Application of TLE4 expression level detection reagent in preparation of melanoma diagnostic kit

Abstract

The invention discloses application of a TLE4 expression level detection reagent in preparation of a melanoma diagnostic kit. Researches find that TLE4 is abnormally low in expression in a malignant melanoma patient and is closely related to prognosis of the patient; rOC curve analysis shows that the area under the curve (AUC) is 0.861, and the 95% confidence interval is 0.833-0.887, which shows that the TLE4 gene has strong ability in distinguishing a tumor sample from a normal sample; therefore, the TLE4 gene can be used as a potential biomarker of the malignant melanoma, and a reference value is provided for clinical diagnosis of the malignant melanoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a detection reagent for TLE4 expression level in the preparation of a melanoma diagnosis kit. Background Art

[0002] Malignant melanoma (also known as melanoma, malignant black) is a malignant tumor that occurs in different parts or tissues such as the skin, mucous membranes, uveal tract, and pia mater. It is manifested as pigmented skin lesions that undergo significant changes over months or years. Although its incidence is low, it is highly malignant, metastasizes early, and has a high mortality rate, so early diagnosis and early treatment are very important.

[0003] On the one hand, in order to achieve early diagnosis of malignant melanoma, Chinese invention patent applications with publication numbers CN113637762A, CN113755601A, CN115992231A, CN117904302A, CN112725451A, CN113637761A, etc. disclose a series of biomarkers that can assist in the early diagnosis of malignant melanoma. However, there is currently no report that the TLE4 gene can be used as a biomarker for malignant melanoma.

[0004] On the other hand, for the treatment of malignant melanoma, the current treatment methods mainly include surgical treatment, immunotherapy, targeted therapy and chemotherapy. For patients in the middle and late stages, chemotherapy is still an important part of the late-line treatment of malignant melanoma. Among them, albumin paclitaxel is an anticancer drug that uses albumin as a nanocarrier to carry paclitaxel, which can improve the therapeutic effect and reduce allergic reactions. There is evidence that albumin paclitaxel shows effectiveness in patients with metastatic melanoma and shows significant advantages in improving progression-free survival and overall survival in patients with metastatic melanoma. However, the development of drug resistance is the main problem limiting the effectiveness of chemotherapy.

[0005] Therefore, enhancing the sensitivity of chemotherapeutic drugs is of great significance for improving the treatment effect of advanced malignant melanoma. Summary of the invention

[0006] The purpose of the present invention is to provide a detection reagent for TLE4 expression level for use in the preparation of a melanoma diagnostic kit, thereby providing a new method for early diagnosis of malignant melanoma.

[0007] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] Application of a TLE4 expression level detection reagent in the preparation of a melanoma diagnostic kit.

[0009] The present invention found that TLE4 was abnormally low expressed in patients with malignant melanoma and was closely related to the patient's prognosis; ROC curve analysis showed that the area under the curve (AUC) was 0.861 and the 95% confidence interval was 0.833-0.887, showing that the TLE4 gene had a strong ability to distinguish tumor samples from normal samples; indicating that the TLE4 gene can be used as a potential biomarker for malignant melanoma and provide reference value for the clinical diagnosis of malignant melanoma.

[0010] Based on this, the present invention also provides the use of a detection reagent for TLE4 expression level in the preparation of a melanoma prognosis assessment kit. Preferably, the detection reagent for TLE4 expression level comprises at least one of the following reagents:

[0011] An oligonucleotide probe that specifically recognizes the TLE4 gene;

[0012] Primers that specifically amplify the TLE4 gene;

[0013] Alternatively, an antibody that specifically binds to the TLE4 protein.

[0014] As further preferred, the primers for specifically amplifying the TLE4 gene include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID No.1 or 5, and the nucleotide sequence of the reverse primer is shown as SEQ ID No.2 or 6.

[0015] In addition, the present invention has found through experiments of expressing and interfering with the TLE4 gene that up-regulating the expression of TLE4 can inhibit tumor cell proliferation and migration and enhance the sensitivity of melanoma cells to albumin-paclitaxel, indicating that the TLE4 gene can be used as a target for melanoma treatment and can assist albumin-paclitaxel in the treatment of tumors such as malignant melanoma, which is beneficial to improving the tumor efficacy and has important significance for the treatment of tumors.

[0016] Therefore, the present invention also provides the use of a TLE4 expression promoter in the preparation of a melanoma therapeutic drug, and the use of a TLE4 expression promoter in the preparation of a reagent for enhancing the sensitivity of melanoma to albumin paclitaxel chemotherapy; at the same time, the present invention also provides the use of a TLE4 expression promoter combined with albumin paclitaxel in the preparation of a melanoma therapeutic drug.

[0017] Preferably, the TLE4 expression promoter comprises at least one of the following agents:

[0018] An agent that promotes the transcription of the TLE4 gene into mRNA;

[0019] Reagents that promote translation of TLE4 mRNA into protein;

[0020] An agent for increasing the activity or content of TLE4 protein. Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) The present invention found that TLE4 was abnormally low expressed in patients with malignant melanoma and was closely related to the patient's prognosis. ROC curve analysis showed that the area under the curve (AUC) was 0.861 and the 95% confidence interval was 0.833-0.887, indicating that the TLE4 gene had a strong ability to distinguish tumor samples from normal samples. This indicated that the TLE4 gene could be used as a potential biomarker for malignant melanoma and provide a reference value for the clinical diagnosis and prognosis evaluation of malignant melanoma.

[0022] (2) The present invention has found through experiments on expressing and interfering with the TLE4 gene that upregulating the expression of TLE4 can inhibit tumor cell proliferation and migration and enhance the sensitivity of melanoma cells to nab-paclitaxel, indicating that the TLE4 gene can be used as a target for melanoma treatment and can also assist nab-paclitaxel in the treatment of tumors such as malignant melanoma, which is beneficial to improving the tumor efficacy and has important significance for the treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of the analysis of the expression levels of TLE4 gene in melanoma tissues and normal tissues;

[0024] Among them, Normal (n=557) indicates normal tissue (557 samples), Tumor (n=469) indicates tumor tissue (469 samples), and TLE4 expression indicates the expression level of TLE4 gene, the same below;

[0025] Figure 2 is the ROC curve used to evaluate the diagnostic efficacy of TLE4 for malignant melanoma;

[0026] Wherein, Specificity means specificity, Sensitivity means sensitivity, AUC means area under the curve (AUC), and 95% CI means 95% confidence interval;

[0027] Figure 3 To analyze the correlation between TLE4 expression and overall survival of patients;

[0028] Among them, Time (years) indicates time (years), TLE4 Levels indicates the expression level of TLE4 gene (High indicates high, low indicates low), and Overall Survival indicates overall survival, the same below;

[0029] Figure 4To analyze the correlation between TLE4 expression and the disease-specific survival of patients;

[0030] Among them, Disease-Specific Survival means disease-specific survival;

[0031] Figure 5 The correlation analysis between TLE4 expression and the progression-free interval of patients' prognosis was performed;

[0032] Among them, Progression-Free Interval means progression-free interval;

[0033] Figure 6 Verification of TLE4 expression levels in A375 cells after TLE4 overexpression and interference experiments;

[0034] Among them, OE-TLE4 is the TLE4 overexpression cell group, OE-NC represents the overexpression test control cell group, Si-TLE4 represents the TLE4 interference cell group, Si-NC represents the interference test control cell group, and Relative TLE4 expression represents the relative expression level of the TLE4 gene, the same below;

[0035] Figure 7 Verification of TLE4 expression levels in B16F10 cells after TLE4 overexpression and interference experiments;

[0036] Figure 8 The effect of TLE4 overexpression on the proliferation of A375 cells and B16F10 cells;

[0037] Among them, Relative Proliferation means relative proliferation, the same below;

[0038] Fig. 9 The effect of TLE4 knockdown on the proliferation of A375 and B16F10 cells;

[0039] Fig.10 The effect of TLE4 overexpression on the migration of A375 cells and B16F10 cells;

[0040] Among them, Migration area means migration area, the same below;

[0041] Fig.11 The effect of TLE4 knockdown on the proliferation of A375 and B16F10 cells;

[0042] Fig.12 The effect of TLE4 overexpression on the sensitivity of malignant melanoma to nab-paclitaxel;

[0043] Among them, Cell Viability means cell viability, the same below;

[0044] Fig.13 Effect of TLE4 knockdown on nab-paclitaxel sensitivity in malignant melanoma. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the present invention more clear, the technical solution of the present invention is further described in detail below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0046] Example 1 Analysis of the difference in expression and diagnostic efficacy of TLE4 in malignant melanoma tissue and normal tissue

[0047] 1. Analysis of differential expression of TLE4 in malignant melanoma tissues and normal tissues

[0048] The malignant melanoma transcriptome data (469 samples) were obtained through the Cancer Genome Atlas (TCGA), and the gene expression data of normal tissues (557 samples) were obtained using the Genotype-Tissue Expression (GTEx) project of UCSC Xena (https: / / xenabrowser.net / datapages / ). The expression levels of TLE4 in tumor tissues and normal tissues were compared using the t-test, and the results were visualized using the 'ggplot2' R package. The results are shown in Figure 2. Figure 1 shown.

[0049] Depend on Figure 1 It can be seen that compared with normal tissues, the expression level of TLE4 in melanoma tissues was significantly reduced.

[0050] 2. Analysis of the diagnostic efficacy of TLE4 for malignant melanoma

[0051] The diagnostic performance was evaluated by constructing a receiver operating characteristic (ROC) curve to analyze the diagnostic efficacy of TLE4 expression levels in distinguishing tumors from normal tissues. Figure 2 shown.

[0052] Depend on Figure 2 From the ROC curve, it can be seen that the area under the curve (AUC) is 0.861 and the 95% confidence interval is 0.833-0.887, showing that the TLE4 gene has a strong ability to distinguish melanoma tissues from normal samples.

[0053] The above results indicate that the TLE4 gene can serve as a potential biomarker for malignant melanoma and provide reference value for clinical diagnosis.

[0054] Example 2 Analysis of the correlation between TLE4 expression level and patient prognosis

[0055] To investigate the correlation between TLE4 expression and the overall survival (OS), disease-specific survival (DSS) and progression-free interval (PFI) of patients, patients were divided into TLE4 high expression group and TLE4 low expression group according to the median value. Kaplan-Meier analysis and log-rank test were used to detect the survival rate. Survival curves were created using the 'survivor' and 'survminer' R packages. The results are shown in Figure 2. Figure 3 , Figure 4 and Figure 5 shown.

[0056] Depend on Figures 3 to 5 It can be seen that high expression of TLE4 was significantly correlated with the extension of OS, DSS and PFI; this suggests that, as a potential biomarker, high expression of TLE4 may have a positive effect on the survival outcomes of patients and indicate a better prognosis.

[0057] Example 3 Effect of abnormal TLE4 expression on malignant biological behavior of malignant melanoma cells

[0058] The nucleotide sequence of the TLE4 gene involved in this example is NCBI Reference Sequence: NM_007005.6, and the amino acid sequence encoding the TLE4 protein is NCBI Reference Sequence: NP_008936.2, both of which are human sequences.

[0059] 1. Construction of TLE4 overexpression plasmid (OE-TLE4)

[0060] First, the TLE4 gene sequence (NCBI Reference Sequence: NM_007005.6) was obtained from the National Center for Biotechnology Information (NCBI) of the United States, and PCR amplification primers were designed and synthesized according to the gene sequence; the TLE4 gene was amplified by PCR, and the XbaI restriction enzyme site was added for cloning.

[0061] The specific information of PCR amplification primers is as follows:

[0062] TLE4 PCR forward primer:

[0063] 5'-TGGCGGCCGCTCGAGTCTAGAATGATTCGCGACCTGAGCAA-3'

[0064] (SEQ ID NO: 1);

[0065] TLE4 PCR reverse primer:

[0066] 5'-GGTTTTAAACGGGGGCCCTCTAGATTAATAAATAACTTCATAAACTGTGGCC-3' (SEQ ID NO: 2).

[0067] Then, the target fragment was connected to the vector pcDNA3.1(+) through seamless cloning technology, and the recombinant plasmid was transformed into Escherichia coli. After antibiotic screening and PCR confirmation of positive clones, plasmid DNA was extracted and sequenced for verification. Finally, the verified plasmid can be frozen for subsequent experimental use.

[0068] 2. Synthesize small interfering RNA of TLE4 (Si-TLE4)

[0069] The sequence of Si-TLE4 is:

[0070] Sense:GUCCCAUCUUCCAAUUAATT(SEQ ID NO: 3);

[0071] Antisense: UUUAAUUGGAAGAUGGGACTT (SEQ ID NO: 4);

[0072] The synthesis was commissioned to Suzhou Genema Co., Ltd.

[0073] 3. Liposome transfection

[0074] First, take 2.5×10 5 Cells (A375, B16F10) were inoculated in a 6-well plate at a density of 100 μg / well per well and cultured in a 5% CO2, 37°C incubator. When the cell density in the well plate reached about 70%, the culture medium was discarded, 1 mL of MEM was added (starvation treatment), and the plate was returned to the 37°C constant temperature incubator.

[0075] Then, prepare working solution A and working solution B:

[0076] Working solution A: OE-NC / OE-TLE4 / Si-NC / Si-TLE4 5μL (20μM) + 245μL MEM;

[0077] Working solution B: 5 μL Lipo2000 solution + 245 μL MEM;

[0078] After preparation, incubate at room temperature for 5 minutes, then mix working solution A and working solution B, blow gently to mix, and continue incubation at room temperature for 20 minutes; add the mixed working solution evenly to each well, place in a 37°C constant temperature incubator for transfection.

[0079] 4. RNA Extraction

[0080] Take the cells after transfection for 48 hours, discard the old cell culture medium, gently wash the cells with PBS 1-2 times, digest the cells with trypsin, and centrifuge at 3000rpm and 4℃ for 5min. Discard the supernatant, add 600μL TRIzol, vortex thoroughly, and let stand on ice for 10min. Add 120μL chloroform, vortex for 20s, let stand at room temperature for 20min, and then centrifuge at 4℃, 12000rpm for 15min. Take the upper aqueous phase to a new EP tube, add isopropanol with the same volume as the absorbed aqueous phase, mix it upside down, let stand at 4℃ for more than 30min, and centrifuge at 4℃, 12000rpm for 15min. Discard the supernatant, add 1ml75% ethanol (DEPC water preparation), turn it upside down and tap the tube wall to allow the ethanol to fully infiltrate the tube wall, tube cover and precipitate, and centrifuge at 4℃, 7500rpm for 5min. Discard the supernatant and let it stand to dry. Add 20μL of DEPC water to dissolve RNA for use.

[0081] 5. cDNA Synthesis

[0082] The sample RNA was reverse transcribed into cDNA using HiScript IIQ RT SuperMix for qPCR. Reverse transcription reaction substrate: 4 μL 5×HiScript II qRT SuperMix reverse transcriptase, 1 μL (500 ng) RNA and 5 μL DEPC water; reaction conditions: pre-denaturation at 50°C for 15 min, denaturation at 85°C for 5 s.

[0083] 6. Perform fluorescent quantitative PCR on cDNA

[0084] During the PCR amplification process, SYBR-Green was used to detect the amplification of TLE4 in real time. 15 μL ChamQ SYBR qPCR Master Mix, 0.8 μL each of qPCR forward and reverse primers (10 μM), 1.8 μL template cDNA and 14 μL deionized water were added to the PCR reaction tube in sequence. The reaction conditions were: 95°C pre-denaturation for 30 s, 95°C denaturation for 5 s, 60°C annealing and extension for 30 s, for a total of 40 cycles.

[0085] The specific information of qPCR amplification primers is as follows:

[0086] TLE4 qPCR forward primer:

[0087] 5'-AGAAGTCGAGCCCTAACTGC-3' (SEQ ID NO: 5);

[0088] TLE4 qPCR reverse primer:

[0089] 5'-TGCGGTCCTTCAGGTAGTCA-3' (SEQ ID NO: 6).

[0090] qPCR results Figure 6 and Figure 7 shown.

[0091] Depend on Figure 6 and Figure 7 It can be seen that compared with the OE-NC group and the Si-NC group, the TLE4 expression level of the cells in the OE-TLE4 group was significantly increased, which verified the effectiveness of the overexpression plasmid we constructed. Compared with the OE-NC group and the Si-NC group, the TLE4 expression level of the cells in the Si-TLE4 group was significantly decreased, indicating that the interference experiment achieved the expected effect.

[0092] 7. Effect of abnormal TLE4 expression on proliferation of malignant melanoma cells

[0093] The treated cells were divided into 1×10 4 Cells / well, 200μL volume were inoculated in a 96-well plate, 6 replicate wells were inoculated in each group, and a zero well was set. The cells inoculated in the 96-well plate were cultured in a 37°C incubator with 5% CO2. Four time points (0, 24, 48, 72h) were selected, 100μL of CCK-8 working solution (CCK-8 reagent: DMEM=1:9) was added in a dark incubator with 5% CO2, incubated for 2h, and the absorbance at 450nm was measured. The OD values ​​detected at different time points were plotted to analyze the proliferation of cells in each group. The results are shown in the figure. Figure 8 and Fig. 9 shown.

[0094] Depend on Figure 8 and Fig. 9 It can be seen that compared with the OE-NC group, the proliferation of cells in the OE-TLE4 group was significantly inhibited; compared with the Si-NC group, the proliferation of cells in the Si-TLE4 group was significantly enhanced; these results were statistically significant (*P<0.05, **P<0.01, ***P<0.001).

[0095] 8. Effect of abnormal TLE4 expression on the migration of malignant melanoma cells

[0096] Before inoculating cells, use a marker pen to draw a marking line on the back of the 6-well plate to facilitate positioning in the same field of view when taking pictures at different time points. The cells were digested with trypsin, and a single-cell suspension was prepared. The cells were inoculated into a 6-well plate and cultured in a 5% CO2, 37°C incubator. After the cells grew to completely cover the bottom of the well plate, liposome transfection was performed and a sterile 200μl gun tip was used to make cell scratches perpendicular to the marking line to ensure that the width of each scratch was uniform. In order to remove the interference of cell debris, 1ml of 1×PBS buffer was added to remove the cell debris, and then 2ml of DMEM culture medium containing 2% serum was added and cultured in a 5% CO2, 37°C incubator. The culture plate was placed in the incubator for continued culture. The culture plate was removed at 0h and 48h, 6 points were selected from each well, and the same magnification (4*10) was selected. Photos were recorded using an inverted microscope. The scratch area was calculated using ImageJ software. The experimental results are shown in the figure. Fig.10 and Fig.11 shown.

[0097] Depend on Fig.10 and Fig.11 It can be seen that overexpression of TLE4 significantly inhibited the migration of A375 and B16F10 cells, while knockdown of TLE4 promoted the migration of cells, and these results were statistically significant (*P<0.05, **P<0.01, ***P<0.001).

[0098] Example 4 Effect of TLE4 on the sensitivity of malignant melanoma to nab-paclitaxel

[0099] The CCK-8 cell activity detection kit was used to detect the cell activity after treatment with different concentrations of albumin-paclitaxel to evaluate the drug sensitivity of the cells.

[0100] After cell counting, cells were divided into 2 × 10 4 Cells / well, 200 μL volume were inoculated in a 96-well plate, 6 duplicate wells were set for each group, and a zero well was set, and the cells were placed in a 5% CO2, 37°C incubator for 24 hours. After the cells adhered to the wall, different concentrations of albumin-bound paclitaxel dilutions (0 μg / ml, 0.001 μg / ml, 0.01 μg / ml, 0.1 μg / ml, 1 μg / ml, 10 μg / ml, 100 μg / ml) were added, and each concentration was repeated 3 wells, and the culture was continued for 48 hours. The cells were taken out of the incubator, and the medium containing albumin-bound paclitaxel was removed with a pipette. Then, 100 μl of DMEM medium containing 10% CCK-8 was added to each well, and the cells were returned to the cell incubator for further incubation for 1 hour. The multifunctional microplate reader was used for on-machine detection, and the wavelength of the microplate reader was set to 450 nm to detect the optical density value of each well.

[0101] The cell viability calculation formula is: cell viability (%) = (OD treatment group - OD zero adjustment group) / (OD control group - OD zero adjustment group) × 100%.

[0102] The analysis results are as follows Fig.12 and Fig.13 shown.

[0103] Depend on Fig.12 and Fig.13 It can be seen that when the expression level of TLE4 increased, the sensitivity of A375 and B16F10 cells to nab-paclitaxel was significantly enhanced; on the contrary, when the expression level of TLE4 decreased, the sensitivity of cells to the drug decreased significantly, and these changes were statistically significant (*P<0.05, **P<0.01, ***P<0.001).

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. As long as the technical effects of the present invention are achieved by the same or equivalent means, they should belong to the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of TLE4 expression level detection reagent in the preparation of melanoma diagnostic kit.

2. The use according to claim 1, It is characterized in that The detection reagent of TLE4 expression level comprises at least one of the following reagents: An oligonucleotide probe that specifically recognizes the TLE4 gene; Primers that specifically amplify the TLE4 gene; Alternatively, an antibody that specifically binds to the TLE4 protein.

3. The use according to claim 2, It is characterized in that The primers for specifically amplifying the TLE4 gene include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID No.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.

2.

4. The use according to claim 2, It is characterized in that The primers for specifically amplifying the TLE4 gene include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID No.5, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.

6.

5. Application of TLE4 expression level detection reagent in the preparation of melanoma prognosis evaluation kit.

6. The use according to claim 5, It is characterized in that The detection reagent of TLE4 expression level comprises at least one of the following reagents: An oligonucleotide probe that specifically recognizes the TLE4 gene; Primers that specifically amplify the TLE4 gene; Alternatively, an antibody that specifically binds to the TLE4 protein.

7. Application of TLE4 expression promoter in the preparation of melanoma therapeutic drugs.

8. The use according to claim 7, It is characterized in that The TLE4 expression promoter comprises at least one of the following reagents: An agent that promotes the transcription of the TLE4 gene into mRNA; Reagents that promote translation of TLE4 mRNA into protein; An agent that increases the activity or content of TLE4 protein.

9. Application of TLE4 expression promoter in the preparation of reagents for enhancing the sensitivity of melanoma to albumin-paclitaxel chemotherapy.

10. Application of TLE4 expression promoter combined with albumin paclitaxel in the preparation of melanoma treatment drugs.

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