A marker for cervical cancer and its application

Abstract

The invention belongs to the field of medicine, discloses a marker for cervical cancer and an application thereof, wherein the markers related to cervical cancer are ZNF695 and ZNF454, and ROC analysis of the expression amount of the marker is performed, and the ROC curve result shows that AUC=0.813 (0.761-0.959), the sensitivity is 70.0%, the specificity is 86.67%, and the p value is less than 0.001, indicating that the combination of ZNF695 and ZNF454 can be used as a diagnostic marker for cervical cancer, can quickly and accurately diagnose cervical cancer, and is helpful for the diagnosis and treatment of cervical cancer.

Description

Technical Field

[0001] The present invention relates to the medical field, and in particular to a marker for cervical cancer and application thereof. Background Art

[0002] Cervical cancer remains the fourth most common and deadly malignancy among women worldwide. Due to limited vaccination and screening in underdeveloped countries or regions, the proportion of advanced cervical cancer at the time of initial diagnosis is still high, with about 52% of patients finding local infiltration or distant metastasis at the time of initial diagnosis. Patients with advanced cervical cancer often lose the opportunity for surgery and respond poorly to radiotherapy and chemotherapy. According to statistics, the 5-year relative survival rate of patients with distant metastasis is only 17.6%. Different targeted therapies, including anti-angiogenic drugs and immune checkpoint inhibitors, have shown certain prospects in persistent, recurrent and metastatic diseases, but they still have not yet met clinical needs. Therefore, it is still necessary to explore new diagnostic and therapeutic targets for cervical cancer. Therefore, identifying markers such as susceptibility genes or pathogenic genes associated with cervical cancer will help in the diagnosis and treatment of cervical cancer. Summary of the invention

[0003] In order to solve the above problems, the first purpose of the present invention is to provide a cervical cancer detection marker, wherein the markers are ZNF695 and ZNF454.

[0004] The second objective of the present invention is to provide the use of the above markers in the preparation of reagents for detecting cervical cancer.

[0005] The third object of the present invention is to provide a primer pair for detecting the above markers, wherein the sequences of the primer pair are shown in SEQ ID NO: 1 to SEQ ID NO: 4.

[0006] The fourth object of the present invention is to provide the use of the above-mentioned primers in the preparation of a kit for detecting cervical cancer.

[0007] The fifth object of the present invention is to provide a detection kit for cervical cancer, which comprises the above-mentioned primers.

[0008] The sixth object of the present invention is to provide the use of the above-mentioned kit in detecting cervical cancer, wherein the use is for non-diagnostic purposes.

[0009] Compared with the prior art, the present invention has at least the following beneficial effects:

[0010] The present invention belongs to the field of medicine, and discloses a marker for cervical cancer and an application thereof. The markers related to cervical cancer are ZNF695 and ZNF454. ROC analysis of the expression amount of the marker is performed, and the ROC curve result shows that AUC=0.813 (0.761-0.959), the sensitivity is 70.0%, the specificity is 86.67%, and the p value is <0.001, indicating that the combination of ZNF695 and ZNF454 can be used as a diagnostic marker for cervical cancer, can quickly and accurately diagnose cervical cancer, and is helpful for the diagnosis and treatment of cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Gene chip was used to detect the expression of ZNF695 and ZNF454 genes in cervical cancer patients and normal controls.

[0012] Figure 2 QPCR was used to detect the expression of ZNF695 and ZNF454 genes in cervical cancer patients and normal controls.

[0013] Figure 3 ROC curves of ZNF695 and ZNF454 for detecting cervical cancer. DETAILED DESCRIPTION

[0014] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0015] Example 1 Screening for markers associated with cervical cancer

[0016] 1. Sample collection: 10 normal cervical tissue samples and 10 cervical cancer tissue specimens admitted to the hospital from 2021 to 2023 were selected. The sample collection obtained the informed consent of the patients, and all of them signed the informed consent form, which was approved by the hospital ethics committee.

[0017] 2. RNA Extraction

[0018] 1) Take the tissue out of the -80℃ freezer, cut the tissue into pieces the size of rice grains and put them into a centrifuge tube containing 1mL Trizol and RNase-free solution. Add 2-3 grinding beads into the centrifuge tube.

[0019] 2) Fix the tissue with a tissue grinder clamp and grind it vigorously for 30 minutes to fully grind the tissue with grinding beads.

[0020] 3) Add 200 μl of chloroform to every 1 mL of Trizol, shake vigorously to mix, and place at room temperature for 5 minutes to allow natural phase separation, and centrifuge at 12,000 rpm and 4°C for 15 minutes. It will be divided into three layers, and the upper aqueous phase is a colorless and transparent layer, which is RNA.

[0021] 4) Pipette the upper aqueous phase into another clean centrifuge tube, add an equal volume of ice-cold isopropanol, mix by inversion for 15 seconds, incubate at room temperature for 20 minutes, centrifuge at 12000 rpm, 4℃ for 15 minutes, the white precipitate at the bottom of the tube is RNA, discard the supernatant, add 1 mL of pre-cooled 75% ethanol to wash the RNA precipitate, centrifuge at 6000 rpm, 4℃ for 2 minutes, discard the supernatant, and repeat.

[0022] 5) Invert the EP tube and leave it at room temperature for 1-2 minutes to dry the precipitate, and add 50-100 μl RNase-free water to fully dissolve the RNA.

[0023] 6) Take 1 μl RNA solution and use a micro-nucleic acid quantitative analyzer to detect the concentration and purity of RNA. The OD260 / OD280 of good quality RNA should be between 1.8-2.2 and stored in a -80℃ refrigerator until use.

[0024] 7) Sequencing: The RNA samples were sent to BGI for sequencing using Solexa sequencing technology and data analysis was commissioned to calculate the differential expression multiples of the gene between different samples based on the gene expression level.

[0025] 8) The results showed that compared with normal controls, the mRNA level of ZNF695 gene in cervical cancer tissues was significantly downregulated, while the mRNA level of ZNF454 gene was significantly upregulated, and the difference was statistically significant (P<0.05). Figure 1 .

[0026] Example 2 QPCR experiment verifies differentially expressed genes between cervical cancer patients and normal subjects

[0027] 1. Research subjects:

[0028] The screening criteria were the same as those in Example 1, and 10 cervical cancer patients and 10 healthy subjects were obtained again.

[0029] 2. The steps for extracting total RNA from blood are the same as those in Example 1.

[0030] 3. Reverse transcription

[0031] Reverse transcription was performed on lμg of total RNA using reverse transcription buffer to synthesize cDNA. A 25μl reaction system was used, and 1μg of total RNA was taken from each sample as template RNA. The following components were added to the PCR tube: DEPC water, 5× reverse transcription buffer, 10mmol / L dNTP, 0.1mmol / l DTT, 30μmmol / l Oligo dT, 200U / μl M-MLV, and template RNA. Incubate at 42℃ for 1h, 72℃ for 10min, and centrifuge briefly.

[0032] 4. QPCR amplification test

[0033] QPCR amplification primers were designed based on the coding sequences of ZNF695, ZNF454 and GAPDH genes in Genebank and synthesized by Wuhan Qingke Company. The specific primer sequences are shown in Table 1:

[0034] Table 1 Primer sequences

[0035]

[0036]

[0037] Reaction system: SYBR Green polymerase chain reaction system 12.5μL, forward and reverse primers (5μM) 1μL each, template cDNA 2.0μL, ddH2O 8.5μL. Reaction conditions: 95℃10min, (95℃15s, 60℃60s)×45 cycles. SYBRGreen was used as a fluorescent marker, and the PCR reaction was performed on a Light Cycler fluorescent quantitative PCR instrument. GAPDH was used as a reference gene, and the target band was determined by melting curve analysis and electrophoresis. ΔΔ The relative quantification was performed by CT method.

[0038] The results are as follows Figure 2 As shown in the data, the expression level of ZNF695 gene was downregulated in cervical cancer tissue (P<0.05), of which 7 samples showed downregulated expression and 3 samples showed no significant difference; the expression level of ZNF454 gene was upregulated in cervical cancer tissue (P<0.05), of which 7 samples showed downregulated expression and 3 samples showed no significant difference, indicating that markers ZNF695 and ZNF454 can be used as diagnostic markers for cervical cancer.

[0039] Example 3 Sample test results

[0040] Another batch of 30 normal cervical tissue samples and 30 cervical cancer tissue specimens admitted to the hospital from 2021 to 2023 were reselected, and the samples were independent of the samples in Example 1. The samples were collected with the informed consent of the patients, all of whom had signed the informed consent form and were approved by the hospital ethics committee for verification of the diagnostic effect of ZNF695 and ZNF454.

[0041] The expression levels of ZNF695 and ZNF454 were used as detection variables to determine the diagnostic results. Analysis method: MedCalcStatistical Software version was used to analyze the ROC of the indicators. Two-sided test, p < 0.05 was considered statistically significant. The ROC curve results are shown in Table 2 and Figure 3 show.

[0042] Table 2 Diagnostic efficacy results of genes

[0043]

[0044]

[0045] The results showed that the combined use of ZNF695 and ZNF454 had high sensitivity and specificity and could be used in the diagnosis of cervical cancer.

[0046] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a primer pair for detecting ZNF695 and ZNF454 in preparing a kit for detecting cervical cancer, characterized in that: The primer pair sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 4, and the primer pair is used for quantitatively detecting the expression levels of ZNF695 and ZNF454.

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