.

It is a further object of the present invention to provide the use of the MARCH8 gene as a target gene in the treatment of pancreatic cancer for the design and manufacture of a medicament and/or biological preparation for the prevention, alleviation or/and treatment of pancreatic cancer.

Preferably, the pharmaceutical and/or biological agent comprises a MARCH8 expression promoter.

More preferably, the pancreatic cancer is pancreatic ductal adenocarcinoma.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a new target for treating pancreatic cancer and provides a new idea for treating pancreatic cancer.

Drawings

FIG. 1 is a graph showing the results of up-regulation of MARCH8 protein expression in example 1.

FIG. 2 is a graph showing the results of MARCH8 protein expression down-regulation in example 2.

FIG. 3 is a graph of MARCH8 ubiquitin ligase expression levels versus patient prognosis in example 3.

Detailed Description

Example 1 evaluation of the Effect of upregulation of MARCH8 protein expression on tumor cell proliferation, survival, migration and invasion

In the embodiment, an inducible CRISPRa gene transcription activation system based on a lentiviral vector is utilized, 5 sgRNA sequences aiming at MARCH8 ubiquitin ligase gene promoters are designed through a public website (http:// crispr-era. Stanford. Edu /), and the CRISPRa transcription activation system aiming at MARCH8 genes is established; the experiment is to select high-metastatic pancreatic cancer cell strains AsPC-1 and PANC-1 with relatively low MARCH8 protein expression background as cell models, and construct a stable cell strain capable of promoting intracellular expression of MARCH8 protein through a CRISPRa system; the effect of upregulation of MARCH8 ubiquitin ligase expression on tumor cell proliferation, survival, in vitro migration and invasiveness was then examined by conventional cell proliferation assay (ATPlaite assay), clonogenic assay (Clonogenic assay), cell Transwell migration, cell streak assay and invasive assay.

The CRISPRa gene transcription activation system used in this example: CRISPR activation (CRISPRa) technology is a novel technology for regulating gene transcription. The system utilizes a Cas9 nuclease mutant (dcas 9) to replace wild Cas9 nuclease and fuse with a VP64 protein region with transcription activator recruitment property to form dcas9-VP64 fusion protein. In contrast to wild-type Cas9, dcas9-VP64 retains DNA binding properties but loses nuclease activity. Thus, dcas9-VP64 was able to bind to a specific gene promoter region (but no longer cleaves DNA) under the direction of sgRNA directed against a specific gene promoter to exert a gene transcriptional activation function (Mol Cell biol.2015nov;35 (22): 3800-9.). To be able to activate expression of functional genes from endogenous levels, the project was based on lentiviral vectors in a pre-work, successfully establishing a CRISPRa gene transcriptional activation system regulated by a tetracycline-inducible expression system (Tet-on system).

The results are seen in fig. 1, where a: detecting the expression of MARCH8 in different cells by using a western blot; b: schematic representation of transcriptional activation technology gene elements of CRISPR; c: QPCR detects MARCH8 expression after different sgRNAs; d: western blot detects MARCH8 expression after different sgRNAs; e: schematic of scratch test cells; f: statistical diagrams of scratch experiments.

Conclusion: the western blot experiment proves that the MARCH8 protein has larger difference in expression level in different cell lines, such as relatively lower expression level in high metastatic AsPC-1 cells and other pancreatic cancer cells with higher malignancy (such as PANC-1) (figure 1A); the transcription activation technology based on CRISPR (figure 1B) is used for promoting the high expression of MARCH8 protein in high metastatic AsPC-1 cells under endogenous conditions (figures 1C and 1D), and the result shows that the protein can obviously inhibit proliferation and migration of tumor cells after the up-regulation of the expression of the protein (figures 1E and 1F), and the MARCH8 ubiquitin ligase is suggested to have potential effect of inhibiting pancreatic cancer. * P <0.05

Example 2 chick embryo allantoic model study of the Effect of MARCH8 differential expression on tumor growth and metastasis

Chick embryo allantoic membrane (Chorioallantoic membrane, CAM) has been developed as an important experimental model for tumor invasion and metastasis and is widely used in research of tumor biology (Palmer TD, et al JVis exp.2011). Based on the above ex vivo cell model, CAM model was used to further identify the effect of MARCH8 ubiquitin ligase differential expression on pancreatic cancer cell PANC-1 growth and metastasis.

The specific experimental scheme is as follows: (1) firstly, respectively regulating the expression of pancreatic cancer cell MARCH8 protein by using CRISPRa technology and RNA interference technology, and then preparing cell suspension; (2) taking an 8-day-old chick embryo, sterilizing eggshells by using iodine and alcohol in advance, and cutting a small hole with the diameter of about 1.5cm at the top of an air chamber by using surgical scissors in a sterile environment; (3) directly dripping tumor cell suspension onto CAM, standing for 10min, sealing the air chamber with sealing film, and culturing in incubator with 37.8deg.C and humidity of 60% -70% for 10-12 days; (4) observing the tumor formation condition, respectively collecting chicken embryo lung and liver, extracting genome DNA, detecting Alu repeated sequence of human genome DNA (taking chicken GAPDH gene as internal reference) by quantitative PCR, and evaluating the influence of MARCH8 ubiquitin ligase differential expression on tumor cell growth and transfer capability.

Conclusion: pre-inhibiting MARCH8 protein expression in pancreatic cancer cells PANC-1 with shRNA, followed by allantoic inoculation of 8 day old chick embryos (fig. 2A); as a result, it was found that the down-regulation of MARCH8 protein expression (FIG. 2D) can promote not only tumor growth (FIG. 2B) but also lung metastasis of tumor cells (FIG. 2C). These results suggest that MARCH8 protein has biological functions of inhibiting pancreatic tumors in vivo. * P <0.05

Example 3 evaluation of MARCH8 ubiquitin ligase expression levels versus patient prognosis

The relation between MARCH8 ubiquitin ligase expression level and pancreatic cancer patients metastasis and prognosis is deeply explored by using statistical methods such as chi-square test and Kaplan-Meier and the like through combining clinical stage, pathological grading and survival period data attached to TCGA cancer database and tissue chips (purchased from US Biomax company and Shanghai core super biotechnology Co., ltd.) of two different queues.

The results are shown in FIG. 3, and FIG. 3 reveals the potential association of MARCH8 protein with pancreatic cancer.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (8)

1. Use of march8 in the manufacture of a medicament for the treatment of pancreatic cancer.
2. 2. The use according to claim 1, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.
3. 3. The use according to claim 1 or 2, wherein the medicament comprises an effective amount of a MARCH8 expression promoter and pharmaceutically acceptable excipients.
4. Use of march8 gene as a drug target for screening, alleviating or/and treating pancreatic cancer.
5. 5. The use of claim 4, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.
6. The MARCH8 gene is used as a target gene in pancreatic cancer treatment and is applied to design and preparation of medicines and/or biological preparations for preventing, relieving or/and treating pancreatic cancer.
7. 7. The use according to claim 6, wherein the pharmaceutical and/or biological agent comprises a MARCH8 expression promoter.
8. 8. The use according to claim 6 or 7, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

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