CN116350755A

CN116350755A - Application of m5C methylation regulatory factors DNMT1 and NSun2 in tumor

Info

Publication number: CN116350755A
Application number: CN202211655495.2A
Authority: CN
Inventors: 唐颢; 邢珺月; 刘词航; 邵东兴; 程晓雷
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-12-22
Filing date: 2022-12-22
Publication date: 2023-06-30

Abstract

The invention provides an application of m5C methylation regulating factors DNMT1 and NSun2 in tumors, wherein the regulating factors comprise DNMT1 and/or NSun2. The invention discovers that DNMT1 mediated methylation of a gene promoter region and NSun2 mediated methylation of mRNA synergistically regulate osteosarcoma cell apoptosis; DNMT1 inhibits NSun2 expression by methylating the promoter region of NSun 2; DNMT1 and NSun2 regulate the anti-apoptotic genes AXL, NOTCH2 and YAP1 by DNA methylation and mRNA methylation, respectively. When exposed to cisplatin or doxorubicin, DNMT1 is elevated and NSun2 is inhibited, promoter methylation of these anti-apoptotic genes is enhanced and mRNA methylation is reduced, greatly reducing expression of these anti-apoptotic genes, and thus causing osteosarcoma cells to undergo apoptosis.

Description

Application of m5C methylation regulatory factors DNMT1 and NSun2 in tumor

Technical Field

The invention relates to the technical field of medicines, in particular to application of m5C methylation regulating factors DNMT1 and NSun2 in tumors.

Background

When cells respond to stress, programmed cell death, known as apoptosis, occurs. Multiple apoptosis signaling pathways have been found and elucidated in tumor therapy and are considered the most promising and effective mechanism in current cancer therapies. Thus, increasing apoptosis sensitivity has become a major strategy for the treatment of multi-drug resistant tumors (e.g., osteosarcoma). The initiation of apoptosis depends to a large extent on the balance of anti-apoptotic and pro-apoptotic gene activation, while regulation of gene expression is multi-layered and orderly.

Methylation modification of genes is taken as an important epigenetic modification means and is involved in expression regulation of genes. Cytosine methylation (m 5C) is a common nucleotide modification in genomic DNA and mRNA, and plays a key role in regulating gene expression at the transcriptional and posttranscriptional levels. DNA methylation often occurs in CpG islands in the promoter region, inactivating gene transcription. DNA methyltransferase I (DNMT 1) methylates the promoter region of many oncogenes and is considered an important target for cancer therapy. In addition, many of the regulatory factors of DNMT1 are involved in DNA methylation activation and recruitment of DNMT1 to the regulation of specific gene sites, suggesting that DNMT1 regulates the functional diversity of gene expression by DNA methylation. Some oncogenes with well known anti-apoptosis have also been shown to be inhibited in transcription by promoter methylation. For example, in Karposi sarcoma cell lines, hypomethylation of CpG islands in the promoter region of the anti-apoptotic gene AXL promotes the binding of transcription factor Sp thereto, thereby up-regulating its expression. Furthermore, it has recently been found that DNMT1 mediated promoter methylation can inhibit the expression of the anti-apoptotic genes Bcl2, bclXL and Bcl6, thereby impeding cell survival and tumor growth.

Modification of RNAm5C determines the fate of mRNA in multiple stages after transcription, including splicing, transport, translation, etc. of mRNA. NSun2, an important RNA cytosine methyltransferase, can maintain the stability of the mitotic spindle, enhance CDK1 translation, and reduce p27 expression, thereby fundamentally promoting tumor cell proliferation. The absence of NSun2 promotes stress-induced tRNA clearance, increasing apoptosis of cortical, hippocampal and striatal neurons. In esophageal squamous cell carcinoma, NSun2 methylated long non-coding RNA (lncRNA) NMR has been reported to inhibit cisplatin-induced apoptosis and promote tumor progression. However, NSun2 is worthy of further study if it regulates expression of apoptosis-related genes through mRNA methylation, thereby affecting the apoptosis process.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of m5C methylation regulating factors DNMT1, NSun2 in tumor.

The technical scheme of the invention is as follows:

use of an m5C methylation modulator comprising DNMT1 and/or NSun2 for modulating apoptosis in a tumor cell.

The DNMT1 regulates the expression of anti-apoptosis genes AXL, NOTCH2 and YAP1 through DNA methylation.

The NSun2 regulates the expression of anti-apoptotic genes AXL, NOTCH2, YAP1 by mRNA methylation.

The DNMT1 inhibits the expression of NSun2 by methylating the promoter region of NSun2.

The tumor is osteosarcoma.

The invention also provides application of the m5C methylation regulating factor in preparing medicines for preventing and treating tumors, wherein the regulating factor comprises DNMT1 and/or NSun2.

The invention also provides an application of the methylation regulating factors DNMT1 and NSun2 in combination with a chemotherapeutic drug in preparing medicines for treating or preventing osteosarcoma.

The chemotherapeutic medicine comprises one or more of cisplatin, carboplatin, oxaliplatin, nedaplatin, leptoplatin and doxorubicin.

The invention also provides an inhibitor of anti-apoptotic gene expression, said inhibitor comprising NSun2.

The anti-apoptosis gene comprises one or more of AXL, NOTCH2 and YAP1.

An increasing number of people recognize that a wide range of nucleotide modifications in eukaryotes play an important role in biological processes. Among hundreds of chemical modifications, 5-methylcytosine is a highly interesting epigenetic modification that has been identified in genomic DNA and various RNA families. The dual nature of m5C in DNA and RNA regulation has prompted researchers to investigate whether there is some correlation or synergy between these two identical methylated components at different levels, and what the relevant mechanisms and results are. We found that DNMT1 reduced expression of NSun2 by methylating the promoter of NSun2, resulting in coupled changes in cytosine methylation on the promoter DNA and mRNA of the mutually targeted anti-apoptotic genes, including, but not likely limited to AXL, NOTCH2 and YAP1. We focused on these genes because of their previously noted marked effects against apoptosis. Through cytosine methylation coupling between their DNA and mRNA, a strong inhibition of gene expression can be achieved in drug-induced osteosarcoma cell apoptosis, which clearly demonstrates the complexity of the apoptosis-related gene regulation mechanism. . The invention can coordinate and determine the expression of anti-apoptosis genes and the resistance of apoptosis on different epigenetic levels, especially on DNA and mRNA, and clearly adds a new level for epigenetic regulation.

In different types of tumors, the promoter regions of a large number of oncogenes are hypermethylated, which directly transcriptionally inactivates them, thus maintaining the survival of tumor cells. DNMT1, which acts as a maintenance enzyme for DNA methylation, is responsible for the methylation of the oncogene promoter. Most of the oncogenes promote apoptosis through hypermethylation of their promoter regions, and our research results indicate that DNMT1 inhibits the expression of anti-apoptotic genes NSun2, AXL, NOTCH2 and YAP1 through promoter methylation, whereas the inhibition of NSun2 further disrupts its translation by attenuating methylation of AXL, NOTCH2 and YAP1mRNA, ultimately enhancing the inhibition of these anti-apoptotic targets by DNMT1 in combination from both transcriptional and posttranscriptional aspects. This finding not only underscores the various modes that DNMT1 may utilize in regulating gene expression, but also expands our understanding of DNMT 1's function in tumor cell apoptosis.

RNAm5C modification is of great importance in the study of malignant solid tumors. Many reports suggest that NSun 2-mediated RNA methylation plays a strong oncogenic role in a variety of cancer pathways, including cancer cell proliferation, migration, invasion and drug resistance. Meanwhile, our study found that the lack of NSun2 impedes tumor growth at early stage of administration, and impairs anti-apoptotic gene expression due to translational inhibition caused by loss of mram 5C, thereby leading to chemotherapy-induced osteosarcoma apoptosis, reiterating the key role of NSun2, and revealing the important role of RNA cytosine methylation in tumor cell progression and drug resistance.

Overall, DNA methyltransferase DNMT1 was found to inhibit expression of RNA methyltransferase NSun2 by methylating NSun2 promoter, thereby establishing a key coupling between DNA and RNA cytosine methylation, which in turn affects osteosarcoma cell apoptosis resistance during chemotherapy. Our findings clearly add a new layer of control of gene expression at different epigenetic levels and provide a new idea for future osteosarcoma treatment.

Compared with the prior art, the invention has the following beneficial effects:

1. apoptosis is the central mechanism of chemotherapy for the treatment of human cancers. 5-methylcytosine (m 5C) modifications on DNA and mRNA have been shown to independently regulate apoptosis. However, their interactions or interactions in apoptosis have not been discovered. Here we found that DNMT1 mediated methylation of the gene promoter region and NSun2 mediated methylation of mRNA synergistically regulated osteosarcoma cell apoptosis. During chemotherapy-induced osteosarcoma apoptosis, DNMT1 expression is increased, while NSun2 expression is inhibited. The present invention found that DNMT1 inhibits NSun2 expression by methylating the NSun2 promoter. DNMT1 and NSun2 regulate the anti-apoptotic genes AXL, NOTCH2 and YAP1 by DNA methylation and mRNA methylation, respectively. When exposed to cisplatin or doxorubicin, DNMT1 is elevated and NSun2 is inhibited, promoter methylation of these anti-apoptotic genes is enhanced and mRNA methylation is reduced, greatly reducing expression of these anti-apoptotic genes, and thus causing osteosarcoma cells to undergo apoptosis. The research results of the invention establish the importance of DNA and RNA cytosine methylation interactions in the anti-apoptosis capability of osteosarcoma during chemotherapy, provide a new view for the treatment of osteosarcoma in the future, and provide new insights for regulating gene expression at different levels of epigenetic inheritance.

2. We disclose an important link in DNA and RNA methylation to modify osteosarcoma cell apoptosis. DNMT1 inhibits the expression of NSun2, thereby altering the methylation coupling of cytosine on the promoters and mRNAs of their downstream common target genes AXL, NOTCH2 and YAP1. In this case, the increase in DNMT1 greatly inhibited the expression of these anti-apoptotic genes, making osteosarcoma cells susceptible to apoptosis under the induction of chemotherapeutic drugs. Thus, we emphasize that the epigenetic modification of m5C, common in genomic DNA and mRNA, regulates expression of anti-apoptotic genes, thus co-determining resistance of osteosarcoma cells to apoptosis-inducing drugs. In summary, these findings will provide new insights into tumor cell apoptosis and open new approaches for the treatment of future osteosarcomas.

Drawings

Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:

FIG. 1 is a graph showing that DNMT1 and NSun2 are involved in chemotherapy drug-induced osteosarcoma apoptosis. Western blotting was used to examine the expression of DNMT1 and NSun2 in cisplatin-or doxorubicin-induced apoptosis of U2OS osteosarcoma cells (panel a), KHOS osteosarcoma cells (panel B), 143B osteosarcoma cells (panel C).

FIG. 2 is a diagram showing the inhibition of NSun2 by DNMT1 through promoter methylation. In U2OS cells knocked down DNMT1, panel A detected changes in DNMT1 and NSun2 proteins using Western blotting, and panel B analyzed DNMT1 and NSun2mRNA levels using a real-time quantitative PCR method. In U2OS cells knocked down with NSun2, panel C detected changes in DNMT1 and NSun2 proteins using Western blotting, and panel D analyzed DNMT1 and NSun2mRNA levels using a real-time quantitative PCR method. Panel E, meDIP-qPCR detects changes in NSun2 promoter methylation in U2OS cells after DNMT1 knockdown. IgG and p21 gene promoters served as controls. * P <0.05; * P <0.01; ns, have no meaning.

FIG. 3 is a graph showing the regulation of anti-apoptotic gene expression by DNMT1 and NSun2. FIG. AWestern blotting shows the detection of protein expression changes of anti-apoptotic genes AXL, NOTCH2, YAP1 in U2OS cells knocked down DNMT 1. Panel B-real-time quantification of pcr detects AXL, NOTCH2, YAP1mRNA levels in U2OS cells knocked down with DNMT 1. FIG. C Western blotting shows the detection of protein expression changes of anti-apoptotic genes AXL, NOTCH2, YAP1 in NSun2 knockdown U2OS cells. Panel D real-time quantification of pcr detects mRNA levels of AXL, NOTCH2 and YAP1 in U2OS cells knocked down by NSun2. * P <0.05; * P <0.01; ns, have no meaning.

FIG. 4 is a graph showing methylation-coupled alterations in the expression of anti-apoptotic genes in DNA and mRNA. Figure A, westernblotting detects changes in anti-apoptotic gene proteins in U2OS cells knockdown DNMT1, NSun2, and both simultaneously. Panel B uses real-time quantitative PCR to analyze the mRNA levels of these anti-apoptotic genes in the U2OS cells described in A. Panel C uses MeDIP-qPCR to detect methylation changes in the promoters of these anti-apoptotic genes in the U2OS cells described in A. Panel D, meRIP-qPCR detects mRNA methylation changes of these anti-apoptotic genes in U2OS cells as described in A. * P <0.05; * P <0.01; ns, have no meaning.

Fig. 5 is a graph showing that DNMT1 and NSun2 determine resistance of osteosarcoma cells to chemotherapy-induced apoptosis. Panel A uses Western blotting to assess changes in protein expression of apoptosis-related genes in U2OS cells with or without DNMT1 and/or NSun2 knockdown under cisplatin (left) or doxorubicin (right) treatment. Panel B flow cytometry examined apoptosis of U2OS cells before and after cisplatin treatment, DNMT1 and/or NSun2 knockout. Panel C flow cytometry examined U2OS cell E apoptosis following doxorubicin treatment with DNMT1 and/or NSun2 knockdown. Panel D evaluation of resistance to xenograft tumors by lentiviral infection stably silencing U2OS cell-derived osteosarcoma cells of DNMT1 and/or NSun2 osteosarcoma tumor images (G) obtained at the time point of end of doxorubicin treatment. * P <0.05; * P <0.01; ns, have no meaning.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that several modifications and improvements can be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.

Example 1

1. Cell culture

Human osteosarcoma cells U2OS,143B and KHOS were cultured in Dulbecco's modified Eagle's Medium (Invitrogen) supplemented with 10% (volume fraction mass concentration) fetal bovine serum and 1% (mass concentration) antibiotics, respectively, at 37℃with 5% CO ₂ (volume fraction) cell culture in a cell incubator. When these cells reach (0.5X10) ⁷ ) At this time, the samples were collected and analyzed after 24h treatment with the chemotherapeutic drugs (300 nmol/L Doxorubicin (DOX) and 30. Mu. Mol/L cisplatin), respectively.

TABLE 1

2. Transfection of sirna

To transiently silence NSUN2 or DNMT1, cells were transfected with NSUN 2-targeted siRNA (AGAUGUUAAGAUACUGUUGACCC as shown in SEQ ID NO. 25) or DNMT 1-targeted siRNA (GGAGAACGGUGCUCAUGCUU as shown in SEQ ID NO. 26) using Lipofectamine RNAiMAX (Invitrogen).

TABLE 2

Test group	Cells	Treatment of
			Test group 10	U2OS of test group 1	Blank control
Test group 11	U2OS of test group 1	SiDNMT1
			Test group 12	U2OS of test group 1	SiNSun2
Test group 13	U2OS of test group 1	SiDNMT1+SiNSun2
			Test group 14	U2OS of test group 1	Blank control + cisplatin treatment
Test group
	15	U2OS of test group 1	SiDNMT1+ cisplatin treatment
Test group 16				U2OS of test group 1	Sinsun2+ cisplatin treatment
	Test group 17	U2OS of test group 1	SiDNMT1+SiNSun 2+cisplatin treatment
Test group 18				U2OS of test group 1	Blank control + doxorubicin treatment
	Test group 19	U2OS of test group 1	SiDNMT1+ Adriamycin treatment
Test group 20				U2OS of test group 1	Treatment with Sinsun2+ Adriamycin
	Test group 21	U2OS of test group 1	Treatment with SiDNMT1+SiNSun 2+Adriamycin

Remarks:

SiDNMT1: transfection NSUN2 as target siRNA (AGAUGUUAAGAUACUGUUGACCC);

SiNSun2: DNMT1 transfected SiDNMT1 was targeted siRNA (GGAGAACGGUGCUCAUGCUU).

Example 2

1. Construction of pHBLV-shdnmt1 and pHBLV-shnsun2 recombinant plasmids

To generate recombinant structures pHBLV-shdnmt1 and pHBLV-shnsun2 of pHBLV, corresponding siRNA of DNMT1 and NSun2 (DNMT 1: GGAGAACGGUGCUCAUGCUU as SEQ ID NO.26; NSun2: AGAUGUUAAGAUACUGUUGACCC as SEQ ID NO. 25) were synthesized, respectively, and inserted between BamHI and EcoRI sites of pHBLV-u6 plasmid, respectively, to obtain recombinant plasmids pHBLV-shdnmt1 and pHBLV-shnsun2 (lentiviral shRNA expression system was purchased from Shanghai HANBIO of China).

2. Lentiviral packaging and transfection

Lipofectamine 3000 reagent (Invitrogen) was incubated with Opti-MEM I reduced serum medium (GIBCO) and HEK239T cells were transfected with pHBLV control (pHBLV-u 6), plasmid (pHBLV-shdnmt 1, pHBLV-shnsun 2) and helper plasmids PMD2G and PsPAX2 (purchased from Shanghai HANBIO, china), respectively, as follows.

TABLE 3 Table 3

After 8 hours of transfection according to Table 2, the medium was replaced with 10ml of fresh DMEM medium. Collecting the supernatant containing each lentivirus at 48h and 72 h; filtered through a 0.45 mm cellulose acetate filter and concentrated for use. To generate stable cell lines, 2.0X10 cells per dish ⁵ Density of individual cells human osteosarcoma cells of the test group prepared in example 1 were inoculated into 6cm dishes, and cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% (volume fraction mass concentration) fetal bovine serum and 1% (mass concentration) antibiotics, and added with 10ng/mL polybrene (Sigma-Aldrich) of each of the supernatants containing lentivirus of Table 2. After 48h the medium was changed (Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% (volume fraction mass concentration) fetal bovine serum and 1% (mass concentration) antibiotics). Cells were cultured with 4. Mu.g/mL puromycin (Invitrogen) for 2 weeks and maintained in 1. Mu.g/mL puromycin, and subsequently used in nude mouse tumor implantation models.

TABLE 4 Table 4

Nude mouse tumor transplantation model:

the breeding process completely accords with the guidelines of the experimental animal center of Zhengzhou university. Cells of test groups 22-25 (1X 10) ⁷ ) Resuspended in 0.1ml pbs and injected subcutaneously on the back of each mouse (six injections per group). After 10 days, mice were injected with doxorubicin (10 mg/kg) via the tail vein and fed for 14d. The length and width of the tumor were measured with a vernier caliper. The tumor volume calculation formula is: volume = length x width ² ×0.52。

Example 3 analysis of results

1. Western blot analysis

Western blot analysis was performed using standard procedures.

Protein was extracted with Radioimmunoprecipitation (RIPA) buffer (25 mM Tris-HCl (pH 7.6), 150mmol/LNaCl,1% NP-40,1% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail test groups 1-21. After homogenization, the supernatant was centrifuged at 4℃and the protein concentration was determined by BCA method, equal amounts of protein were separated by SDS-PAGE and transferred onto a pdf membrane (Millipore) after blocking, each incubated with the following one:

DNMT1 from Abcam (cat. No. ab13537, dilution ratio 1:1000), pro-caspase3 (cat. No. ab32150, dilution ratio 1:1000), NOTCH2 (cat. No. ab8926, dilution ratio 1:1000).

Caspase3 of Cell Signaling Technology (cat# 9661, dilution ratio 1:1000), AXL (cat# 8661, dilution ratio 1:1000), YAP1 (cat# 14074, dilution ratio 1:1000), tubulin (cat# 5666, dilution ratio 1:1000).

Protein NSUN2 (cat. No. 20854-1-AP, dilution ratio 1:1000).

The immunocomplexes were observed with chemiluminescent reagents and tested for AI800 (GE).

RNA extraction, reverse Transcription (RT) and quantitative real-time qPCR analysis:

RNA was extracted from test group 10.11.12.13 with Trizol (TIANGEN, beijin, china) and used

Reverse transcriptase treatment was used for cDNA synthesis (Transgen, beijing, china). Two-step real-time qPCR amplification reactions were performed using a general SYBR qPCR Master Mix (Vazyme, nanj, china) and Bio-Rad CFX 96. TM. Real-time system. To quantify the levels of different mRNAs, RT-qPCR analysis was performed using the following primer pairs:

TABLE 5

2. Methylated RNA immunoprecipitation (MeRIP)

RNA (20. Mu.g) from test group 10-13 was isolated and incubated with anti-m5C antibody (1. Mu.g) and RNasin (1U/. Mu.L) in immunoprecipitation reaction (150 nmol/L NaCl,0.1% NP-40, 10mmol/LTris-HCl (pH 7.4) at 4℃for 12h, then protein a/g agarose resin (30. Mu.L) was added and incubation continued at 4℃for 2h after centrifugation 5 times with IP buffer, m5C modified RNA was enriched and real-time qPCR analysis was performed using the primer pair consisting of rabbit IgG as negative control.

3. Methylated DNA immunoprecipitation (MeDIP)

DNA of test group 10-13 was extracted using TaKaRa MiniBEST Universal genomic DNA extraction kit Ver.5.0 (TaKaRa BIO, JAPAN). DNA methylation levels were assessed by methylation DNA immunoprecipitation (MeDIP) using MagMeDIP kit (dienode, denville, NJ).

The following primers were used for PCR:

4. flow cytometry assessment of apoptosis

The test groups 14-21 were analyzed for apoptosis rate using flow cytometry, and cells were collected by centrifugation at 300 Xg for 5min at 4 ℃. Cells were rinsed 2 times with PBS pre-chilled to 4℃and resuspended in 250. Mu.L of 1X Annexin V binding buffer to adjust the cells to 1X 106cells/ml. After Annexin V binding buffer, 5. Mu.l of Annexin V-fitc conjugated antibody and 5. Mu.l of 7-amino actinomycin D (7-AAD) were added. After 15min of standing at room temperature, 400. Mu.L of 1 XAnexin V binding buffer was added. Samples were analyzed by LRSFortessa (BD Biosciences) over 1 hour.

Bioinformatics analysis:

statistical analysis was performed using SPSS 18.0 software (SPSS, inc., chicago, IL, USA). The single comparison uses student t test, and the multiple comparison uses Tukey-Kramer multiple comparison after single factor analysis of variance. The P value <0.05 is statistically significant for the difference. Data are expressed as mean ± standard deviation unless otherwise indicated. * P <0.05; * P <0.01; ns, have no meaning.

The results were as follows:

fig. 1 shows that DNMT1 and NSun2 are involved in chemotherapy-induced osteosarcoma apoptosis. Westernblotting was used to examine the expression of DNMT1 and NSun2 in cisplatin-or doxorubicin-induced apoptosis of U2OS osteosarcoma cells (panel A), KHOS osteosarcoma cells (panel B), 143B osteosarcoma cells (panel C).

FIG. 2DNMT1 inhibits NSun2 expression by promoter methylation. In U2OS cells knocked down DNMT1, panel A detected changes in DNMT1 and NSun2 proteins using Western blotting, and panel B analyzed DNMT1 and NSun2mRNA levels using a real-time quantitative PCR method. In U2OS cells knocked down with NSun2, panel C detected changes in DNMT1 and NSun2 proteins using Western blotting, and panel D analyzed DNMT1 and NSun2mRNA levels using a real-time quantitative PCR method. Panel E, meDIP-qPCR detects changes in NSun2 promoter methylation in U2OS cells after DNMT1 knockdown. IgG and p21 gene promoters served as controls. * P <0.05; * P <0.01; ns, have no meaning.

FIG. 3DNMT1 and NSun2 regulate anti-apoptotic gene expression. FIG. AWestern blotting shows the detection of protein expression changes of anti-apoptotic genes AXL, NOTCH2, YAP1 in U2OS cells knocked down DNMT 1. Panel B-real-time quantification of pcr detects AXL, NOTCH2, YAP1mRNA levels in U2OS cells knocked down with DNMT 1. FIG. CWesternblotting assay detects changes in protein expression of anti-apoptotic genes AXL, NOTCH2, YAP1 in NSun2 knockdown U2OS cells. Panel D real-time quantification of pcr detects mRNA levels of AXL, NOTCH2 and YAP1 in U2OS cells knocked down by NSun2. * P <0.05; * P <0.01; ns, have no meaning.

FIG. 4 methylation coupling of DNA and mRNA alters expression of anti-apoptotic genes. Panel A, western blotting detects changes in anti-apoptotic gene proteins in U2OS cells knocked down by DNMT1, NSun2, respectively, and both. Panel B uses real-time quantitative PCR to analyze the mRNA levels of these anti-apoptotic genes in the U2OS cells described in A. Panel C uses MeDIP-qPCR to detect methylation changes in the promoters of these anti-apoptotic genes in the U2OS cells described in A. Panel D, meRIP-qPCR detects mRNA methylation changes of these anti-apoptotic genes in U2OS cells as described in A. * P <0.05; * P <0.01; ns, have no meaning.

Conclusion:

1. DNMT1 and NSun2 are both involved in chemotherapy-induced osteosarcoma cell apoptosis

To induce osteosarcoma apoptosis, the present invention treats osteosarcoma with two conventional chemotherapeutics cisplatin (Cpt) and doxorubicin (Dox). Both drugs successfully induced apoptosis of U2OS cells, and changes in the expression of pro-caspase3 and clear-caspase 3 were shown (FIG. 1A). The expression of DNA methyltransferase DNMT1 and RNA methyltransferase NSun2 in these apoptotic cells was inversely altered, indicating a significant increase in DNMT1 and a significant decrease in NSun2 (fig. 1A). In addition, DNMT1 and NSun2 also have similar expression patterns in apoptotic KHOS and 143B cells (fig. 1B,1 c), suggesting that DNMT1 and NSun2 may regulate osteosarcoma cell apoptosis, independent of the osteosarcoma cell type and the choice of apoptosis inducer.

2. DNMT1 inhibits NSun2 expression by methylation of the NSun2 promoter

DNMT1 is thought to inactivate gene transcription by promoter methylation, while NSun2 regulates posttranscriptional gene expression by methylation of mRNA. To verify the possibility of DNMT1 and NSun2 modulating each other during osteosarcoma cell apoptosis, we knockdown DNMT1 and NSun2, respectively, alone in U2OS cells. As shown in fig. 2A, silencing of DNMT1 significantly increased NSun2 protein levels. In contrast, expression of DNMT1 remained unchanged by knocking down NSun2 (fig. 2C). Meanwhile, the mRNA level thereof was detected by real-time quantitative PCR. Consistent with the protein changes, DNMT1 knockdown significantly increased expression of NSun2mRNA (fig. 2B), but NSun2 knockdown did not affect DNMT1mRNA levels (fig. 2D). From these observations, DNMT1 may inhibit expression of NSun2, whereas NSun2 does not affect expression of DNMT 1. We hypothesize that DNMT1 most likely inhibits expression of NSun2 by promoter methylation-mediated transcriptional inactivation. When DNMT1 was silenced in U2OS cells, cytosine methylation of the NSun2 gene promoter was significantly reduced (fig. 2E). Thus, DNMT1 can methylate the NSun2 promoter in osteosarcoma cells.

3. DNMT1 and NSun2 regulate anti-apoptotic gene expression

Next, we continued to investigate the effect of DNMT1 promoter methylation and NSun2mRNA methylation on the expression of these anti-apoptotic genes AXL, NOTCH2 and YAP1 in osteosarcoma cells. In U2OS cells, DNMT1 knockdown significantly increased not only the amount of expression of the protein, but also the amount of expression of AXL, NOTCH2, and YAP1mRNA (fig. 4A and 4B), suggesting that DNMT1 may inhibit transcription of these anti-apoptotic genes by promoter methylation. In contrast, NSun2 knockdown significantly inhibited protein expression of these target genes, but did not reduce their mRNA levels (fig. 4C and 4D). Overall, DNMT1 inhibition while NSun2 promotes expression of the anti-apoptotic genes AXL, NOTCH2 and YAP1, likely through epigenetic modification at different regulatory levels.

4. Methylation-coupled changes in expression of anti-apoptotic genes in DNA and mRNA

Based on the above findings, we found that DNMT 1-mediated promoter methylation and NSun 2-mediated mRNA methylation are coupled to each other to coordinate the expression of the above anti-apoptotic genes. To confirm this, a series of experiments were performed aimed at deciphering the coordinated regulation at different epigenetic levels. The study found that NSun2 knockdown significantly blocked DNMT 1-induced upregulation of AXL, NOTCH2 and YAP1 proteins (fig. 5A), but had no effect on their mRNA (fig. 5B), reiterating the difference in the level of DNA and RNA m5C modification. The corresponding methylation changes in the promoters and mRNAs of these genes were assessed using MeDIP-qPCR and MeRIP-qPCR, respectively. Unexpectedly, promoter methylation of these genes was significantly reduced in DNMT 1-deleted cells compared to control cells, while disruption of NSun2 did not affect promoter methylation levels at all (fig. 5C). Likewise, mRNA methylation of these anti-apoptotic genes was inhibited alone in NSun 2-deleted cells (fig. 5D). Notably, DNMT1 knockdown theoretically increased expression of NSun2, but did not enhance methylation of these target gene mrnas, possibly due to the simultaneous increase in nascent substrate transcripts lacking cytosine methylation.

According to these results, the promoter cytosine methylation of DNMT1 and mRNA cytosine methylation of NSun2 regulate expression of anti-apoptotic genes AXL, NOTCH2 and YAP1 in combination.

5. DNMT1 and NSun2 determine resistance of osteosarcoma cells to chemotherapy-induced apoptosis

To our knowledge, proliferation of apoptosis resistance is considered the culprit of tumor cells to develop resistance during chemotherapy. Thus, we studied the contribution of DNMT1 and NSun2, alone or in combination, to the resistance of osteosarcoma cells to chemotherapy-induced apoptosis. Silencing DNMT1 inhibited induction of the apoptosis marker caspase3 in cisplatin-or doxorubicin-treated U2OS cells; in contrast, disruption of NSun2 enhanced an increase in caspase3 expression. However, by combined silencing of DNMT1 and NSun2, changes in expression of caspase3 and other related apoptotic proteins (pro-caspase 3, AXL, NOTCH2 and YAP 1) were completely abolished, suggesting that DNMT1 and NSun2 have a synergistic effect in regulating anti-apoptotic gene expression.

In addition, apoptosis of U2OS cells transfected with DNMT1 and/or NSun2siRNA was further assessed by flow cytometry following cisplatin or doxorubicin treatment. DNMT1 loss reduced the rate of apoptosis, while NSun2 loss significantly increased apoptosis. Also, these changes were not observed in cells in which DNMT1 and NSun2 were knocked down simultaneously, emphasizing that DNMT1 and NSun2 may act on osteosarcoma cell apoptosis in an interconnected manner. Subsequently, DNMT1 and/or NSun2 were stably silenced by lentivirus, and the obtained cells were injected into nude mice, respectively, to generate osteosarcoma xenograft models, and then the resistance of tumors to doxorubicin was determined. DNMT1 knockdown cell-derived tumor xenograft tumors exhibited chemotherapy resistance, as evidenced by tumor size at the endpoint of the study. In contrast, tumors produced by NSun2 knockout cells significantly shrink following doxorubicin treatment. Likewise, the simultaneous deletion of DNMT1 and NSun2 abrogates these observed size variation differences, reiterating the important ability of DNMT1 and NSun2 to modulate osteosarcoma resistance. Overall, DNMT1 and NSun2 are considered synergistic, playing a central role in determining resistance of osteosarcoma to apoptosis during chemotherapy.

Apoptosis is the central mechanism of chemotherapy for the treatment of human cancers. 5-methylcytosine (m 5C) modifications on DNA and mRNA have been shown to independently regulate apoptosis. However, their interactions or interactions in apoptosis have not been discovered. We found that DNMT 1-mediated methylation of the gene promoter region and NSun 2-mediated methylation of mRNA synergistically regulate osteosarcoma cell apoptosis. During chemotherapy-induced osteosarcoma apoptosis, DNMT1 expression is increased, while NSun2 expression is inhibited. We found that DNMT1 inhibited expression of NSun2 by methylating the promoter region of NSun2. DNMT1 and NSun2 regulate the anti-apoptotic genes AXL, NOTCH2 and YAP1 by DNA methylation and mRNA methylation, respectively. When exposed to cisplatin or doxorubicin, DNMT1 is elevated and NSun2 is inhibited, promoter methylation of these anti-apoptotic genes is enhanced and mRNA methylation is reduced, greatly reducing expression of these anti-apoptotic genes, and thus causing osteosarcoma cells to undergo apoptosis. In conclusion, our results established the importance of DNA and RNA cytosine methylation interactions in osteosarcoma's ability to resist apoptosis during chemotherapy, providing a new view for future osteosarcoma treatment, and new insight into the regulation of gene expression at different levels of epigenetic.

The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.

Claims

1. Use of an m5C methylation modulator for modulating apoptosis in a tumor cell, wherein the modulator comprises DNMT1 and/or NSun2.

2. The use according to claim 1, wherein the DNMT1 regulates expression of the anti-apoptotic genes AXL, NOTCH2, YAP1 by DNA methylation.

3. The use according to claim 1, wherein NSun2 modulates the expression of anti-apoptotic genes AXL, NOTCH2, YAP1 by mRNA methylation.

4. The use according to claim 1, wherein said DNMT1 inhibits expression of NSun2 by methylating the promoter region of NSun2.

5. The use according to claim 1, wherein the tumor is osteosarcoma.

6. Use of an m5C methylation regulator in the manufacture of a medicament for the prevention and treatment of a tumor, the regulator comprising DNMT1 and/or NSun2.

7. Use of methylation regulating factors DNMT1, NSun2 in combination with a chemotherapeutic agent for the preparation of a medicament for the treatment or prevention of osteosarcoma.

8. The use according to claim 7, wherein the chemotherapeutic agent comprises one or more selected from cisplatin, carboplatin, oxaliplatin, nedaplatin, cisplatin, doxorubicin.

9. An inhibitor of anti-apoptotic gene expression, wherein said inhibitor comprises NSun2.

10. The inhibitor of anti-apoptotic gene expression according to claim 9, wherein said anti-apoptotic gene comprises AXL, NOTCH2, YAP1.