TERC promoter methylation and application thereof in non-small cell lung cancer diagnosis and prognosis

Abstract

The invention discloses TERC promoter methylation and application thereof in non-small cell lung cancer diagnosis and prognosis, and relates to the technical field of biological medicine. It is found that the methylation level of the TERC promoter is reduced in non-small cell lung cancer tissues, TERC methylation modification is remarkably changed in most of the non-small cell lung cancer tissues, and universal detection of tumors in different stages can be achieved; the low methylation state of a key site in a promoter region is specifically captured, so that cancer tissues and normal lung tissues are effectively distinguished, and a reliable basis is provided for clinical definite diagnosis. The kit for detecting the methylation of the DNA sequence of the TERC promoter can specifically detect the DNA methylation level of the TERC promoter, and can also specifically and quantitatively detect the DNA methylation level of the TERC promoter; the detection result can provide an evaluation method for the condition and individualized medication monitoring of non-small cell lung cancer patients, the early diagnosis of tumors, and the prognosis detection of the patients on drug therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to TERC promoter methylation and its application in the diagnosis and prognosis of non-small cell lung cancer. Background Technology

[0002] Lung cancer is one of the most common malignant tumors in the world and has become the leading cause of cancer death among urban residents in my country. Non-small cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers, and about 75% of patients are diagnosed at an advanced stage, with a very low 5-year survival rate. In-depth research into the molecular mechanisms of immune escape in NSCLC, and the discovery and identification of new biomarkers and therapeutic targets, are of great significance for the prevention, early diagnosis, and treatment of lung cancer.

[0003] Epigenetics regulates cell and tissue-specific gene expression through DNA methylation and histone modifications, participating in numerous life activities such as development, differentiation, function, and metabolism. Unlike gene mutations, the progressive phenotypic changes caused by epigenetic alterations can be reversed under certain conditions. This characteristic has opened up new avenues for the prevention and treatment of some diseases, especially tumors. In particular, abnormal changes in gene expression and function caused by abnormal hypermethylation of CpG islands in the promoter regions of tumor suppressor genes and abnormal histone modifications are not only used as epigenetic markers and targets in the molecular diagnosis, treatment, and prognostic assessment of tumors, but are also considered one of the main factors initiating disease and the formation of treatment resistance.

[0004] Currently, there is a lack of epigenetic markers for non-small cell lung cancer that can be associated with tumor stage (such as T3 / T4, M1 metastasis) and survival. There is an urgent need to develop early diagnosis and precise prognostic tools to improve clinical decision-making. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide TERC methylation markers and their application in the diagnosis and prognosis of non-small cell lung cancer.

[0006] To address the above problems, the present invention proposes the following technical solution:

[0007] This invention provides the use of reagents for detecting TERC promoter methylation modification levels in the preparation of products for the diagnosis, targeted therapy monitoring, and / or prognostic assessment of non-small cell lung cancer.

[0008] Furthermore, the TERC promoter CpG site includes at least one of cg25090302, cg24333189, and cg12615982.

[0009] Furthermore, the methylation level of the TERC promoter was downregulated in patients with non-small cell lung cancer.

[0010] Furthermore, the methylation level of the TERC promoter decreased with the progression of non-small cell lung cancer tumor stages.

[0011] The present invention also provides the use of pharmaceutical compositions that increase the methylation level of the TERC promoter in the preparation of products for the treatment and / or prevention of non-small cell lung cancer.

[0012] Furthermore, the pharmaceutical composition inhibits the progression of non-small cell lung cancer by increasing the methylation modification level of at least one of cg25090302, cg24333189 and cg12615982.

[0013] The present invention also provides a kit for the diagnosis, targeted therapy monitoring and / or prognostic assessment of non-small cell lung cancer, the kit comprising reagents for detecting the methylation modification level of the TERC promoter.

[0014] Furthermore, the kit includes primer pairs that specifically amplify at least one of the CpG sites cg25090302, cg24333189, and cg12615982.

[0015] Compared with the prior art, the technical effects achieved by the present invention include:

[0016] This invention reveals that TERC promoter methylation levels are reduced in non-small cell lung cancer (NSCLC) tissues, and TERC methylation modification shows significant changes in the vast majority of NSCLC tissues, enabling universal detection of tumors at different stages. By specifically capturing the hypomethylation state of key sites in the promoter region, it effectively distinguishes cancerous tissue from normal lung tissue, providing a reliable basis for clinical diagnosis. The kit for detecting TERC promoter DNA sequence methylation can specifically detect TERC promoter DNA methylation levels, and can also perform specific and quantitative detection of TERC promoter DNA methylation levels. The detection results can provide a method for monitoring the condition and individualized medication of NSCLC patients, early tumor diagnosis, and prognostic assessment of drug treatment. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1The TERC promoter methylation level is reduced in non-small cell lung cancer tissues: In the figure, (A) shows the DNA methylation level (β value) of the TERC promoter region and gene CpG sites in NSCLC patients of the TCGA NSCLC cohort. (B) is a box plot of DNA methylation levels at nine CpG sites (cg25090302, cg24333189, cg12615982, cg23036508, cg17527574, cg01389761, cg15599946, cg15494117, and cg20839041) in the TERC promoter and gene body in NSCLC (n=503) and normal lung (n=32) tissues. The p value was determined by Student's test, and p<0.05 indicated a statistically significant difference. (C) shows the correlation between TERC promoter methylation and TERC RNA expression. (D) Pearson correlation analysis of TERC RNA expression at CpG sites cg25090302, cg24333189, and cg12615982 in NSCLC tissues from the TCGA cohort.

[0019] Figure 2 Results showing altered methylation levels of TERC in non-small cell lung cancer: In the figure, (A) verifies the efficiency of 5-Aza treatment and sulfite conversion. Genomic DNA was extracted from 5-Aza-treated A549 and H1299 cells and further treated with (+) or (-) bisulfite to convert unmethylated cytosine to uracil, but not methylated cytosine. A 165 bp region in the TERC promoter was amplified using methylation-specific PCR (MSP) primers designed using MethPrimer.32. (B) is a representative image of the MSP product. DNA was prepared from A549 and H1299 cells treated with 5-Aza or DMSO. After bisulfite conversion, a 165 bp region in the TERC promoter was amplified using primer pairs specific to methylated (M) and unmethylated (U) states. (C) shows the relative RNA levels of TERC in NSCLC cells treated with or without 5-azacytidine (5-Aza). A549 and H1299 cells were treated with 5 mM 5-Aza or DMSO (as controls) for 48 h, RNA was extracted, and RT-qPCR was performed. Data are expressed as mean ± SD (n = 3). (D) Methylation levels of the TERC promoter in 10 pairs of adjacent (P) and tumor (T) tissues from patients in the SYSUCC cohort. (CD) p-values ​​were determined by Student's test, p < 0.05 indicated statistical significance, and ns indicated no statistical significance.

[0020] Figure 3Results showing the correlation between TERC promoter methylation and stage, prognosis, and classification of non-small cell lung cancer (NSCLC): In the figure, (AC) represents Kaplan-Meier survival analysis of NSCLC patients with high and low methylation at cg25090302, cg24333189, and cg12615982 sites. p-values ​​were calculated using the log-rank test. (DF) is a box plot of cg25090302 methylation levels in NSCLC tissues from different stages (D), T stages (E), and distant metastases (F). p-values ​​were calculated using the Student's test; p < 0.05 was considered statistically significant, and "*" represents p < 0.05.

[0021] Figure 4 Results showing that TERC knockdown inhibits TERT nuclear function: In the figure, (A) shows gene expression analysis of A549 cell xenograft tumors in control or stably knocked-down TERC (TERC-sh) cells. Data are presented as single points and mean ± SD (n = 4), and p-values ​​were determined using Student's test. "*" indicates p < 0.05, and "**" indicates p < 0.01. (BC) shows Pearson correlation analysis of TERC RNA (B), TERT RNA (C), and c-Myc, cyclin D1, and VEGF RNA expression in NSCLC samples in the GSE32863 dataset.

[0022] Figure 5 Results showing that TERC promoter methylation levels can regulate TERT nuclear function: In the figure, (AB) shows the Pearson correlation analysis of TERC promoter methylation (A) and TERC DNA (B) levels with c-Myc, cyclin D1, and VEGFRNA expression in NSCLC samples in the TCGA dataset.

[0023] Figure 6 The protein expression results of cyclin D1: The level of cyclin D1 in A549 cells was detected by Western blotting. Histone H3 was used as an internal control protein. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0025] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0026] It should also be understood that the terminology used in this specification of embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. As used in this specification of embodiments of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0027] This invention relates to 374 lung cancer tissue samples, all obtained from the specimen bank of Sun Yat-sen University Cancer Center. These specimens were collected between 2009 and 2015, and this study was approved by the Ethics Review Committee of Sun Yat-sen University Cancer Center (Ethics No.: GZR2019-077). RNA was extracted from the obtained tissue samples using a kit, and tissue RNA samples were prepared using a DNA / RNA co-extraction kit (TIANGEN DP422). Detection was performed by RT-qPCR. The tissue microarrays of 129 lung cancer cases involved in this study were also obtained from the specimen bank of Sun Yat-sen University Cancer Center, collected between 2008 and 2010. This study was approved by the Ethics Review Committee of Sun Yat-sen University Cancer Center (Ethics No.: SL-B2023-467-01). The obtained microarrays were subjected to immunohistochemical staining. All microarray samples were confirmed as tumor specimens and adjacent normal specimens by pathologists, and the immunohistochemical results were scored under the guidance of pathologists. The staging and classification of lung cancer patients were determined according to the 8th edition of the American Joint Committee on Cancer (AJCC) staging manual. All experiments complied with the principles of the Helsinki WMA Declaration and the Belmont Report of the Department of Health and Human Services.

[0028] The animal experiments in this embodiment of the invention were conducted by the Laboratory Animal Ethics Committee of Sun Yat-sen University Cancer Center, which has approved the animal experiments used in this study (No.: L102012019005N; L102012023050D). Four-week-old female C57BL / 6 mice and nude mice were purchased from the Guangdong Provincial Medical Laboratory Animal Center and subsequently housed in an SPF-grade environment at the Animal Experiment Center of Sun Yat-sen University Cancer Center, 7th Floor, Building 39, North Campus, Sun Yat-sen University. Nude mice were subcutaneously injected with 1×10 6 WT and TERCknockdown A549 cells were sacrificed on day 28, and the tumors were measured, photographed, and then subjected to immunohistochemical staining. C57BL / 6 mice were subcutaneously injected with 5×10⁶ cells. 5LLC cells with WT, TERC knockdown, SENP3 knockdown, or TERC knockdown-SENP3 overexpression were sacrificed on day 21. After sacrifice, the tumor was dissected and measured. Volume was calculated as: Volume = π / 6 × Tumor long diameter × Tumor width. 2 Then, the cells were dissociated to form a single-cell suspension for staining and flow cytometry analysis, and the remaining tissue was subjected to immunohistochemical staining.

[0029] The cell culture method involved in this invention includes: cell lines A549, H322, H460, H1299, and LLC were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; HEK293T and Jurkat cells were purchased from the American Type Culture Collection (ATCC); and some human peripheral blood T cells were provided by Professor Zeng Musheng's team at Sun Yat-sen University Cancer Center. H322, H460, H1299, and Jurkat cells were cultured in RPMI-1640 medium; A549, LLC, and HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM); and T cells were cultured in ImmunoCult-XF T Cell Exp Medium. The medium contained 10% fetal bovine serum (FBS) and 100 U / mL of penicillin-streptomycin antibiotics. The ImmunoCult-XF T Cell Exp Medium also required the addition of IL-2 cytokine. All cells were cultured at 37°C in a humidified incubator with 5% CO2.

[0030] The cell resuscitation method of the present invention includes: taking the cells out of a liquid nitrogen tank or a -80°C freezer, thawing them in a 37°C constant temperature water bath for 2 minutes, then centrifuging them at 1000 rpm for 5 minutes, discarding the supernatant in the cryopreservation tube, adding the corresponding complete culture medium to resuspend them, then adding the cell suspension to a culture flask or culture dish, adding complete culture medium to 4 mL, and placing it in a 37°C, 5% CO2 constant temperature incubator. The medium is changed the next day.

[0031] The cell digestion method of this invention includes: when the cells reach about 90% confluence, discard the cell culture medium, wash once with 1 mL of autoclaved PBS, discard the PBS, add 1 mL of trypsin, shake the culture dish or flask, and place it in an incubator at 37°C and 5% CO2 for 1-3 minutes to digest. After removing it, add complete culture medium at a ratio of 1:3 to stop the digestion, pipette the mixture evenly, and aspirate it into a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 minutes. After centrifugation, discard the supernatant, resuspend the cells in complete culture medium, and perform plating and subsequent experiments as needed.

[0032] The cell cryopreservation method of the present invention includes: adding a prepared cryopreservation solution to the digested and centrifuged cell pellet. The cryopreservation solution is prepared with 90% serum + 10% DMSO or directly using serum-free cryopreservation solution of purchased NCM. The cryopreservation tubes are placed in a gradient cooling box and placed in a -80°C freezer for gradient cooling. After 3-5 days, they are transferred to a liquid nitrogen tank for storage.

[0033] Unless otherwise specified, the experimental methods described in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] This invention examined TERC expression in a cohort from Sun Yat-sen University Cancer Center (SYSUCC) containing 20 pairs of non-small cell lung cancer (NSCLC) and adjacent tissue samples, along with 354 additional tumor tissue samples. Pairwise analysis revealed significantly elevated TERC levels in all (20 / 20) tumor samples compared to adjacent normal tissues, while only half (10 / 20) of the tumor samples showed higher TERT mRNA levels than their matched adjacent normal tissue counterparts. Overall, TERC RNA levels were significantly elevated in tumor tissues, a finding not observed in TERT mRNA expression in NSCLC and adjacent normal tissue samples. The findings from the SYSUCC specimen bank cohort were validated on the public dataset GEO:GSE32863, showing elevated TERC levels in 26 out of 45 tumor pairs, but elevated TERT mRNA levels in less than half (20 / 45) of the paired samples.

[0035] To elucidate the clinical relevance of TERC expression to NSCLC progression, this invention further analyzed data from a cohort at Sun Yat-sen University Cancer Center. This invention found that elevated TERC levels were significantly associated with cancer stage (I vs III+IV, p = 0.0420), and also with T stage (T1 vs T3+4, p = 0.0506; T2 vs T3+4, p = 0.0323) and distant metastasis (M0 vs M1, p = 0.0346). Kaplan-Meier survival analysis showed that higher TERC levels were closely associated with shorter overall survival (p = 9.86 x 10⁻⁵). These results indicate that TERC expression is upregulated in primary non-small cell lung cancer and is associated with disease progression and poor prognosis.

[0036] To explore the regulatory mechanism of TERC expression in non-small cell lung cancer, this invention analyzes DNA methylation data from the Human Methylation 450 BeadChip database from the TCGA-NSCLC database. Nine probes targeting the TERC promoter region and gene body CpG sites (cg25090302, cg24333189, cg12615982, cg23036508, cg17527574, cg01389761, cg15599946, cg15494117, and cg20839041) were used. Figure 1 A) Methylation detection was performed. Results showed that cg25090302, cg24333189, and cg12615982 in the TERC promoter region exhibited significant hypermethylation under normal conditions, but their methylation levels were significantly reduced in NSCLC tissues. Figure 1 B). Furthermore, DNA methylation at these three CpG sites was significantly negatively correlated with TERC RNA levels (B). Figure 1 The results (C and 1D) indicate that hypomethylation of the TERC promoter may lead to increased TERC expression in NSCLC.

[0037] To confirm this, A549 and H1299 cells were treated with the DNA methylation inhibitor 5-azacytidine (5-Aza). Methylation-specific PCR (MSP) showed that TERC promoter methylation levels decreased after 5-Aza treatment. Figure 2 A and 2B), RT-qPCR confirmed increased TERC expression ( Figure 2C). This invention also compared the methylation levels of the TERC promoter in paired NSCLC and adjacent tissues, and found that TERC promoter methylation showed a decreasing trend in tumor tissues.

[0038] Furthermore, this invention investigated whether TERC promoter methylation might be a prognostic factor for non-small cell lung cancer. The results showed that high methylation of cg25090302, cg24333189, and cg12615982 was associated with longer overall survival. Figure 3 A, 3B, 3C). Furthermore, cg25090302 methylation level was correlated with tumor stage (I vs III / IV, p = 0.0315); Figure 3 D), T stages (T1 vs T2, p = 0.0345; T1 vs T3+4, p = 0.0469); Figure 3 E) and distant migration (M0 vs M1, p = 0.0453; Figure 3 F) showed a negative correlation.

[0039] Alterations in DNA copy number can also lead to dysregulation of RNA expression. Analysis of data from the Sun Yat-sen University Cancer Center and the TCGA cohort showed a positive correlation between TERC gene copy number and RNA levels, but no clear association was found between TERC DNA abundance and overall survival in NSCLC patients. Therefore, the data from the embodiments of this invention suggest that hypomethylation of the TERC promoter is the main reason for increased TERC expression in NSCLC, indicating it as an indicator of disease progression and poor prognosis.

[0040] This invention demonstrates through in vitro experiments that knocking down TERC (TERC-sh) inhibits the growth of non-small cell lung cancer cells. Simultaneously, data from A549 xenograft tumor RNA extracts also indicate that knocking down TERC inhibits the growth of non-small cell lung cancer cells. Figure 4 A). Furthermore, gene expression correlation analysis of NSCLC patient samples in the GEO:GSE32863 dataset showed that TERC RNA levels were positively correlated with c-Myc, Cyclin D1, and VEGF mRNA levels. Figure 4 B). Similarly, TERT mRNA expression was positively correlated with c-Myc, Cyclin D1, and VEGF. Figure 4 C). Furthermore, in the TCGA cohort, DNA methylation of the TERC promoter was negatively correlated with the mRNA expression of c-Myc, cyclin D1, and VEGF. Figure 5 A). However, there was no correlation between TERC gene copy number and c-Myc, cyclin D1, and VEGF mRNA levels. Figure 5B). Furthermore, immunoblotting experiments showed that cyclin D1 protein levels were reduced in the absence of TERC. Figure 6 This is consistent with G1 phase cell cycle arrest. These data indicate that TERC promoter hypomethylation leads to increased TERC expression, enhancing TERT function in the nucleus of NSCLC cells.

[0041] In summary, this invention discovered that hypomethylation of the TERC promoter is associated with elevated TERC levels, and elevated TERC levels are associated with disease progression and poor prognosis. This invention collected tissue samples from 374 NSCLC patients and used RT-qPCR to measure the abundance of different RNA species. First, it was found that TERC levels were consistently higher in NSCLC tissues than in matched normal lung tissues, and the increase in TERC expression in NSCLC tissues was significantly more pronounced than the increase in TERT. Furthermore, elevated TERC expression was associated with disease progression and shorter overall survival. Further analysis of the methylation status of the TERC gene and its flanking DNA showed that the CpG sites cg25090302, cg24333189, and cg12615982 in the TERC promoter were normally hypermethylated. However, in NSCLC tissues, the methylation levels at these sites were significantly reduced and negatively correlated with TERC gene expression, consistent with current epigenetic regulatory models that DNA methylation in this region typically inhibits gene transcription. Furthermore, we observed that decreased methylation of cg25090302, cg24333189, and cg12615982 was associated with shorter overall survival, and hypomethylation of cg25090302 was associated with advanced cancer stage, T type, and distant metastasis. This suggests that TERC promoter methylation may play a dominant role in the regulation of TERC expression. The results of this invention indicate that TERC DNA methylation expression may serve as a biomarker for the diagnosis and prognosis of non-small cell lung cancer.

[0042] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0043] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Application of reagents for detecting TERC promoter methylation modification levels in the preparation of products for the diagnosis, targeted therapy monitoring and / or prognostic assessment of non-small cell lung cancer.

2. The application as described in claim 1, characterized in that, The TERC promoter CpG site includes at least one of cg25090302, cg24333189, and cg12615982.

3. The application as described in claim 1 or 2, characterized in that, TERC promoter methylation levels are downregulated in patients with non-small cell lung cancer.

4. The application as described in claim 1 or 2, characterized in that, The methylation level of the TERC promoter decreased with the progression of non-small cell lung cancer tumor stage.

5. Use of pharmaceutical compositions that increase the methylation level of the TERC promoter in the preparation of products for the treatment and / or prevention of non-small cell lung cancer.

6. The pharmaceutical composition of claim 5, characterized in that the pharmaceutical composition inhibits the progression of non-small cell lung cancer by increasing the methylation modification level of at least one of cg25090302, cg24333189 and cg12615982.

7. A kit for the diagnosis, targeted therapy monitoring, and / or prognostic assessment of non-small cell lung cancer, characterized in that, The kit includes reagents for detecting the methylation modification level of the TERC promoter.

8. The kit of claim 7, characterized in that the kit comprises a primer pair for specifically amplifying at least one of the CpG sites cg25090302, cg24333189, and cg12615982.

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