Application of reagent for intervening circTACC3 to form R-loop structure in preparation of medicine for treating MASH-HCC

Abstract

The invention discloses an application of a reagent for intervening circTACC3 to form an R-loop structure in preparation of a medicine for treating MASH-HCC. The invention verifies that the circR-loop structure exists in an in-situ cancer tissue of human MASH-HCC, an organ-like model and an HCC cell model under a lipid overload condition, and further proves that circTACC3 with a positioning characteristic in a cell nucleus can form an R-loop structure at a DNA double-chain breakage site; therefore, malignant proliferation and adaptive survival of MASH-HCC tumor cells in a lipid overload environment are promoted, and finally generation and development of tumors are promoted. The malignant proliferation and the adaptive survival of the MASH-HCC tumor cells can be obviously inhibited by intervention aiming at the R-loop structure; and a new target spot and a medication strategy are provided for treatment of MASH-HCC.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of a reagent that intervenes in the formation of an R-loop structure by circTACC3 in the preparation of a drug for treating MASH-HCC. Background Art

[0002] R-loop refers to a special structure formed by DNA:RNA hybrid chains and free single-stranded DNA in the genome. With the progress of genomic research, more and more evidence has revealed that R-loop structure plays a vital role in gene expression, chromatin structure regulation, DNA damage response (DDR) and DNA replication.

[0003] According to the difference in the generation mechanism, R-loop can be divided into two forms: physiological and pathological. Among them, pathological R-loop is widely reported to cause DNA damage and genomic instability. The accumulation and regulation of pathological R-loop are closely related to the occurrence and development of cancer and are considered to be potential targets for molecular diagnosis and treatment of tumors.

[0004] Non-coding RNA (NcRNA) plays an important role in a variety of biological processes. Circular RNAs (circRNAs) are a type of NcRNA that is abundant and stable in cells. CircRNAs are not evenly distributed throughout the cell, but have specific subcellular localizations that match their functions. At present, the functions of nuclear-localized circRNAs are still relatively less studied. Previous studies have reported the existence of R-loop (circR-loop) structures formed by circRNAs in the genome of leukemia cells. However, whether the circR-loop structure also exists in solid tumor cells and its biological function in the occurrence and development of solid tumors remains to be studied.

[0005] Due to the increasing prevalence of obesity and metabolic syndrome, the global incidence of metabolic-associated steatotic liver disease (MASLD) has risen to 20-25% in the past decade. Metabolic dysfunction-associated steatohepatitis (MASH) is a more severe manifestation of MASLD, causing approximately 2% of hepatocellular carcinoma (HCC) each year; by 2030, MASH is expected to become the main cause of HCC. Compared with other causes, MASH-associated HCC (MASH-HCC) has unique molecular and immune characteristics, in which lipid overload caused by excess lipids in hepatocytes induces oxidative stress, replication stress, and nucleotide pool imbalance, further promoting DNA damage and mutation accumulation, which is an important precancerous event of MASH-HCC. However, whether circR-loop exists in MASH-HCC and whether it is mechanistically associated with DNA damage and mutation accumulation during the malignant progression of MASH-HCC remains to be explored. Summary of the invention

[0006] The purpose of the present invention is to address the deficiencies in the prior art and provide an application of a reagent for intervening in the formation of an R-loop structure of circTACC3 in the preparation of a drug for treating MASH-HCC.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] Provided is the use of a reagent that intervenes in the formation of an R-loop structure of circTACC3 in the preparation of a drug for treating MASH-HCC.

[0009] Furthermore, the reagent includes a vector for doxycycline-inducible expression of RNase H1, and a CRISPR-Cas9 guide RNA sequence designed for a circTACC3-specific sequence.

[0010] Furthermore, the drug for treating MASH-HCC also includes pharmaceutically acceptable excipients.

[0011] Furthermore, the reagent can inhibit the survival of MASH-HCC tumor cells in a lipid overload environment by intervening in the formation of an R-loop structure by 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 confirms that the existence of the circR-loop structure is verified in human MASH-HCC tissues, organoid models and lipid overload-induced HCC models, and further proves that circTACC3 with intranuclear localization characteristics can form an R-loop structure at DNA double-strand breaks (DSBs) sites, thereby promoting the survival of MASH-HCC tumor cells in a lipid overload environment; and intervention targeting the R-loop structure can significantly inhibit the survival of MASH-HCC tumor cells in a lipid overload environment; providing new targets and medication strategies for the treatment of MASH-HCC. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It was shown that circTACC3 localized at DSBs sites promoted the survival of MASH-HCC tumor cells in a lipid-overloaded environment.

[0015] Figure 2 It was shown that circTACC3 formed R-loop structures at DSBs sites in the genome of MASH-HCC tumor cells.

[0016] Figure 3 It was shown that interfering with R-loop structure formation inhibited the localization of circTACC3 at DSBs sites in the genome of MASH-HCC tumor cells.

[0017] Figure 4 It was shown that intervention of circTACC3 could inhibit the survival of MASH-HCC tumor cells in a lipid overload environment. DETAILED DESCRIPTION

[0018] 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.

[0019] Example 1 DSBs site-localized circTACC3 promotes the survival of MASH-HCC tumor cells in a lipid overload environment

[0020] The present invention first identified circRNA-circTACC3 with increased expression and nuclear localization from MASH-HCC, and characterized its binding to DSBs sites in MASH-HCC tumor cells, promoting the survival of MASH-HCC tumor cells in a lipid overload environment. The specific steps are as follows:

[0021] (1) The circTACC3(h) in situ hybridization kit was used according to the manufacturer's instructions to perform in situ hybridization experiments to detect the expression and localization of circTACC3 in human MASLD and MASH-HCC tumors and paired adjacent tissues. It was confirmed that in MASH-HCC tumors, circTACC3 expression was increased and showed nuclear localization; while in paired adjacent tissues and MASLD tissues with NAS ≥ 6, circTACC3 expression was low and mainly localized in the cytoplasm ( Figure 1 A).

[0022] (2) Fluorescence in situ hybridization experiments were performed using the circTACC3(h) in situ hybridization kit according to the manufacturer's instructions to detect the expression and localization of circTACC3 in human MASH-HCC tumors and paired adjacent cancer organoids. The results showed that circTACC3 expression was elevated only in MASH-HCC tumor organoids and showed nuclear localization ( Figure 1 B).

[0023] (3) Lipid overload was induced in 9 HCC cell lines and 2 normal liver cell lines, respectively. The specific steps are as follows: The cells were seeded into culture dishes with a cell confluence of 50%. 1% BSA (free fatty acid-free) 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 palmitic acid (PA) + oleic acid (OA) group, and 1 / 1000 DMSO was added to the culture medium of the MOCK group. RT-qPCR technology was used to detect the changes in the nuclear / cytoplasmic expression of circTACC3 in the above 11 cell lines before and after lipid overload induction. The results showed that among the HCC cell lines, the nuclear expression level of circTACC3 in HepG2 and HCCLM3 was most significantly increased after lipid overload induction; while the nuclear expression level of circTACC3 in Huh7 and Hep3B in the HCC cell lines and the normal liver cell lines did not increase significantly after lipid overload induction ( Figure 1 C).

[0024] (4) Using 3-OH BrDU probe, the DNA damage-induced apoptosis levels of the above 11 cell lines were detected before and after lipid overload induction. The results showed that: among HCC cell lines, the apoptosis levels of HepG2 and HCCLM3 did not increase significantly after lipid overload induction; while the apoptosis levels of Huh7 and Hep3B in HCC cell lines and normal liver cell lines increased significantly after lipid overload induction ( Figure 1 D).

[0025] (4) Using an antibody targeting the specific marker of DSBs, γH2AX (phosphorylation of theSer139residue of the histone variant H2AX), and a circTACC3(h) in situ hybridization kit, the co-localization of DSBs sites and circTACC3 in the above 11 cell lines before and after lipid overload induction was detected. The results showed that among HCC cell lines, there was a large amount of co-localization of circTACC3 and DSBs in the nuclei of HepG2 and HCCLM3 cells; while there was a lack of co-localization of circTACC3 and DSBs in the nuclei of Huh7 and Hep3B in HCC cell lines and normal liver cell lines ( Figure 1 E).

[0026] Example 2 circTACC3 forms R-loop structure at DSBs sites in the genome of MASH-HCC tumor cells

[0027] The present invention first discovered that in the genome of MASH-HCC-related tumor cells and the genome of HCC cells induced by lipid overload, there is a special R-loop structure formed by circTACC3. And this structure mainly exists at the genomic DSBs site. The specific steps are as follows:

[0028] (1) Fluorescence in situ hybridization and immunofluorescence experiments were performed according to the manufacturer's instructions using the circTACC3(h) in situ hybridization kit and the S9.6 antibody that specifically targets the R-loop structure to detect the co-localization of circTACC3 and the R-loop structure in human MASLD and MASH-HCC tumors and paired adjacent tissues. The results showed that circTACC3 co-localized with the R-loop signal only in MASH-HCC tumor tissues ( Figure 2 A).

[0029] (2) Fluorescence in situ hybridization and immunofluorescence experiments were performed in combination with the circTACC3(h) in situ hybridization kit and the S9.6 antibody specifically targeting the R-loop structure according to the manufacturer's instructions to detect the co-localization of circTACC3 and R-loop structures in human MASH-HCC tumors and paired adjacent cancer organoids. The results showed that circTACC3 co-localized with the R-loop signal only in MASH-HCC tumor organoids ( Figure 2 B).

[0030] (3) Fluorescence in situ hybridization and immunofluorescence experiments were performed in combination with the circTACC3(h) in situ hybridization kit, the S9.6 antibody specifically targeting the R-loop structure, and the γH2AX antibody specifically targeting the DSBs marker according to the manufacturer's instructions to detect the co-localization of circTACC3 with the R-loop structure and DSBs sites in lipid overload-induced HCC cells. The results showed that circTACC3 formed an R-loop structure at the genomic DSBs site of lipid overload-induced HCC ( Figure 2 C).

[0031] Example 3 Intervention of R-loop structure formation to inhibit the localization of circTACC3 at DSBs sites in the genome of MASH-HCC tumor cells

[0032] The present invention is the first to discover that intervention of the R-loop structure can affect the nuclear localization of circTACC3 and the binding of circTACC3 to the DSBs site of the HCC cell genome. The specific steps are as follows:

[0033] (1) Construct a doxycycline (Dox)-induced RNase H1 expression vector and use lentivirus to transfect HCC cell lines ( Figure 3 A).

[0034] (2) Using the S9.6 antibody that specifically targets the R-loop structure, we detected that after 24 hours of Dox-induced RNase H1 expression, the R-loop structure in HCC cells basically disappeared ( Figure 3 B).

[0035] (3) HCC cells were subjected to nuclear and cytoplasmic separation, and the nuclear / cytoplasmic expression of circTACC3 in cells induced by RNase H1 and in the control group was detected by RT-qPCR. The results showed that after induced expression of RNase H1 to clear the intracellular R-loop structure, the nuclear distribution ratio of circTACC3 was significantly reduced ( Figure 3 C).

[0036] (4) Fluorescence in situ hybridization and immunofluorescence experiments were performed in combination with the circTACC3(h) in situ hybridization kit and the S9.6 antibody specifically targeting the R-loop structure according to the manufacturer's instructions to detect the co-localization of circTACC3 and the R-loop structure in cells induced by RNase H1 expression and in the control group. The results showed that after inducible expression of RNase H1 to eliminate the intracellular R-loop structure, the R-loop structure formed by circTACC3 was significantly reduced ( Figure 3 D, 3E).

[0037] (5) Fluorescence in situ hybridization and immunofluorescence experiments were performed in combination with the circTACC3(h) in situ hybridization kit, the S9.6 antibody specifically targeting the R-loop structure, and the γH2AX antibody specifically targeting the DSBs marker according to the manufacturer's instructions to detect the co-localization of circTACC3 with the R-loop structure and DSBs sites in cells induced by RNase H1 expression and control groups. The results showed that after inducible expression of RNase H1 to clear the intracellular R-loop structure, the binding of circTACC3 to the DSBs site and the R-loop structure formed at the DSBs site were significantly reduced ( Figure 3 F, 3G).

[0038] Example 4 Intervention of circTACC3 can inhibit the survival of MASH-HCC tumor cells in a lipid overload environment

[0039] The present invention first discovered that intervention of circTACC3 can affect the survival of MASH-HCC tumor cells in a lipid overload environment. The specific steps are as follows:

[0040] (1) Detection of circTACC3 expression in human MASH-HCC tumors and paired adjacent organoids using RT-qPCR Figure 4 A), and observed the growth of organoids with different circTACC3 expression levels under lipid overload culture conditions. The results showed that under lipid overload culture conditions, tumor organoids with high circTACC3 expression had larger volume and more complete morphology ( Figure 4 B).

[0041] (2) The proliferation of MASH-HCC tumor organoids was detected using Ki-67 antibody, and the apoptosis level of MASH-HCC tumor organoids was detected using 3-OH BrDU probe. The results showed that under lipid overload culture conditions, tumor organoids with high expression of circTACC3 had high proliferation levels and lower apoptosis levels ( Figure 4 C).

[0042] (2) Nile blue oxazone staining was used to detect lipid deposition in MASH-HCC tumor organoids under lipid overload culture conditions. The results showed that MASH-HCC tumor organoids with high expression of circTACC3 had more significant lipid deposition ( Figure 4 D).

[0043] (3) CRISPR-Cas9 technology was used to knock out circTACC3 in HCC cells, and the circTACC3(h) in situ hybridization kit was used according to the manufacturer's instructions to perform in situ hybridization experiments to detect the knockout effect of circTACC3 in HCC cells. The results showed that circTACC3 in HCC cells was completely knocked out ( Figure 4 E).

[0044] (4) CCK-8 assay showed that after knocking out circTACC3 in HCC cells, the proliferation of HCC cells in a lipid overload culture environment was significantly weakened ( Figure 4 F). The proliferation of HCC cells was detected by Ki-67 antibody, and the apoptosis level of HCC cells was detected by 3-OH BrDU probe. The results showed that after knocking out circTACC3 in HCC cells, the proliferation signal of HCC cells in the lipid overload culture environment was significantly weakened, and the apoptosis signal was significantly enhanced ( Figure 4 G, 4H).

[0045] 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. Application of reagents that interfere with the formation of R-loop structure of circTACC3 in the preparation of drugs for the treatment of MASH-HCC.

2. The use according to claim 1, characterized in that: The reagents include a vector for expressing RNaseH1 inducibly by doxycycline, and a CRISPR-Cas9 guide RNA sequence designed for a circTACC3 specific sequence.

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

4. The use according to claim 2, characterized in that: The reagent can inhibit the survival of MASH-HCC tumor cells in a lipid overload environment by intervening in the formation of an R-loop structure by circTACC3.

Images