Use of an agent blocking the interaction between circ-cdyland hrnr in the preparation of a medicament for treating hepatocytes

Abstract

The application provides application of a reagent for blocking the interaction between Circ-CDYL and HRNR in preparation of a drug for treating hepatocytes and improving the effect of anti-PD-L1 immunotherapy. + The interaction between Circ-CDYL and HRNR is verified for the first time, and the interaction between Circ-CDYL and HRNR induces PD-L1

Description

TECHNICAL FIELD

[0001] The application relates to the field of biological medicines, and in particular to application of an agent for blocking interaction between Circ-CDYL and HRNR in preparation of a hepatocyte treatment drug. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) accounts for 75-85% of primary liver cancer and is the sixth most common cancer in the world and the fourth leading cause of cancer-related death. Because the early clinical symptoms of HCC are not obvious and there are insufficient diagnostic biomarkers, two-thirds of HCC patients are in the advanced stage at the time of first diagnosis. Because most HCCs are insidious in onset and there are insufficient diagnostic biomarkers, most patients are in the middle and advanced stages at the time of diagnosis, missing the most effective surgical treatment opportunity. At present, there are very limited effective treatment methods for advanced liver cancer, and the overall survival (OS) and prognosis of patients are very poor. After successful liver resection, the 5-year survival rate of early HCC (BCLC0 and a) patients is between 70% and 90%, while the 5-year survival rate of advanced HCC patients is only 10%. At the same time, HCC shows high genetic heterogeneity in its occurrence and development, which to some extent limits the application of existing molecular targeted drugs.

[0003] More and more studies have shown that circular RNA (CircRNA) as a kind of non-coding RNA has tissue specificity in expression and is closely related to the occurrence and development of cancer. In our previous study, it was reported that Circ-CDYL was significantly increased in early HCC and played the function of miR-892a and miR-318-3p sponge, thereby promoting the occurrence and development of early HCC. However, in recent years, studies have shown that CircRNA may play its biological function by interacting with downstream proteins in addition to participating in the occurrence and development of tumors as a microRNA sponge.

[0004] Current studies have shown that S100 family member HRNR has single nucleotide site variation in HCC tissues, and its high expression is related to poor prognosis of HCC. However, the specific mechanism by which HRNR promotes the occurrence and development of HCC and whether the biological function is related to the interaction of Circ-CDYL is still unclear. SUMMARY

[0005] In order to overcome the defects in the prior art, the application provides application of an agent for blocking interaction between Circ-CDYL and HRNR in preparation of a hepatocyte treatment drug.

[0006] To achieve the above object, the application adopts the following technical scheme:

[0007] The first aspect of the present application provides the use of an agent blocking the interaction between Circ-CDYL and HRNR in the preparation of a medicament for treating hepatocytes.

[0008] Further, the agent blocking the interaction between Circ-CDYL and HRNR is selected from one or more of a Circ-CDYL inhibitor, a HRNR inhibitor, and an expression promoter of E3 ubiquitin ligase SYVN1.

[0009] Further, the Circ-CDYL inhibitor is selected from one or more of the following:

[0010] a small molecule compound specifically inhibiting Circ-CDYL;

[0011] an interfering molecule specifically interfering with the expression of Circ-CDYL gene;

[0012] a gene editing agent specifically knocking out Circ-CDYL gene.

[0013] Further, the HRNR inhibitor is selected from one or more of the following:

[0014] a small molecule compound specifically inhibiting HRNR;

[0015] an interfering molecule specifically interfering with the expression of HRNR gene;

[0016] a gene editing agent specifically knocking out HRNR gene;

[0017] an antibody or ligand specifically binding to the protein encoded by HRNR gene.

[0018] Further, the interfering molecule specifically interfering with the expression of HRNR gene is SiRNA, and the sequence is SEQ ID NO. 3-4.

[0019] The second aspect of the present application provides the use of an agent blocking the interaction between Circ-CDYL and HRNR in the preparation of a medicament for improving the effect of anti-PD-L1 immunotherapy.

[0020] Further, the effect of anti-PD-L1 immunotherapy is the effect of anti-PD-L1 agent in treating hepatocellular carcinoma.

[0021] Further, the agent blocking the interaction between Circ-CDYL and HRNR is selected from one or more of a Circ-CDYL inhibitor, a HRNR inhibitor, and an expression promoter of E3 ubiquitin ligase SYVN1.

[0022] Further, the Circ-CDYL inhibitor is selected from one or more of the following:

[0023] Small molecule compounds that specifically inhibit Circ-CDYL;

[0024] Interfering molecules that specifically interfere with the expression of the Circ-CDYL gene;

[0025] Gene editing reagents that specifically knock out the Circ-CDYL gene.

[0026] Furthermore, the aforementioned HRNR inhibitors are selected from one or more of the following:

[0027] Small molecule compounds that specifically inhibit HRNR;

[0028] Interfering molecules that specifically interfere with HRNR gene expression;

[0029] Gene editing reagents that specifically knock out the HRNR gene;

[0030] Antibodies or ligands that specifically bind to the protein encoded by the HRNR gene.

[0031] Furthermore, the interfering molecule that specifically interferes with HRNR gene expression is siRNA, and its sequence is SEQ ID NO. 3-4.

[0032] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0033] This invention is the first to demonstrate that the interaction between Circ-CDYL and HRNR induces PD-L1. + Exosomes inhibit the efficacy of anti-PD-L1 immunotherapy, providing a reliable and effective research basis for developing strategies to treat hepatocellular carcinoma. Attached Figure Description

[0034] Figure 1 The circular RNA Circ-CDYL was shown to interact with the HRNR protein; among which, Figure 1 A and Figure 1 B represents the results of mass spectrometry identification of proteins enriched in Circ-CDYL-RAP precipitates and statistical results, respectively. Figure 1 C represents the further verification by western blotting experiments of the enrichment of HRNR in the Circ-CDYL-RAP precipitate; Figure 1 D shows the results of the RNA immunoprecipitation experiment and the results of RT-qPCR detection of the precipitated RNA;

[0035] Figure 2 The study showed that the circular RNA Circ-CDYL inhibits the degradation of HRNR protein in HCC; among which, Figure 2A shows the HRNR protein levels in each treatment group; Figure 2 B shows the HRNR mRNA levels in HCC cell lines overexpressing Circ-CDYL and negative control cell lines; Figure 2 C shows the HRNR protein levels in HCC cell lines overexpressing Circ-CDYL and negative control cell lines after treatment with CHX and MG132; Figure 2 D shows the Western blot results and corresponding statistical results of HRNR protein levels in HCC cell lines overexpressing Circ-CDYL and negative control cell lines at different time points after CHX treatment;

[0036] Figure 3 The circular RNA Circ-CDYL was shown to inhibit ubiquitination of HRNR protein in HCC; among which, Figure 3 A shows the ubiquitin signaling of HRNR protein in HCC cell lines overexpressing Circ-CDYL and negative control cell lines; Figure 3 B shows the results of predictions for E3 ubiquitin ligases that interact with HRNR using the "Ubibrowser" database; Figure 3 C shows the results of protein enrichment using anti-HRNR antibody and Co-IP detection using anti-SYVN1 antibody in each treatment group;

[0037] Figure 4 The interaction between Circ-CDYL and HRNR was shown to have a pro-cancer effect on HCC; among which, Figure 4 A shows the inhibitory effect of small interfering RNAs with different sequences on HRNR expression; Figure 4 B shows the changes in cell proliferation in each treatment group at different time points; Figure 4 C shows the changes in HCC self-renewal capacity in HCC cell lines overexpressing Circ-CDYL and negative control cell lines after downregulation of HRNR expression; Figure 4 D shows the changes in HCC monoclonal formation ability in HCC cell lines overexpressing Circ-CDYL and negative control cell lines after downregulation of HRNR expression;

[0038] Figure 5 This demonstrates that the interaction between Circ-CDYL and HRNR induces PD-L1. + Exosomes inhibit the efficacy of anti-PD-L1 immunotherapy; Figure 5 A showed that the interaction between Circ-CDYL and HRNR limited the response to anti-PD-L1 immunotherapy, and this effect could be partially reversed by HRNR inhibition; Figure 5B shows the size of tumors in each treatment group, indicating that tumors with suppressed HRNR expression all exhibited smaller volumes, and the reduction in subcutaneous tumor volume was more pronounced in the ATE-injected group. Figure 5 C shows the expression of downstream signaling pathways after inhibiting HRNR expression in HCC cell lines that stably overexpress Circ-CDYL and negative control cell lines; Figure 5 D showed that HCC cells overexpressing Circ-CDYL produced higher levels of PD-L1. + Exosomes, PD-L1 + Exosomes decreased accordingly after HRNR expression was inhibited. Detailed Implementation

[0039] This invention provides the application of reagents that block the interaction between Circ-CDYL and HRNR in the preparation of drugs for treating hepatocellular carcinoma. It is the first to clarify that Circ-CDYL participates in the regulation of phenotypes such as malignant transformation of HCC and resistance to immunotherapy through its interaction with HRNR protein, thereby providing a new target for developing new strategies for the treatment of HCC.

[0040] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings to enable a better understanding of the invention. However, the following embodiments do not limit the scope of the invention.

[0041] Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared according to conventional methods, unless otherwise specified.

[0042] Example 1

[0043] This embodiment explores the relationship between the circular RNA Circ-CDYL and the HRNR protein. The specific steps and results are as follows:

[0044] 1. Circular RNA Circ-CDYL interacts with HRNR protein.

[0045] (1) 2 × 10⁻⁶ cells from the HCCLM3 cell line 8 Cells were lysed, and the lysate was purified using the RNA antisense purification kit (BersinBio) according to the instructions. Mass spectrometry was then used to identify proteins enriched in the Circ-CDYL-RAP precipitates. Among the 52 proteins enriched in the Circ-CDYL-RAP precipitates, HRNR was one of the top ten proteins in terms of enrichment. Figure 1 A, B). Subsequently, western blotting experiments further confirmed the enrichment of HRNR in the Circ-CDYL-RAP precipitate ( Figure 1 C).

[0046] (2) RNA immunoprecipitation was performed according to the instructions of the RNA immunoprecipitation kit (Magna, Millipore), and the precipitated RNA was detected by RT-qPCR. The results showed that, compared to the negative control IgG antibody, the HRNR antibody could enrich Circ-CDYL (… Figure 1 D).

[0047] 2. Circular RNA Circ-CDYL inhibits the degradation of HRNR protein in HCC.

[0048] Based on the above findings, the following further clarifies that the interaction between circular RNA Circ-CDYL and HRNR in HCC cells inhibits the degradation of HRNR protein, thereby enhancing the stability of HRNR protein. The specific steps are as follows:

[0049] (1) Using a lentiviral infection system, the Circ-CDYL overexpression plasmid was transfected into the HCC cell line SMMC-7701 to construct a stable HCC cell line overexpressing Circ-CDYL (Ov-Circ-CDYL) and a negative control cell line (Ov-NC). Figure 2 B), and transfected the Circ-CDYL inhibitory expression plasmid into the HCC cell line HCCLM3 to construct a stable HCC cell line with inhibited Circ-CDYL expression (Sh-Circ-CDYL) and a negative control cell line (Sh-NC). The results showed that the HRNR protein level was increased in Circ-CDYL overexpressing cells, while the HRNR protein level was correspondingly decreased in Circ-CDYL inhibited cells. Figure 2 A), while HRNR mRNA levels did not change significantly ( Figure 2 B). Therefore, we hypothesize that Circ-CDYL may regulate the expression of HRNR protein at the posttranscriptional level.

[0050] (2) Cells were treated with cyclohexanone (CHX) at a final concentration of 80 μg / mL for 12 hours to inhibit protein synthesis. Without additional protein supplementation, HRNR protein expression was relatively decreased in the blank control group, while high HRNR protein expression was still detectable in the Circ-CDYL overexpressing HCC line. Simultaneous treatment with the proteasome inhibitor MG132 at a final concentration of 10 μM significantly restored HRNR protein levels in Circ-CDYL low-expression cells. Figure 2 C).

[0051] (3) Cells were treated with CHX at a final concentration of 80 μg / mL, and cells were collected at 0, 4, 8, and 12 hours to detect HRNR protein expression levels. The results showed that the degradation rate of HRNR in Circ-CDYL overexpressing cells was significantly slower. Figure 2 D)

[0052] These results indicate that circ-CDYL increases its protein levels by preventing the degradation of HRNR.

[0053] 3. Circular RNA Circ-CDYL inhibits ubiquitination of HRNR protein in HCC.

[0054] The interaction between the circular RNA Circ-CDYL and HRNR enhances the stability of the HRNR protein by inhibiting HRNR protein degradation mediated by the ubiquitin-proteasome pathway. The specific steps are as follows:

[0055] (1) Anti-HRNR antibody (MBS2026014, Mybiosource) and anti-IgG antibody (abCAM) were added at 12 μg to MAg25K Protein A / G bead suspension for binding, followed by incubation overnight at 4°C with cell lysis buffer from SMMC7701 and HCCLM3. After washing with DPBST buffer, the protein was eluted with SDS, and the results were detected using anti-ubiquitination antibody. The results showed that the ubiquitin signal of HRNR protein was significantly weakened in Circ-CDYL overexpression cells, while the ubiquitin signal of HRNR protein was significantly enhanced in Circ-CDYL downregulated cells. Figure 3 A).

[0056] (2) The "Ubibrowser" database (http: / / ubibrowser.bio-it.cn / ) was used to predict E3 ubiquitin ligases that interact with HRNR. Among the 23 candidate E3 ubiquitin ligases, SYVN1 had the highest confidence score. Figure 3 B).

[0057] (3) Proteins were enriched using anti-HRNR antibodies as described above, and Co-IP detection was performed using anti-SYVN1 antibody (sc-293484, Santa Cruz). The results showed that there was a relationship between SYVN1 and HRNR, and that overexpression of Circ-CDYL inhibited the interaction between HRNR and SYVN1, while downregulation of Circ-CDYL expression enhanced the interaction between HRNR and SYVN1. Figure 3 C).

[0058] In summary, these findings suggest that Circ-CDYL increases the stability of HRNR by blocking the degradation of HRNR by the ubiquitin ligase SYVN1.

[0059] Example 2

[0060] This embodiment verifies the pro-cancer effect of the interaction between Circ-CDYL and HRNR on HCC and studies the specific mechanism. The specific experimental steps are as follows:

[0061] 1. The interaction between Circ-CDYL and HRNR on the pro-cancer effect of HCC

[0062] (1) In the HCC cell line SMMC-7701, the inhibitory effect of three small interfering RNAs (si-RNAs) targeting different sequences on HRNR expression was verified. The sequences of si-RNAs and negative controls are shown in Table 1 below. Si-RNA No. 2, which showed the most significant effect, was selected to inhibit HRNR expression in the previously constructed stable HCC cell line overexpressing Circ-CDYL and the negative control cell line. Figure 4 A).

[0063] Table 1. Sequence information of si-RNA

[0064]

[0065]

[0066] (2) After cell counting, 2000 cells / well were seeded into 96-well cell culture plates, with 6 replicates per group. The final volume of complete culture medium per well was 200 μl. After seeding, the 96-well plates were placed in a cell culture incubator at 37℃ and 5% CO2. At 0, 24, 48, 72 and 96 hours after seeding, the culture medium was aspirated, and CCK8 reagent was mixed with DMEM culture medium at a ratio of 1:9. The final volume of each well was 100 μl, and the plates were added to the 96-well plates. The plates were incubated at 37℃ for 2 hours, and the absorbance of each sample well at 450 nm was measured using a microplate reader. After all time points were measured, a line graph was plotted with culture time as the X-axis and absorbance at 450 nm as the Y-axis to reflect the proliferation rate of the cell line. The results showed that Circ-CDYL overexpression significantly promoted HCC cell proliferation, and the ability of Circ-CDYL to promote HCC proliferation was partially inhibited after HRNR expression interference. Figure 4 B).

[0067] (3) After cell counting, 2000 cells / well were seeded in 6-well low-adhesion cell culture plates and cultured in a cell culture incubator at 37℃ and 5% CO2. Dynamic observation was conducted until day 10, and the number and size of cell spheroids were observed under a light microscope to reflect cell self-renewal capacity. The results showed that Circ-CDYL overexpression significantly promoted HCC self-renewal capacity, and the effect of Circ-CDYL on HCC self-renewal capacity was partially inhibited after downregulation of HRNR expression. Figure 4 C).

[0068] (4) Cell lines were seeded at 1000, 2000, and 3000 cells / well in 6-well cell culture plates, with 3 replicates per group, and cultured in a 37℃, 5% CO2 cell culture incubator. Dynamic observation continued until day 14. After fixation with 4% paraformaldehyde for 10 min, crystal violet staining was performed for 5 min, and colony formation was recorded by photograph. The results showed that Circ-CDYL overexpression significantly promoted HCC monoclonal formation, and this monoclonal formation-promoting ability was partially inhibited after HRNR expression downregulation. Figure 4 D).

[0069] 2. The interaction between Circ-CDYL and HRNR induces PD-L1 + Exosome-mediated inhibition of anti-PD-L1 immunotherapy efficacy

[0070] The interaction between Circ-CDYL and HRNR can promote PD-L1 expression and induce PD-L1. + Exosome production limits the response to anti-PD-L1 immunotherapy both in vivo and in vitro; while targeted inhibition of HRNR can eliminate the inhibitory effect of Circ-CDYL-HRNR interaction on anti-PD-L1 immunotherapy, as follows:

[0071] (1) 2×10 5HCC cells were seeded into 6-well plates. After adhesion, activated peripheral blood mononuclear cells (PBMCs) were centrifuged at 400g and resuspended in RPMI-1640 medium. PBMCs were counted and added to HCC cell culture medium at a ratio of 8:1 (HCC cells:PBMCs), followed by the addition of DMSO or anti-PD-L1 antibody Atezolizumab (ATE). After 48 hours of treatment, the culture medium and the suspended PBMCs and HCC cells were aspirated. The remaining attached cells were washed three times with PBS and fixed with 4% paraformaldehyde, followed by treatment with 0.5% Triton X-100. Cells were incubated with a mixture of TdT enzyme, TdT buffer, and TAM-dUTP at 37°C in the dark for 2 hours. Apoptotic cells were observed under a fluorescence microscope and counted using ImageJ software. The results showed that in a co-culture system consisting of PBMCs and HCC cells, overexpression of Circ-CDYL in HCCs significantly reduced the number of apoptotic cells, a phenomenon that was more pronounced when ATE was added. Conversely, inhibiting HRNR expression with siRNA significantly increased the number of apoptotic cells during ATE immunotherapy. Figure 5 A). These data suggest that the interaction between Circ-CDYL and HRNR limits the response to anti-PD-L1 immunotherapy, and this effect can be partially reversed by HRNR inhibition.

[0072] (2) In HCC cell lines stably overexpressing Circ-CDYL, siRNA inhibiting HRNR expression and its negative control group were transfected. The cells were digested with trypsin and resuspended in Matrixgel:DMEM = 1:9 solution to prepare 1×10⁶ cells / year. 4 Cell suspension of 10 cells / μl, at 1×10 6 100 μl of PBMCs per mouse was subcutaneously injected into the flank of 4-week-old male NCG mice (NOD-Prkdcem26Cd52Il2rgem26Cd22 / NjuCrl), with each mouse receiving the same volume. Activated PBMCs were injected into the NCG mice via the tail vein one day prior to the subcutaneous injection. ATE or DMSO was administered intraperitoneally every 3 days after tumor cell formation as a control, for a total of 12 days. Mice were sacrificed after the injection period, and tumor tissue was collected for observation and measurement. The results showed that, regardless of whether ATE or DMSO was injected, tumors with inhibited HRNR expression exhibited smaller volumes, with a more significant reduction in subcutaneous tumor volume observed in the ATE-injected group. Figure 5 B) This indicates that downregulating HRNR expression significantly increases the killing effect of ATE immunotherapy on HCC cells while inhibiting the malignant proliferation of HCC cells in vivo.

[0073] (3) As described above, HRNR expression was inhibited in HCC cell lines stably overexpressing Circ-CDYL and in negative control cell lines, and the expression of downstream signaling pathways was detected by Western blot. The results showed that Circ-CDYL could activate the mTOR-p70s6k signaling pathway through interaction with HRNR. Simultaneously, Circ-CDYL overexpression increased PD-L1 expression in HCC cells, and HRNR interference partially reversed the high expression of PD-L1. Figure 5 C). The above results indicate that the interaction between Circ-CDYL and HRNR regulates PD-L1 expression through the mTOR-p70s6k signaling pathway, thereby affecting the mechanism of HCC immunotherapy response.

[0074] (4) The above cell lines were divided into 2*10 7 One cell / dish was seeded into a 15cm cell culture dish, and 16ml of DMEM containing 10% exosome-free fetal bovine serum was added. After culturing for 48 hours, exosomes were purified from the cell supernatant using ultracentrifugation. The specific steps are as follows: a) Collect the supernatant; centrifuge at 300g, 4℃ for 10 minutes, and discard the supernatant; c) centrifuge the supernatant at 12000g, 4℃ for 90 minutes, and the precipitate is the exosome.

[0075] Exosomes purified from the culture medium were incubated with 0.5% bovine serum albumin (BSA, GIBCO) in PBS for 30 minutes to block nonspecific antigens. The purified exosomes were then co-incubated overnight at 4°C in the dark with biotin-labeled anti-CD63 monoclonal antibody (353018, Biolegend, San Diego, CA). Streptococcus-coated magnetic beads were then added, and after incubation at room temperature for 2 hours, the mixture was co-incubated overnight at 4°C in the dark with fluorescein-conjugated monoclonal anti-PD-L1 antibody (557924, BD Pharmingen, New Jersey) or isotype control antibody (555749, BD Pharmingen, New Jersey). Beckman MoFloXDP was used. TM Samples were analyzed using flow cytometry (Beckman Coulter, USA). A positive threshold of <2% was set in the negative control. Results are expressed as relative fluorescence values ​​(%). The results showed that HCC cells overexpressing Circ-CDYL produced higher levels of PD-L1. + Exosomes, PD-L1 + Exosomes decreased accordingly after HRNR expression was inhibited. Figure 5D). This indicates that the interaction between Circ-CDYL and HRNR induces PD-L1. + The production of exosomes weakens the effect of anti-PD-L1 immunotherapy.

[0076] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. The use of a reagent that blocks the interaction between Circ-CDYL and HRNR in the preparation of a medicament for the combined treatment of hepatocellular carcinoma with an anti-PD-L1 antibody, characterized in that, The reagent used to block the interaction between Circ-CDYL and HRNR is an HRNR inhibitor; the HRNR inhibitor is any one of three pairs of siRNAs targeting HRNR, named Si-HRNR-1, Si-HRNR-2 and Si-HRNR-3 respectively; wherein, Si-HRNR-1 consists of two complementary strands as shown in SEQ ID NO: 1 and SEQ ID NO: 2, Si-HRNR-2 consists of two complementary strands as shown in SEQ ID NO: 3 and SEQ ID NO: 4, and Si-HRNR-3 consists of two complementary strands as shown in SEQ ID NO: 5 and SEQ ID NO: 6.

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