A target gene for the treatment of ovarian cancer and its application

Abstract

The present invention relates to a target gene for the treatment of ovarian cancer and its application. The present invention discovers that the expression level of lnc-GPAT3-1 in the blood of ovarian cancer patients is significantly higher than that of healthy people, confirming that the lnc-GPAT3-1 gene has good application prospects in the diagnosis of ovarian cancer patients. In addition, it is found that knocking down lnc-GPAT3-1 will significantly inhibit the expression level of lnc-GPAT3-1 in ovarian cancer patients and can effectively inhibit the proliferation ability of ovarian cancer, which can be used as a feasible treatment plan for ovarian cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of ovarian cancer treatment, and particularly relates to a target gene for ovarian cancer treatment and its application. Background Art

[0002] Ovarian cancer is a malignant tumor of ovarian tumors, referring to malignant tumors growing on the ovaries. Among them, 90% - 95% are primary ovarian cancers, and the other 5% - 10% are cancers metastasized from other primary sites to the ovaries. Since there are few symptoms in the early stage of ovarian cancer, even if there are symptoms, they are not specific, and the role of screening is limited. Therefore, early diagnosis is relatively difficult. When seeking medical treatment, 60% - 70% of the cases are already in the advanced stage, and the curative effect of advanced cases is not good. Therefore, although the incidence rate of ovarian cancer is lower than that of cervical cancer and endometrial cancer, ranking the third among gynecological malignancies, the mortality rate exceeds the sum of cervical cancer and endometrial cancer, ranking first among gynecological cancers, and it is the greatest disease seriously threatening women's health.

[0003] Currently, the research on lncRNAs (long non-coding RNAs) has become a hot topic in many cancers. Initially, it was found that lncRNAs are abnormally expressed in the occurrence and development of many cancers: the expression of some lncRNAs is overexpressed as if the control has been lifted, while the expression of some lncRNAs is downregulated as if it has been inhibited. This finding indicates that lncRNAs may be participants in the process of tumorigenesis and development. Subsequently, many experimental results have also shown that lncRNAs have the ability to promote or inhibit tumor development and progression. Based on the role of lncRNAs in regulating gene expression and the high possibility that lncRNAs are involved in tumorigenesis, lncRNAs are considered to be a promising cancer biomarker based on tissues or blood, and new ways of cancer treatment will be developed on this basis.

[0004] In summary, it is necessary to develop new lncRNA markers for clinical diagnosis and targeted treatment of ovarian cancer. Summary of the Invention

[0005] Aiming at the technical problems existing in the prior art, the present invention provides a target gene for ovarian cancer treatment and its application. In particular, it relates to the application of lnc-GPAT3-1 as an auxiliary diagnostic biomarker and a potential therapeutic target for ovarian cancer. The present invention discovers that lnc-GPAT3-1 serves as an auxiliary diagnostic biomarker for ovarian cancer, and lnc-GPAT3-1 can be used as a therapeutic target. Targeted inhibition of its expression can achieve the treatment of ovarian cancer patients, and it has a broad application prospect.

[0006] One of the objectives of the present invention is to provide the use of lnc-GPAT3-1 as a diagnostic marker in the preparation of reagents for the auxiliary diagnosis and / or evaluation of ovarian cancer, and the nucleotide sequence of the lnc-GPAT3-1 is as shown in SEQ ID No.1.

[0007] Preferably, the expression level of lnc-GPAT3-1 in ovarian cancer patients is detected to assist in the diagnosis and / or evaluation of ovarian cancer.

[0008] Another objective of the present invention is a kit for the auxiliary diagnosis and / or evaluation of ovarian cancer, which is characterized by containing primers for specifically amplifying lnc-GPAT3-1.

[0009] Preferably, the primers for specifically amplifying lnc-GPAT3-1 are: the upstream primer sequence is SEQ ID No.2, and the downstream primer sequence is SEQ ID No.3.

[0010] Preferably, the test sample in the kit is blood.

[0011] One of the objectives of the present invention is the use of lnc-GPAT3-1 in the preparation of a kit for the auxiliary diagnosis and / or evaluation of ovarian cancer.

[0012] One of the objectives of the present invention is the use of lnc-GPAT3-1 as a therapeutic target in the preparation of drugs for the treatment of ovarian cancer.

[0013] Preferably, the use of lnc-GPAT3-1 siRNA in the preparation of drugs for the treatment of ovarian cancer, the drug targets lnc-GPAT3-1, and the lnc-GPAT3-1 siRNA includes any one or more of siRNA1-3, and their sequences are as shown in SEQ ID No.4-9 in sequence.

[0014] One of the objectives of the present invention is a therapeutic drug for ovarian cancer, and the drug contains a nucleic acid, a bioactive functional fragment or a variant having at least one of the sequences shown in SEQ ID No.4-9.

[0015] Furthermore, the drug includes a pharmaceutically acceptable carrier or excipient.

[0016] The advantages of the present invention are as follows: The present invention discovers that the expression level of lnc-GPAT3-1 in the blood of ovarian cancer patients is significantly higher than that of healthy people. Through calculation and analysis, the area under the AUC curve is 0.857, its diagnostic sensitivity can reach 90.5%, and the specificity is as high as 77.8%. It is confirmed that the lnc-GPAT3-1 gene has good application prospects in the diagnosis of ovarian cancer patients. In addition, it is found that knocking down lnc-GPAT3-1 will significantly inhibit the expression level of lnc-GPAT3-1 in ovarian cancer patients and can effectively inhibit the proliferation ability of ovarian cancer, which can be used as a feasible treatment plan for ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 . Expression of lnc-GPAT3-1 gene in ovarian cancer patients;

[0018] Figure 2 . Analysis of the amount of lnc-GPAT3-1 gene in ovarian cancer cells treated with interfering siRNA;

[0019] Figure 3 . Analysis of the proliferation ability of ovarian cancer cells treated with interfering siRNA. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0021] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0022] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well-known to those skilled in the art.

[0023] Example 1 Screening and determination of lncRNA biomarkers in ovarian cancer

[0024] Extract ovarian cancer samples from the TCGA database, screen all lncRNAs on the above website, select the mRNA Expression z-Scores (RNA Seq V2 RSEM) of the gene expression profiles of ovarian cancer, sort the generated results, and select the lncRNAs with the largest change rate in ovarian cancer genetics as alternative biomarkers. Compare the results preliminarily screened from the TCGA database with the microarray sequencing results of ovarian cancer in the GEO database, and remove some results with no obvious differential expression. Finally, determine the target lnc-GPAT3-1, whose sequence is shown in SEQ ID NO.1.

[0025] Example 2 Expression analysis of lnc-GPAT3-1 gene in clinical ovarian cancer patients

[0026] I. Preparation of experimental materials:

[0027] All experimental subjects were patients diagnosed with ovarian cancer by clinicians from January 2021 to December 2022. Blood samples of 80 patients were collected for standby; at the same time, blood samples of 80 healthy people were collected as controls.

[0028] II. Test methods

[0029] 1) Total RNA extraction of blood genes: The extraction steps were carried out according to the instructions of the miRNeasy-SerumPlasma-217184 kit, and the serum specimen was 200 μL.

[0030] 2) Obtain the specific primers of the above lnc-GPAT3-1 gene using primer design software. Among them, the specific amplification primers are as follows:

[0031] Forward primer: 5'-GCTGGCTGCAGTGAAGATGA-3' (SEQ ID NO.2);

[0032] Reverse primer: 5'-GTCTGCTCTGGCATGCTACT-3' (SEQ ID NO.3).

[0033] 3) Complete the reverse transcription of the total RNA in step 1) using the M-MLV reverse transcription kit.

[0034] 4) PCR quantitative detection and analysis of the expression of lnc-GPAT3-1 gene, using the 2 -ΔΔCt method for calculation.

[0035] Among them, the 25 μL PCR reaction system includes: 10 μL of 10×PCR Buffer, 2 μL of SYBR Green I fluorescent dye, 2 μL of dNTP, 2 μL each of the upstream and downstream primers of lnc-GPAT3-1, 2 μL of Taq DNA polymerase, and 5 μL of cDNA;

[0036] Among them, the PCR reaction program is pre-denaturation at 94°C for 10 min; 30 s at 94°C, 45 s at 58°C, and extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 10 min.

[0037] The results are shown in Figure 1 , compared with the healthy population, the expression level of lnc-GPAT3-1 detected in 80 pairs of blood samples of ovarian cancer patients was significantly up-regulated. And through calculation and analysis, the area under the AUC curve was 0.857, its diagnostic sensitivity could reach 90.5%, and the specificity was as high as 77.8%. Further experimental data analysis confirmed that the lnc-GPAT3-1 gene has good application prospects in the diagnosis of ovarian cancer patients.

[0038] Example 3 Analysis of the improvement of ovarian cancer by inhibiting the expression level of lnc-GPAT3-1

[0039] I. Preparation of experimental materials:

[0040] Using ovarian cancer OVCAR-3 cells as the research object, they were cultured using a special medium for OVCAR-3 cells (purchased from Beijing NaChuangLian Biotechnology Co., Ltd., No. BNCC360841), and were used after stable passage.

[0041] II. Test methods

[0042] 1) Design of lnc-GPAT3-1 interference fragments: 3 groups of siRNAs were synthesized by Shanghai GenePharma, and the sequences are shown as siRNA1 (SEQ ID NO.4-5), siRNA2 (SEQ ID NO.6-7), and siRNA3 (SEQ ID NO.8-9).

[0043] 2) Verification of interference efficiency: The OVCAR-3 cells were divided into four parts, and three of them were transfected with siRNA1-3 respectively, and the fourth part was not transfected with siRNA during the same period.

[0044] 3) After 24 h of transfection, the relative expression level of lnc-GPAT3-1 was quantitatively analyzed by the method described in Example 2, and the proliferation change of OVCAR-3 cells was detected by CCK8.

[0045] As shown in the result diagram, all three groups of siRNAs could significantly reduce the expression of lnc-GPAT3-1. Moreover, compared with the control group, the proliferation ability of OVCAR-3 cells in the experimental group was significantly weakened, showing a significant difference compared with the control group. This further confirmed that targeting the downregulation of the lnc-GPAT3-1 gene is used for the treatment of ovarian cancer.

[0046] The role of the above embodiments is to illustrate the substantial content of the present invention, but does not limit the protection scope of the present invention. Those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and protection scope of the technical solutions of the present invention.

Claims

1. Use of primers for specific amplification of lnc-GPAT3-1 in the preparation of a reagent for diagnosing ovarian cancer, wherein the nucleotide sequence of the lnc-GPAT3-1 is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The primers for specifically amplifying lnc-GPAT3-1 are: the upstream primer sequence is SEQ ID No. 2, and the downstream primer sequence is SEQ ID No.

3.

3. Use of siRNA targeting lnc-GPAT3-1 in the preparation of a drug for treating ovarian cancer, characterized in that: The drug takes lnc-GPAT3-1 as a therapeutic target, and the siRNA targeting lnc-GPAT3-1 is any one or more of siRNA1, 2 or 3, wherein the sequence of siRNA1 is shown in SEQ ID NO.4-5, the sequence of siRNA2 is shown in SEQ ID NO.6-7, and the sequence of siRNA3 is shown in SEQ ID NO.8-9.

4. The use according to claim 3, characterized in that The medicine also includes a pharmaceutically acceptable carrier or excipient.

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