CN116676391B - Application of USP21-MOF (universal serial bus-metal-oxide-fiber) regulation shaft in preparation of drug for targeted treatment of esophageal squamous cell carcinoma - Google Patents

Abstract

The invention relates to an application of a USP21-MOF regulatory shaft in preparing a drug for targeted therapy of esophageal squamous cell carcinoma. The USP21-MOF regulatory axis includes USP21 and MOF genes. The mRNA nucleotide sequence of the USP21 gene is shown as SEQ ID NO.1, and the mRNA nucleotide sequence of the MOF gene is shown as SEQ ID NO. 2. The invention discovers that the proliferation, migration and invasion of esophageal squamous cell carcinoma cells can be effectively inhibited by inhibiting the USP21-MOF regulation axis through interference for the first time, and designs si-USP21, si-MOF and si-UM which specifically knock down the expression level of USP21 and MOF mRNA. The invention also discovers that the effect of simultaneously knocking down the expression level of USP21 and MOF mRNA on the USP21-MOF regulation shaft is optimal, the effect of inhibiting the proliferation and metastasis of esophageal squamous cell carcinoma is optimal, and the method can be applied to the research and the preparation of targeted therapeutic drugs for esophageal squamous cell carcinoma.

Description

Application of USP21-MOF (universal serial bus-metal-oxide-fiber) regulation shaft in preparation of drug for targeted treatment of esophageal squamous cell carcinoma

Technical Field

The invention relates to an application of a USP21-MOF regulation shaft in preparing a drug for targeted treatment of esophageal squamous cell carcinoma, belonging to the technical field of biomedicine.

Background

The malignant degree of the esophageal cancer is extremely high, and the malignant degree brings serious threat to human health. About half of cases of confirmed esophageal cancer worldwide are concentrated in China, and the pathological types of esophageal cancer in China are mainly squamous cell carcinoma. The traditional esophageal cancer treatment mode mainly comprising operation and radiotherapy and chemotherapy is difficult to further improve the prognosis of patients due to lack of individual accurate treatment guidance. In recent years, with the development of sequencing technology, many genes driving cancer progression have been found, and some related targeted drugs have been applied to clinical treatment of cancer patients. Esophageal cancer currently lacks an effective therapeutic target, and the high tumor heterogeneity of esophageal cancer often results in treatment failure with a single targeted drug. Thus, elucidating a new regulatory pathway axis that drives malignant progression of esophageal cancer, and simultaneously conducting interventions for multiple genes in that pathway axis, would help to drive the development of clinical targeted therapeutic strategies for esophageal cancer and patient prognosis improvement.

The RNA-induced silencing complex (RNA-induced silencing complex, RISC) is a main protein complex for regulating and controlling the degradation of mRNA in cells, the complex is assembled with siRNA and degrades one sense strand of the mRNA, and then is combined with target mRNA in a base complementary pairing mode under the guidance of an antisense strand of the siRNA, and the target mRNA is sheared through ribonuclease II in the complex, so that the aim of down-regulating gene expression is fulfilled. siRNA is a double-stranded RNA composed of 20-25 pairs of nucleotides, and is aimed at the specificity and effectiveness of mRNA of a target gene, which is the key point of RNA interference technology. Therefore, the design of siRNA with high knocking-down efficiency and high specificity aiming at the target gene lays an important foundation for exploring the biological function of the target gene in the malignant progress of esophageal cancer and the research and development of targeted drugs thereof.

Disclosure of Invention

Aiming at the defects of the prior art, the USP21-MOF regulating shaft is applied to preparing the medicine for targeted treatment of esophageal squamous cell carcinoma.

The technical scheme of the invention is as follows:

the application of USP21-MOF regulation shaft as a drug target in preparing a drug for targeted treatment of esophageal squamous cell carcinoma.

According to a preferred embodiment of the present invention, the USP21-MOF regulatory shaft comprises USP21 and MOF genes.

According to the invention, preferably, the mRNA nucleotide sequence of the USP21 gene is shown as SEQ ID NO.1, and the mRNA nucleotide sequence of the MOF gene is shown as SEQ ID NO. 2.

According to the invention, preferably, the medicine for treating esophageal squamous cell carcinoma takes a USP21-MOF regulatory axis as an action target point, and can efficiently and specifically inhibit the USP21-MOF regulatory axis based on the expression of interference inhibition USP21 and MOF genes.

According to a preferred aspect of the invention, the medicament for treating esophageal squamous cell carcinoma is siRNA, shRNA, microRNA or a USP21-MOF regulatory shaft chemistry inhibitor.

Further preferred, the siRNA is si-USP21, si-MOF or si-UM;

the nucleotide sequence of the si-USP21 is shown as SEQ ID NO.3, and the nucleotide sequence of the si-MOF is shown as SEQ ID NO. 4;

the si-UM is a mixture of si-USP21 and si-MOF mixed in a molar ratio of 1:1.

A medicament for treating esophageal squamous cell carcinoma comprising a USP21-MOF regulatory axis inhibitor.

The beneficial effects are that:

1. the invention discovers that the proliferation, migration and invasion of esophageal squamous cell carcinoma cells can be effectively inhibited through inhibiting a USP21-MOF regulation shaft by interference for the first time. The invention designs si-USP21, si-MOF and si-UM which specifically knock down the expression level of USP21 and MOF mRNA, and the USP21-MOF regulation shaft is inhibited by knocking down the expression level of USP21 and MOF mRNA, so that proliferation, migration and invasion of esophageal squamous cell carcinoma cells are successfully inhibited, and therefore, the USP21-MOF regulation shaft can be used as a treatment target to prepare targeted medicine for treating esophageal squamous cell carcinoma.

2. The invention also discovers that the si-UM using the mixture of si-USP21 and si-MOF specifically knocks down the expression level of USP21 and MOF mRNA simultaneously, compared with the application of the same dosage of si-USP21 and si-MOF to knock down the expression level of USP21 and MOF mRNA independently, the invention has more remarkable inhibition effect on the proliferation, migration and invasion capacity of esophageal squamous cell carcinoma cells, which indicates that the inhibition effect of the expression level of USP21 and MOF mRNA on the regulatory axis of USP21-MOF is optimal, the effect of inhibiting the proliferation and the metastasis of esophageal squamous cell carcinoma is best, and the invention can be applied to the research and the development of esophageal squamous cell carcinoma targeted therapeutic drugs.

Drawings

FIG. 1 shows the statistical significance of the clear group differences obtained by transfection of si-UM into KYSE-150 cells, detection of USP21mRNA and MOF mRNA levels using reverse transcription-real time fluorescent quantitative PCR, and detection using Student's t.

FIG. 2 shows the statistical significance of differences between groups as determined by the transfection of si-USP21, si-MOF, si-NC and si-UM into KYSE-150 cells, respectively, with the same dose of each siRNA, using CCK-8 experiments to assess the proliferation potency of KYSE-150 cells.

FIG. 3 shows the statistical significance of differences between groups determined by selecting KYSE-150 cells, using Transwell experiments to detect changes in KYSE-150 cell migration and invasion capacity, and using one-way variance tests, with the same transfection agent amounts of si-UM, si-USP21, si-MOF and si-NC, respectively.

Detailed Description

The technical scheme of the present invention will be further described with reference to specific experimental examples, but the scope of the present invention is not limited thereto. The reagents and materials referred to in the examples are all commercially available products unless otherwise specified.

Human esophageal squamous cell carcinoma cell line KYSE-150 cells, available from Shanghai Fuchong biosciences Inc.

Example 1

Previous studies by the inventors found that USP21 and MOF expression levels were abnormally up-regulated in esophageal squamous cell carcinoma and were closely related to poor prognosis in patients; further investigation of its mechanism then found that USP21 was able to bind and stabilize MOF protein, thereby promoting malignant progression of esophageal squamous cell carcinoma. Thus, the inventors believe that the USP21-MOF regulatory axis can be targeted for use in the preparation of a medicament for targeted treatment of esophageal squamous cell carcinoma.

Example 2

Based on mRNA nucleotide sequences of USP21 and MOF genes, designing si-USP21 and si-MOF which specifically knock down expression levels of USP21 and MOF mRNA, wherein the nucleotide sequence of the si-USP21 is shown as SEQ ID NO.3, the nucleotide sequence of the si-MOF is shown as SEQ ID NO.4, and then mixing the si-USP21 and the si-MOF according to a molar ratio of 1:1 to obtain si-UM. The same doses of si-UM and si-NC were transfected into KYSE-150 cells, respectively, and USP21 and MOF mRNA levels were detected using reverse transcription-real-time fluorescent quantitative PCR, the specific results are shown in FIG. 1.

As can be seen from FIG. 1, si-UM is capable of effectively knocking down USP21 and MOF mRNA levels in KYSE-150 cells, interfering with the inhibition of the USP21-MOF regulatory axis.

The specific implementation process is as follows:

(1) KYSE-150 cells were cultured in 6-well plates, and when the cell growth density exceeded 60%, the same dose (volume 3. Mu.L, concentration 10. Mu.M) of si-UM and si-NC was transfected into KYSE-150 cells, respectively, using transfection reagent Lipofectamine RNAiMAX Reagent (Life technologies, 13778-150), and the cells were cultured for a further 48 hours;

(2) Extracting RNA from the cells obtained in the step (1) by using an RNA-Quick Purification Kit (China fir, RN 001) kit, detecting the concentration of the extracted RNA by using a NanoDrop2000, and evaluating the integrity of the extracted RNA by using agarose gel electrophoresis;

(3) Using the RNA extracted in the step (2) as a template and applying a reverse transcription kit LunaScript ^TM cDNA products were synthesized from RT Supermix Kit (NEB, E3010) using PowerGreen Master Mix (Thermo Fisher Scientific, 4367659) kit and Quantum studio ^TM Fluorescent quantitative PCR was performed by a 5System (Thermo Fisher Scientific) PCR apparatus using ACTB as a reference, according to 2 ^-ΔΔCT The formula calculates the relative expression levels of USP21 and MOF mRNA.

Example 3

KYSE-150 cells were selected and transfected with si-UM, si-USP21, si-MOF and si-NC, respectively, and the effect of the siRNA on the proliferation potency of KYSE-150 cells was examined by CCK-8 assay, and the specific results are shown in FIG. 2.

As can be seen from FIG. 2, transfection of si-UM significantly attenuated the proliferative capacity of KYSE-150 cells compared to the same dose of si-USP21 transfected alone. And it was also found that the use of si-UM in combination with si-USP21, si-MOF mixture specifically knockdown USP21 and MOF mRNA expression levels, resulted in a more pronounced inhibition of KYSE-150 cell proliferation capacity than the use of the same dosage of si-USP21, si-MOF alone to knock down USP21, MOF mRNA expression levels. It shows that the simultaneous knocking down of the expression level of USP21 and MOF mRNA achieves the best effect of inhibiting the USP21-MOF regulating shaft and the best effect of inhibiting the proliferation of esophageal squamous cell carcinoma.

The specific implementation process is as follows:

(1) The siRNA doses used were the same (volume 3. Mu.L, concentration 10. Mu.M) for each of si-UM, si-USP21, si-MOF and si-NC transfected into KYSE-150 cells, as detailed in example 2, the transfected cells were collected and the different transfected cells were re-cultured in 96-well plates at a concentration of 2,000 cells/well, 4 groups of four transfected cells each comprising si-UM, si-USP21, si-MOF, si-NC, 5 multiplex wells each;

(2) After the cells are attached (about 2-4 hours), adding CCK-8 reagent (TargetMol, C0005) (10 mu L/hole) into the 1 st group, placing in a 37 ℃ incubator for 1 hour, and measuring absorbance value (450 nm) in an enzyme-labeled instrument after simple shaking, namely the absorbance value of the initial planted cells;

(3) And (3) after the measurement of the absorbance values of the initial cells is completed, measuring the absorbance values of the rest three groups of cell pore plates at 450nm by using CCK-8 reagent respectively, and calculating the cell activities of different transfected cells in each group according to the same method.

Example 4

The same doses of si-UM, si-USP21, si-MOF, si-NC were transfected into KYSE-150 cells and the change in cell migration and invasiveness was examined using a Transwell assay, the specific results of which are shown in FIG. 3.

As can be seen from FIG. 3, the migration and invasion capacity of KYSE-150 cells was significantly inhibited by si-UM compared to si-USP21, si-MOF alone at the same transfection agent amount. And it has also been found that the use of si-USP21 and si-MOF mixture si-UM simultaneously specifically knocks down USP21 and MOF mRNA expression levels, compared with the use of the same dosage of si-USP21 and si-MOF alone to knock down USP21 and MOF mRNA expression levels, the inhibition effect on the migration and invasion capacity of KYSE-150 cells is more remarkable, which means that the inhibition effect of simultaneously knocking down USP21 and MOF mRNA expression levels on the regulatory axis of USP21-MOF is optimal, and the effect of inhibiting esophageal squamous cell carcinoma migration and invasion is best.

The specific implementation process is as follows:

(1) KYSE-150 cells were selected and transfected with si-UM, si-USP21, si-MOF and si-NC to KYSE-150 cells, respectively, using the same siRNA dose (volume 3. Mu.L, concentration 10. Mu.M) as described in example 2;

(2) Selecting a Transwell chamber (Corning, 3422), pre-spreading matrigel for detecting the invasive capacity of cells, and not spreading matrigel for detecting the migration capacity of cells, placing the Transwell chamber into a 24-well plate (600 mu L of complete culture medium is added in advance to each well);

(3) Transfected cells were collected, resuspended in serum-free medium to a cell concentration of 2 ten thousand cells/200. Mu.L, 200. Mu.L of the cell suspension was added to a transwell chamber at 37℃with 5% CO ₂ Culturing cells in a incubator for 48 hours;

(4) The transwell cells were removed and washed clean, cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, then stained with 0.1% crystal violet overnight, the non-migrated, invasive cells on the inside of the cells were carefully removed with a cotton swab, and the cells on the outside of the cells were observed and counted using an inverted microscope.

Claims (3)

1. The application of the si-RNA of the specific interference USP21 gene and the si-RNA of the specific interference MOF gene in preparing medicaments for targeted treatment of esophageal squamous cell carcinoma;

the nucleotide sequence of the si-RNA of the specific interference USP21 gene is shown as SEQ ID NO.3, and the nucleotide sequence of the si-RNA of the specific interference MOF gene is shown as SEQ ID NO. 4.

2. The use of claim 1, wherein the molar ratio of si-RNA that specifically interferes with the USP21 gene to si-RNA that specifically interferes with the MOF gene in the medicament is 1:1.

3. A medicament for treating esophageal squamous cell carcinoma, comprising si-RNA that specifically interferes with USP21 gene and si-RNA that specifically interferes with MOF gene;

the nucleotide sequence of the si-RNA of the specific interference USP21 gene is shown as SEQ ID NO.3, and the nucleotide sequence of the si-RNA of the specific interference MOF gene is shown as SEQ ID NO. 4.