Application of LncRNA as ovarian cancer biomarker and therapeutic target
By using LncRNA SLC8A1-AS1 as a biomarker for ovarian cancer and designing Smart Silencer RNA inhibitors, the challenges of early diagnosis and targeted therapy for ovarian cancer have been solved, achieving highly efficient diagnostic and therapeutic effects.
Patent Information
- Application Number
- CN202511665232.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-13
- Publication Date
- 2026-02-06
AI Technical Summary
Early diagnosis of ovarian cancer is difficult, prognostic assessment methods are limited, and targeted therapy resistance exists. Existing treatment methods are highly invasive, have adverse reactions, and are prone to drug resistance. More effective diagnostic markers and therapeutic targets are needed.
Using LncRNA SLC8A1-AS1 as a biomarker for ovarian cancer, we developed targeted therapeutic drugs by designing a specific and highly effective inhibitor, Smart Silencer RNA, to suppress its expression.
It provides a highly specific early diagnostic tool, identifies potential targets for targeted therapy of ovarian cancer, significantly inhibits the proliferation of ovarian cancer cells, and improves treatment efficacy.
Smart Images
Figure CN121472407A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of biotechnology and medicine, and mainly relates to application of LncRNA as an ovarian cancer biomarker and a therapeutic target. BACKGROUND
[0002] Ovarian cancer is a collection of heterogeneous malignancies that can originate from any tissue part of the ovary. The incidence of ovarian cancer is 3.4%, and the mortality rate is 4.7%. More than 300,000 women suffer from ovarian cancer every year, and about 207,000 women die of ovarian cancer every year, which is the eighth most common and the fifth most deadly cancer among women worldwide. Because there is no specificity in the early symptoms of ovarian cancer, and the anatomical position is special, most of the clinical patients are in the middle and late stages when they are diagnosed.
[0003] The current clinical treatment method is surgical treatment and platinum-taxol maintenance chemotherapy, but because surgical treatment is highly traumatic, and the chemotherapy drugs have certain adverse reactions and drug resistance, patients still have different degrees of recurrence risk, and the prognosis is poor. The maintenance treatment of bevacizumab or poly (ADP-ribose) polymerase inhibitors (PARPi) shows the effect of prolonging the progression-free survival, but cannot prolong the overall survival, which shows that more effective maintenance treatment is needed.
[0004] In summary, the clinical diagnosis and treatment of ovarian cancer still faces many challenges such as difficulty in early diagnosis, limited means of prognosis evaluation, and drug resistance of targeted therapy. Therefore, it is an urgent need of current research to explore a new ovarian cancer diagnostic marker and therapeutic target with high diagnostic value and prognosis prediction ability. SUMMARY
[0005] In view of the above problems, the application provides application of LncRNA as an ovarian cancer biomarker and a therapeutic target.
[0006] In a first aspect, the application provides application of an ovarian cancer biomarker LncRNA in preparation of a product for diagnosing ovarian cancer, wherein the LncRNA positively regulates occurrence and development of ovarian cancer, and the ovarian cancer biomarker is LncRNA SLC8A1-AS1.
[0007] Further, the product comprises a diagnostic reagent or reagent for detecting the ovarian cancer biomarker.
[0008] In a second aspect, the application provides application of a primer pair for detecting an ovarian cancer biomarker LncRNA SLC8A1-AS1 in preparation of a product for diagnosing ovarian cancer, wherein the primer pair comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO. 1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO. 2.
[0009] In a third aspect, the present application provides application of the LncRNA SLC8A1-AS1 in preparation of a product for preventing and / or treating ovarian cancer.
[0010] Further, the expression of the LncRNA SLC8A1-AS1 is inhibited.
[0011] In a fourth aspect, the present application provides a LncRNA SLC8A1-AS1 expression inhibitor, wherein the LncRNA SLC8A1-AS1 expression inhibitor is a Smart silencer RNA, and the nucleotide sequence of the Smart silencer RNA is any one of SEQ ID NO. 3-8.
[0012] In a fifth aspect, the present application provides a pharmaceutical composition comprising the inhibitor.
[0013] In a sixth aspect, the present application provides a pharmaceutical preparation comprising the pharmaceutical composition and a pharmaceutically acceptable carrier.
[0014] In a seventh aspect, the present application provides application of the inhibitor, the pharmaceutical composition or the pharmaceutical preparation in preparation of a product for preventing and / or treating ovarian cancer.
[0015] The present application has the following beneficial effects: (1) The LncRNA SLC8A1-AS1 is innovatively used in the research of ovarian cancer, and it is found that the increase of the expression level is significantly related to the decrease of the survival rate of patients, thereby establishing the potential of the LncRNA SLC8A1-AS1 as a biomarker of ovarian cancer and laying a solid foundation for developing a high-specificity early diagnosis product of ovarian cancer.
[0016] (2) The positive regulation role of the LncRNA SLC8A1-AS1 in the occurrence and development of ovarian cancer is further clarified, and a new potential target for the targeted treatment of ovarian cancer is provided.
[0017] (3) Based on the above target, a specific and efficient inhibitor, Smart Silencer RNA, is designed, which can effectively inhibit the expression of SLC8A1-AS1, and provides a core candidate molecule for the development of a targeted therapeutic drug. BRIEF DESCRIPTION OF DRAWINGS
[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows.
[0019] Figure 1 is the survival curve result of the LncRNA SLC8A1-AS1 in the Kaplan-Meier Plotter database; Figure 2 The expression of LncRNA SLC8A1-AS1 in different databases; Figure 3 The expression level of LncRNA SLC8A1-AS1 in human normal ovarian epithelial cells and ovarian cancer cells; Figure 4 The efficiency of small interfering RNA knockdown of LncRNA SLC8A1-AS1 in ovarian cancer cells ES2 and SKOV3 is shown in the graph; Figure 5 The microphotograph of the plate cloning result is shown; Figure 6 The microphotograph of the EdU experiment result is shown. DETAILED DESCRIPTION
[0020] The present application will be further described in detail below with reference to examples, so that those skilled in the art can implement the present application according to the description.
[0021] Example 1 Survival curve analysis of LncRNA SLC8A1-AS1 Enter the Kaplan-Meier Plotter database (https: / / kmplot.com / analysis / index.php?p=home) and select the ovarian cancer panel, input the gene name as SLC8A1-AS1, and select the survival curve of OS and PSF respectively. The results are shown in Figure 1 The left graph is the OS survival curve of LncRNA SLC8A1-AS1, and the expression of LncRNA SLC8A1-AS1 increases, and the survival rate of the patient decreases; vice versa. The right graph is the PSF survival curve of LncRNA SLC8A1-AS1, and the expression of LncRNA SLC8A1-AS1 increases, and the survival rate of the patient decreases; vice versa.
[0022] Example 2 Analysis of the expression of LncRNA SLC8A1-AS1 in patients Enter the Kaplan-Meier Plotter database (https: / / kmplot.com / analysis / index.php?p=home) and select the compare normal and tumor panel, input the gene name and cancer name, and analyze. The results are shown in Figure 2The expression level of LncRNA SLC8A1-AS1 in the ovarian cancer patients was significantly increased compared with the normal patients, as shown in the left graph in FIG. 1. The UALCAN database (https: / / ualcan.path.uab.edu / analysis.html) was entered, TCGA lncRNA was selected, the gene name was input, ovarian cancer was selected, and the expression level of the patients of different ages was analyzed. The results are shown in the right graph in FIG. 1. Figure 2 As shown in the right graph in FIG. 1, the expression level of LncRNA SLC8A1-AS1 was significantly increased with the increase of the age of the patients, by measuring the gene expression level by FPKM value.
[0023] Example 3 Expression of LncRNA SLC8A1-AS1 in ovarian cancer cell lines and human ovarian normal epithelial cells 1. Resuscitation and culture of cells Add 3 mL complete medium (containing 10% FBS, 100 units / mL penicillin + streptomycin) to a 15 mL centrifuge tube; After taking the cells out of the liquid nitrogen, quickly place them in a 37°C water bath to thaw; Transfer the thawed cells to a 15 mL centrifuge tube and place it in a benchtop centrifuge, 1500 rpm, room temperature, centrifuge for 5 min; Discard the supernatant, add 1 mL complete medium to resuspend the cells; Transfer the cells to a T25 cell culture flask, the culture system is 4 mL, and shake it in an "8" shape; Place the cells in a cell culture incubator (37°C, containing 5% CO2) for culture, and subculture when the cell density reaches 80%-90%.
[0024] 2. Cell subculture Take out the cells with a cell density of 80-90%, and discard the old culture medium; Add 2 mL PBS along the wall, and gently shake it once; Discard the original PBS and repeat the PBS rinse once; Add 1 mL 0.25% trypsin-EDTA to the T25 cell culture flask, gently shake the liquid to distribute it evenly on the bottom of the flask, and place it in a 37°C incubator for 1 min 30 s of digestion; After the cells are completely digested, add 3 mL complete medium to terminate the digestion; Blow the cells with a pipette gun, collect the cell suspension in a 15 mL centrifuge tube, and place it in a benchtop centrifuge, 1500 rpm, room temperature, centrifuge for 5 min; Discard the supernatant, add 1 mL complete medium to resuspend the cells; Transfer the cells to a T25 cell culture flask, and shake it in an "8" shape; Cells were incubated in a cell incubator (37°C, 5% CO2) and subcultured when the cell density reached 80-90%.
[0025] 3. Cell plating Cells in logarithmic growth phase were taken, the old culture medium was discarded, 2 mL PBS was used to rinse twice, and then 1 mL 0.25% trypsin-EDTA was added for digestion in a 37°C incubator. The cells were blown with a pipette gun, and the cell suspension was collected in a 15 mL centrifuge tube and centrifuged in a table centrifuge at 1500 rpm and room temperature for 5 min. The supernatant was discarded, and 1 mL complete medium was added to resuspend the cells. A hemocytometer was used to count the cells under an inverted microscope. The appropriate cell suspension and complete medium were added to the centrifuge tube and mixed well. The appropriate diluted cell suspension was added to the six-well plate, mixed well, and then incubated in a cell incubator.
[0026] 4. RNA extraction When the cell density reached 80-90%, the cells were washed twice with PBS. 1 mL of RNAiso Plus was added to each plate, and the plate was shaken to distribute the reagent evenly and digest thoroughly. The cells were collected with a cell scraper and transferred to a 1.5 mL RNA-free centrifuge tube with a pipette gun, and mixed well by blowing. Room temperature for 5 min. Chloroform was added in a ratio of Trizol:chloroform=5:1, and vortexed vigorously for 30 s, and then stood for 5 min.
[0027] Centrifuge at 4°C, 12000 rpm, 15 min to extract RNA, and transfer the upper clear liquid to a new 1.5 mL RNA-free centrifuge tube. Add an equal volume of isopropanol and mix well immediately.
[0028] After standing for 10 min, centrifuge at 4°C, 12000 rpm, 10 min, remove the supernatant, and leave the white solid at the bottom.
[0029] Add 1 mL of 75% alcohol to wash the RNA, centrifuge at 8000 rpm for 5 min, and remove the supernatant.
[0030] Air dry the excess alcohol, and when the RNA appears translucent, add an appropriate amount of DEPC water to dissolve the RNA and mix well with the gun head.
[0031] RNA concentration determination: Open the NanoDrop 2000 spectrophotometer software on the computer, and set the nucleic acid mode to RNA. After cleaning the base arm and setting the baseline, set the sample information to be detected on the software, add 1 μL of mixed sample to the base, and click Measure. Repeat the steps to detect the next sample, clean the base after detection, analyze the detection results, and import the results into the Excel table.
[0032] 5. Reverse transcription (1) According to the first step and the second step of the reverse transcription kit instructions, 1 μg of RNA was used to reverse transcribe cDNA. The cDNA can be stored at -20°C, and the reverse transcription process needs to be completed on ice.
[0033] Table 1 gDNA removal reaction system (10 μL)
[0034] (2) After the above system configuration is completed, vortex thoroughly and centrifuge, and place the sample in the PCR instrument. The program is set to 42°C, 2 min.
[0035] Table 2 Reverse transcription reaction system (20 μL)
[0036] (3) After the above system configuration is completed, vortex thoroughly and centrifuge, and place the sample in the PCR instrument. The program is set to 42°C 15 min, 85°C 5 s, for subsequent experiments or -20°C storage.
[0037] 6. Fluorescent real-time quantitative PCR (qRT-PCR) (1) Dissolve the cDNA, SYBR Green MasterMix (2x), and 10 μM Primers on ice. Dilute the 20 μL cDNA obtained by reverse transcription to 200 μl as needed. After the following system is configured, add it to the 96-well plate.
[0038] Table 3 qRT-PCR reaction system (10 μL)
[0039] GAPDH as an internal reference gene for qPCR to detect the relative expression of LncRNA SLC8A1-AS1; the primers of the LncRNA SLC8A1-AS1 are as shown in SEQ ID NO. 1 and SEQ ID NO. 2; the primers of the GAPDH are as shown in SEQ ID NO. 9 and SEQ ID NO. 10: LncRNA SLC8A1-AS1-F (SEQ ID NO. 1): 5'-ACACTCCAAGGTAATGCCACT-3'; LncRNA SLC8A1-AS1-R (SEQ ID NO. 2): 5'-CCAGAGAGACAGTTTGGTGGA-3'; GAPDH-F (SEQ ID NO: 9): 5'-GGAGCGAGATCCCTCCAAAAT-3'; GAPDH-R (SEQ ID NO: 10): 5'-GGCTGTTGTCATACTTCTCATGG-3'.
[0040] (2) After all samples are added to the 96-well plate, centrifuge for a few seconds. Place the 96-well plate in the PCR instrument, set the program, and run the experiment. The program is set as follows: Table 4 qRT-PCR reaction program
[0041] (3) After the experiment is completed, process, analyze, and export the data for storage.
[0042] The results are shown in Figure 3 Compared with human normal ovarian epithelial cells IOSE80, the expression of LncRNA SLC8A1-AS1 in ovarian cancer cells Hey, A2780, SKOV3, and ES2 was significantly increased, and the expression in ES2 was increased by 100 times. Note: *** indicates that the data are significantly different, p<0.001.
[0043] Example 4 Cell transfection Guangzhou Ribo Biological Technology Co., Ltd. was commissioned to design and synthesize SmartSilencer RNA including 3 siRNAs and 3 ASOs for LncRNA SLC8A1-AS1; the nucleotide sequences are as follows: si-LncRNA SLC8A1-AS1-1 (SEQ ID NO. 3): 5'-CAGCAATGCAGAACTAAGT-3'; si-LncRNA SLC8A1-AS1-2 (SEQ ID NO. 4): 5'-ACATACACTCCAAGGTAAT-3'; si-LncRNA SLC8A1-AS1-3 (SEQ ID NO. 5): 5'-GAGTCTTCCATTCCTTCTA-3'; ASO-LncRNA SLC8A1-AS1-1 (SEQ ID NO. 6): 5'-GACAGGTCATCAGAAAACAT-3'; ASO-LncRNA SLC8A1-AS1-2 (SEQ ID NO. 7): 5'-CAGATCCACCAAACTGTCTC-3'; ASO-LncRNA SLC8A1-AS1-3 (SEQ ID NO. 8): 5'-CTCTATGTCTGCGCTTAAGA-3'; Cells were cultured and seeded in 96-well plates at 1 x 10 5 When the confluence reached 30-50%, transfection was performed; The transfection complex was prepared and incubated at room temperature for 0-15 min; The transfection complex was added dropwise to the cells containing the complete culture medium without secondary antibody, and mixed gently; Incubate for a period of time; When the confluence reached 80%, RNA extraction and qRT-PCR experiments were performed.
[0044] The results are shown in Figure 4 Compared with the Smart Silencer control RNA si-NC, the expression level of LncRNA SLC8A1-AS1 in ES2 and SKOV3 cells was significantly reduced after the addition of si-SLC8A1-AS1. Note: *** indicates that the data are significantly different, p<0.001.
[0045] Example 5 Cells were cultured and seeded in six-well plates at 1 x 10 3 After one week of cell culture, the transfection complex was added and the culture was continued; When the cells were cultured to the naked eye, the six-well plate was removed, the original culture medium was discarded, and 1 mL of 4% paraformaldehyde was added to each well for fixation for 15 min; The original solution was discarded, and 0.1% crystal violet solution was added to each well and left at room temperature for 30 min; The original solution was discarded, and 0.1% crystal violet solution was added to each well and left at room temperature for 30 min; The original solution was discarded, and 0.1% crystal violet solution was added to each well and left at room temperature for 30 min;
[0046] The results are shown in Figure 5 The left is a microscopic photograph of the plate clone, and the right is a statistical diagram of the number of clone cells. Compared with the Smart Silencer control RNA si-NC, the colony formation ability of ES2 and SKOV3 cells was significantly reduced after the addition of si-SLC8A1-AS1. Note: *** indicates that the data are significantly different, p<0.001.
[0047] Example 6 1. Cell culture, and inoculate 5x10 5 cells per hole in a 24-well plate, and the cell climbing sheet has been laid in the hole; 2. When the cell confluence reaches 30-50%, add the transfection complex, and continue to culture; 3. When the cell confluence of the control group reaches 80%, add the prepared 2x EdU working solution, and continue to culture for 2h; 4. Take out the six-hole plate, discard the original culture medium, and add 1 mL of 4% paraformaldehyde to each hole for fixation for 15 min; 5. Discard the original solution, and add 1 mL of 3% BSA to each hole for washing 3 times, each time for 5 min; 6. Discard the original solution, and add 1 mL of 0.3% Triton X-100 to each hole, and stand for 15 min at room temperature; 7. Discard the original solution, and add 1 mL of 3% BSA to each hole for washing 3 times, each time for 5 min; 8. Discard the original solution, and add 0.5 mL of the prepared Click reaction solution to each hole, and avoid light for 30 min; 9. Discard the original solution, and add 1 mL of 3% BSA to each hole for washing 3 times, each time for 5 min; 10. Discard the original solution, and add 1 mL of the prepared Hoechst 33342 reaction solution to each hole, and avoid light for 10 min; 11. Discard the original solution, and add 1 mL of 3% BSA to each hole for washing 3 times, each time for 5 min; 12. Take out the cell climbing sheet and perform the mounting treatment; 13. Take pictures under the laser confocal microscope, and perform calculation and analysis.
[0048] EdU marks proliferating cells, Hoechst marks living cells, and Merge is the superimposed graph of EdU and Hoechst. The results are shown in Figure 6 the left EdU microphotograph and the right EdU quantity statistical graph. Compared with the Smart Silencer control RNA si-NC, after adding si-SLC8A1-AS1, the proliferation abilities of ES2 and SKOV3 cells are significantly decreased. Note: *** indicates that the data are significantly different, p<0.001.
[0049] The above results show that the Smart Silencer RNA of LncRNA SLC8A1-AS1 described in the application can significantly inhibit the proliferation of human ovarian cancer cells. It is proved that knocking down LncRNA SLC8A1-AS1 can effectively kill ovarian cancer cells.
[0050] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.
Claims
1. The application of an ovarian cancer biomarker, lncRNA, in the preparation of products for diagnosing ovarian cancer, characterized in that, The LncRNA positively regulates the occurrence and development of ovarian cancer, and the ovarian cancer biomarker is LncRNASLC8A1-AS1.
2. The application according to claim 1, characterized in that, The product includes diagnostic reagents or reagents for detecting the ovarian cancer biomarkers of claim 1.
3. The application of a primer pair for detecting the ovarian cancer biomarker LnRNA SLC8A1-AS1 in the preparation of products for ovarian cancer diagnosis, characterized in that... The primer pair includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.
2.
4. Application of LnCRNA SLC8A1-AS1 in the preparation of products for the prevention and / or treatment of ovarian cancer.
5. The application according to claim 4, characterized in that, Inhibit the expression of LncRNA SLC8A1-AS1.
6. A lncRNA SLC8A1-AS1 expression inhibitor, characterized in that, The LnCRNA SLC8A1-AS16 expression inhibitor is a Smart silencer RNA, and the nucleotide sequence of the Smart silencer RNA is any one of those shown in SEQ ID NO. 3-8.
7. A pharmaceutical composition, characterized in that, It includes the inhibitor as described in claim 6.
8. A pharmaceutical preparation, characterized in that, It comprises the pharmaceutical composition of claim 7 and a pharmaceutically acceptable carrier.
9. The use of the inhibitor of claim 6, the pharmaceutical composition of claim 7, or the pharmaceutical preparation of claim 8 in the preparation of products for the prevention and / or treatment of ovarian cancer.