Application of SDHA in the diagnosis of cervical cancer and as a therapeutic target

Abstract

The present invention discloses the use of SDHA as a biomarker in the preparation of products for diagnosis or auxiliary diagnosis of cervical cancer. The present invention also discloses the use of SDHA gene or protein as a target in screening drugs for treatment or prevention of cervical cancer. The present invention also discloses a method for detecting the functional damage of cervical cancer cells caused by overexpression of SDHA using a cervical cancer cell model overexpressing SDHA. The present invention found that SDHA is abnormally highly expressed in cervical cancer cells, and overexpression of SDHA can activate the expression of downstream proteins 4E‑BP1 and P70s6K of the mTOR signaling pathway in human cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa, causing an increase in mitochondrial membrane potential and intracellular calcium ion levels, and inhibiting autophagy and apoptosis, promoting the occurrence and progression of cervical cancer. SDHA can be used as a molecular biomarker for diagnosing cervical cancer, and can also be used as a potential therapeutic target for cervical cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of crude drug medicine, and in particular to the application of SDHA in the diagnosis of cervical cancer and as a therapeutic target. Background Art

[0002] Cervical cancer is the fourth most common female cancer worldwide. Squamous cell carcinoma accounts for 90% of cervical cancer cases, and adenocarcinoma accounts for 10% of cases. However, current cervical cancer diagnosis and treatment methods still have certain limitations, and more accurate and personalized strategies are needed to improve patient survival and treatment outcomes. Therefore, finding new therapeutic targets and indicators is of great significance for the diagnosis and treatment of cervical cancer.

[0003] Succinate dehydrogenase (SDH) is located in the inner membrane of mitochondria, also known as mitochondrial respiratory chain complex II. It consists of four subunits, SDHA flavoprotein and SDHB iron-sulfur protein as the catalytic core, combined with SDHC and SDHD proteins, anchoring the entire protein complex in the inner membrane of mitochondria. SDH is involved in the conversion of succinate to fumarate. In the context of cancer, SDH is often affected by loss-of-function mutations, resulting in the accumulation of succinate, mainly in mitochondria, followed by leakage into the cytoplasm. Increasing evidence indicates the key role of mitochondrial function in tumor progression. However, the specific role and molecular mechanism of SDHA in tumor progression, especially in cervical cancer, are still unclear. Summary of the invention

[0004] Based on the technical problems existing in the background technology, the present invention proposes an application of SDHA in the diagnosis of cervical cancer and as a therapeutic target. The present invention finds that the expression of SDHA gradually increases with the degree of intracervical neoplasia, and is abnormally highly expressed in cervical cancer. The present invention establishes human cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa that overexpress SDHA, evaluates the effect of overexpression of SDHA on a series of functions of the two cervical cancer cells, and finds that overexpression of SDHA can activate the expression of two key proteins 4E-BP1 and P70s6K downstream of the mTOR signaling pathway in human cervical cancer cells HeLa (cervical adenocarcinoma cell line) and SiHa (cervical squamous cell carcinoma cell line), causing an increase in mitochondrial membrane potential and intracellular calcium ion levels, and inhibiting autophagy and apoptosis, promoting the occurrence and progression of cervical cancer.

[0005] The present invention proposes an application of SDHA as a biomarker in the preparation of a product for diagnosing or assisting in the diagnosis of cervical cancer.

[0006] The present invention also proposes the use of a reagent for detecting the expression level of SDHA gene or protein in the preparation of a product for diagnosing or assisting in the diagnosis of cervical cancer.

[0007] Preferably, the diagnostic product or auxiliary diagnostic product is used to detect the expression level of SDHA gene or protein in cervical tissue.

[0008] The above-mentioned products for diagnosing or assisting in the diagnosis of cervical cancer may be diagnostic kits or diagnostic reagents.

[0009] The present invention also proposes an application of SDHA gene or protein as a target in screening drugs for treating or preventing cervical cancer.

[0010] The present invention also provides a cervical cancer cell model overexpressing SDHA, wherein the cell model is a human cervical adenocarcinoma cell line HeLa overexpressing SDHA or a human cervical squamous cell carcinoma cell line SiHa overexpressing SDHA.

[0011] The present invention also proposes a method for establishing the above-mentioned SDHA-overexpressing cervical cancer cell model, comprising the following steps: transfecting the SDHA encoding gene into cervical cancer cells, and detecting the SDHA protein expression of the transfected cervical cancer cells, and screening to obtain a cervical cancer cell model that overexpresses SDHA protein.

[0012] Preferably, the SDHA encoding gene is transfected into cervical cancer cells using Polyplusjet PRIME transfection reagent.

[0013] The present invention also proposes a method for detecting the functional damage of cervical cancer cells caused by overexpression of SDHA using the above-mentioned cervical cancer cell model overexpressing SDHA, comprising:

[0014] a. Detect the effect of overexpression of SDHA on the mitochondrial membrane potential level of cervical cancer cells;

[0015] b. Detecting the effect of overexpression of SDHA on the calcium ion level of cervical cancer cells;

[0016] c. Detect the effect of overexpression of SDHA on the autophagy level of cervical cancer cells;

[0017] d. Detect the effect of overexpression of SDHA on the apoptosis level of cervical cancer cells;

[0018] e. Detect the effect of overexpression of SDHA on key proteins of the mTOR signaling pathway in cervical cancer cells.

[0019] Preferably, the mitochondrial membrane potential level is reflected by detecting the fluorescence intensity of cells by staining with TMRE.

[0020] Preferably, the calcium ion level is reflected by detecting the fluorescence intensity of cells by staining with Rhod2.

[0021] Preferably, the level of autophagy is reflected by detecting the protein content of autophagy-related proteins Beclin1 and LC3II / I or detecting the mRNA level of autophagy-related proteins Beclin1 and LC3II.

[0022] Preferably, the apoptosis level is reflected by detecting the content of anti-apoptotic protein BCL2 and pro-apoptotic protein Bax, or detecting the mRNA level of anti-apoptotic protein BCL2 and pro-apoptotic protein Bax.

[0023] Preferably, the key proteins of the mTOR signaling pathway include: 4E-BP1 protein, phosphorylated 4E-BP1 protein, P70s6k protein or phosphorylated P70s6k protein.

[0024] Beneficial effects:

[0025] The present invention found that SDHA is abnormally highly expressed in cervical cancer, and the expression level gradually increases with the degree of cervical neoplasia. The present invention establishes human cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa that overexpress SDHA, and evaluates the effects of overexpression of SDHA on a series of functions of the two cervical cancer cells, including the effects caused by mitochondrial membrane potential, calcium ion level, cell autophagy, cell apoptosis level, etc.

[0026] The present invention found that overexpression of SDHA can activate the expression of 4E-BP1 and P70s6K, the downstream proteins of the mTOR signaling pathway, in human cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa, causing an increase in mitochondrial membrane potential and intracellular calcium ion levels, inhibiting autophagy and apoptosis, and promoting the occurrence and progression of cervical cancer.

[0027] The above findings indicate that SDHA can be used as a molecular biomarker for the diagnosis of cervical cancer and as a potential therapeutic target for cervical cancer, and has great clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figure 2 is the expression of SDHA in cervical squamous cell carcinoma tissue, cervical adenocarcinoma tissue, LSIL cervical tissue, and HSIL cervical tissue, where A is the result image of WB detection, B is the data quantitative statistical chart of the WB detection results, C is the representative image of IHC detection, D is the IHC score of SDHA, CON is normal cervical tissue, LSIL is cervical tissue with low-grade cervical intraepithelial neoplasia, HSIL is cervical tissue with high-grade cervical intraepithelial neoplasia, SCC is cervical squamous cell carcinoma tissue, and AC is cervical adenocarcinoma tissue.

[0029] Figure 2These are the detection results of SDHA protein expression levels in adenocarcinoma Hela cells overexpressing SDHA and squamous cell carcinoma SiHa cells overexpressing SDHA, wherein A is the result image of SDHA protein detected in Hela cells overexpressing SDHA by WB method, B is the data quantitative statistical graph of A, C is the result image of SDHA protein detected in SiHa cells overexpressing SDHA by WB method, and D is the data quantitative statistical graph of C.

[0030] Figure 3 The figures show the effect of overexpression of SDHA on autophagy-related proteins (Beclin1 and LC3II / I) in cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa, wherein A is the WB detection result of HeLa cells overexpressing SDHA, B and C are the quantitative analysis results of the protein in A, D is the WB detection result of SiHa cells overexpressing SDHA, E and F are the quantitative analysis results of the protein in D, G and H are the mRNA levels of Beclin1 and LC3B in HeLa cells overexpressing SDHA, and I and J are the mRNA levels of Beclin1 and LC3B in SiHa cells overexpressing SDHA.

[0031] Figure 4 The figures show the effect of overexpression of SDHA on apoptosis-related proteins (BCL2 and BAX) in cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa, wherein A is the WB detection result of HeLa cells overexpressing SDHA, B and C are the quantitative analysis results of the protein in A, D is the WB detection result of SiHa cells overexpressing SDHA, E and F are the quantitative analysis results of the protein in D, G and H are the mRNA levels of BAX and BCL2 in HeLa cells overexpressing SDHA, and I and J are the mRNA levels of BAX and BCL2 in SiHa cells overexpressing SDHA.

[0032] Figure 5 The figures are the results of TMRE staining method to detect the effect of overexpression of SDHA on mitochondrial membrane potential level in cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa, wherein A is the TMRE staining result of cervical adenocarcinoma cells HeLa overexpressing SDHA, and B is the fluorescence intensity quantitative analysis result of A; C is the TMRE staining result of cervical squamous cell carcinoma cells SiHa overexpressing SDHA, and D is the fluorescence intensity quantitative analysis result of C.

[0033] Figure 6The figures are the results of detecting the effect of overexpression of SDHA on calcium ion levels in cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa using Rhod-2 staining method, wherein A is the Rhod-2 staining result of cervical adenocarcinoma cells HeLa overexpressing SDHA, and B is the fluorescence intensity quantitative analysis result of A; C is the Rhod-2 staining result of squamous cell carcinoma cells SiHa overexpressing SDHA, and D is the fluorescence intensity quantitative analysis result of C.

[0034] Figure 7 The figure shows the effect of overexpression of SDHA on the expression of key downstream proteins of the mTOR signaling pathway in cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa, wherein A is the WB detection result of adenocarcinoma cells HeLa overexpressing SDHA, BE are the quantitative analysis results of the proteins in A, F are the WB detection results of squamous cell carcinoma cells SiHa overexpressing SDHA, and GJ are the quantitative analysis results of the proteins in F. DETAILED DESCRIPTION

[0035] The technical solutions of the present invention are described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustration only and are not to be construed as limiting the scope of the present invention.

[0036] Example 1

[0037] Western Blot (WB) and immunohistochemistry (IHC) were used to detect the expression of SDHA in patients with cervical cancer, low-grade cervical intraepithelial neoplasia (LSIL) and high-grade cervical intraepithelial neoplasia (HSIL).

[0038] 1) Collect human cervical tissue samples

[0039] Eighteen cases of cervical neoplasia tissues (9 LSIL, 9 HSIL), 19 cases of cervical cancer tissues (12 squamous cell carcinomas, 7 adenocarcinomas), and 9 cases of normal cervical tissues were collected. All patients were clinically staged according to the FIGO guidelines. Clinical specimens were obtained from the First Affiliated Hospital of Anhui Medical University. The patients signed the informed consent form, and the Ethics Committee of the First Affiliated Hospital of Anhui Medical University approved this study.

[0040] 2) WB method to detect the expression of SDHA in cervical cancer tissue

[0041] ① Extract protein from normal cervical tissue, LSIL cervical tissue, HSIL cervical tissue, and cervical cancer tissue (squamous cell carcinoma, adenocarcinoma):

[0042] Take cervical tissue from each group, add pre-cooled protein extraction reagent (protein extraction reagent is a mixture containing protease inhibitors, PMSF and phosphatase inhibitors), lyse on ice for 20 minutes, and then centrifuge at 12000rpm for 15 minutes in a pre-cooled centrifuge, discard the precipitate, take the supernatant and immediately transfer it to a new centrifuge tube, boil at 100℃ for 10 minutes to obtain protein samples, and store at -80℃ for later use.

[0043] ②WB method was used to detect the expression of SDHA in the tissues of cervical cancer patients:

[0044] Protein samples from each group were separated on a 12% SDS-polyacrylamide gel electrophoresis gel, then transferred to a nitrocellulose membrane and detected with an antibody against SDHA, with GAPDH as an internal reference. One-way ANOVA was used for statistical analysis, and graphPad was used for plotting; the results are shown in Figure 1 shown.

[0045] 3) IHC method to detect the expression of SDHA in cervical cancer tissues

[0046] The cervical tissues of each group were washed with 0.9% saline solution, fixed in 4% paraformaldehyde, and embedded in paraffin blocks to cut into 4 mm thick tissue layers. These layers were fixed on slides and then dewaxed in xylene. After gradient hydration, permeabilization and antigen retrieval in ethanol, they were incubated with SDHA antibody and blocked overnight at 4°C. The next day, they were washed three times with PBS, each for 5 min; then incubated with secondary antibodies at 37°C for 2 h, and finally stained with DAB and hematoxylin. Images were taken with a fully automatic high-resolution panoramic imaging analysis system and data were analyzed with Image J. One-way ANOVA was used for statistical analysis, and graphPad was used for plotting. The results are shown in Figure 2. Figure 1 shown.

[0047] Figure 1 Figure 2 is the expression of SDHA in cervical squamous cell carcinoma tissue, cervical adenocarcinoma tissue, LSIL cervical tissue, and HSIL cervical tissue, where A is the result image of WB detection, B is the data quantitative statistical chart of the WB detection results, C is the representative image of IHC detection, D is the IHC score of SDHA, CON is normal cervical tissue, LSIL is cervical tissue with low-grade cervical intraepithelial neoplasia, HSIL is cervical tissue with high-grade cervical intraepithelial neoplasia, SCC is cervical squamous cell carcinoma tissue, and AC is cervical adenocarcinoma tissue.

[0048] Figure 1 Data are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0049] Depend on Figure 1It can be seen that compared with normal cervical tissue, the expression of SDHA in LSIL and HSIL cervical tissue increased, and the expression of SDHA in squamous cell carcinoma and adenocarcinoma cervical tissue increased significantly.

[0050] Example 2

[0051] Construction of SDHA-overexpressing cervical cancer cell line

[0052] 1) In vitro culture of human cervical adenocarcinoma cell line HeLa and squamous cell carcinoma cell line SiHa

[0053] The cervical adenocarcinoma cell line HeLa and the squamous cell carcinoma cell line SiHa were maintained in DMEM medium supplemented with 10% fetal bovine serum, and 0.1 mg / L streptomycin and 100 units / ml penicillin G were added at 4.0×10 5 The cells were added to a 100 mm culture dish at a density of 10 cells / ml and cultured at 37°C in a humidified 5% CO2 incubator;

[0054] 2) Cell transfection

[0055] According to the manufacturer's recommendation, the SDHA coding gene was transfected into cervical cancer cells (HeLa, SiHa) using Vector-EGFP and SDHA-EGFP plasmids using Polyplus jetPRIME transfection reagent (purchased from Polyplus-transfection). After 48 hours of transfection, the expression levels of Vector-EGFP and SDHA-EGFP were determined by laser confocal microscopy; and the expression of SDHA in the transfected HeLa and SiHa cells was verified by WB method (GAPDH as an internal reference). Adenocarcinoma cells HeLa overexpressing SDHA (denoted as HeLa-SDHA), adenocarcinoma cells HeLa transfected with empty plasmid (denoted as HeLa-N1), squamous cell carcinoma cells SiHa overexpressing SDHA (denoted as SiHa-SDHA), and squamous cell carcinoma cells SiHa transfected with empty plasmid (denoted as SiHa-N1) were obtained. Untransfected adenocarcinoma cells HeLa (denoted as HeLa-Con) and untransfected squamous cell carcinoma cells SiHa (denoted as SiHa-Con) were used as blank control groups, respectively. The results are shown in FIG. Figure 2 shown.

[0056] Figure 2 These are the detection results of SDHA protein expression levels in adenocarcinoma Hela cells overexpressing SDHA and squamous cell carcinoma SiHa cells overexpressing SDHA, wherein A is the result image of SDHA protein detected in Hela cells overexpressing SDHA by WB method, B is the data quantitative statistical graph of A, C is the result image of SDHA protein detected in SiHa cells overexpressing SDHA by WB method, and D is the data quantitative statistical graph of C.

[0057] Figure 2 Statistical analysis was performed using one-way ANOVA and graphed using GraphPad; data are mean ± SEM, *p < 0.05, ***p < 0.001.

[0058] Depend on Figure 2 It can be seen that there is no significant difference in the SDHA protein content between the untransfected adenocarcinoma cells HeLa (HeLa-Con) and the HeLa cells transfected with the empty plasmid (HeLa-N1), while the SDHA protein content of the HeLa cells overexpressing SDHA (HeLa-SDHA) is significantly higher than that of the first two cells, indicating that the adenocarcinoma cells HeLa overexpressing SDHA were successfully obtained;

[0059] There was no significant difference in the SDHA protein content between untransfected squamous cell carcinoma cells SiHa (SiHa-Con) and SiHa cells transfected with an empty plasmid (SiHa-N1), while the SDHA protein content of SiHa cells overexpressing SDHA (SiHa-SDHA) was significantly higher than that of the former two cells, indicating that SiHa cells overexpressing SDHA were successfully obtained.

[0060] Example 3

[0061] Detection of the effect of overexpression of SDHA on the autophagy level of cervical adenocarcinoma and squamous cell carcinoma cells

[0062] Untransfected adenocarcinoma cells HeLa (HeLa-Con), adenocarcinoma cells HeLa transfected with an empty plasmid (HeLa-N1), adenocarcinoma cells HeLa overexpressing SDHA (HeLa-SDHA), untransfected squamous cell carcinoma cells SiHa (SiHa-Con), squamous cell carcinoma cells SiHa transfected with an empty plasmid (SiHa-N1), and squamous cell carcinoma cells SiHa overexpressing SDHA (SiHa-SDHA) were extracted with proteins according to the method of step ① in Example 1 to obtain protein samples of each group, and all were stored at -80°C for use;

[0063] The protein samples of each group were separated on 12% SDS-polyacrylamide gel electrophoresis gel, then transferred to nitrocellulose membrane, and detected with antibodies of autophagy-related proteins Beclin1 and LC3II / I, GAPDH was used as an internal reference, and the indicated western blot detection bands were quantitatively analyzed using Image J.

[0064] Q-PCR was used to detect the mRNA levels of Beclin1 and LC3B (LC3II) in each group of cells. The results are as follows: Figure 3 shown.

[0065] Figure 3The figures show the effect of overexpression of SDHA on autophagy-related proteins (Beclin1 and LC3II / I) in cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa, wherein A is the WB detection result of HeLa cells overexpressing SDHA, B and C are the quantitative analysis results of the protein in A, D is the WB detection result of SiHa cells overexpressing SDHA, E and F are the quantitative analysis results of the protein in D, G and H are the mRNA levels of Beclin1 and LC3B in HeLa cells overexpressing SDHA, and I and J are the mRNA levels of Beclin1 and LC3B in SiHa cells overexpressing SDHA.

[0066] Figure 3 One-way ANOVA was used for statistical analysis, and graphs were drawn using GraphPad. Data are mean ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001.

[0067] Depend on Figure 3 It can be seen that compared with untransfected adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa (HeLa-Con, SiHa-Con) and adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa transfected with empty plasmid (HeLa-N1, SiHa-N1), the protein expression and mRNA levels of autophagy-related proteins Beclin1 and LC3II / I in adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa overexpressing SDHA (HeLa-SDHA, SiHa-SDHA) were significantly reduced, indicating that overexpression of SDHA would inhibit the autophagy level of cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa.

[0068] Example 4

[0069] Detection of the effect of overexpression of SDHA on apoptosis of cervical adenocarcinoma and squamous cell carcinoma cells

[0070] Untransfected adenocarcinoma cells HeLa (HeLa-Con), adenocarcinoma cells HeLa transfected with an empty plasmid (HeLa-N1), adenocarcinoma cells HeLa overexpressing SDHA (HeLa-SDHA), untransfected squamous cell carcinoma cells SiHa (SiHa-Con), squamous cell carcinoma cells SiHa transfected with an empty plasmid (SiHa-N1), and squamous cell carcinoma cells SiHa overexpressing SDHA (SiHa-SDHA) were extracted with proteins according to the method of step ① in Example 1 to obtain protein samples of each group, and all were stored at -80°C for use;

[0071] The protein samples of each group were separated on 12% SDS-polyacrylamide gel electrophoresis gel, and then transferred to nitrocellulose membranes and detected with antibodies against cell anti-apoptotic protein BCL2 and cell pro-apoptotic protein Bax, with GAPDH as an internal reference, and the indicated protein blot detection bands were quantitatively analyzed using Image J.

[0072] Q-PCR was used to detect the mRNA levels of BCL2 and BAX in each group of cells. Figure 4 shown.

[0073] Figure 4 The figures show the effect of overexpression of SDHA on apoptosis-related proteins (BCL2 and BAX) in cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa, wherein A is the WB detection result of HeLa cells overexpressing SDHA, B and C are the quantitative analysis results of the protein in A, D is the WB detection result of SiHa cells overexpressing SDHA, E and F are the quantitative analysis results of the protein in D, G and H are the mRNA levels of BAX and BCL2 in HeLa cells overexpressing SDHA, and I and J are the mRNA levels of BAX and BCL2 in SiHa cells overexpressing SDHA.

[0074] Figure 4 One-way ANOVA was used for statistical analysis, and graphs were drawn using GraphPad. Data are mean ± SEM, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0075] Depend on Figure 4 It can be seen that compared with untransfected adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa (HeLa-Con, SiHa-Con) and adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa transfected with empty plasmid (HeLa-N1, SiHa-N1), the expression level and mRNA level of the anti-apoptotic protein BCL2 in adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa overexpressing SDHA (HeLa-SDHA, SiHa-SDHA) were significantly increased, and the expression level and mRNA level of the pro-apoptotic protein Bax were significantly decreased, indicating that overexpression of SDHA would inhibit the apoptosis level of cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa.

[0076] Example 5

[0077] Effect of overexpression of SDHA on mitochondrial membrane potential in cervical adenocarcinoma and squamous cell carcinoma cells detected by TMRE staining

[0078] Adenocarcinoma cells HeLa transfected with empty plasmid (HeLa-N1), adenocarcinoma cells HeLa overexpressing SDHA (HeLa-SDHA), squamous cell carcinoma cells SiHa transfected with empty plasmid (SiHa-N1), and squamous cell carcinoma cells SiHa overexpressing SDHA (SiHa-SDHA) were taken, and each group of cells was exposed to 100nM TMRE for 20min at 37°C; each group of cells was then washed with PBS to remove surface fluorescence, and analyzed using a laser confocal microscope (LSM 980, Zeiss, Germany), and the fluorescence intensity in each group of cells was measured. Image J was used to quantitatively analyze the fluorescence intensity shown. The parameters of each group of cells during image acquisition were consistent, and the experiment was repeated 3 times independently. The test results are shown in Figure 5 shown.

[0079] Figure 5 The figures are the results of TMRE staining method to detect the effect of overexpression of SDHA on mitochondrial membrane potential level in cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa, wherein A is the TMRE staining result of cervical adenocarcinoma cells HeLa overexpressing SDHA, and B is the fluorescence intensity quantitative analysis result of A; C is the TMRE staining result of cervical squamous cell carcinoma cells SiHa overexpressing SDHA, and D is the fluorescence intensity quantitative analysis result of C.

[0080] Figure 5 Statistical analysis was performed using t-test, and graphs were prepared using graphPad; data are mean ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001; Figure 5 The scale bars in A and C are 50 μm.

[0081] Depend on Figure 5 It can be seen that compared with adenocarcinoma HeLa cells and squamous cell carcinoma SiHa cells transfected with empty plasmid (HeLa-N1, SiHa-N1), the mitochondrial membrane potential levels in adenocarcinoma HeLa cells and squamous cell carcinoma SiHa cells overexpressing SDHA (HeLa-SDHA, SiHa-SDHA) were significantly increased. This indicates that overexpression of SDHA will increase the mitochondrial membrane potential levels of cervical cancer adenocarcinoma HeLa cells and squamous cell carcinoma SiHa cells.

[0082] Example 6

[0083] Effect of overexpression of SDHA on calcium ion levels in cervical adenocarcinoma and squamous cell carcinoma cells detected by Rhod2 staining

[0084] Adenocarcinoma cells HeLa transfected with empty plasmid (HeLa-N1), adenocarcinoma cells HeLa overexpressing SDHA (HeLa-SDHA), squamous cell carcinoma cells SiHa transfected with empty plasmid (SiHa-N1), and squamous cell carcinoma cells SiHa overexpressing SDHA (SiHa-SDHA) were taken, and each group of cells was exposed to 3μM rhod-2 at 37°C for 30 minutes; each group of cells was then washed with PBS to remove surface fluorescence, and continued to incubate in PBS for 20 minutes; and analyzed using a laser confocal microscope (LSM 980, Zeiss, Germany), and the fluorescence intensity in each group of cells was measured. Image J was used to quantitatively analyze the fluorescence intensity shown. The parameters of each group of cells during image acquisition were consistent, and the experiment was repeated 3 times independently. The test results are shown in Figure 2. Figure 6 shown.

[0085] Figure 6 The figures are the results of detecting the effect of overexpression of SDHA on calcium ion levels in cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa using Rhod-2 staining method, wherein A is the Rhod-2 staining result of cervical adenocarcinoma cells HeLa overexpressing SDHA, and B is the fluorescence intensity quantitative analysis result of A; C is the Rhod-2 staining result of squamous cell carcinoma cells SiHa overexpressing SDHA, and D is the fluorescence intensity quantitative analysis result of C.

[0086] Figure 6 Statistical analysis was performed using t-test, and graphs were prepared using graphPad; data are mean ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001; Figure 5 The scale bars in A and C are 50 μm.

[0087] Depend on Figure 6 It can be seen that compared with adenocarcinoma HeLa cells and squamous cell carcinoma SiHa cells transfected with empty plasmid (HeLa-N1, SiHa-N1), the calcium ion levels in adenocarcinoma HeLa cells and squamous cell carcinoma SiHa cells overexpressing SDHA (HeLa-SDHA, SiHa-SDHA) were significantly increased, indicating that overexpression of SDHA would increase the calcium ion levels of cervical cancer cell adenocarcinoma HeLa cells and squamous cell carcinoma SiHa cells.

[0088] Example 7

[0089] Detection of the effect of overexpression of SDHA on key proteins of mTOR signaling pathway in cervical adenocarcinoma and squamous cell carcinoma cells

[0090] Untransfected adenocarcinoma cells HeLa (HeLa-Con), adenocarcinoma cells HeLa transfected with an empty plasmid (HeLa-N1), adenocarcinoma cells HeLa overexpressing SDHA (HeLa-SDHA), untransfected squamous cell carcinoma cells SiHa (SiHa-Con), squamous cell carcinoma cells SiHa transfected with an empty plasmid (SiHa-N1), and squamous cell carcinoma cells SiHa overexpressing SDHA (SiHa-SDHA) were extracted with proteins according to the method of step ① in Example 1 to obtain protein samples of each group, and all were stored at -80°C for use;

[0091] The protein samples of each group were separated on 12% SDS-polyacrylamide gel electrophoresis gel, and then transferred to nitrocellulose membrane, and detected with antibodies against 4E-BP1, phosphorylated 4E-BP1 (referred to as p4E-BP1), P70s6k, and phosphorylated P70s6k (referred to as P-P70s6k), the key proteins in the downstream of the mTOR signaling pathway, GAPDH as an internal reference, and the protein blot detection bands shown were quantitatively analyzed using Image J. The results are shown in Figure 7 shown.

[0092] Figure 7 The figure shows the effect of overexpression of SDHA on the expression of key downstream proteins of the mTOR signaling pathway in cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa, wherein A is the WB detection result of adenocarcinoma cells HeLa overexpressing SDHA, BE are the quantitative analysis results of the proteins in A, F are the WB detection results of squamous cell carcinoma cells SiHa overexpressing SDHA, and GJ are the quantitative analysis results of the proteins in F.

[0093] Figure 7 One-way ANOVA was used for statistical analysis, and graphs were drawn using GraphPad. Data are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0094] Depend on Figure 7 It can be seen that compared with untransfected adenocarcinoma HeLa and squamous cell carcinoma SiHa (HeLa-Con, SiHa-Con), and adenocarcinoma HeLa and squamous cell carcinoma SiHa transfected with empty plasmid (HeLa-N1, SiHa-N1), the expression levels of phosphorylated 4EBP1 and phosphorylated p70s6k in adenocarcinoma HeLa and squamous cell carcinoma SiHa overexpressing SDHA (HeLa-SDHA, SiHa-SDHA) were significantly increased. This indicates that overexpression of SDHA activates the mTOR / 4E-BP1 / P70s6k signaling pathway in cervical adenocarcinoma HeLa and squamous cell carcinoma SiHa.

[0095] In summary, overexpression of SDHA can activate the expression of 4E-BP1 and P70s6k, downstream proteins of the mTOR signaling pathway, in human cervical adenocarcinoma cells HeLa and squamous cell carcinoma cells SiHa, increase mitochondrial membrane potential and intracellular calcium ion levels, inhibit autophagy and apoptosis, and promote the occurrence and progression of cervical cancer. SDHA can be used as a molecular biomarker for the diagnosis of cervical cancer and a potential therapeutic target for cervical cancer.

[0096] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Use of a reagent for detecting the expression level of SDHA protein in the preparation of a product for diagnosing or assisting in the diagnosis of cervical cancer progression; the product is used to detect the expression level of SDHA protein in cervical tissue.

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