Application of lnc81 related reagents in ovarian function impairment related products

Abstract

The invention relates to the technical field of biological medicine, in particular to application of an lnc81 related reagent in ovarian function impairment related products. The invention provides an application of an lnc81 related reagent in ovarian function impairment related products, and provides a new biomarker for detecting ovarian function impairment, the biomarker is lnc81, and by using the marker, the false positive risk can be reduced, the detection accuracy is improved, the trauma of tissue biopsy is avoided, and the risk of ovarian function impairment is reduced. The method is suitable for dynamically monitoring curative effect or relapse.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of lnc81 related reagent in the ovarian function damage related product. BACKGROUND

[0002] A biomarker refers to a characteristic that can be objectively detected and rated, and can be used as an indicator molecule of a normal biological process, a pathological process or a pharmacological response of a therapeutic intervention. In a broad sense, a biomarker can be anatomical, histological, imaging, or genetic, protein, metabolic, etc., as long as it meets the two characteristics of objective measurement and evaluation of the human body process. In a narrow sense, a biomarker refers to a biological chemical molecule derived from human tissue, such as blood, body fluid or tissue, which can be used to assist in disease diagnosis, efficacy prediction, prognosis evaluation, etc.

[0003] The existing markers for detecting ovarian function mainly include anti-Mullerian hormone, estradiol, follicle-stimulating hormone, and follicle-stimulating hormone, etc. Among them, anti-Mullerian hormone is abbreviated as AMH, estradiol is abbreviated as E2, follicle-stimulating hormone is abbreviated as FSH, and follicle-stimulating hormone is abbreviated as Inhibin B. However, these methods have certain limitations. First, individual differences affect: factors such as race, BMI, smoking, and living environment may interfere with the test results. Second, dynamic function evaluation is insufficient, and it is difficult to fully reflect the response of the ovary to ovulation stimulation, such as ovarian hyporesponsiveness or excessive stimulation risk. Third, disease specificity is limited, and gynecological diseases such as endometriosis and ovarian surgery history may change the marker level, which needs to be combined with clinical judgment.

[0004] Long-chain non-coding RNA, abbreviated as lncRNA, is defined as a non-coding transcript with a length of more than 200 nucleotides, which is involved in the regulation of multiple important reproductive physiological processes such as follicular development, ovarian development, and placental development. Due to its high specificity, stability and easy detection, lncRNA has been proven to be a new diagnostic and prognostic biomarker for predicting, early diagnosing and prognosticating various tumors such as prostate cancer, thyroid cancer and breast cancer. So far, there has been no report on the prediction of lncRNA on ovarian function damage. SUMMARY

[0005] In order to solve the above technical problems, the present application provides the application of lnc81 related reagent in the ovarian function damage related product.

[0006] The application of lnc81 related reagent in the ovarian function damage related product includes at least one of the following: The application of a reagent for detecting the expression amount of the lnc81 in the preparation of a diagnostic product for ovarian function damage; Use of a reagent for overexpressing the lnc81 in the preparation of a therapeutic product for ovarian function impairment;

[0007] Preferably, the reagent for detecting the expression level of lnc81 is a reagent used in ELISA technology or a reagent used in q-PCR technology.

[0008] Preferably, the reagents used in the q-PCR technology include primer sequences shown as SEQ ID NO.22 to SEQ ID NO.23.

[0009] Preferably, the reagents used in the q-PCR technology also include an RNA extraction kit and a reverse transcription kit.

[0010] Preferably, the diagnostic product is a kit.

[0011] Preferably, the reagent for overexpressing lnc81 is an lnc81 overexpressing lentiviral vector.

[0012] Preferably, the therapeutic product increases the total follicle count.

[0013] Preferably, the therapeutic product reduces atretic follicles.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This study has developed a novel biomarker, lnc81, for detecting ovarian dysfunction. Using lnc81 reduces the risk of false positives and improves detection accuracy. It avoids the trauma of a tissue biopsy and is suitable for dynamic monitoring of treatment efficacy or recurrence. lnc81 expression changes early in the course of the disease, even before clinical symptoms or traditional biomarkers are detected, making it a promising early warning indicator of ovarian dysfunction. Therefore, this study proposes the use of a reagent for detecting lnc81 expression in the preparation of diagnostic products for ovarian dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Figure 2 shows the changes in the number and proportion of ovarian follicles in each group of mice. A shows HE staining of mouse ovarian sections. The scale bar is 200 μm. B shows the number of primordial follicles, primary follicles, secondary follicles, antral follicles, atretic follicles, and total follicles in the mouse ovaries. C shows the ratio of primordial follicles and atretic follicles to the total number of follicles. * indicates comparison with the control group. P <0.05, ** represents comparison with the control group P <0.01, *** represents comparison with the control group P <0.001, # represents comparison with CY-BUS group P <0.05, ## represents comparison with CY-BUS group P <0.01, ### represents comparison with CY-BUS group P <0.001.

[0016] Figure 2 Apoptosis level of each group of mice ovaries, scale 100 pm.

[0017] Figure 3 Expression changes of lnc81, ovarian function related indicators, Hippo signaling pathway and downstream mRNA in each group of mouse ovaries, wherein A is the expression change of lnc81 in each group of mouse ovaries, B is the expression change of TEAD2 mRNA in each group of mouse ovaries, C is the expression change of CCN1 mRNA in each group of mouse ovaries, D is the expression change of CCN2 mRNA in each group of mouse ovaries, E is the expression change of OCT4 mRNA in each group of mouse ovaries, F is the expression change of MVH mRNA in each group of mouse ovaries, G is the expression change of PCNA mRNA in each group of mouse ovaries, * represents comparison with the control group P <0.05, ** represents comparison with the control group P <0.01, *** represents comparison with the control group P <0.001, # represents comparison with CY-BUS group P <0.05, ## represents comparison with CY-BUS group P <0.01, ### represents comparison with CY-BUS group P <0.001.

[0018] Figure 4 Expression levels of Hippo signaling pathway and downstream proteins in each group of mouse ovaries, wherein A is the expression level of CCN1 protein in each group of mouse ovaries, B is the relative expression amount of CCN1 protein in each group of mice, C is the expression level of p-ERK1 / 2 and ERK1 / 2 protein in each group of mouse ovaries, D is the relative expression amount of ERK1 / 2 and P-ERK1 / 2 protein in each group of mice, n=3, E is the expression level of OCT4 protein in each group of mouse ovaries, F is the relative expression amount of OCT4 protein in each group of mice, G is the expression level of Bax and Bcl-2 protein in each group of mouse ovaries, * represents comparison with the control group P <0.05, ** represents comparison with the control group P <0.01, *** represents comparison with the control group P <0.001, # represents comparison with CY-BUS group P <0.05, ## represents comparison with CY-BUS group P <0.01, ### represents comparison with CY-BUS group P <0.001.

[0019] Figure 5Figure 2 is the diagnostic value of lnc81 for ovarian dysfunction. A shows the expression changes of lnc81 in mice with ovarian dysfunction caused by CTX treatment compared with normal mice. B shows the expression changes of TEAD2 mRNA in mice with ovarian dysfunction caused by CTX treatment compared with normal mice. C shows the correlation analysis between the expression levels of lnc81 and TEAD2 mRNA. D shows the classification and diagnostic effect of lnc81 on normal mice and mice with ovarian dysfunction. *** represents P <0.001.

[0020] Figure 6 This is a map of the lnc81 overexpression lentiviral vector. DETAILED DESCRIPTION

[0021] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0022] Experimental materials: BALB / c mice Experimental reagents: Normal saline, cyclophosphamide, busulfan, and lnc81 overexpression lentiviral vector were purchased from Cyagen. Figure 6 .

[0023] Instruments and equipment: centrifuge, gas anesthesia machine, real-time fluorescence quantitative PCR instrument, inverted microscope, microplate reader, electrophoresis instrument, chemiluminescence imaging system.

[0024] Example 1 Experimental animal handling ① Establish an infertility mouse model. Six- to eight-week-old female mice were intraperitoneally injected with normal saline. Two weeks later, normal saline was injected again intraperitoneally to establish control mice. Intraperitoneally, 120 mg / kg cyclophosphamide plus 30 mg / kg busulfan was injected. Two weeks later, 60 mg / kg cyclophosphamide plus 15 mg / kg busulfan were injected again intraperitoneally to establish an infertility model. This pathological aging mouse model was used to simulate chemotherapy damage.

[0025] ② Microinjection of mouse ovaries. BALB / c mice were anesthetized with gas anesthetic, their hair was removed, and their skin was disinfected with iodine. The skin on both sides of the waist and back was cut open layer by layer to expose the ovaries. 25 μL of 4.22×10 8 TU / mL of blank control lentiviral vector or 25 μL of lentiviral vector with a concentration of 4.71×10 8TU / mL of lnc81 overexpression lentiviral vector was injected into the ovaries of control mice and infertile model pathological aging mice, respectively. The muscles and skin of the mice were sutured layer by layer, disinfected with iodine tincture, and protected with a bandage. The mice were then returned to their cages for breeding, resulting in a normal saline + blank control lentiviral vector group, designated as the Control group; a normal saline + lnc81 overexpression lentiviral vector group, designated as the OElnc81 group; an infertile model + blank control lentiviral vector group, designated as the CY-BUS group; and an infertile model + lnc81 overexpression lentiviral vector group, designated as the CY-BUS+OElnc81 group. In the four groups of model mice, ovarian tissue treated with DNase I was used as a positive control.

[0026] Example 2 Follicular development detection The following indicators were used to detect the growth, differentiation and apoptosis of primordial follicles: (1) The percentage of primordial follicles, primary follicles and secondary follicles at each stage; (2) Follicle survival rate: the ratio of viable follicles to atretic follicles; (3) A digital image analysis system measures the diameters of follicles and eggs and the number of eggs per square millimeter; (4) Immunohistochemical determination of PCNA in follicular cells to determine the state of follicular proliferation; (5) Western Blot and Realtime-PCR were used to detect the changes in the expression of steroid regulatory proteins and FSH receptors, and Tunel was used to determine the apoptosis of follicles. Steroid regulatory proteins include steroidogenic factor-1, steroidogenic sensitive regulatory protein and cytochrome P450 aromatase.

[0027] See the results Figures 1 to 4 .

[0028] Figure 1 HE staining results of A in Figure 2 showed that compared with the control group, the ovarian tissue morphology of the OElnc81 group remained basically unchanged, while the corpus luteum in the ovarian tissue of the CY-BUS group increased significantly and the number of normal growing follicles decreased significantly. Compared with the CY-BUS group, the number of growing follicles in the ovarian tissue of the CY-BUS+OElnc81 group increased significantly. Figure 1 The follicle count results in Figure B showed that the total follicle count in the CY-BUS group was significantly decreased compared with the control group, while the total follicle count in the CY-BUS+OElnc81 group was significantly increased compared with the CY-BUS group. Figure 1The ratio of C in the total follicle number shows that, compared with the Control group, the ratio of primordial follicles in the OElnc81 group is increased, and the ratio of atretic follicles is decreased. The ratio of primordial follicles in the CY-BUS group is significantly decreased, and the ratio of atretic follicles is significantly increased. Compared with the CY-BUS group, the ratio of primordial follicles in the CY-BUS+OElnc81 group is increased, and the ratio of atretic follicles is decreased.

[0029] Figure 2 The TUNEL staining results show that, compared with the Control group, the apoptosis level of the ovarian tissue in the OElnc81 group is decreased, and the apoptosis level of the ovarian tissue in the CY-BUS group is increased. Compared with the CY-BUS group, the apoptosis level of the ovarian tissue in the CY-BUS+OElnc81 group is decreased.

[0030] Figure 3 The q-PCR results of A in the total follicle number show that, compared with the Control group, the expression level of lnc81 in the ovarian tissue of the OElnc81 group is increased, and the expression level of lnc81 in the ovarian tissue of the CY-BUS group is increased. Compared with the CY-BUS group, the expression level of lnc81 in the ovarian tissue of the CY-BUS+OElnc81 group is increased. The ovarian microinjection of the lnc81 lentivirus vector indeed up-regulates the expression level of lnc81 in the mouse ovarian tissue. Figure 3 The q-PCR results of B~G in the total follicle number show that, compared with the Control group, the mRNA expression levels of TEAD2, CCN1, CCN2, OCT4, MVH and PCNA in the ovarian tissue of the OElnc81 group are increased, and the mRNA expression levels of TEAD2, CCN1, CCN2, OCT4, MVH and PCNA in the ovarian tissue of the CY-BUS group are decreased. Compared with the CY-BUS group, the mRNA expression levels of TEAD2, CCN1, CCN2, OCT4, MVH and PCNA in the ovarian tissue of the CY-BUS+OElnc81 group are increased. We can reasonably speculate that the CY-BUS treatment will cause the increase of the expression level of lnc81 and the decrease of the expression level of TEAD2 mRNA. The overexpression of lnc81 will cause the increase of the expression level of TEAD2 mRNA in the ovarian tissue, which will cause the increase of the expression levels of downstream genes CCN1 and CCN2 mRNA, and CCN1 and CCN2 as cell junction factors will affect the ovarian function and thus affect the expression levels of OCT4, MVH and PCNA mRNA, so that the overexpression of lnc81 will restore the damage of the ovarian function.

[0031] Figure 4Western blot results showed that compared with the control group, the expression levels of CCN2 and OCT4 proteins in the ovaries of the OElnc81 group were increased, while those in the CY-BUS group were decreased. Compared with the CY-BUS group, the expression levels of CCN2 and OCT4 proteins in the ovaries of the CY-BUS+OElnc81 group were increased. Compared with the control group, the relative expression levels of p-ERK1 / 2 / ERK1 / 2 and Bax / Bcl-2 proteins in the ovaries of the OElnc81 group were decreased, while those in the CY-BUS group were increased. Compared with the CY-BUS group, the relative expression levels of p-ERK1 / 2 / ERK1 / 2 and Bax / Bcl-2 proteins in the ovaries of the CY-BUS+OElnc81 group were decreased.

[0032] Example 3 A mouse model of ovarian dysfunction was established. Six- to eight-week-old female mice were intraperitoneally injected with normal saline to establish control mice. Two weeks later, 120 mg / kg of cyclophosphamide (CTX) was injected intraperitoneally. Serum was collected from mice for the detection of sex hormone levels using ELISA, and ovarian ovaries were collected for the detection of lnc81 expression using q-PCR. Receiver-operating characteristic (ROC) curves were used to validate the efficacy of lnc81 in diagnosing ovarian dysfunction.

[0033] The primers used in the q-PCR of the present invention are shown in Table 1 below.

[0034] Table 1 Primer sequences

[0035] Mouse ovaries were harvested, placed in DNA- and RNase-free centrifuge tubes, and stored at -80°C. lnc81 expression levels in the ovaries were determined using an RNA extraction kit, reverse transcription kit, and q-PCR kit, strictly following the manufacturer's instructions.

[0036] See the results Figure 5 , Figure 5 q-PCR results in Figures A–C show that compared with normal mice, CTX-treated mice with ovarian dysfunction showed increased lnc81 expression and decreased TEAD2 mRNA expression. A linear relationship was observed between lnc81 and TEAD2 expression levels. Pearson correlation analysis demonstrated a statistically significant association between lnc81 and TEAD2 mRNA expression. CTX treatment resulted in increased lnc81 expression and decreased TEAD2 mRNA expression, mirroring the trend seen in ovarian dysfunction caused by CY-BUS treatment. Figure 5The area under the ROC curve (AUC) of D in the figure was > 0.9, indicating that lnc81 has high value and excellent performance in predicting ovarian function damage caused by CTX.

[0037] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0038] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0039] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. The use of lnc81-related reagents in products related to ovarian function damage, characterized in that: The application includes at least one of the following: Use of a reagent for detecting the expression level of lnc81 in the preparation of a diagnostic product for ovarian function damage; Use of a reagent that overexpresses lnc81 in preparing a therapeutic product for ovarian dysfunction; The nucleotide sequence of lnc81 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The reagent for detecting the expression level of lnc81 is a reagent used in ELISA technology or a reagent used in q-PCR technology.

3. The use according to claim 2, characterized in that The reagents used in the q-PCR technology include primer sequences shown as SEQ ID NO.22 to SEQ ID NO.

23.

4. The use according to claim 3, characterized in that The reagents used in the q-PCR technology also include RNA extraction kits and reverse transcription kits.

5. The use according to claim 1, characterized in that The diagnostic product is a kit.

6. The use according to claim 1, characterized in that The lnc81 overexpression reagent is an lnc81 overexpression lentiviral vector.

7. The use according to claim 6, characterized in that The therapeutic product increases the total follicle count.

8. The use according to claim 6, characterized in that The therapeutic product reduces atretic follicles.

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