A marker group for cervical cancer screening and diagnosis and use thereof

Abstract

The application discloses a marker group for cervical cancer screening and diagnosis and application thereof, and belongs to the technical field of gynecological tumor diagnosis. The marker group is composed of CIDEC and TMEM82. CIDEC and TMEM82 are significantly up-regulated in cervical cancer tissues and cervical cancer cell lines, and the high expression of the two is closely related to the occurrence and development of cervical cancer. After the expression of CIDEC and TMEM82 is inhibited by specific siRNA, the proliferation, migration and invasion ability of cervical cancer cells are obviously inhibited. Clinical sample verification shows that when the expression amount of CIDEC and TMEM82 is jointly detected for diagnosing cervical cancer, the area under the ROC curve (AUC) can reach more than 0.86, the sensitivity is 86.67%, the specificity is 76.67%, and P<0.001, indicating that the marker group can be used as an effective index for screening and diagnosing cervical cancer. The application provides a new reliable way for early discovery and accurate diagnosis of cervical cancer, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostics, and more specifically, to a set of biomarkers for cervical cancer screening and diagnosis and their uses. Background Technology

[0002] Cervical cancer is the fourth most common malignant tumor among women worldwide, posing a serious threat to women's lives and health. The main causative factor of cervical cancer is persistent infection with high-risk human papillomavirus (HPV), particularly HPV types 16 and 18. However, not all HPV-infected individuals will develop cervical cancer. The progression from HPV infection to cervical intraepithelial neoplasia (CIN) and eventually invasive carcinoma typically takes years or even decades. Therefore, early detection, early diagnosis, and timely intervention are crucial for reducing cervical cancer mortality.

[0003] Currently, the most commonly used cervical cancer screening methods in clinical practice include liquid-based thin-layer cytology (TCT) and HPV DNA testing. TCT relies on the pathologist's experience, which introduces subjectivity and a potential for missed diagnoses. While HPV DNA testing has high sensitivity, its specificity is insufficient, potentially leading to overdiagnosis and overtreatment, placing unnecessary psychological and financial burdens on patients. Furthermore, although colposcopy and histopathological biopsy are considered the "gold standard" for diagnosis, they are invasive procedures unsuitable for large-scale population screening. In recent years, while some protein biomarker-based detection methods have emerged, such as squamous cell carcinoma antigen (SCC-Ag) and carcinoembryonic antigen (CEA), the sensitivity and specificity of these biomarkers in early cervical cancer or precancerous lesions are often unsatisfactory, failing to meet the clinical demand for high-precision, non-invasive, or minimally invasive screening. Therefore, finding novel, efficient, and highly specific molecular biomarkers and constructing more accurate cervical cancer screening and diagnostic models is of significant scientific and clinical value for improving cervical cancer prevention and control. Summary of the Invention

[0004] To address the aforementioned issues, this invention first provides a biomarker set for cervical cancer screening and diagnosis, characterized in that the biomarker set consists of CIDEC and TMEM82.

[0005] The present invention also provides a detection primer pair for CIDEC in the above-mentioned biomarker group, the nucleotide sequences of the primer pair being shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0006] The present invention also provides a detection primer pair for TMEM82 in the biomarker group as described in claim 1, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0007] The present invention also provides a kit for cervical cancer screening and diagnosis, the kit comprising the above-mentioned CIDEC detection primer pair and / or the above-mentioned TMEM82 detection primer pair.

[0008] In some embodiments, the kit further comprises one or more of reverse transcriptase, DNA polymerase, dNTPs, and reaction buffer.

[0009] The present invention also provides the application of the above-mentioned biomarker group in the preparation of cervical cancer screening or diagnostic reagents.

[0010] The present invention also provides the application of the above-mentioned detection primer pair in the preparation of cervical cancer screening or diagnostic kits.

[0011] The present invention also provides an application of the above-described kit in cervical cancer screening or diagnosis.

[0012] The present invention also provides an siRNA molecule for inhibiting the proliferation, migration or invasion of cervical cancer cells, wherein the siRNA molecule targets the CIDEC and / or TMEM82 genes, wherein the siRNA sequence targeting CIDEC is shown in SEQ ID NO: 7 to SEQ ID NO: 10, and the siRNA sequence targeting TMEM82 is shown in SEQ ID NO: 11 to SEQ ID NO: 14.

[0013] Finally, this invention provides the application of the above-mentioned siRNA molecule in the preparation of cervical cancer treatment drugs.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: (1) This invention is the first to discover that the biomarker group composed of CIDEC and TMEM82 is synergistically highly expressed in cervical cancer tissues. The combined detection of the two can significantly improve the sensitivity and specificity of cervical cancer diagnosis and make up for the limitations of single biomarker detection.

[0015] (2) The biomarker detection of the present invention is based on molecular biology technology. Compared with traditional cytological examination and histopathological examination, it has the advantages of being relatively simple to operate, highly objective, and less affected by human factors, making it suitable for screening large-scale clinical samples.

[0016] (3) Gene silencing experiments confirmed that inhibiting the expression of CIDEC and TMEM82 can effectively inhibit the proliferation, migration and invasion of cervical cancer cells, suggesting that this biomarker group not only has diagnostic value, but may also become a potential therapeutic target for cervical cancer.

[0017] (4) The detection primers provided by this invention have high specificity and high amplification efficiency. Based on this, the detection kit developed can quickly and accurately detect the expression levels of CIDEC and TMEM82 in samples, providing strong support for the early diagnosis, disease monitoring and prognosis assessment of cervical cancer.

[0018] (5) The biomarker group of the present invention has excellent diagnostic efficacy. ROC curve analysis shows that the AUC value is high, and the sensitivity and specificity are ideal. It has significant clinical application value and broad market prospects. Attached Figure Description

[0019] Figure 1 Comparison of TMEM82 mRNA expression levels in cervical cancer tissue and normal cervical tissue.

[0020] Figure 2 A comparison of CIDEC mRNA expression levels in cervical cancer tissue and normal cervical tissue.

[0021] Figure 3 Comparison of TMEM82 mRNA expression levels in different cervical cancer cell lines (HeLa, SiHa, Caski) and normal cervical epithelial cells (H8, control group).

[0022] Figure 4 A comparison of CIDEC mRNA expression levels in different cervical cancer cell lines (HeLa, SiHa, Caski) and normal cervical epithelial cells (H8, control group).

[0023] Figure 5 A graph showing the silencing efficiency of different siRNAs on TMEM82 mRNA expression in cervical cancer cells.

[0024] Figure 6 A graph showing the silencing efficiency of different siRNAs on CIDEC mRNA expression in cervical cancer cells.

[0025] Figure 7 ROC curve of combined detection of CIDEC and TMEM82 expression for diagnosing cervical cancer. Detailed Implementation

[0026] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0027] Example 1: Screening for diagnostic biomarkers associated with cervical cancer 1. Sample collection Fifteen cases of cervical squamous cell carcinoma surgically removed at the gynecology department of a collaborating hospital, along with paired adjacent normal cervical tissue samples (>2cm from the cancerous lesion margin), were collected. All patients had not received radiotherapy, chemotherapy, or immunotherapy prior to surgery, and the diagnosis was confirmed by pathology. Simultaneously, cervical exfoliated cell samples were collected from 30 healthy women undergoing outpatient physical examinations during the same period as normal controls. Informed consent was obtained from all patients or their families for all sample collection, and the process was reviewed and approved by the hospital's medical ethics committee.

[0028] 2. High-throughput transcriptome sequencing Total RNA was extracted from tissue samples using the TRIzol method (Invitrogen). RNA concentration and purity (OD260 / 280 ratios between 1.8 and 2.1) were measured using a Nanodrop 2000 spectrophotometer, and RNA integrity was assessed by agarose gel electrophoresis. Samples with RIN values ​​> 8.0 were sent to a sequencing company for library construction. cDNA libraries were constructed using the Illumina TruSeq RNA Sample Preparation Kit, and paired-end sequencing (PE150) was performed on the Illumina NovaSeq 6000 sequencing platform.

[0029] 3. Bioinformatics analysis and biomarker screening The raw sequencing data underwent quality control to remove adapter sequences and low-quality reads. Clean reads were aligned to the human reference genome (GRCh38). Differentially expressed genes were analyzed using DESeq2 software. The screening threshold was set as follows: |log2 Fold Change|>1 and corrected P-value<0.05. Results showed that hundreds of genes were significantly differentially expressed in cervical cancer tissue compared to normal cervical tissue. Among them, CIDEC and TMEM82 showed significantly upregulated expression in cancer tissue, with the highest fold-up regulation, and were therefore selected as candidate biomarkers in this embodiment.

[0030] Example 2: qPCR validation of differential expression of CIDEC and TMEM82 in tissue samples 1. Sample and RNA extraction The sample size was expanded to include 40 cervical cancer tissue samples, 40 adjacent normal tissue samples, and 40 healthy cervical exfoliated cell samples. RNA extraction and quality testing methods were the same as in Example 1.

[0031] 2. Reverse transcription and qPCR detection 1 μg of total RNA was used for reverse transcription to synthesize cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). Specific primers were designed using Primer Premier 5.0 software and synthesized by Beijing Qingke Biotechnology Co., Ltd., based on the mRNA sequences of CIDEC, TMEM82, and the internal reference gene GAPDH from GenBank. The primer sequences are as follows:

[0032] CIDEC: Forward primer (SEQ ID NO: 1): 5'-GAGCCAGGGGATGAGAAACA-3'; Reverse primer (SEQ ID NO: 2): 5'-CTCCAGCACCAGGAAGAAGG-3'; TMEM82: Forward primer (SEQ ID NO: 3): 5'-GGGCTGCCATGTTCTCTCTT-3'; Reverse primer (SEQ ID NO: 4): 5'-GGAGGCTGTTCAGGACCAAG-3'; GAPDH: Forward primer (SEQ ID NO: 5): 5'-CACCAGGGCTGCTTTTAACTC-3'; Reverse primer (SEQ ID NO: 6): 5'-TTCCCGTTCTCAGCCATGT-3'.

[0033] qPCR reaction system (20μL): 10μL SYBR Premix Ex Taq II (2×), 0.8μL each of forward and reverse primers (10μM), 2.0μL cDNA template, 0.4μL ROX Reference Dye II, and ddH2O to bring the total to 20μL.

[0034] Reaction conditions: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing and extension for 34 s, for a total of 40 cycles. Each sample was set up in 3 replicates. GAPDH was used as the internal control gene, and the relative expression level of the target gene was calculated using the 2^(-ΔΔCt) method.

[0035] 3. Results The results are as follows Figure 1 and Figure 2As shown, compared with normal cervical tissue and healthy exfoliated cells, the mRNA expression levels of CIDEC and TMEM82 in cervical cancer tissue were significantly increased (P<0.001). This indicates that high expression of CIDEC and TMEM82 is closely related to the occurrence of cervical cancer and has the potential to serve as diagnostic biomarkers for cervical cancer.

[0036] Example 3: Validation of CIDEC and TMEM82 expression in cervical cancer cell lines 1. Cell Culture Human cervical cancer cell lines HeLa, SiHa, and Caski, as well as the normal human cervical epithelial cell line H8, were all purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. Cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics, and routinely cultured in a cell culture incubator at 37°C and 5% CO2 saturated humidity. Cells were passaged every 2-3 days, and cells in the logarithmic growth phase were used for experiments.

[0037] 2. qPCR detection The expression levels of CIDEC and TMEM82 mRNA in each cell line were detected according to the method in Example 2.

[0038] 3. Results The results are as follows Figure 3 and Figure 4 As shown, compared with normal cervical epithelial cells H8 (HPV16-immortalized human cervical epithelial cells), the expression levels of CIDEC and TMEM82 were significantly upregulated in all three cervical cancer cell lines (HeLa, SiHa, and Caski) (P<0.01). This further confirms the high expression characteristics of CIDEC and TMEM82 at the cervical cancer cell level.

[0039] Example 4: Gene silencing (siRNA transfection) targeting CIDEC and TMEM82 1. siRNA design and transfection Two pairs of specific siRNAs (si-CIDEC#1 / #2, si-TMEM82#1 / #2) and a negative control siRNA (si-NC) were designed and synthesized targeting the mRNA sequences of CIDEC and TMEM82. The sequences are as follows:

[0040] si-CIDEC#1: (SEQ ID NO: 7): 5'-GGAGUUUGCAAUAAAUUAUUG-3'; (SEQ ID NO: 8): 5'-AUAAUUUAUUGCAAACUCCCU-3'; si-CIDEC#2: (SEQ ID NO: 9): 5'-GUUUGCAAUAAAUUAUUGAGG-3'; (SEQ ID NO: 10): 5'-UCAAUAAUUUAUUGCAAACUC-3'.

[0041] si-TMEM82#1: (SEQ ID NO: 11): 5'-CCAUGAACAAUAAAGACGAUG-3'; (SEQ ID NO: 12): 5'-UCGUCUUUAUUGUUCAUGGAG-3'; si-TMEM82#2: (SEQ ID NO: 13): 5'-CAAUAAAGACGAUGUCUUUCU-3'; (SEQ ID NO: 14): 5'-AAAGACAUCGUCUUUAUUGUU-3'.

[0042] HeLa cells in the logarithmic growth phase were harvested at a concentration of 2 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 6-well plates. After 24 hours of culture, transfection was performed when cell confluence reached 50%-60%. Using Lipofectamine RNAiMAX transfection reagent (Invitrogen), following the manufacturer's instructions, si-NC, si-CIDEC#1, si-CIDEC#2, si-TMEM82#1, and si-TMEM82#2 were transfected. The final transfection concentration was 50 nM. A blank control group (untransfected cells) was also included.

[0043] 2. qPCR knockdown efficiency 48 hours after transfection, cells from each group were collected, total RNA was extracted and reverse transcribed into cDNA, and qPCR was performed according to the method in Example 2.

[0044] 3. Results The results are as follows Figure 5 and 6As shown, compared with the Blank group and the si-NC group, the expression level of TMEM82 mRNA was significantly reduced in the experimental groups transfected with si-TMEM82#1 and si-TMEM82#2 (P<0.001). CIDEC expression was also significantly inhibited. This indicates that the selected siRNAs can effectively silence the expression of CIDEC and TMEM82 in HeLa cells. Subsequent experiments were conducted using si-TMEM82#1 and si-CIDEC#2.

[0045] Example 5: Effects of CIDEC and TMEM82 gene silencing on malignant phenotypes of cervical cancer cells 1. Cell proliferation capacity assay (CCK-8 assay) Transfected HeLa cells (Blank group, si-NC group, si-CIDEC#2 group, and si-TMEM82#1 group) were digested with trypsin and seeded into 96-well plates at a density of 3000 cells per well, with 5 replicates per group. At 24h, 48h, 72h, and 96h post-transfection, 10μL of CCK-8 solution (Dojindo) was added to each well, and the cells were incubated for another 2h. The absorbance (OD) value at 450nm was measured using a microplate reader.

[0046] 2. Cell migration ability assay (Transwell assay) Cells from each group transfected for 48 h were resuspended in serum-free DMEM medium and the cell density was adjusted to 2 × 10^5 cells / mL. 200 μL of cell suspension was added to the upper chamber of a Transwell (8 μm pore size, Corning), and 600 μL of DMEM medium containing 20% ​​FBS was added to the lower chamber. After 24 h of culture, the chambers were removed, and unmigrated cells on the upper chamber surface were wiped away with a cotton swab. Cells on the lower chamber surface were fixed with methanol for 15 min and stained with 0.1% crystal violet for 20 min. Cell counts were performed in five randomly selected fields of view under a microscope.

[0047] 3. Cell invasion ability detection (Matrigel invasion assay) The Matrigel (BD Biosciences) was diluted beforehand and coated onto the polycarbonate membrane in the upper chamber of the Transwell. The remaining steps were the same as in the migration experiment, but the incubation time was extended to 48 hours.

[0048] 4. Results Proliferation: Compared with the Blank group and the si-NC group, the proliferation rate of HeLa cells was significantly slowed down after silencing CIDEC or TMEM82 expression, especially at 72h and 96h after transfection, and the OD value was significantly reduced (P<0.01).

[0049] Migration: The number of transmembrane cells in the si-CIDEC#2 group and the si-TMEM82#1 group was significantly reduced compared with the control group (P<0.001), indicating that inhibiting CIDEC or TMEM82 can effectively weaken the migration ability of cervical cancer cells.

[0050] Invasion: After silencing CIDEC or TMEM82, the number of cells crossing the matrix gel decreased significantly (P<0.001), demonstrating that CIDEC and TMEM82 are involved in the invasion process of cervical cancer cells.

[0051] In summary, high expression of CIDEC and TMEM82 promotes the proliferation, migration, and invasion of cervical cancer cells, and they represent potential therapeutic targets.

[0052] Example 6: ROC curve analysis of biomarker group for diagnosing cervical cancer 1. Sample testing Independent clinical cohort samples were collected: cervical tissues from 30 newly diagnosed patients with cervical cancer and 30 normal cervical tissues obtained from hysterectomy for uterine fibroids were collected (as controls). The expression levels of CIDEC and TMEM82 in all samples were detected using the qPCR method established in Example 2, and the ΔCt values ​​were obtained.

[0053] 2. ROC Curve Plotting and Analysis Using pathological diagnosis as the gold standard, receiver operating characteristic (ROC) curves were plotted using MedCalc software. The diagnostic efficacy for cervical cancer was analyzed using CIDEC single index, TMEM82 single index, and a combination of both (predictive probability of a logistic regression model fit) as test variables.

[0054] 3. Results The results are as follows Figure 7 As shown, the AUC for CIDEC alone was 0.772 (0.646 to 0.871); the AUC for TMEM82 alone was 0.811 (0.689 to 0.900); while the AUC for the combined diagnosis of CIDEC and TMEM82 reached 0.866 (0.753 to 0.940), significantly higher than that of single tests (P<0.05). The optimal cutoff value for the combined diagnosis corresponded to a sensitivity of 86.67% and a specificity of 76.67%, with a P value <0.001. These results fully demonstrate that the biomarker group composed of CIDEC and TMEM82 has extremely high accuracy in the diagnosis of cervical cancer, superior to single biomarkers, and is suitable for clinical cervical cancer screening and auxiliary diagnosis.

[0055] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles 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 biomarker set for cervical cancer screening and diagnosis, characterized in that, The group of markers consists of CIDEC and TMEM82.

2. A primer pair for detecting CIDEC in the biomarker set as described in claim 1, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

3. A primer pair for detecting TMEM82 in the biomarker set as described in claim 1, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO:3 and SEQ ID NO:

4.

4. A reagent kit for cervical cancer screening and diagnosis, characterized in that, The kit contains the CIDEC detection primer pair as described in claim 2 and / or the TMEM82 detection primer pair as described in claim 3.

5. The reagent kit according to claim 4, characterized in that, The kit also contains one or more of the following: reverse transcriptase, DNA polymerase, dNTPs, and reaction buffer.

6. The use of the biomarker group of claim 1 in the preparation of cervical cancer screening or diagnostic reagents.

7. The use of the detection primer pair according to claim 2 or 3 in the preparation of a cervical cancer screening or diagnostic kit.

8. The use of the kit according to claim 4 in cervical cancer screening or diagnosis.

9. A siRNA molecule for inhibiting the proliferation, migration, or invasion of cervical cancer cells, characterized in that, The siRNA molecule targets the CIDEC and / or TMEM82 genes, wherein the siRNA sequence targeting CIDEC is shown in SEQ ID NO: 7 to SEQ ID NO: 10, and the siRNA sequence targeting TMEM82 is shown in SEQ ID NO: 11 to SEQ ID NO:

14.

10. The use of the siRNA molecule of claim 9 in the preparation of a cervical cancer treatment drug.

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