Application of TGM2 as cervical cancer diagnosis and prognosis biomarker and treatment target

Abstract

The invention discloses application of TGM2 as a cervical cancer diagnosis and prognosis biomarker and a treatment target. The invention provides a novel cervical cancer diagnosis and prognosis marker TGM2, which can clearly characterize the occurrence and development of cervical cancer; the marker shows a specific high expression phenomenon in human clinical cervical cancer tissues compared with normal tissues; high expression is also shown in a human cervical cancer cell line. Knock-down of TGM2 can inhibit proliferation and migration ability of cervical cancer cells. As an enzyme, the TGM2 has higher diagnosis and treatment target application potential. The invention provides a new marker and target for diagnosis, prognosis and treatment of cervical cancer, and can provide a new strategy and direction for treatment of cervical cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of TGM2 as a diagnostic and prognostic biomarker and therapeutic target for cervical cancer. Background Art

[0002] Cervical cancer (CC) is the fourth most common malignant tumor in women worldwide and the fourth leading cause of cancer death in women. Persistent infection with high-risk HPV is the main factor in the occurrence of cervical cancer, but its mechanism is not clear. The current HPV vaccine can only prevent viral infection and has no effect on patients with cervical cancer. At present, the status of clinical treatment of cervical cancer is still not optimistic. Traditional surgical treatment, radiotherapy and chemotherapy cannot completely control and eliminate the occurrence and metastasis of cervical cancer. Not only that, treatment methods for cervical cancer patients, such as surgery, will also seriously affect the quality of life of patients, and there are few targeted drugs on the market.

[0003] The current tests for cervical cancer, such as the Pap smear test, have poor sensitivity; current studies have also shown that the sensitivity and specificity of liquid-based cytology for cervical cancer precursor lesions and early cervical cancer screening are not statistically significant. Therefore, actively exploring the molecular mechanism of the occurrence and development of cervical cancer is of great significance for the development of new diagnostic markers and therapeutic targets.

[0004] Transglutaminase 2 (TGM2) is a calcium-dependent enzyme that mediates post-translational modification of proteins by incorporating lysine residues or polyamines into protein-bound glutamate residues. TGM2 expression and activity are often dysregulated in a variety of cancer types, including breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer. TGM2 has been shown to participate in inflammatory pathways and regulate the tumor microenvironment by enhancing the stability of the extracellular matrix and promoting cell adhesion and migration. However, there are no reports on the correlation between TGM2 and cervical cancer. Summary of the invention

[0005] The purpose of the present invention is to address the above problems and provide an application of TGM2 as a diagnostic and prognostic biomarker and therapeutic target for cervical cancer.

[0006] In order to achieve its purpose, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a use of a reagent for detecting the expression of the biomarker TGM2 in the preparation of a product for auxiliary diagnosis, diagnosis or prognosis of a subject with cervical cancer.

[0008] In the above-mentioned application technology scheme, the diagnostic method includes: obtaining a test sample from a subject suspected of having cervical cancer, determining the expression level of TGM2 in the test sample, and if the expression of TGM2 is higher than that of the normal control, it indicates that the subject is a high-risk group for cervical cancer or a cervical cancer patient.

[0009] In the above-mentioned application technology scheme, the prognosis method includes: obtaining a test sample from a subject suffering from cervical cancer, and determining the expression level of TGM2 in the test sample. High expression of TGM2 indicates a poor prognosis for the patient.

[0010] The second aspect of the present invention provides the use of TGM2 as a target in screening drugs for treating cervical cancer.

[0011] The drug inhibits the expression of TGM2, thereby inhibiting the viability, proliferation, invasion and migration of cervical cancer cells.

[0012] The third aspect of the present invention provides use of a TGM2 expression inhibitor in the preparation of a medicament for treating cervical cancer.

[0013] The expression inhibitors include nucleic acid molecules, proteins, and compounds.

[0014] Preferably, the nucleic acid molecule comprises shRNA.

[0015] Preferably, the shRNA is shTGM2#2 or shTGM2#4,

[0016] The sense strand sequence of shTGM2#2 is: 5′-CCGGACAGCAACCTTCTCATC-3′;

[0017] The antisense sequence of shTGM2#2 is: 5′-AGAAGGTTGCTGTTTTTTT-3′;

[0018] The positive strand sequence of shTGM2#4 is: 5'-CCGGGCAGTGACTTTGACGTC-3'

[0019] The antisense sequence of shTGM2#4 is: 5'-GTCAAAGTCACTGCTTTTTT-3'.

[0020] The beneficial effects of the present invention are: providing a new cervical cancer diagnosis and prognosis marker TGM2, which can clearly characterize the occurrence and development of cervical cancer; the marker shows a specific high expression phenomenon in human clinical cervical cancer tissue compared with normal tissue; and it also shows high expression in human cervical cancer cell lines. Knocking down TGM2 will inhibit the proliferation and migration ability of cervical cancer cells; TGM2, as an enzyme, has a high potential for use as a diagnostic and therapeutic target. The present invention provides new markers and targets for the diagnosis, prognosis and treatment of cervical cancer, and can provide new strategies and directions for the treatment of cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of immunohistochemistry (IHC) analysis of 12 human cervical tissue samples using TGM2-specific antibodies, including 4 normal, 4 precancerous lesions and 4 cervical cancer tissue samples.

[0022] Figure 2 The results of western blot were used to detect the expression of TGM2 in several cervical cancer cell lines (SiHa, HeLa).

[0023] Figure 3 The results of RT-qPCR detection of TGM2 expression in several cervical cancer cell lines (SiHa, HeLa).

[0024] Figure 4 The Kaplan-MeierPlotter database was used to analyze the overall survival results of patients with cervical cancer with high TGM2 expression.

[0025] Figure 5 Western blot and RT-qPCR were used to detect the knockdown effect of shRNA on TGM2 in SiHa cells.

[0026] Figure 6 The results of CCK-8 cell proliferation assay to determine the effect of TGM2 on the proliferation of cervical cancer cells are shown.

[0027] Figure 7 The results of Transwell migration and invasion assays were used to detect the effect of TGM2 on the migration and invasion ability of cervical cancer cells.

[0028] Figure 8 The results showed that GEPIA database analysis indicated that there was a high correlation between IL-32 and TGM2 in cervical cancer tissues.

[0029] Fig. 9 The results of RT-qPCR detection of IL-32 levels in cervical cancer cells after TGM2 knockdown.

[0030] Fig.10The results showed that knocking down TGM2 had an inhibitory effect on the proliferation, migration and invasion of cervical cancer cells after replenishing IL-32, as detected by CCK-8 proliferation assay and Transwell assay.

[0031] Fig.11 GEPIA database analysis showed that IL-32 was highly expressed in cervical cancer tissues.

[0032] Fig.12 This is the result of CCK-8 detection of cell proliferation rate after adding IL-32 to SiHa cells.

[0033] Fig.13 The results of Transwell migration assay were used to detect the migration ability of cervical cancer cells with the addition of IL-32.

[0034] Fig.14 The results are the effects of knocking down TGM2 on tumor size and weight in a mouse xenograft tumor model. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0037] Example 1

[0038] 1 Materials and Methods

[0039] 1.1 Cell culture

[0040] This experiment used two human cervical cancer cell lines SiHa and HeLa cells and an immortalized normal human cervical epithelial cell line H8. SiHa cells (Procell, Wuhan) were cultured in MEM-α medium (L570KJ, Basalmedia) containing 10% fetal bovine serum, HeLa cells (Procell, Wuhan) were cultured in DMEM medium (C11995500BT, Gibco) containing 10% fetal bovine serum; H8 cells (Qingqi) were cultured in DMEM medium supplemented with 10% fetal bovine serum. All these cell lines were cultured in a standard environment of 37°C and 5% CO2.

[0041] 1.2 Lentiviral packaging and construction of TGM2 knockdown cells

[0042] In order to obtain cells with knockdown of TGM2 expression, shRNA virus targeting TGM2 was prepared by lentiviral packaging. The process was as follows: using a lentiviral packaging three-plasmid system, the lentiviral packaging plasmids pMD2G, psPAX2, and shTGM2 (Tsingke Biotech, China) or pLK0.1-TRC vector (shTGM2 control (NC) plasmid, Tsingke Biotech, China) were packaged with lentivirus at a mass ratio of 1:4:4. When HEK293T cells were 50% confluent, they were transfected into cells by PEI (24765-1, Polysciences) according to the plasmid product instructions. The viral supernatant was collected after 48 hours. When the cells were 50% confluent, the shRNA virus targeting TGM2 was transfected into SiHa cells, and infection was mediated by polybrene reagent (C0351, Beyotime) according to the product instructions. After 48 hours, puromycin (ST551, Beyotime) was selected. The shTGM2 sequence is shown in Table 1.

[0043] Table 1 shRNA targeting sequences

[0044]

[0045] 1.3 CCK-8 experiment

[0046] The cells were cultured at 2.5 × 10 3 Cells were inoculated into 96-well plates at a density of 10 cells / well, with 5 replicate wells per group. Subsequently, 10uL of CCK-8 solution was added to each well at 0, 24, 48, 72, and 96h, respectively, and incubated at 37°C for 1h. The absorbance at a wavelength of 450nm was measured using an ELISA reader. Cell viability curves were drawn based on the absorbance values.

[0047] 1.4 Immunohistochemistry (IHC)

[0048] Paraffin sections were stained with IHC. Paraffin sections were dewaxed in xylene and different concentrations of ethanol. After antigen retrieval, endogenous peroxidase was blocked, and TGM2 antibody (AWA58126, abiowell, China) was incubated at 4°C overnight. Enzyme-labeled goat anti-mouse / rabbit IgG polymer secondary antibody (PV-6000, zsbio, China) was incubated at room temperature for 20 min. Then DAB color development, hematoxylin staining, dehydration with different concentrations of ethanol, and neutral resin sealing were performed.

[0049] 1.5 Real-time quantitative polymerase chain reaction (RT-qPCR)

[0050] Total RNA from cervical cancer cell lines was extracted using RNAiso Plus (9109, Takara, Beijing) and reverse transcribed into cDNA template using ABScriptⅢRTMaster Mix for qPCR with gDNARemover (RK20429, ABclonal, Wuhan). The NCBI Gene ID of human gene TGM2 is 7052, and the designed specific primer sequences (shown in Table 2) were synthesized by Tsingke Company (China). Subsequently, the cDNA template, specific primers, and SYBR Green Fast qPCR Mix reagent (RK21203, ABclonal, China) were mixed to amplify the target gene, and RT-qPCR reaction was performed on Roche LightCycler480Ⅱsystem. TGM2 expression was normalized with GADPH. Relative expression was calculated using the 2-△△CT method.

[0051] Table 2 RT-qPCR primer sequences

[0052]

[0053]

[0054] The cDNA obtained by reverse transcription was used as a template, 3 duplicate wells were set up for each group of samples, and the reaction system was prepared on ice according to the components listed below.

[0055] Reagents Addition amount SYBRGreenFastqPCRMix(RK21203,ABclonal) 10uL Forward primer (10uM) 0.4uL Reverse primer (10uM) 0.4uL cDNA 2uL <![CDATA[RNase-FreeH20]]> 7.2uL

[0056] Perform qPCR reaction according to the following conditions:

[0057]

[0058] Calculation results: The relative mRNA expression of the target gene was calculated according to the Ct value of the target gene and the Ct value of the internal reference gene obtained by RT-qPCR reaction using the 2-△△CT method.

[0059] 1.6 Transwell assay

[0060] Transwell assay was used to evaluate the migration and invasion ability of cells. 4 cells / well) were inoculated in the upper chamber of Transwell (8um, labselect), and bubbles were removed and added slowly. For invasion experiments, cell suspension (8×10 4Cells / well) were seeded in the upper chamber of Transwell, and Matrigel (BD Biosciences, USA) was diluted 1:8 with serum-free medium and pre-coated on the upper chamber 2 hours in advance. Medium containing 20% ​​fetal bovine serum was added to the lower chamber. After incubation for 48 hours, the Transwell chamber was removed and rinsed twice with PBS. The cells were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (C0121, Beyotime). The number of migrating or invading cells in the lower layer was observed using an upright fluorescence microscope, and 3 fields of view were randomly selected in each image for counting.

[0061] 1.7 Immunoblotting

[0062] The total protein of cervical cancer cells was lysed using RIPA lysis buffer (P0013B, Beyotime) containing protease inhibitors, and protein quantification was performed using a BCA detection kit (P0010, Beyotime). Then, an equal amount was loaded on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, and electrophoresis was continued at 80V for 30min and then at 120V for 60min. Then, the membrane was transferred to a PVDF membrane (ISEQ00010, Millipore). After blocking with 5% skim milk powder at room temperature for 1h, the membrane was incubated with the following specific antibodies overnight at about 4°C: anti-TGM2 antibody (), GADPH (1:10000; 60004-1-Ig, Proteintech). Subsequently, HRP-conjugated goat anti-rabbit antibody (1:10000, 5220-0336, Seracare, USA) or HRP-conjugated goat anti-mouse antibody (1:10000; 5220-0341, Seracare) was incubated with the membrane at room temperature for 1 h. The membrane was developed and photographed using an ECL kit and a Bio-Rad multifunctional imaging system.

[0063] 1.8 Xenograft tumor model establishment

[0064] This study was approved by the Ethics Committee of Chongqing Medical University. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Balb / c nude mice (4-5 weeks old) were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. and housed in standard conditions. sh-TGM2 or sh-NC (control) cells were stably transfected with puro antibiotics. 6 SiHa cells stably transfected with sh-TGM2 or sh-NC were subcutaneously injected into the left side of the back of mice at a density of 10 cells. 3 )=(length×width 2 The tumor volume was measured every 3-5 days using a vernier caliper using the formula ) / 2. The mice were sacrificed after 27 days and the tumor weight was measured.

[0065] 1.9 Statistical analysis

[0066] The above data are expressed as mean ± standard deviation and analyzed using GraphPad Prism software (San Diego, California, USA). The t-test after Bonferroni test or one-way analysis of variance was used for comparison, and p < 0.05 was considered significant.

[0067] 2. Results

[0068] 2.1 Abnormal upregulation of TGM2 expression in cervical cancer

[0069] We first examined the expression of TGM2 in cervical cancer and its effect on the overall survival of cervical cancer patients. We used TGM2-specific antibodies to perform immunohistochemistry (IHC) analysis on 12 human cervical tissue samples, including 4 normal, 4 precancerous lesions, and 4 cervical cancer tissue samples. The results showed that the staining intensity of TGM2 in cancer tissues was higher than that in normal tissues ( Figure 1 ), indicating that TGM2 is significantly upregulated in cervical cancer tissues. Then, the expression of TGM2 was detected in several cervical cancer cell lines (SiHa, HeLa) by western blot and RT-qPCR. Compared with the immortalized normal cervical epithelial cell line (H8), cervical cancer cells showed higher TGM2 protein and mRNA levels, which is consistent with the observation levels in tissues ( Figure 2 , 3 ). Kaplan-Meier Plotter database analysis showed that patients with cervical cancer with high TGM2 expression had a shorter overall survival rate than those with low TGM2 expression ( Figure 4 ). This suggests that TGM2 may affect the occurrence and development of cervical cancer.

[0070] 2.2 Knockdown of TGM2 inhibits proliferation, migration and invasion of cervical cancer cells

[0071] To further determine whether TGM2 regulates the occurrence and development of cervical cancer cells, shRNA was used to knock down TGM2 and its effect on cell proliferation was detected. Knockdown cells were generated from SiHa cells, which were transduced with two different lentiviruses expressing TGM2-shRNA and stably selected by puromycin. The western blot and RT-qPCR in the figure show that TGM2 has a good knockdown efficiency. The CCK-8 cell proliferation assay was used to determine the effect of TGM2 on the proliferation of cervical cancer cells. Our results ( Figure 5 , 6 ) showed that the growth rate of the knockdown group was lower than that of the control group cells, indicating that silencing TGM2 inhibited the proliferation of cervical cancer cells.

[0072] In addition to exploring the effect of TGM2 on the proliferation of cervical cancer cells, we also determined its effects on cell migration and invasion. The effects of TGM2 on the migration and invasion ability of cervical cancer cells were detected using Transwell migration and invasion assays. Figure 7 The results showed that fewer cells migrated in the TGM2 knockdown group compared with the control group. In addition, fewer cervical cancer cells invaded after TGM2 knockdown. These results indicate that TGM2 promotes the proliferation, migration, and invasion of cervical cancer cells.

[0073] 2.3 Knockdown of TGM2 inhibits the expression of IL-32 in cervical cancer cells

[0074] like Figure 8 As shown in the GEPIA database, there is a high correlation between IL-32 and TGM2 in cervical cancer tissues. Therefore, we speculate that TGM2 can affect the occurrence and development of cervical cancer by regulating IL-32. RT-qPCR experiments confirmed that knocking down TGM2 reduced the level of IL-32 in cervical cancer cells ( Fig. 9 ). Then, a replenishment experiment was conducted, in which IL-32 (30 ng / ml) was added to the cervical cancer cells with TGM2 knockdown. Through CCK-8 proliferation assay and Transwell assay, it was found that after replenishment of IL-32, the inhibitory effect of TGM2 knockdown on the proliferation, migration and invasion of cervical cancer cells was weakened ( Fig.10 ). These data suggest that TGM2 regulates IL-32 to affect the occurrence and development of cervical cancer.

[0075] 2.4IL-32 promotes the proliferation and migration of cervical cancer cells

[0076] IL-32 (interleukin-32) is highly expressed in most solid tumors, such as gastric cancer tissues, and is associated with poor prognosis. However, there are reports and studies on IL-32 in cervical cancer. Therefore, we intend to explore whether IL-32 affects the occurrence and development of cervical cancer. The GEPIA database shows that IL-32 is highly expressed in cervical cancer tissues ( Fig.11 ). To explore the effect of IL-32 on the proliferation of cervical cancer cells, CCK-8 assay found that after adding IL-32 (30ng / ml) to SiHa cells, the cell proliferation rate was slightly enhanced compared with the control group ( Fig.12 ). The Transwell migration experiment also showed that the migration ability of cervical cancer cells with the addition of IL-32 was enhanced compared with the control group ( Fig.13 ). In short, these experiments show that IL-32 enhances the proliferation and migration of cervical cancer cells.

[0077] 2.5 Knockdown of TGM2 inhibits xenograft tumor formation

[0078] To test the effect of TGM2 on tumor growth in vivo, SiHa cells were stably transfected with shTGM2 and injected subcutaneously into the left back of nude mice. Fig.14 As shown, downregulation of TGM2 can inhibit tumor size and weight.

[0079] 3. Conclusion

[0080] The present study shows that: 1) TGM2 expression is increased in cervical cancer tissues and is associated with poor prognosis. 2) Specific knockdown of TGM2 inhibits the malignant progression of cervical cancer in vitro and in vivo. 3) TGM2 regulates the expression of IL-32 and thus affects the malignant progression of cervical cancer.

Claims

1. Use of a reagent for detecting the expression of the biomarker TGM2 in the preparation of a product for auxiliary diagnosis, diagnosis or prognosis of cervical cancer subjects.

2. The use according to claim 1, characterized in that: The diagnostic method comprises: obtaining a test sample from a subject suspected of having cervical cancer, determining the expression level of TGM2 in the test sample, and if the expression level of TGM2 is higher than that of a normal control, it indicates that the subject is a high-risk group for cervical cancer or a cervical cancer patient.

3. The use according to claim 1, characterized in that: The prognosis method comprises: obtaining a test sample from a subject suffering from cervical cancer, and determining the expression level of TGM2 in the test sample. High expression of TGM2 indicates a poor prognosis for the patient.

4. Application of TGM2 as a target in screening drugs for the treatment of cervical cancer.

5. The use according to claim 4, characterized in that: The drug inhibits the expression of TGM2, thereby inhibiting the viability, proliferation, invasion and migration of cervical cancer cells.

6. Application of TGM2 expression inhibitors in the preparation of drugs for the treatment of cervical cancer.

7. The use according to claim 6, characterized in that: The expression inhibitors include nucleic acid molecules, proteins, and compounds.

8. The use according to claim 7, characterized in that: The nucleic acid molecule includes shRNA.

9. The use according to claim 8, characterized in that: The shRNA is shTGM2#2 or shTGM2#4, The sense strand sequence of shTGM2#2 is: 5′-CCGGACAGCAACCTTCTCATC-3′; The antisense sequence of shTGM2#2 is: 5′-AGAAGGTTGCTGTTTTTTT-3′; The positive strand sequence of shTGM2#4 is: 5'-CCGGGCAGTGACTTTGACGTC-3' The antisense sequence of shTGM2#4 is: 5'-GTCAAAGTCACTGCTTTTTT-3'.

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