Application of m6A modified inhibitor for intervening circTACC3 in preparation of medicine for treating MASH-related HCC

Abstract

The invention discloses an application of an m6A modified inhibitor for intervening circTACC3 in preparation of a medicine for treating MASH-related HCC (Hepatocellular Carcinoma). The invention verifies that the m6A modified inhibitor can reduce the intranuclear localization and aggregation of circTACC3 by inhibiting m6A to modify circTACC3, thereby inhibiting the survival and proliferation of tumor cells in MASH-related HCC, and providing a new target and medication strategy for the treatment of MASH-related HCC.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to m 6 Application of A modification inhibitors in the preparation of drugs for the treatment of MASH-related HCC. Background Art

[0002] Due to the increasing incidence of obesity and metabolic syndrome, the global incidence of metabolic-associated steatotic liver disease (MASLD, formerly known as non-alcoholic fatty liver disease) has risen to 20-25% in the past decade. Metabolic dysfunction-associated steatohepatitis (MASH) is a serious manifestation of metabolic-associated fatty liver disease, and approximately 2% of hepatocellular carcinoma (HCC) is caused by it each year. It is expected that by 2030, MASH will become the main cause of HCC.

[0003] MASH-related HCC has unique molecular and immune features. For example, excessive lipids in the microenvironment and hepatocytes lead to lipid overload, inducing oxidative stress, replication stress, and nucleotide pool imbalance, which further leads to DNA damage and mutation accumulation. This may be the reason why traditional liver cancer treatments are not effective for this type of liver cancer.

[0004] Noncoding RNA plays a key role in a variety of biological processes. Circular RNA (circRNA) is a unique type of RNA that is abundant and stable in cells. CircRNA plays an important role in the progression of HCC and can be used as a diagnostic biomarker or therapeutic target. 6 A) is the most abundant RNA modification in eukaryotes and affects the functions of multiple circRNAs, including their production, transport, and degradation. 6 A modification is crucial in physiological and pathological liver immune responses, lipid metabolism, hepatitis virus infection, and HBV-related HCC. 6 The application of A modification in the treatment of MASH-related HCC has not been reported. Summary of the invention

[0005] The purpose of the present invention is to provide a method for intervening circTACC3 in order to address the deficiencies in the prior art. 6 Application of A modification inhibitors in the preparation of drugs for the treatment of MASH-related HCC.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] Provide m 6 Application of A modification inhibitors in the preparation of drugs for the treatment of MASH-related HCC.

[0008] Furthermore, the m 6 The A modification inhibitor is one or a combination of the METTL3 inhibitor STM2457, the METTL3-METTL14 heterodimer complex inhibitor S-adenosylhomocysteine ​​(SAH), and the SAH hydrolase inhibitor 3-deazaadenosine (DAA).

[0009] Furthermore, the drug for treating MASH-related HCC also includes pharmaceutically acceptable excipients.

[0010] Furthermore, the m 6 A modification inhibitors inhibit m 6 A modified circTACC3 and inhibited the survival and proliferation of tumor cells in MASH-related HCC.

[0011] Furthermore, the m 6 A modification inhibitors inhibit m 6 A modification of circTACC3 can reduce the nuclear localization and aggregation of circTACC3.

[0012] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0013] The present invention verifies that 6 A modification inhibitors inhibit m 6 A modification of circTACC3 can reduce the nuclear localization and aggregation of circTACC3, thereby inhibiting the survival and proliferation of tumor cells in MASH-related HCC, providing a new target and medication strategy for the treatment of MASH-related HCC. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 (Example 1) showed that m was identified from MASH-related HCC 6 The circRNA with the highest modification level was circTACC3. The subcellular localization and mRNA expression of circTACC3 in MASH-related HCC tumor cells were also shown. 6 A modification characteristics.

[0015] Figure 2 (Example 2) shows the m 6A. Identification results of modification-related features.

[0016] Figure 3 (Example 3) shows m 6 A modification regulates the nuclear localization and aggregation of circTACC3 and inhibits m 6 A modification can inhibit the survival and proliferation of tumor cells in MASH-related HCC.

[0017] Figure 4 (Example 4) shows that m 6 A. Modification regulates the nuclear localization and aggregation mechanism of circTACC3.

[0018] Figure 5 (Example 5) shows that the mRNA expression of circTACC3 is inhibited by point mutation 6 A modification can inhibit the aggregation of circTACC3 and its interaction with NONO / p54 nrb interaction. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0020] Example 1

[0021] The present invention identifies m from MASH-related HCC 6 The circRNA with the highest modification level was circTACC3, and its subcellular localization and mRNA expression in MASH-related HCC tumor cells were characterized. 6 A modification features, the specific steps are as follows:

[0022] 1.1 Using m 6 A-circRNA epitranscriptome chip simultaneously detected the relative expression levels, mRNAs, and mRNAs of circular RNAs in 5 human MASH-related HCC tumors and paired adjacent adjacent tissues. 6 A modification ratio and m 6 A absolute modification level ( Figure 1 A). The results show that m 6 The absolute modification levels of A were most significantly different between MASH-related HCC tumors and paired adjacent adjacent tissues ( Figure 1 B) There are 1105 circular RNA m 6 A The absolute modification level was significantly increased (fold difference > 2, P value < 0.05) ( Figure 1 B right, 1C). The m 6The A modification ratio was positively correlated with the expression level (Pearson's R = 0.67, p value < 1 × 10 -10 ; Figure 1 D) m of 38 genes 6 A modification ratio and expression level were significantly increased (difference fold>2, P value<0.05; Figure 1 E). Among them, the absolute m 6 The A modification level was the most significantly increased, and its function has not yet been reported ( Figure 1 E).

[0023] 1.2 Methylated RNA immunoprecipitation experiments were performed using a methylated RNA immunoprecipitation kit according to the manufacturer's instructions to detect 3 human MASLD tissues with NAS (NASH activity score) ≥ 6 and 3 pairs of human MASH-related HCC tumors and paired adjacent cancer tissues. The results showed that in MASH-related HCC tumors, m 6 A-modified circTACC3 had a higher proportion, but no obvious m-modified circTACC3 was observed in its paired adjacent paracancerous tissues or MASLD tissues with NAS ≥ 6. 6 A modification ( Figure 1 F, 1G).

[0024] 1.3 Fluorescence in situ hybridization experiments were performed using the circTACC3 in situ hybridization kit according to the manufacturer's instructions to detect human MASLD tissues and human MASH-related HCC tumors and paired adjacent cancer tissues. The results further confirmed that in MASH-related HCC tumors, m 6 The proportion of circTACC3 modified by A was higher, and circTACC3 was mainly located in the nucleus; while in paired paracancerous tissues and MASLD tissues with NAS≥6, circTACC3 was mainly located in the cytoplasm ( Figure 1 H).

[0025] Example 2

[0026] This example identified the m 6 A modification related features, as follows:

[0027] 2.1 Using the F2-circTACC3 pulldown kit, the F2-pulldown assay was performed according to the manufacturer’s instructions, and the proteins bound to circTACC3 were detected by western blot assay. 6 The components of the writer complex, METTL3, METTL14, and KIAA1429, can interact with circTACC3 ( Figure 2 A).

[0028] 2.2 Western blot technology was used to detect the expression levels of METTL3 and METTL14 proteins in liver cancer cell lines. The results showed that the expression levels of METTL3 and METTL14 in HCCLM3 and HepG2 cells were higher than those in Hep3B and Huh7 cells ( Figure 2 B).

[0029] 2.3 Using the RIP kit, the meRIP experiment was performed according to the manufacturer's instructions to detect the mRNA expression of circTACC3 in HCCLM3 and HepG2 cells. 6 A modification level. The results showed that m of circTACC3 in HCCLM3 and HepG2 cells 6 A modification is higher ( Figure 2 C. Figure 2 D).

[0030] 2.4 Using the RIP kit, the meRIP experiment was performed according to the manufacturer's instructions to detect the mRNA expression of circTACC3 in the nuclear and cytoplasmic components of HCCLM3 cells. 6 A modification level. The results showed that circTACC3 in the nucleus showed higher m 6 A modification ( Figure 2 E), indicating that its m 6 There is a correlation between A modification and intracellular localization.

[0031] 2.5 Using the SRAMP database (http: / / www.cuilab.cn / sramp) to predict possible mRNAs on circTACC3 6 The results showed that there may be 5 sites on circTACC3 that are affected by m 6 A modification: 153, 317, 763, 918 and 1030 ( Figure 2 F).

[0032] 2.6 Using the single motif level to analyze nuclear circTACC3 6 The A modified analysis technique detected the modification ratios of the above five predicted modification sites. The results showed that the modification rates of sites 317, 763, 918 and 1030 were >95%, and the modification rate of site 153 was approximately 70% ( Figure 2 G).

[0033] Example 3

[0034] This example demonstrates that 6 A modification regulates the nuclear localization and aggregation of circTACC3 and inhibits m 6A modification can inhibit the survival and proliferation of tumor cells in MASH-related HCC as follows:

[0035] 3.1 Dotblot kit was used to detect the expression of palmitic acid (PA) + oleic acid (OA) in the presence of three m 6 A modification inhibitors: METTL3 inhibitor STM2457, METTL3-METTL14 heterodimer complex inhibitor S-adenosylhomocysteine ​​(SAH) and SAH hydrolase inhibitor 3-deazaadenosine (DAA) inhibit m 6 After A modification, the overall m 6 A modification level. The PA+OA induction steps are as follows: cells were seeded into culture dishes with a cell confluence of 50%. 1% BSA (free fatty acids) culture medium was prepared with DMEM culture medium. PA+OA (PA final concentration 120 μM, OA final concentration 240 μM) was added to the culture medium of the PA+OA group, and 1 / 1000 DMSO was added to the culture medium of the MOCK group. m 6 The steps of A inhibitor treatment are as follows: cells were seeded in culture dishes with a cell confluence of 50%. STM2457 (final concentration 10 μM), SAH (final concentration 20 μM), and DAA (final concentration 20 μM) were added to the culture medium, and 1 / 1000 DMSO was added to the MOCK group. 6 The results showed that PA+OA-induced lipid overload increased the intracellular m 6 The overall level of A modification ( Figure 3 A).

[0036] 3.2 The meRIP experiment was performed using the RIP kit according to the manufacturer’s instructions. The results showed that after PA+OA treatment, the m 6 A modification level increased ( Figure 3 B) Under normal culture and lipid overload conditions, the three m 6 A inhibitors can reduce the total amount of intracellular Figure 3 A) and circTACC3-specific m 6 A modification level ( Figure 3 B).

[0037] 3.3 Using STM2457, SAH, and DAA to inhibit m 6 A modification, nuclear and cytoplasmic separation combined with qPCR method was used to detect the nuclear and cytoplasmic distribution of circTACC3 in HCCLM3 and HepG2 cells induced by PA+OA. 6A inhibitor reduced the nuclear localization of circTACC3 induced by lipid overload ( Figure 3 C)

[0038] 3.4 Using STM2457, SAH, and DAA to inhibit m 6 A modification, and the intracellular distribution of circTACC3 in HCCLM3 and HepG2 cells induced by PA+OA was detected by in situ hybridization. 6 A inhibitor reduced the nuclear localization and nuclear aggregation of circTACC3 induced by lipid overload ( Figure 3 D).

[0039] 3.5 Using STM2457, SAH, and DAA to inhibit m 6 A modification, the nuclear and cytoplasmic distribution of circTACC3 in MASH-related HCC organs after 12 days of PA+OA high-fat culture was detected by in situ hybridization. 6 A inhibitor reduced the nuclear localization and aggregation of circTACC3 induced by lipid overload in liver cancer cells and MASH-related HCC organs ( Figure 3 E).

[0040] 3.6 Using STM2457, SAH, and DAA to inhibit m 6 A modification, and Nile blue oxazone staining was used to detect lipid deposition in MASH-related HCC organs induced by PA+OA. 6 A modification alleviates the accumulation of lipid droplets in MASH-related HCC organ tissues ( Figure 3 F).

[0041] 3.7 Using STM2457, SAH, and DAA to inhibit m 6 The growth of MASH-related HCC organs after PA+OA high-fat culture for 12 days was detected by proliferation marker Ki-67, apoptosis marker 3-OH BrDU, and HE staining. The results showed that under normal and PA+OA culture conditions, the inhibition of m 6 A modification leads to inhibition of MASH-related HCC tissue proliferation ( Figure 3 G), increased apoptosis ( Figure 3 H), reduced size, damaged morphology ( Figure 3 I).

[0042] These results indicate that inhibition of m 6 A modification inhibited the survival and proliferation of tumor cells in MASH-related HCC by reducing the nuclear localization and accumulation of circTACC3.

[0043] Example 4

[0044] This example demonstrates m 6 The mechanism by which A modification regulates the nuclear localization and aggregation of circTACC3 is as follows:

[0045] 4.1 Using the RIP kit, the expression of circTACC3 and the nuclear marker protein NONO / p54, which may regulate RNA nuclear localization and aggregation, in HCCLM3 cells was detected according to the manufacturer's instructions. nrb The results showed that circTACC3 interacted with NONO / p54 nrb There is interaction ( Figure 4 A).

[0046] 4.2 CLIP-qPCR assay was performed using CLIP kit according to the manufacturer’s instructions to detect circTACC3 and nuclear marker protein NONO / p54 nrb The results showed that circTACC3 interacted with NONO / p54 nrb There are interaction sites in the range of 150-232, 296-351, 592-640, and 719-790 of circTACC3. 6 Among the modified sites, sites 153, 317, and 763 were located between circTACC3 and the nuclear marker protein NONO / p54. nrb Interaction fragments ( Figure 4 B).

[0047] 4.3 Detection of m using 3D in situ hybridization combined with immunofluorescence technology 6 A modification inhibitor affects circTACC3 and NONO / p54 in MASH-related HCC tumor organoids nrb The results show that m 6 A modification inhibitor can significantly inhibit lipid overload-induced circTACC3 and NONO / p54 nrb Aggregation and interaction ( Figure 4 C).

[0048] 4.4 Detection of m using RIP and in situ hybridization combined with immunofluorescence 6 Effects of A modification inhibitor on lipid-induced circTACC3 and NONO / p54 in HCCLM3 and HepG2 cells nrb The results show that m 6 A modification inhibitor can significantly inhibit lipid overload-induced circTACC3 and NONO / p54nrb Aggregation and interaction ( Figure 4 D, 4E).

[0049] Example 5

[0050] This example demonstrates that point mutations can be used to inhibit the mRNA expression of circTACC3. 6 A modification can inhibit the aggregation of circTACC3 and its interaction with NONO / p54 nrb The interactions are as follows:

[0051] 5.1 Knockout of circTACC3 in cells using CRISPR-Cas9 technology and overexpression of m 6 A modified site mutated circTACC3 to construct circTACC3m 6 A cell line with mutations in the modification site. RIP technique was used to detect m in HCCLM3 and HepG2 cells induced by lipids 6 Effects of A modification site mutation on circTACC3 and NONO / p54 nrb The results show that m 6 Mutations in A modification sites 153, 317, and 763 significantly inhibited the interaction between circTACC3 and NONO / p54 nrb The interaction ( Figure 5 A, 5B).

[0052] 5.2 Detection of lipid-induced mRNA expression in HCCLM3 and HepG2 cells using in situ hybridization 6 Effects of A modification site mutation on circTACC3 and NONO / p54 nrb The results show that m 6 Mutations in A modification sites 153, 317, and 763 significantly inhibited the interaction between circTACC3 and NONO / p54 nrb interaction and intracellular aggregation ( Figure 5 C).

[0053] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Intervention of circTACC3 m 6 Application of A modification inhibitors in the preparation of drugs for the treatment of MASH-related HCC.

2. The use according to claim 1, characterized in that: The m 6 The A modification inhibitor is one or a combination of the METTL3 inhibitor STM2457, the METTL3-METTL14 heterodimer complex inhibitor S-adenosylhomocysteine, and the SAH hydrolase inhibitor 3-deazaadenosine.

3. The use according to claim 1, characterized in that: The drug for treating MASH-related HCC further comprises a pharmaceutically acceptable excipient.

4. The use according to claim 1, characterized in that: The m 6 A modification inhibitors inhibit m 6 A modified circTACC3 and inhibited the survival and proliferation of tumor cells in MASH-related HCC.

5. The use according to claim 1, characterized in that: The m 6 A modification inhibitors inhibit m 6 A modification of circTACC3 can reduce the nuclear localization and aggregation of circTACC3.

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