Application of MATN1-AS1 in preparation of medicine for treating endometrial cancer

Abstract

The invention provides application of MATN1-AS1 in preparation of a medicine for treating endometrial cancer. The MATN1-AS1 is used for regulating proliferation of endometrial cancer cells by regulating expression of miR-200b. It is verified that MATN1-AS1 promotes growth of endometrial cancer through an E2F1 / miR-200b signal axis, the regulation mechanism of MATN1-AS1 for promoting development of endometrial cancer is clarified, a new theoretical basis is provided for pathogenesis of endometrial cancer, and a new potential action target is provided for treatment of endometrial cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medicine, and particularly relates to application of MATN1-AS1 in preparation of a drug for treating endometrial carcinoma. BACKGROUND

[0002] Endometrial carcinoma (EC) originates from the endometrium and is one of the most common cancers in women worldwide, accounting for 7% of all newly diagnosed cancers and 4% of all estimated cancer deaths in 2022. Given that EC mainly affects perimenopausal and postmenopausal women, the cancer burden of EC is likely to continue to increase due to the increase in adult and aging populations. Surgery is the main treatment for EC, supplemented by adjuvant therapy such as chemotherapy, followed by pelvic external beam radiotherapy and vaginal brachytherapy. Most EC patients who receive surgery and adjuvant therapy according to clinical pathological characteristics have a good prognosis, with a 5-year survival rate of 76-95%. However, the pathogenesis of EC has not been fully elucidated.

[0003] In recent years, the role of ncRNA in cancer has attracted increasing attention, including studies in endometrial carcinoma. More and more studies have shown that lncRNA, miRNA or the regulatory network composed of them affect EC biological processes. ncRNA can be used as a biomarker for EC diagnosis, predicting disease mechanisms and evaluating treatment effects, and as a therapeutic target for EC, through the design of interfering RNA (siRNA) or small molecule drugs targeting ncRNA, which can interfere with related signaling pathways and inhibit the malignant growth of EC tumors.

[0004] MATN1 antisense RNA 1 (MATN1-S1) is a newly discovered long non-coding RNA located at 1p35.2, which was first reported to be abnormally expressed in ischemic stroke. Subsequent studies have shown that MATN1-S1 is abnormally expressed in glioma and osteosarcoma, and it participates in the malignant growth of glioma and osteosarcoma cells by absorbing miR-200b / c / 429 and miR-1299. However, the expression and role of MATN1-S1 in endometrial carcinoma have not been reported. SUMMARY

[0005] Therefore, the application aims to overcome the defects in the prior art and provide application of MATN1-AS1 in preparation of a drug for treating endometrial carcinoma.

[0006] To achieve the above-mentioned purpose, the technical scheme of the application is as follows: Application of MATN1-AS1 in preparation of a drug for treating endometrial carcinoma, wherein MATN1-AS1 regulates the proliferation of endometrial carcinoma cells by regulating the expression of miR-200b.

[0007] Further, MATN1-AS1 positively regulates the expression of miR-200b.

[0008] Further, MATN1-AS1 up-regulates the expression of miR-200b by binding with E2F1.

[0009] Further, MATN1-AS1 promotes the proliferation of endometrial cancer cells through the E2F1 / miR-200b signaling axis.

[0010] Further, knocking out MATN1-AS1 inhibits the proliferation of endometrial cancer cells.

[0011] Further, knocking out MATN1-AS1 inhibits the expression of β-catenin protein, c-Myc gene and Cyclin D1 protein.

[0012] Further, the MATN1-AS1 / miR-200b axis regulates the proliferation of endometrial cancer cells through the Wnt / β-catenin pathway.

[0013] Further, knocking out MATN1-AS1 and overexpressing miR-200b positively regulate the expression of β-catenin protein, c-Myc gene and Cyclin D1 protein.

[0014] Compared with the prior art, the present application has the following advantages: The present application verifies that MATN1-AS1 promotes the growth of endometrial cancer through the E2F1 / miR-200b signaling axis, elucidates the regulatory mechanism of MATN1-AS1 in promoting the development of endometrial cancer, provides a new theoretical basis for the pathogenesis of endometrial cancer, and provides a new potential target for the treatment of endometrial cancer. The present application also verifies that the MATN1-AS1 / miR-200b axis can promote the progression of endometrial cancer, and its involvement in the molecular mechanism of endometrial cancer development is related to the activation of the Wnt / β-catenin pathway. This finding provides new insights into the molecular mechanism of endometrial cancer and lays a foundation for subsequent transformation research. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The miRNA-200b and MATN1-AS1 described in the embodiments of the present application are analyzed for expression in endometrial cancer. Figure 2 RT-PCR is used to detect the expression of MATN1-AS1 and miR-200b as described in the embodiments of the present application: A-B are the expression of MATN1-AS1 and miR-200b in tissues, and C-D are the expression of MATN1-AS1 and miR-200b in cells. Figure 3RT-PCR detection of MATN1-AS1 and miR-200b expression described in the embodiments of the present application: A-B is the detection of miR-200b expression in EC cells after MATN1-AS1 gene intervention, C-D is the detection of MATN1-AS1 expression in EC cells after miR-200b gene intervention; Figure 4 MATN1-AS1 affects EC cell viability by regulating miR-200b expression described in the embodiments of the present application; Figure 5 MATN1-AS1 affects EC cell proliferation by regulating miR-200b expression described in the embodiments of the present application; Figure 6 MATN1-AS1 upregulates miR-200b expression by binding to E2F1 described in the embodiments of the present application: A-B is the JASPAR database and PROMO database showing that E2F1 is one of the transcription factors that bind to the miR-200b promoter, C is the RIP experiment, and D is the luciferase reporter gene experiment; Figure 7 Protein expression electrophoretogram of β-catenin, AXIN2, and Cyclin D1 described in the embodiments of the present application; Figure 8 Continuous culture of si-MATN1-AS1 in human endometrial carcinoma organoid model described in the embodiments of the present application. DETAILED DESCRIPTION

[0016] Unless otherwise defined, the technical terms used in the following examples have the same meanings as generally understood by those skilled in the art to which the present application belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0017] The present application will be described in detail below with reference to the examples.

[0018] Example 1 Materials and Methods 1. Bioinformatics analysis (1) Ualcan database analysis of the expression difference of miR-200b in normal tissues and UCEC tissues (https: / / ualcan.path.uab.edu / cgi-bin / TCGA-miR-Result.pl?genenam=hsa-mir-200b&ctype=UCEC) (2) Heatmap expression of miR-200b in UCEC tissues analyzed by ualcan database (https: / / ualcan.path.uab.edu / cgi-bin / TCGA-miR-HeatMap.pl?cancer=UCEC&subt=&index=3) (3) R language was used to perform KEGG pathway enrichment analysis of MATN1-AS1 2. Clinical tissue collection: Collect EC and para-cancer tissues from patients who underwent surgery in our hospital from January 2023 to January 2024. The inclusion criteria are as follows: (1) diagnosed as EC; (2) no preoperative radiotherapy or chemotherapy; (3) available clinical information. If a patient has other tumors and serious diseases, and their clinical data are incomplete, they are excluded. All patients signed informed consent before surgery. Grouped by pathological grade or clinical stage.

[0019] 3. RT-PCR detection of target gene expression: Total RNA was extracted from tissues or cells using TRIzol method. According to the instructions of RNAPCR Kit reverse transcription kit, the reverse transcription system was prepared, and the cDNA was obtained after reaction; according to the operation of SYBR Premix Ex TaqII kit instructions, the PCR reaction system was prepared, and the fluorescent quantitative PCR reaction was carried out; according to the method of calculating the relative expression of each gene, GAPDH was used as an internal reference. -ΔΔCt

[0020] 4. CCK8 detection of cell viability: Collect the treated cells, dilute them to a certain proportion, and add them to the 96-well plate (5000 cells / 100ul). Set up blank wells (with medium, no cells) and control wells (medium without drug, with cells), 5 replicates for each group. Incubate at 37°C, 5% CO2 overnight. The next day, add 10 μl CCK-8 solution to each well, and incubate at 37°C for 1 hour. Measure the absorbance of each well at 450 nm. Cell viability% = (treated cell OD-blank OD) / (control cell OD-blank OD) x 100%. Organoid cell viability detection: harvest the organoids and dissociate them into single cells, then follow the same method.

[0021] 5. EDU detection of cell proliferation: The cells to be detected were cultured in medium containing EDU, and the cells were allowed to uptake EDU during growth. Collect the treated cells and deposit them on glass slides or culture dishes by centrifugation. Fix the cells, penetrate the treatment, then combine EDU with fluorescently labeled dye. Observe the stained cells under a fluorescence microscope to detect the number and location of stained cells. Count the number of EDU-positive cells to assess cell proliferation.

[0022] ​6、Western blot: The tissue or cells were placed in pre-cooled RIPA lysis buffer, lysed on ice for 30 minutes, centrifuged at 4°C, 12,000 x g for 15 minutes, the supernatant was collected, the protein concentration was determined by BCA method, and the concentration was adjusted to the same. Take 20-30 μg of protein sample, add SDS loading buffer, mix well, and boil at 95°C for 5 minutes to denature the protein. SDS-PAGE gel electrophoresis was performed for protein separation, and wet transfer method (Bio-Rad) was used to transfer the protein to PVDF membrane, and blocked in 5% BSA blocking solution at room temperature for 2h. The primary antibody (1:1000) was incubated at 4°C overnight, and the membrane was washed with TBST for 3 times. The secondary antibody (1:5000) was incubated at room temperature for 1 hour, and the membrane was washed with TBST for 3 times. ECL chemiluminescence reagent was used for development, and the signal was collected by chemiluminescence imaging system. The gray value of each group of bands in the Western blot image was quantified by Image Pro Plus 6.0, and GAPDH was used as an internal reference.

[0023] Results and discussion of example 2 1, Bioinformatics analysis of MATN1-AS1 and miRNA-200b Through UALCAN database analysis, it was found that miRNA-200b was significantly highly expressed in UCEC tissue (p<0.001, Figure 1 A-C) KEGG pathway enrichment analysis of MATN1-AS1 was performed using R language, and it was found that it was significantly enriched in Wnt signaling pathway. Figure 1 D) 2, Abnormal expression of MATN1-AS1 and miR-200b in EC tissue and cells Some EC tissues and para-cancer tissues were collected, and it was found that the expression of MATN1-AS1 and miR-200b in EC tissues was abnormally high compared with para-cancer tissues. Figure 2 Further, we found that the expression of MATN1-AS1 and miR-200b in EC cell lines was also abnormally high compared with normal endometrial epithelial cells.

[0024] 3, MATN1-AS1 can positively regulate the expression of miR-200b In order to clarify the upstream and downstream regulation relationship between MATN1-AS1 and miR-200b, MATN1-AS1 or miR-200b was performed in EC cells (Ishikawa, the following experiments were all used this cell). As shown in Figure 3 MATN1-AS1 can positively regulate the expression of miR-200b, while miR-200b has no significant effect on the expression of MATN1-AS1. The above results suggest that miR-200b is a downstream molecule of MATN1-AS1.

[0025] 4. MATN1-AS1 affects EC cell viability by regulating miR-200b expression. MATN1-AS1 gene intervention in EC cells, such as Figure 4 As shown, overexpression of MATN1-AS1 increases cell viability, while knockout of MATN1-AS1 inhibits cell viability. Simultaneous treatment of EC cells with MATN1-AS1 knockout and miR-200b overexpression showed that miR-200b overexpression reversed the inhibitory effect of MATN1-AS1 knockout on EC cell viability, indicating that MATN1-AS1 may affect EC cell viability by regulating miR-200b expression.

[0026] 5. MATN1-AS1 affects EC cell proliferation by regulating miR-200b expression. MATN1-AS1 gene intervention in EC cells, such as Figure 5 As shown, knockout of MATN1-AS1 inhibits cell proliferation, while overexpression of MATN1-AS1 promotes cell proliferation. EC cells were treated with MATN1-AS1 knockout and simultaneously treated with miR-200b overexpression. EDU analysis showed that overexpression of miR-200b reversed the inhibitory effect of MATN1-AS1 knockout on EC cell proliferation, indicating that MATN1-AS1 may affect EC cell proliferation by regulating miR-200b expression.

[0027] 6. MATN1-AS1 may upregulate miR-200b expression by binding to E2F1. JASPAR database ( Figure 6 A) and the PROMO database ( Figure 6 B) shows that E2F1 can bind to the miR-200b promoter. RIP experiments demonstrate that MATN1-AS1 binds to E2F1. Figure 6 C). Simultaneously, luciferase reporter gene assays confirmed that E2F1 plays a role in miR-200b promoter transcriptional activation. Figure 6 D).

[0028] 7. Western blot analysis validates the molecular mechanism by which the MATN1-AS1 / miR-200b axis regulates endometrial cancer progression through the Wnt / β-catenin pathway. Gene intervention was performed in EC cells, and the results of Western blot analysis were as follows: Figure 7As shown, the difference in the protein levels of β-catenin, c-Myc and Cyclin D1 in the Control group and the si-NC group was not statistically significant (p>0.05). After the EC cells were subjected to MATN1-AS1 knockout treatment, the protein levels of β-catenin, c-Myc and Cyclin D1 were significantly reduced (p<0.01). After the EC cells were subjected to MATN1-AS1 knockout treatment and miR-200b overexpression treatment at the same time, the protein levels of β-catenin, c-Myc and Cyclin D1 were increased (p<0.01). This indicates that the MATN1-AS1 / miR-200b axis has a certain regulatory effect on the occurrence of EC through the Wnt / β-catenin pathway.

[0029] 8. Functional role of RNA MATN1-AS1 in the occurrence and development of endometrial cancer We constructed small interfering RNA (si-MATN1-AS1) targeting MATN1-AS1 and performed functional loss experiments in a human endometrial cancer organoid model. The organoids were divided into two groups: a control group (transfected with negative control siRNA, i.e., a knockout blank group) and an experimental group (transfected with si-MATN1-AS1). After transfection, the morphology of the organoids was observed every 48 hours and the growth was recorded, and the continuous culture lasted for 16 days.

[0030] The results are shown in Figure 8 Compared with the blank control group, the proliferation ability of the si-MATN1-AS1 group of organoids was significantly decreased. Since the 8th day, the average diameter growth rate of the organoids in the experimental group was significantly slowed down, and by the 16th day, the volume of the organoids in the experimental group was significantly reduced by about 50% compared with the control group. In addition, the budding structure of the organoids in the si-MATN1-AS1 group was reduced, the overall structure tended to be loose, the cell density was reduced, suggesting that the self-renewal and proliferation potential was impaired. In summary, in the human endometrial cancer organoid model, silencing MATN1-AS1 can significantly inhibit the growth and expansion ability of the organoids, indicating that MATN1-AS1 may play an important role in maintaining the stemness and proliferative activity of tumor organoids.

[0031] In conclusion, the present application finds that long non-coding RNA (lncRNA) MATN1-AS1 and its downstream molecule miR-200b are significantly highly expressed in EC tissues and cell lines through experimental verification. Function experiments (CCK-8, EdU) confirm that overexpression of MATN1-AS1 significantly enhances the viability and proliferation ability of EC cells, and its knockout has the opposite effect, suggesting that MATN1-AS1 has a pro-cancer effect. The present application finds that overexpression of miR-200b can reverse the inhibition of EC cell viability and proliferation caused by MATN1-AS1 knockout, indicating that MATN1-AS1 at least partly exerts its effect by regulating miR-200b.

[0032] The present application finds that MATN1-AS1 has a high possibility of binding to transcription factor E2F1 through RPISeq analysis. Through JASPAR database and PROMO database, we find that E2F1 can bind to the miR-200b promoter. Therefore, it is speculated that MATN1-AS1 may activate miR-200b by binding to E2F1, and then promote the malignant growth of EC.

[0033] In order to further prove the above speculation, the present application proves that MATN1-AS1 binds to E2F1 through RIP experiment, and at the same time, verifies that E2F1 has the effect of transcriptional activation of miR-200b promoter through luciferase reporter gene. In addition, EC cells are subjected to MATN1-AS1 knockout treatment, and then subcutaneously injected to construct nude mouse xenograft tumor. The tumor growth curve results show that knockout of MATN1-AS1 can significantly inhibit the growth of EC tumor.

[0034] The present application finds that MATN1-AS1 is significantly enriched in Wnt pathway through KEGG enrichment analysis. The present application finds that the expression of MATN1-AS1 silencing causes the down-regulation of the expression of β-catenin, c-Myc and Cyclin D1 in EC cells through WB experiment, and β-catenin is the core effector molecule of Wnt pathway, the accumulation of which can promote nuclear translocation and activate downstream target genes (such as c-Myc and Cyclin D1), and then drive cell proliferation and tumor progression, suggesting that silencing MATN1-AS1 can induce WNT / β-catenin pathway inactivation. EC cells are subjected to MATN1-AS1 knockout treatment while being subjected to miR-200b overexpression treatment, and the protein levels of β-catenin, c-Myc and Cyclin D1 are obviously reversed to increase, which can partially rescue the influence of MATN1-AS1 silencing, indicating that the MATN1-AS1 / miR-200b / WNT axis has a certain regulatory effect in the process of EC occurrence. MATN1-AS1 plays a pro-cancer role, which may remove the inhibition of miR-200b on downstream target genes, thereby activating the pro-cancer Wnt / β-catenin signaling pathway. The knockout of MATN1-AS1 inhibits the pathway, suggesting that it may enhance Wnt signaling by stabilizing β-catenin or inhibiting its degradation complex, and MATN1-AS1 can positively regulate Wnt / β-catenin pathway. The down-regulation of c-Myc and Cyclin D1 may explain the phenomenon that the proliferation of EC cells is inhibited after MATN1-AS1 silencing. However, future research still needs to further explore how WNT / β-catenin regulates the specific mechanism of EC occurrence and development through c-Myc and Cyclin D1, especially the interaction of the pathway with other oncogenic signals.

[0035] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of MATN1-AS1 in the preparation of a drug for treating endometrial cancer, characterized in that: MATN1-AS1 regulates the proliferation of endometrial cancer cells by modulating the expression of miR-200b.

2. The use of MATN1-AS1 according to claim 1 in the preparation of a medicament for treating endometrial cancer, characterized in that: MATN1-AS1 positively regulates the expression of miR-200b.

3. The use of MATN1-AS1 according to claim 2 in the preparation of a medicament for treating endometrial cancer, characterized in that: MATN1-AS1 upregulates miR-200b expression by binding to E2F1.

4. The use of MATN1-AS1 according to claim 3 in the preparation of a medicament for treating endometrial cancer, characterized in that: MATN1-AS1 promotes the proliferation of endometrial cancer cells through the E2F1 / miR-200b signaling axis.

5. The use of MATN1-AS1 according to claim 1 in the preparation of a medicament for treating endometrial cancer, characterized in that: Knocking out MATN1-AS1 inhibits the proliferation of endometrial cancer cells.

6. The use of MATN1-AS1 according to claim 5 in the preparation of a medicament for treating endometrial cancer, characterized in that: Knocking out MATN1-AS1 inhibits the expression of β-catenin protein, c-Myc gene and Cyclin D1 protein.

7. The use of MATN1-AS1 according to claim 1 in the preparation of a medicament for treating endometrial cancer, characterized in that: The MATN1-AS1 / miR-200b axis regulates the proliferation of endometrial cancer cells through the Wnt / β-catenin pathway.

8. The use of MATN1-AS1 according to claim 7 in the preparation of a medicament for treating endometrial cancer, characterized in that: Knockout of MATN1-AS1 and overexpression of miR-200b positively regulate the expression of β-catenin, c-Myc, and Cyclin D1 proteins.

Images