Cervical cancer prognosis evaluation kit based on ITM2A mRNA expression level and application thereof

Abstract

The invention provides a cervical cancer prognosis evaluation kit based on ITM2A mRNA expression level and application of the cervical cancer prognosis evaluation kit, and belongs to the technical field of molecular detection. The kit disclosed by the invention comprises a primer pair capable of specifically amplifying a human ITM2A gene sequence; through detection of expression levels of ITM2A genes and encoded proteins in normal cervical tissues and tumor tissues, it is found that expression of ITM2A in the tumor tissues of cervical cancer patients is remarkably reduced, and a kit for prognosis judgment of cervical cancer is developed based on the discovery. The method has extremely important significance on risk assessment and prognosis prediction of cervical cancer patients.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, and in particular to a cervical cancer prognostic assessment kit based on ITM2A mRNA expression levels and its application. Background Technology

[0002] Cervical cancer is one of the most serious malignant tumors threatening women's health worldwide. Although widespread human papillomavirus (HPV) vaccination and cervical cancer screening have significantly reduced its incidence and mortality, significant differences remain in the clinical prognosis of cervical cancer patients. Some patients have a good prognosis and high long-term survival rate after receiving standard treatment; while others are prone to recurrence, metastasis, or even treatment resistance, leading to poor clinical outcomes. Therefore, accurately assessing the prognostic risk of cervical cancer patients is of crucial clinical significance for guiding individualized treatment decisions.

[0003] Currently, prognostic assessment of cervical cancer mainly relies on clinicopathological features, such as tumor stage and lymph node metastasis status. However, these indicators have limitations in predicting long-term survival and disease progression, and cannot fully meet the needs of personalized treatment. In recent years, with the development of molecular biology techniques, more and more studies have begun to focus on molecular changes in the tumor microenvironment and their relationship with prognosis. However, existing biomarkers still have shortcomings in terms of specificity and sensitivity, and there is an urgent need to discover new and reliable molecular biomarkers.

[0004] Integral Membrane Protein 2A (ITM2A) is a member of the BRICHOS domain protein family. Recent studies have shown that ITM2A is not simply a structural protein, but plays important roles in various biological processes, including cell differentiation, autophagy, and extracellular matrix interactions. However, research on the role and clinical significance of ITM2A in cervical cancer remains lacking. To date, no studies have clearly revealed the correlation between ITM2A mRNA expression levels and clinicopathological features and survival prognosis in cervical cancer patients. Whether ITM2A can serve as a stable and reliable prognostic molecular marker for cervical cancer, and what predictive value its expression level has for progression-free survival (PFS) and overall survival (OS) in cervical cancer patients, remain unexplored and unanswered.

[0005] Therefore, there is an urgent need in this field to discover a molecular biomarker that is closely related to the prognosis of cervical cancer, and to develop a test kit that can objectively, accurately, and conveniently assess the prognostic risk of cervical cancer patients, so as to make up for the shortcomings of existing clinicopathological parameters in prognostic stratification accuracy, and provide strong molecular evidence for clinicians to formulate more reasonable individualized treatment plans. Summary of the Invention

[0006] In view of this, the present invention provides a cervical cancer prognostic assessment kit based on ITM2A mRNA expression level and its application, in order to solve the above problems.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a kit for prognostic assessment of cervical cancer, the kit containing a detection reagent for specifically detecting the expression level of the human ITM2A gene.

[0009] Preferably, the detection reagent includes primer pairs capable of specifically amplifying the human ITM2A gene sequence.

[0010] Preferably, the nucleotide sequences of the primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0011] Preferably, the kit is a real-time PCR kit, and the detection kit further includes a fluorescent dye.

[0012] Preferably, the kit further includes an internal reference gene detection reagent, wherein the internal reference gene is selected from GAPDH.

[0013] Preferably, the kit further includes reagents for extracting total RNA from the sample, reverse transcription reagents, and / or PCR reaction buffers.

[0014] Preferably, the sample is cervical tissue or paraffin-embedded tissue from a cervical cancer patient.

[0015] The present invention also provides a primer pair for prognostic assessment of cervical cancer, wherein the primer pair is capable of specifically amplifying the sequence of the human ITM2A gene, the sequence of which is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0016] This invention provides the use of the kit or primer pair described herein in the preparation of products for assessing the prognosis of patients with cervical cancer.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects: The kit of the present invention includes primer pairs that can specifically amplify the human ITM2A gene sequence. By detecting the expression level of ITM2A gene and encoded protein in normal cervical tissue and tumor tissue, it was found that ITM2A expression was significantly downregulated in the tumor tissue of cervical cancer patients. Based on the above findings, a kit for cervical cancer prognosis assessment was developed, which is of great significance for risk assessment and prognosis prediction of cervical cancer patients. Attached Figure Description

[0018] Figure 1This is a graph showing the ITM2A mRNA levels in cervical cancer tissue and the corresponding adjacent normal tissue.

[0019] Figure 2 The difference in ITM2A expression between normal and tumor tissues (A) and the ROC curve of the relationship between ITM2A expression level and prognosis (B).

[0020] Figure 3 The figure shows the Kaplan-Meier survival analysis results of ITM2A expression levels and patients' progression-free survival (PFS) and disease-specific survival (DSS), where A represents the results of the progression-free survival (PFS) analysis and B represents the results of the disease-specific survival (DSS) analysis. Detailed Implementation

[0021] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] Example 1

[0023] Patients with pathologically confirmed cervical cancer who underwent surgery at Gansu Cancer Hospital between January 2023 and March 2025 were selected as the study subjects. Postoperative tumor tissue samples were collected, treated with paraffin, and stored at room temperature. The excised white slides were stored in a refrigerator at 4°C.

[0024] Inclusion criteria: newly diagnosed cervical cancer patients with a single tumor, who had not received radiotherapy or chemotherapy before surgery, patients with FIGO stage I, II, or III who underwent radical surgery, and whose surgical specimens contained both cervical cancer tissue and adjacent normal tissue (the area of ​​normal cervical tissue >5cm from the tumor margin).

[0025] All cervical cancer patients included in the study were staged according to the FIGO 2018 staging system. Patient-related data were obtained from medical records, and follow-up data were obtained from the hospital's follow-up department. Collected clinicopathological data included age, sex, tumor location (cervical squamocolumnar junction, cervical canal, external cervical os, etc.), tumor size, pathological type (squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, etc.), depth of invasion, intraoperative lymph node status and pathological results, distant metastasis, FIGO / TNM staging, preoperative SCC-Ag, CA125, and CEA levels, and postoperative treatment regimen (radiotherapy, chemotherapy, concurrent chemoradiotherapy, etc.).

[0026] All surgical patients included in the study signed informed consent forms, and the study protocol has been approved by the Ethics Committee of Gansu Provincial Cancer Hospital. (Approval No.: A202212180067)

[0027] Survival indicators are calculated as follows: Progression-free survival (PFS) is calculated from the date of surgery to the date of tumor recurrence, metastasis, or the last follow-up (for those without recurrence or metastasis); Disease-specific survival (DSS) is calculated from the date of surgery to the date of death related to cervical cancer, and for those who die from non-cervical cancer-related causes, the date of the last follow-up is used.

[0028] The expression level of ITM2A mRNA in cervical cancer tissue was detected by quantitative PCR, including the following steps:

[0029] (1) The cDNA sequences of ITM2A and GAPDH (internal reference) were obtained by querying the NCBI Gene database. PCR primers were designed based on the sequences as shown in Table 1:

[0030] Table 1 Primer Sequences

[0031]

[0032] (2) Total RNA was extracted from 16 fresh tumor tissues and 13 paired adjacent normal tissues (controls) using the miRNeasy Mini Kit (Qiagen). RNA purity was measured using a NanoDrop spectrophotometer, and the A260 / A280 ratio was controlled between 1.8 and 2.1. 1 μg of total RNA was taken from each sample and reverse transcribed into cDNA using a reverse transcription kit according to the instructions. The cDNA was then stored at -20℃ for later use.

[0033] (3) Real-time quantitative PCR detection

[0034] Reaction system preparation: Prepare the following reaction system in a PCR reaction plate or tube, with a total reaction volume of 20 μL:

[0035] 10 μL of 2× real-time quantitative PCR premix (containing SYBR Green I dye);

[0036] 0.4 μL of ITM2A-specific forward primer (10 μM);

[0037] 0.4 μL of ITM2A specific reverse primer (10 μM);

[0038] Template cDNA 2 μL;

[0039] Add RNase-free water to a final volume of 20 μL.

[0040] At the same time, parallel reaction wells for the internal reference gene GAPDH need to be set up for each sample. The system is the same as above, only the primers for the internal reference gene are replaced.

[0041] PCR amplification procedure: Run the following procedure on a real-time quantitative PCR instrument:

[0042] Stage 1: Pre-denaturation, 95°C, 30s;

[0043] Phase 2: Denaturation at 95°C for 5 seconds, annealing / extending at 60°C and acquiring fluorescence signal for 34 seconds; 40 cycles;

[0044] Phase 3: Melting curve analysis to confirm the specificity of the amplified products.

[0045] (4) Data analysis and prognostic judgment

[0046] Use 2 −ΔΔCt The relative expression level of the ITM2A gene relative to the internal reference gene in each sample was calculated using a method that calibrates the expression level against the average expression level of a group of adjacent normal tissues or specified calibration samples. The results are as follows: Figure 1 As shown. Figure 1 The results showed that the relative expression level of ITM2A mRNA in adjacent normal tissues was significantly higher than that in tumor tissues, suggesting that downregulation of ITM2A expression is closely related to tumorigenesis.

[0047] (5) Prognostic risk assessment

[0048] The relative expression level of ITM2A in the test samples was compared with a predetermined cutoff value. This cutoff value was the median ITM2A expression level in the patient cohort, 2.38, as the dividing point.

[0049] Judgment criteria: If the relative expression level of ITM2A in a sample is lower than or equal to the critical value of 2.38, it is judged as "low expression of ITM2A", which indicates that the patient has a poor prognosis and a higher risk of relapse and disease-specific death.

[0050] If the relative expression level of ITM2A in a sample is higher than the critical value of 2.38, it is judged as "high expression of ITM2A", indicating that the patient has a better prognosis.

[0051] Example 2. Clinical Sample Validation

[0052] To verify the effectiveness of the kit, a blinded assay was performed on 50 archived paraffin-embedded tissue samples from cervical cancer patients with known clinical follow-up information. The ITM2A mRNA expression level of all samples was detected using the kit, following the same method as in Example 1. The samples were divided into high and low expression groups based on a pre-set median cutoff value. ROC curves were plotted based on overall survival (OS) and progression-free survival (PFS) data. The expression differences of ITM2A between normal tissue and cervical cancer tissue are shown below. Figure 2 As shown in Figure A, the ROC curve of the relationship between ITM2A expression level and prognosis is as follows. Figure 2 As shown in B in the diagram.

[0053] The analysis results showed that the 3-year progression-free survival rate of patients in the low ITM2A expression group was significantly lower than that in the high expression group (Log-rank test, P<0.05), which is consistent with previous research findings and confirms the feasibility and effectiveness of this kit for cervical cancer prognostic assessment.

[0054] Example 3

[0055] To investigate the relationship between ITM2A expression and prognosis in cervical cancer patients and to verify the effectiveness of the kit of this invention, data analysis was conducted based on the TCGA public database. Cervical cancer patients (n=306) with complete clinical follow-up information were selected from the database, and their ITM2A mRNA expression levels were obtained. Patients were divided into high-expression and low-expression groups based on the median cutoff value of expression level (2.38). Based on follow-up data of progression-free survival (PFS) and disease-specific survival (DSS), patients were further divided into high-expression and low-expression groups according to the median ITM2A expression level. Kaplan-Meier analysis in SPSS software was used to study the correlation between ITM2A expression in tumor tissue and prognosis. Results (…) Figure 3 The results showed that cases with high ITM2A expression in tumor tissue (H-score ≥ 2.38) had significantly longer progression-free survival (PFS; P < 0.001) and disease-specific survival (DSS; P < 0.001) than cases with low ITM2A expression in tumor tissue (H-score < 2.38).

[0056] As can be seen from the above embodiments, the present invention provides a cervical cancer prognostic assessment kit based on ITM2A mRNA expression levels and its application. The kit of the present invention can be used for prognostic assessment of cervical cancer.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for prognostic assessment of cervical cancer, characterized in that, The kit contains ingredients for specific human testing. ITM2A Reagents for detecting gene expression levels.

2. The reagent kit according to claim 1, characterized in that, The detection reagent includes primer pairs capable of specifically amplifying the human ITM2A gene sequence.

3. The reagent kit according to claim 2, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO.1 and SEQ ID NO.

2.

4. The reagent kit according to claim 3, characterized in that, The kit is a real-time PCR kit, and the detection kit also includes a fluorescent dye.

5. The reagent kit according to claim 4, characterized in that, The kit also includes an internal reference gene detection reagent, wherein the internal reference gene is selected from GAPDH.

6. The reagent kit according to any one of claims 1 to 5, characterized in that, The kit also includes reagents for extracting total RNA from samples, reverse transcription reagents, and / or PCR reaction buffers.

7. The reagent kit according to claim 6, characterized in that, The samples were cervical tissue or paraffin-embedded tissue from patients with cervical cancer.

8. A primer pair for prognostic assessment of cervical cancer, characterized in that, The primer pair is capable of specifically amplifying the sequence of the human ITM2A gene, as shown in SEQ ID NO: 1 and SEQ ID NO:

2.

9. The use of the kit according to any one of claims 1 to 7 or the primer pair according to claim 8 in the preparation of a product for assessing the prognosis of patients with cervical cancer.

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