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

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 effect of exosome on the anti-PD-L1 immunotherapy is inhibited, so that a reliable and effective research basis is provided for developing a strategy for treating hepatocellular carcinoma.

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 is a lack of 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 is a lack of diagnostic biomarkers, most patients are in the middle and advanced stages at the time of diagnosis, and have lost the opportunity for the most effective surgical treatment. 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 CircRNAs, as a kind of non-coding RNA, have tissue-specific expression and are 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 a miR-892a and miR-318-3p sponge function, 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 this 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] a small molecule compound specifically inhibiting Circ-CDYL;

[0024] an interference molecule specifically interfering with the expression of Circ-CDYL gene;

[0025] a gene editing reagent specifically knocking out Circ-CDYL gene.

[0026] Further, the HRNR inhibitor is selected from one or more of the following:

[0027] a small molecule compound specifically inhibiting HRNR;

[0028] an interference molecule specifically interfering with the expression of HRNR gene;

[0029] a gene editing reagent specifically knocking out HRNR gene;

[0030] an antibody or ligand specifically binding to the protein encoded by HRNR gene.

[0031] Further, the interference molecule specifically interfering with the expression of HRNR gene is SiRNA, and the sequence is SEQ ID NO. 3-4.

[0032] Compared with the prior art, the above technical scheme has the following technical effects:

[0033] The present application first verifies that the interaction between Circ-CDYL and HRNR induces PD-L1 + Exosome inhibits the effect of anti-PD-L1 immunotherapy, and provides a reliable and effective research basis for developing a strategy for treating hepatocellular carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 shows that the circular RNA Circ-CDYL interacts with the HRNR protein; wherein, Figure 1 A and Figure 1 B are the results of mass spectrometry for identifying the proteins enriched in the Circ-CDYL-RAP precipitate and the statistical results, respectively; Figure 1 C is the western blotting experiment for verifying the enrichment of HRNR in the Circ-CDYL-RAP precipitate again; Figure 1 D shows the results of RNA immunoprecipitation experiment and the results of RT-qPCR detection of the precipitated RNA;

[0035] Figure 2 shows that the circular RNA Circ-CDYL inhibits the degradation of HRNR protein in HCC; wherein, Figure 2A shows the HRNR protein level in each treatment group; Figure 2 B shows the HRNR mRNA level in HCC cell lines overexpressing Circ-CDYL and negative control cell lines; Figure 2 C shows the HRNR protein level in HCC cell lines overexpressing Circ-CDYL and negative control cell lines after CHX and MG132 treatment; Figure 2 D shows the western blot results and corresponding statistical results of HRNR protein level in HCC cell lines overexpressing Circ-CDYL and negative control cell lines at different time periods after CHX treatment;

[0036] Figure 3 It is shown that the circular RNA Circ-CDYL inhibits the ubiquitination modification of HRNR protein in HCC; wherein, Figure 3 A shows the ubiquitin signal of HRNR protein in HCC cell lines overexpressing Circ-CDYL and negative control cell lines; Figure 3 B shows the results of predicting E3 ubiquitin ligases that interact with HRNR using the "Ubibrowser" database; Figure 3 C shows the results of using anti-HRNR antibody to enrich proteins and using anti-SYVN1 antibody for Co-IP detection in each treatment group;

[0037] Figure 4 It is shown that the interaction of Circ-CDYL with HRNR has a pro-cancer effect on HCC; wherein, Figure 4 A shows the inhibitory effect of small interfering RNA of different sequences on HRNR expression; Figure 4 B shows the changes in cell proliferation of cells in each treatment group at different time points; Figure 4 C shows the changes in HCC self-renewal ability in HCC cell lines overexpressing Circ-CDYL and negative control cell lines after HRNR expression is down-regulated; Figure 4 D shows the changes in HCC monoclonal formation ability in HCC cell lines overexpressing Circ-CDYL and negative control cell lines after HRNR expression is down-regulated;

[0038] Figure 5 It is shown that the interaction of Circ-CDYL with HRNR induces PD-L1 + Exosomes inhibit the effect of anti-PD-L1 immunotherapy; Figure 5 A shows that the interaction between Circ-CDYL and HRNR limits the anti-PD-L1 immunotherapy response, and this effect can be partially reversed by HRNR inhibition; Figure 5B shows the size of tumors in each treatment group, indicating that HRNR expression inhibition in tumor-bearing mice showed smaller volume, and the volume of subcutaneous tumor in mice injected with ATE was reduced more obviously; Figure 5 C shows the expression of HRNR and downstream signaling pathways in HCC cell lines stably overexpressing Circ-CDYL and negative control cell lines; Figure 5 D shows that HCC cells overexpressing Circ-CDYL produce higher levels of PD-L1 + Exosomes, PD-L1 + Exosomes are correspondingly reduced after HRNR expression inhibition. DETAILED DESCRIPTION

[0039] The application provides a reagent for blocking the interaction between Circ-CDYL and HRNR in the preparation of a drug for treating hepatocellular carcinoma. The application first determines that Circ-CDYL participates in the regulation of malignant transformation and immunotherapy resistance phenotypes of HCC through interaction with HRNR protein, thereby providing a new target for developing a new strategy for treating HCC.

[0040] The application will be described in detail and specifically below through specific examples and drawings, so that the application can be better understood. However, the following examples do not limit the scope of the application.

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

[0042] Example 1

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

[0044] 1. Circular RNA Circ-CDYL and HRNR protein interact

[0045] (1) 2x10 8 cells from the HCCLM3 cell line were lysed, and the lysate was subjected to RNA antisense purification according to the steps in the RNA antisense purification kit (BersinBio) instructions, and then the proteins enriched in the Circ-CDYL-RAP precipitate were identified by mass spectrometry. Among the 52 proteins enriched in the Circ-CDYL-RAP precipitate, HRNR was one of the top ten enriched proteins (A, B). Subsequently, western blotting experiments again verified the enrichment of HRNR in the Circ-CDYL-RAP precipitate (C). Figure 1 Figure 1 ​​

[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 with 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, the expression level of HRNR protein in the blank control group decreased relatively, 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 the HRNR protein level in Circ-CDYL low-expression cells. Figure 2 C).

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

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

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

[0054] The interaction between circular RNA Circ-CDYL and HRNR mediates the degradation of HRNR protein through the ubiquitin-proteasome pathway, thereby increasing the stability of HRNR protein. The specific steps are as follows:

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

[0056] (2) The E3 ubiquitin ligase interacting with HRNR was predicted using the "Ubibrowser" database (http: / / ubibrowser.bio-it.cn / ). Among the 23 candidate E3 ubiquitin ligases, SYVN1 had the highest confidence score Figure 3 B)。

[0057] (3) The proteins were enriched using anti-HRNR antibody as described above, and anti-SYVN1 antibody (sc-293484, Santa Cruz) was used for Co-IP detection. The results showed that there was a connection between SYVN1 and HRNR, and Circ-CDYL overexpression inhibited the interaction between HRNR and SYVN1, while after Circ-CDYL down-regulation, the interaction between HRNR and SYVN1 was enhanced Figure 3 C)。

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

[0059] Example 2

[0060] This example verifies the pro-cancer effect of the interaction between Circ-CDYL and HRNR on HCC and specific mechanism research, and the specific experimental steps are as follows:

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

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

[0063] Table 1 Sequence information of Si-RNA

[0064]

[0065] (2) After cell counting, 2000 cells / well were inoculated in a 96-well cell culture plate, 6 replicate wells were set for each group, and the final volume of complete medium in each well was 200 μl. After inoculation, the 96-well plate was placed in a cell incubator at 37°C, 5% CO2, and the medium was aspirated at 0, 24, 48, 72, and 96 hours after inoculation. CCK8 reagent was mixed with DMEM medium at a ratio of 1:9, and the final volume in each well was 100 μl. The above 96-well plates were added, and incubated at 37°C for 2 hours. The absorbance value of each sample well at 450 nm was determined by a microplate reader. After all time points were determined, the incubation time was taken as the X-axis and the absorbance value at 450 nm as the Y-axis, and a line graph was drawn 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 was interfered. Figure 4 B).

[0066] (3) After cell counting, 2000 cells / well were inoculated in a low-adhesion 6-well cell culture plate and placed in a cell incubator at 37°C, 5% CO2. Dynamic observation was performed until day 10, and the number and size of cell spheres were observed under an optical microscope to reflect the self-renewal ability of cells. The results showed that Circ-CDYL overexpression significantly promoted the self-renewal ability of HCC, and the effect of Circ-CDYL on promoting the self-renewal ability of HCC was partially inhibited after HRNR expression was down-regulated Figure 4 C).

[0067] (4) Cell lines were inoculated in 6-well cell culture plates at 1000, 2000, and 3000 cells / well, respectively, with 3 replicate wells in each group, and were placed in a cell incubator at 37°C, 5% CO2. Dynamic observation was performed until day 14, and then 4% polyformaldehyde was used for fixation for 10 min, followed by crystal violet staining for 5 min. The cloning formation was recorded by taking pictures. The results showed that Circ-CDYL overexpression significantly promoted the monoclone formation ability of HCC, and the effect of Circ-CDYL on promoting the monoclone formation ability was partially inhibited after HRNR expression was down-regulated Figure 4 D).

[0068] 2, The interaction between Circ-CDYL and HRNR induces PD-L1 + Exosomes inhibit the effect of anti-PD-L1 immunotherapy

[0069] The interaction between Circ-CDYL and HRNR can promote the expression of PD-L1 and induce the production of PD-L1 + exosomes, thereby limiting the response of anti-PD-L1 immunotherapy in vivo and in vitro; and targeted inhibition of HRNR can eliminate the inhibitory effect of Circ-CDYL-HRNR interaction on anti-PD-L1 immunotherapy, as follows:

[0070] (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.

[0071] (2) In HCC cell lines that stably overexpress Circ-CDYL, si-RNA 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 a solution of 1×10⁻⁶ 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.

[0072] (3) As described above, HRNR expression was inhibited in HCC cell lines overexpressing Circ-CDYL and negative control cell lines, respectively, 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 by interacting with HRNR. At the same time, overexpression of Circ-CDYL could increase the expression of PD-L1 in HCC cells, and HRNR interference partially reversed the high expression of PD-L1 Figure 5 C). The above results show that the interaction between Circ-CDYL and HRNR regulates the expression of PD-L1 through the mTOR-p70s6k signaling pathway, thereby affecting the mechanism of HCC immunotherapy response.

[0073] (4) The above cell lines were inoculated into 15 cm cell culture dishes at 2*10 7 cells / dish, and 16 ml of DMEM containing 10% exosome-free fetal bovine serum was added. After 48 hours of culture, exosomes were purified from the cell supernatant by ultracentrifugation, and the specific steps were as follows: a) collect the supernatant; 300g, 4°C, centrifuge for 10 minutes, take the supernatant and discard the precipitate; centrifuge the supernatant at 2000g, 4°C for 20 minutes, take the supernatant and discard the precipitate; centrifuge the supernatant at 12000g, 4°C for 30 minutes, take the supernatant and discard the precipitate; c) centrifuge the supernatant at 120000g, 4°C for 90 minutes, and the precipitate is the exosome.

[0074] The exosomes purified from the culture medium were incubated with 0.5% bovine serum albumin (BSA, GIBCO) in PBS for 30 minutes to block non-specific antigens. Then the purified exosomes were incubated with biotin-labeled anti-CD63 monoclonal antibody at 4°C in the dark overnight (353018, Biolegend, San Diego, CA). Then streptavidin-coated magnetic beads were added, and after incubation at room temperature for 2 hours, they were incubated with fluorescein-conjugated monoclonal anti-PD-L1 antibody (557924, BD Pharmingen, New Jersey) or isotype control antibody (555749, BD Pharmingen, New Jersey) at 4°C overnight. The samples were analyzed using a Beckman MoFlo XDP TM flow cytometer (Beckman Coulter, USA). The positive threshold was set at <2% positive in the negative control. The results are expressed as relative fluorescence value (%). The results show that HCC cells overexpressing Circ-CDYL produce higher levels of PD-L1 + exosomes, PD-L1 + exosomes correspondingly decreased after HRNR expression was inhibited Figure 5D). This indicates that the interaction of Circ-CDYL with HRNR induces PD-L1 + Exosome production thus weakens the effect of anti-PD-L1 immunotherapy.

[0075] The above detailed description merely illustrates the specific implementation of the present application, but it is only an example, and the present application is not limited to the above described specific implementation. Any equivalent modification and substitution of the present application for those skilled in the art is also within the scope of the present application. Therefore, any equivalent transformation and modification made without departing from the spirit and scope of the present application should be covered within the scope of the present application.

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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