Application of 18S rRNA m7G modified inhibitor in sensitization of PD1 in treatment of liver cancer

Abstract

The invention relates to an application of an 18S rRNA m7G modified inhibitor in sensitization of PD1 in treatment of liver cancer. Through a large number of in-vivo and in-vitro experiments, it is clear that WBSCR22-mediated 18S rRNA m7G modification serves as a cancer promoting factor of liver cancer, and high expression and high modification level of the WBSCR22-mediated 18S rRNA m7G modification are closely related to tumor metastasis and poor prognosis. Through targeting WBSCR22 mediated 18S rRNA m7G modification, tumor migration can be inhibited, an immune microenvironment can be remodeled, generation of mtROS is promoted, and the expression level of inflammatory factors is improved, so that I-type interferon response caused by a cGAS-STING signal channel is activated, and the curative effect of anti-PD-1 treatment is remarkably improved. The invention has important practical significance for solving the problems of clinical curative effect difference and prognosis evaluation blank among individuals and better realizing accurate treatment. A new drug treatment target is provided for human to treat liver cancer, so that a new direction is provided for subsequent drug research and development, clinical treatment and the like, and extremely high social value and market application prospects are achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of 18S rRNA m7G modification inhibitors in enhancing the sensitization of PD1 in liver cancer treatment. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide, and its treatment faces the dual challenges of high postoperative recurrence rates and limited efficacy of advanced-stage treatments. In recent years, immune checkpoint inhibitors (ICIs), represented by PD-1 antibodies, have become a significant breakthrough in the treatment of liver cancer. From postoperative adjuvant therapy to advanced systemic therapy, PD-1 antibodies have demonstrated significant clinical value at different stages.

[0003] In adjuvant therapy, PD-1 antibodies can effectively reduce the risk of recurrence after radical surgery, especially in VETC-positive patients (5-year OS rate increased to 36%, HR=0.48), and short-term (<6 months) regimens show efficacy comparable to long-term regimens. In first-line treatment of advanced disease, PD-1 antibody combination strategies have replaced traditional targeted therapy as the new standard: targeted immunotherapy combination regimens (such as fenolinumab + bevacizumab) have extended median OS to 22.1 months (55% higher than sorafenib monotherapy), and dual immunotherapy blocking regimens (nivolumab + ipilimumab) have further increased the objective response rate to 36.1%. For PD-1 / L1 resistant patients, the novel TIGIT / PD-1 dual antibody regimen has achieved an objective response rate of 17.2% and a median PFS of 4.4 months in second-line treatment, providing a new option for patients with primary resistance.

[0004] However, this field still faces multiple challenges. The primary issue is the lack of a precise biomarker system: while VETC vascular patterns can predict adjuvant therapy benefit (HR=0.48 in positive patients), negative patients show no significant response; PD-1-positive patients have an ORR of approximately 35%, while negative patients still have a 10% response rate; dynamic monitoring of circulating tumor DNA (ctDNA) (e.g., decreased PIK3CA / FGFR2 mutations) is associated with efficacy, but a standardized testing procedure has not yet been established. The complexity of drug resistance mechanisms restricts the improvement of efficacy. The "immune desertification" phenotype caused by Wnt / β-catenin pathway activation and the suppressive immune microenvironment (Treg / MDSCs infiltration and TGF-β overexpression) constitute the main causes of primary drug resistance, while epigenetic regulation mediates acquired drug resistance. Safety management also presents potential risks. The incidence of grade III-IV hepatotoxicity with PD-1 monotherapy is 5-8%, while the incidence of grade III or higher adverse events with combination therapy (such as atezolizumab + bevacizumab) is as high as 41%. Special attention should be paid to the risk of viral reactivation and bleeding in special populations (Child-Pugh B, portal vein tumor thrombosis or HBV / HCV infected individuals).

[0005] Future development of HCC treatment will focus on three main areas: First, developing next-generation immune checkpoint inhibitors (such as LAG-3 / TIM-3 targeted drugs) and Fc-silencing antibodies (domvanalimab) to enhance anti-tumor activity and reduce toxicity; second, optimizing combination therapy strategies, exploring a triple therapy model of PD-1 antibody + anti-angiogenic drugs + local therapy (TACE / radiotherapy), and implementing sequential therapy based on dynamic ctDNA monitoring; and third, constructing multi-dimensional predictive models that integrate VETC genotyping, ctDNA evolution profiles, and immune microenvironment characteristics to achieve personalized medication, while establishing dose adjustment pathways for specific populations. Solving these problems will drive liver cancer immunotherapy towards precision and efficiency, providing patients with more sustainable survival benefits. Therefore, it is urgent to identify molecules that inhibit immunotherapy during the development and progression of HCC, clarify potential inhibitory mechanisms, develop effective immunotherapy methods, and improve the prognosis of HCC patients. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the targeted therapy and immunotherapy for liver cancer in the prior art, and to clarify that 18S rRNA m7G modification is a key reason for the occurrence and development of liver cancer and immunotherapy tolerance; by assessing the level of 18S rRNA m7G modification in patients, the prognosis of patients after immunotherapy can be effectively evaluated; in addition, inhibiting 18S rRNA m7G modification helps to enhance the therapeutic effect of anti-PD-1 antibody and improve the prognosis of patients.

[0007] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution.

[0008] The first aspect of this invention provides the use of 18S rRNA m7G modification inhibitors in the preparation of drugs that improve the sensitivity of liver cancer to immunotherapy.

[0009] Preferably, the 18S rRNA m7G modification inhibitor is selected from shRNAs that can specifically reduce the level of m7G-modified 18S rRNA.

[0010] Preferably, the shRNA is selected from one or more of sh#1 (sequence as shown in SEQ ID NO: 1, 5'-GCCAAAGCAAAGAAATTCTAC-3') and sh#2 (sequence as shown in SEQ ID NO: 2, 5'-GGAGGTTCCCATTAAGGATGT-3').

[0011] Preferably, the immunotherapy is anti-PD-1 antibody therapy.

[0012] The second aspect of this invention provides the use of 18S rRNA m7G inhibitors and anti-PD-1 antibodies in the preparation of medicaments for treating liver cancer.

[0013] Preferably, the 18S rRNA m7G modification inhibitor is selected from shRNAs that can specifically reduce the level of m7G-modified 18S rRNA.

[0014] Preferably, the shRNA is selected from one or more of sh#1 and sh#2.

[0015] A third aspect of the present invention provides a pharmaceutical composition for treating liver cancer, comprising an 18S rRNA m7G modification inhibitor and an anti-PD-1 antibody.

[0016] Preferably, the 18S rRNA m7G modification inhibitor is selected from shRNAs that can specifically reduce the level of m7G-modified 18S rRNA.

[0017] Preferably, the shRNA is selected from one or more of sh#1 and sh#2.

[0018] Preferably, the pharmaceutical composition may optionally contain a pharmaceutically acceptable carrier.

[0019] Preferably, the pharmaceutically acceptable carrier includes one or more of fillers, disintegrants, binders, lubricants, flavoring agents, preservatives, antioxidants, and colorants.

[0020] The fourth aspect of this invention provides the use of a reagent for detecting 18S rRNA m7G modification levels in the preparation of products for predicting the efficacy and / or prognostic assessment of immunotherapy for liver cancer.

[0021] Preferably, the reagent for detecting the 18S rRNA m7G modification level includes primers for detecting the 18S rRNA level and / or reagents for detecting the m7G level.

[0022] Preferably, the primers for detecting 18S rRNA levels are selected from the following primer pairs:

[0023] The upstream sequence of the primer pair is shown in SEQ ID NO: 3 (5'-TGAGAAGACGGTCGAACTTGACT-3'), and the downstream sequence is shown in SEQ ID NO: 4 (5'-GCATTGTTCCAAAGGCATCCACT-3').

[0024] Preferably, the reagent for detecting m7G levels is selected from anti-m7G antibodies; for example, RN017M from MBL can be selected.

[0025] Preferably, the immunotherapy is anti-PD-1 antibody therapy.

[0026] It should be understood that, unless otherwise specified, in the context of this invention, the 18S rRNA m7G modification inhibitor refers to a substance capable of specifically downregulating the level of 18S rRNA m7G modification and / or its activity. This downregulation can be direct or indirect. For example, WBSCR22, which introduces m7G at position 1639 of 18S rRNA to promote its m7G modification, as demonstrated in this invention, indirectly downregulates the level of 18S rRNA m7G modification by inhibiting the level of WBSCR22. The inhibition methods can be varied, such as using antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNA erasers, target masking, and / or multi-target methods to downregulate the level and / or activity of 18S rRNA m7G, as long as it can reduce the level and / or activity of 18S rRNA m7G. The primers and / or primer pairs refer to the primers used for analyzing the expression level of 18S rRNA m7G in PCR. In addition to the primers and / or primers listed in this invention, those skilled in the art are fully capable of designing corresponding primers and / or primer pairs based on the 18S rRNA sequence using conventional methods and techniques in the art, including but not limited to molecular biology, and screening the designed primers and / or primer pairs using conventional experimental methods, as long as they can specifically detect the 18S rRNA level; the 18S rRNA m7G protein level can also be detected using conventional reagents and methods in the art; the same applies to other genes / proteins.

[0027] Ribosome (rRNA) biogenesis is a fundamental cellular process regulated at multiple levels, including rRNA transcription, precursor rRNA processing, and assembly with ribosomal proteins into mature macromolecular ribonucleoprotein complexes. Because tumor cells are characterized by nucleolar enlargement during ribosomal biogenesis and require higher protein levels than normal cells, in-depth research into the mechanisms of these processes is crucial for developing beneficial intervention strategies for cancer treatment. Although overactivation of ribosomal biogenesis plays a key role in supporting oncogenic protein synthesis and stress responses, current technologies lack a deep understanding of how this process influences cancer progression. Ribosomal RNA modification has become a key regulator of translation and cancer progression; however, how dysregulated rRNA modifications at different sites maintain tumor cell survival remains a mystery. This invention, through extensive research, has discovered that WBSCR22, a conserved transspecies methyltransferase initially identified as a gene-deleted methyltransferase in Williams-Beuren syndrome, plays an important and highly conserved role in translation, catalyzing the addition of n7-methylguanosine (m7G) at position 1639 of 18S rRNA, and is highly expressed in various tumors. Although ICBs provide an ideal window for HCC treatment and have become one of the main treatment options, their response rate is limited due to the immunosuppressive nature of the liver tumor microenvironment. This invention observed significantly upregulated expression levels of inflammatory factors, including IL6, CCL5, ISG15, and OAS1, in WBSCR22 knockdown HCC, in addition to increased mtROS production. Specifically, silencing WBSCR22 activated the type I interferon response induced by the cGAS-STING signaling pathway, which can be blocked by MitoQ treatment to eliminate mtROS. Therefore, overexpression of WBSCR22 may confer a growth advantage to HCC in terms of anti-ferrode deposition and immune evasion. Considering the key role of cGAS-STING in promoting lymphocyte infiltration in solid tumors and CD8... + This invention explores the effect of T cell-induced tumor cell ferroptosis and discovers that targeting WBSCR22 can significantly improve the efficacy of anti-PD-1 therapy in a mouse liver cancer model. Notably, specific knockout of WBSCR22 has minimal impact on the physiological state of mice and normal hepatocytes, indicating that WBSCR22-mediated 18S rRNA m7G modification is a potential and safe therapeutic target with no adverse effects on normal tissues.

[0028] In summary, this invention, through extensive in vitro and in vivo experiments, reveals that HCC exhibits a tumor-promoting effect dependent on WBSCR22-mediated 18S rRNA m7G modification, deepening our understanding of the interaction between rRNA epigenetic control of mRNA translation and ferroptosis defense. WBSCR22-mediated 18S rRNA m7G modification, as a tumor-promoting factor in liver cancer, is closely associated with tumor metastasis and poor prognosis due to its high expression and modification levels. Mechanistically, WBSCR22 weakens the anti-tumor immune response by mediating 18S rRNA m7G modification. Targeting WBSCR22-mediated 18S rRNA m7G modification not only inhibits tumor migration but also reshapes the immune microenvironment, promotes mtROS generation, and increases the expression levels of inflammatory factors including IL6, CCL5, ISG15, and OAS1, thereby activating the type I interferon response induced by the cGAS-STING signaling pathway and significantly enhancing the efficacy of anti-PD-1 therapy. This invention reveals the association between WBSCR22-mediated 18S rRNA m7G modification and the development and progression of liver cancer, as well as anti-PD-1 antibody therapy. This finding has significant practical implications for addressing the challenges of inter-individual variability in clinical efficacy and the lack of prognostic assessment, thereby enabling more precise treatment. It provides a novel drug target for conquering liver cancer, offering a new direction for subsequent drug development and clinical treatment, and possesses immense social value and market application prospects.

[0029] The present invention has the following advantages over the prior art:

[0030] (1) Bioinformatics analysis and clinical sample detection of the present invention revealed that WBSCR22 was significantly highly expressed in liver cancer tissue and liver metastases. Its high expression was significantly positively correlated with poor prognosis of liver cancer patients. High expression of WBSCR22 and its mediated 18S rRNA m7G modification can significantly promote liver cancer cell metastasis, which can be used to assess the metastasis risk and prognosis of liver cancer patients and guide the formulation of treatment plans.

[0031] (2) This study, through a series of in vivo and in vitro experiments, clarified that WBSCR22-mediated 18S rRNA m7G modification can inhibit immune infiltration and reduce the therapeutic efficiency of immune checkpoint inhibitors. Targeting WBSCR22 with 18S rRNA m7G modification can improve the efficacy of immunotherapy, providing effective support for the treatment of liver cancer. This invention, by revealing the correlation between WBSCR22-mediated 18S rRNA m7G modification and liver cancer immunotherapy, has significant practical implications for achieving precision medicine. This invention provides a new drug target for conquering liver cancer, offers new immunotherapy strategies and scientific basis for the treatment and prognostic assessment of liver cancer, and provides a new direction for subsequent drug development and clinical treatment, possessing extremely high social value and market application prospects. Attached Figure Description

[0032] Figure 1 This diagram illustrates the differences in WBSCR22 mRNA expression between different types of tumor tissues and surrounding tissues.

[0033] Figure 2 This is a schematic diagram of the Kaplan-Meier survival analysis results for patients (n=365) with different WBSCR22 expressions in the TCGA-LIHC dataset.

[0034] Figure 3 This is a schematic diagram showing the results of gene dependence analysis of WBSCR22 in different tumor cell lines based on the DepMap dataset.

[0035] Figure 4 This is a schematic diagram showing the results of WBSCR22 expression level analysis in different hepatocellular carcinoma cells.

[0036] Figure 5 This is a schematic diagram showing the results of IP-qRT-PCR analysis of 18S rRNA levels in normal THLE-2 and HCC cell lines.

[0037] Figure 6 This is a schematic diagram of the IHC analysis results of clinical samples of liver cancer (scale bar: top image is 200μm; bottom image is 100μm).

[0038] Figure 7 This is a schematic diagram showing the efficiency of shRNA knockdown of WBSCR22.

[0039] Figure 8 This is a schematic diagram illustrating the effects of WBSCR22 knockdown on the levels of inflammation-related genes in HCC using gene enrichment analysis (GSEA).

[0040] Figure 9This is a schematic diagram illustrating the effect of knocking down WBSCR22 on the expression levels of inflammatory factors in liver cancer cells.

[0041] Figure 10 This is a schematic diagram showing the effect of knocking down WBSCR22 on the phosphorylation levels of STING, TBK1, and IRF3.

[0042] Figure 11 A schematic diagram (200 μm) showing the effect of IHC analysis on WBSCR22 protein expression level and STING S366 phosphorylation level on clinical liver cancer samples.

[0043] Figure 12 This is a schematic diagram showing the correlation analysis results of WBSCR22 and STING pS366 protein expression in clinical samples of liver cancer (n=90).

[0044] Figure 13 This is a schematic diagram showing the results of the analysis of WBSCR22 expression levels in patients who received immune checkpoint blockade (ICB) therapy.

[0045] Figure 14 A morphological diagram illustrating the effect of WBSCR22 knockout combined with anti-PD-1 antibody therapy on liver volume in mice with hepatocellular carcinoma.

[0046] Figure 15 This diagram illustrates the changes in liver mass and liver / body weight ratio in mice with hepatocellular carcinoma after WBSCR22 knockout combined with anti-PD-1 antibody therapy.

[0047] Figure 16 This is a schematic diagram of HE staining and IHC analysis results of liver cancer tumor tissue from mice (scale bar: HE image: 200 μm; IHC image: 50 μm).

[0048] Figure 17 This is a schematic diagram showing the quantitative analysis results of tumor number and maximum tumor diameter in HE staining of liver cancer mouse tumor tissue.

[0049] Figure 18 This is a schematic diagram showing the quantitative analysis results of the expression levels of Ki67 and 4-HNE in IHC analysis of liver cancer mouse tumor tissue.

[0050] Figure 19 To investigate the activity of NK cells and CD8+ cells in liver cancer mouse tumor tissue. + Schematic diagram of quantitative analysis results of T cells, TNFα, and IFNγ. Detailed Implementation

[0051] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0052] Unless otherwise specified, the cell lines listed in this invention, including HUH7 and SNU449, were purchased from the Manassas Culture Collection (USA) and cultured according to existing techniques. All cell lines were identified by short tandem repeat analysis at the China Center for Type Culture Collection (Wuhan), and the presence of mycoplasma contamination was verified using a PCR detection kit (Shanghai Biothrive Sci). They were also cryopreserved in liquid nitrogen for subsequent experiments. All reagents used in this invention were commercially available.

[0053] In this invention, informed consent was obtained from all patients using clinical samples. The relevant procedures and methods were approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University, complying with medical ethics requirements and the Good Clinical Practice (GCP) for drug clinical trials. All experimental procedures adhered to the Declaration of Helsinki. The experimental methods used in this invention, such as bioinformatics analysis, molecular biology experiments, cell biology experiments, immunohistochemistry, and animal experiments, are all conventional methods and techniques in the field. Representative results from repeated biological experiments are presented in the accompanying figures, with data displayed as mean ± SD and mean ± SEM as specified in the figures. All in vitro experiments were repeated at least three times, and animal experiments were repeated twice. Data were analyzed using GraphPad Prism 8.0 software. Conventional medical statistical methods such as t-tests, chi-square tests, and analysis of variance were used to compare the differences in means between two or more groups. *p < 0.05 was considered a significant difference.

[0054] Example 1

[0055] To determine the potential function of 18S rRNA m7G modification in cancer, WBSCR22 expression was first evaluated in the Cancer Genome Atlas (TCGA) database. The results showed that WBSCR22 was significantly upregulated in most common cancers, including liver cancer (see [link to relevant documentation]). Figure 1 Survival analysis showed that HCC patients with high WBSCR22 expression had a poorer prognosis (see [link to study]). Figure 2 This indicates that WBSCR22 plays a crucial role in promoting HCC development. To investigate whether WBSCR22 is also critical for maintaining cancer cell survival, DepMap analysis was used on most tumor cell lines, including HCC. The results showed that the dependence score was below -1, indicating a significant correlation between WBSCR22 and cancer cell survival (see [link to DepMap analysis]). Figure 3Next, the protein level of WBSCR22 was detected in various hepatocellular carcinoma cell lines using Western blot. The specific steps are as follows:

[0056] (1) Digest and collect liver cancer cells, add lysis buffer, and lyse on ice for 1 hour.

[0057] (2) Centrifuge at 15000×g at 4℃ for 15 min, take the supernatant and add loading buffer (to 1×), then incubate at 95℃ for 5 min.

[0058] (3) Take the sample prepared in step (2) and perform protein gel electrophoresis.

[0059] (4) After electrophoresis, the protein gel was transferred to a membrane (PVDF membrane, 200mA constant current transfer for 2h).

[0060] (5) After the transfer is completed, the PVDF membrane is removed and placed in the sealing solution and sealed at room temperature for 2 hours on a vertical shaker at 10 rpm.

[0061] (6) After sealing, clean the PVDF membrane, immerse it in the primary antibody, and incubate it overnight on a vertical shaker at 10 rpm and 4°C.

[0062] (7) After the primary antibody incubation is complete, wash the PVDF membrane and then immerse it in the secondary antibody (HRP-linked anti-rabbit IgG) and incubate it at room temperature for 2 hours at 10 rpm on a vertical shaker.

[0063] (8) After the secondary antibody incubation is completed, the PVDF membrane is washed and the target protein is detected using a chemiluminescence analyzer (α-tubulin is used as an internal control).

[0064] Test results as follows Figure 4 As shown, the results are consistent with the DepMap analysis, indicating that the expression level of WBSCR22 is significantly higher in liver cancer cells.

[0065] Subsequently, the intracellular 18S rRNA m7G modification level was analyzed using immunoprecipitation-qPCR. The specific steps were as follows: 2 μg of total RNA was mixed with 4 μg of anti-m7G antibody (MBL, RN017M) or anti-IgG antibody (Biolegend) and 1 mg of Protein Abeads (Invitrogen, 1001D) in 500 μL of IP buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% NP-40) containing 10 U RNase inhibitor, and incubated at 4°C for 4 hours. After washing three times with IP buffer, the RNA was resuspended in 200 μL of IP buffer. Finally, the m7G-containing RNA component was purified by elution with TRIzol reagent. qRT-PCR analysis was performed on m7G-enriched RNA and total RNA using 18S rRNA primers (the upstream sequence of the primer pair is shown in SEQ ID NO: 3 (5'-TGAGAAGACGGTCGAACTTGACT-3'), and the downstream sequence is shown in SEQ ID NO: 4 (5'-GCATTGTTCCAAAGGCATCCACT-3')). The analytical results are as follows: Figure 5 As shown in the figure. The results showed that the level of m7G-modified 18S rRNA was significantly increased in the HUH7 and SNU449 cell lines.

[0066] Furthermore, tumor tissue samples were obtained from liver cancer patients at the First Affiliated Hospital of Sun Yat-sen University (n=90). The inclusion criteria for patients were as follows: (1) age 18 years and above (including 18 years), both male and female were acceptable; (2) patients were confirmed by histology or pathology to have liver cancer, and stages I-IV were acceptable; (3) paraffin sections were prepared from fresh tumor biopsy tissue; (4) expected survival was greater than or equal to 3 months. Immunohistochemical (IHC) analysis was performed on them, and the specific steps were as follows:

[0067] (1) Sample fixation and embedding: After the tissue sample obtained from the biopsy is fixed, it is embedded in paraffin and cut into white slices for later use.

[0068] (2) Baking the slides: Place the paraffin slides in a 60°C oven and bake for about 2 hours to ensure that the slides adhere firmly to the glass slides.

[0069] (3) Dewaxing and hydration: The sections were immersed in xylene I and xylene II for 10 minutes each, and then treated in anhydrous ethanol I, anhydrous ethanol II, 90% ethanol, 80% ethanol and 70% ethanol for 5 minutes each. Finally, they were washed three times with PBS for 3 minutes each time.

[0070] (4) Antigen retrieval: Immerse the slide in EDTA antigen retrieval solution and heat it in a microwave oven on high for 15 minutes to perform antigen retrieval. After cooling naturally to room temperature, wash with PBS three times for 3 minutes each time.

[0071] (5) Organize the circle: Use the grouping pen to organize the circle, ensuring that the circle is complete for subsequent operations.

[0072] (6) Endogenous enzyme blocking: Place the slide in a humidified chamber, add 3% hydrogen peroxide to cover the tissue, and incubate at room temperature for 20 minutes to remove endogenous peroxidase. Then wash with PBS three times for 3 minutes each time.

[0073] (7) Blocking treatment: Add goat serum blocking solution to cover the tissue, place it in a humidified box and incubate at room temperature for 30 minutes to reduce non-specific binding of the primary antibody.

[0074] (8) Primary antibody incubation: After removing the blocking solution, add primary antibody to cover the tissue and place it in a humidified chamber for overnight incubation at 4°C (determine the primary antibody concentration according to the antibody instructions).

[0075] (9) Washing and warming: On the second day, the slides were removed from the wet box, warmed to room temperature for 30 minutes, and then washed three times with PBS for 3 minutes each time.

[0076] (10) Secondary antibody incubation: Add biotin-labeled secondary antibody, incubate at room temperature for 30 minutes, and then wash three times with PBS for 3 minutes each time.

[0077] (11) DAB staining: Add DAB solution for staining, observe the staining effect under a microscope, control the staining time to 3–10 minutes, and terminate the reaction with tap water when the staining is appropriate.

[0078] (12) Counterstaining and differentiation: After counterstaining with hematoxylin for 3 minutes, place in water to stop the staining; then add 1% hydrochloric acid alcohol for differentiation for 3 seconds, and rinse with tap water for 3 minutes. Rinse in running water for 30 minutes until the color is stable, in order to perform the reverse blue treatment.

[0079] (13) Gradient dehydration and mounting: The slides were immersed in 70%, 80%, 90%, anhydrous ethanol No. 1, anhydrous ethanol No. 2, xylene No. 1 and xylene No. 2 in sequence for 5 minutes each time to complete the dehydration treatment, and finally mounted for observation.

[0080] Two independent researchers scored the clinicopathological immunohistochemical slides of the patients without prior knowledge of their condition. The staining score was determined based on staining intensity and the proportion of positive cells. Staining intensity was scored as follows: 0 (no staining), 1 (light), 2 (moderate), and 3 (strong); the proportion of positive cells was scored as follows: 1 (<25%), 2 (25-50%), 3 (50-75%), and 4 (75-100%). The final score was obtained by multiplying the staining intensity by the proportion of positive cells. The results are shown below. Figure 6 As shown (due to content limitations, only a portion of the detection results are randomly displayed in this invention). The results revealed that patients with high WBSCR22 expression were significantly associated with advanced pathological grade and advanced cancer stage (T3-T4 classification, stage III-IV disease), and had lower overall survival than patients with low expression (see...). Figure 6 The above results collectively indicate that the level of 18S rRNA m7G modification plays a crucial role in the occurrence and development of HCC.

[0081] Example 2

[0082] The foregoing examples demonstrate the upregulation of WBSCR22 in HCC. To clarify the crucial role of WBSCR22 expression levels in liver cancer immunotherapy, overexpression plasmids of shRNAs designed to inhibit intracellular WBSCR22 expression levels were first developed, and their respective activities were verified (shNC was used as a blank control). The shRNA construction steps are as follows:

[0083] (1) Design shRNA knockdown sequences on the Sigma website, design and synthesize primers based on the hairpin structure of shRNA, dilute the synthesized shRNA primers with water to 100 μM, take 10 μL of each of the upstream and downstream primers, mix them evenly, and use a PCR instrument to cool down from 100℃ to room temperature and anneal to synthesize double strands; In this example, two shRNAs were used as examples, namely shW22-1 (sh#1) (the sequence is shown in SEQ ID NO: 1, which is 5'-GCCAAAGCAAAGAAATTCTAC-3') and shW22-2 (sh#2) (the sequence is shown in SEQ ID NO: 2, which is 5'-GGAGGTTCCCATTAAGGATGT-3').

[0084] (2) The pLKO.1 lentiviral vector was digested with EcoRI and AgeI restriction enzymes. After digestion, two single bands were obtained by gel running. The larger band was cut and recycled. Then, the annealed double strands were ligated into the vector after gel recycling.

[0085] The liver cancer cells were then transfected, and the specific steps are as follows:

[0086] (1) Logarithmic growth phase 293T cells were seeded in 10cm culture dishes at a seeding density of 60%-70%; 6000ng of target plasmid loaded with shW22-1, shW22-2 or shNC, 4500ng of PSPAX2 and 1500ng of PMD2.G were transfected into 293T cells and cultured for 48h.

[0087] (2) After 48 hours, collect the culture medium and filter out the cell debris with a 0.45 μm filter to obtain the virus solution, which is then aliquoted and stored at -80℃.

[0088] (3) One day before infection, the logarithmic growth phase liver cancer cells (HUH7, SNU449) to be infected were inoculated into a 6cm culture dish, the original culture medium was discarded, 3mL of virus solution and 3mL of fresh culture medium were added, and 6ul of polybrene was added at the same time to promote virus infection (the ratio of polybrene to culture medium was 1:1000). After 24 hours, the virus solution was discarded and fresh culture medium was replaced to continue the culture.

[0089] (4) Discard the culture medium 48 hours after infection and add a culture medium with an appropriate concentration of puromycin. At this time, cells that have successfully integrated the exogenous gene are resistant to puromycin and can survive. After continuous screening for 7-10 days, a stable cell line can be obtained. The expression level of WBSCR22 can be detected by the aforementioned WB method to determine whether the knockdown was successful. The detection results are as follows: Figure 7 As shown, the results indicate that both shRNAs designed according to this invention can significantly inhibit WBSCR22 and reduce the expression level of WBSCR22 protein in liver cancer cells.

[0090] Gene enrichment analysis (GSEA) was used to study its transcriptomic information, and the results showed that knockdown of WBSCR22 significantly upregulated the levels of genes related to the inflammation response in HCC (see [link to study]. Figure 8 Subsequently, HUH7 cells were used for qRT-PCR analysis. The specific steps are as follows:

[0091] (1) Collect HUH7 cells in the logarithmic growth phase into 1.5 mL EP tubes after digestion. Add NucleoZol (740404.200, biofive) to each sample for cell lysis, and then add ddH2O at a ratio of 1 mL: 200 µL (NucleoZol: ddH2O). Centrifuge the lysis buffer at 12,000 rpm for 15 minutes, and transfer the supernatant to a fresh tube. Then add an equal volume of isopropanol, and centrifuge again at 12,000 rpm for 10 minutes. Discard the supernatant, wash the RNA twice with 500 µL of 75% ethanol, centrifuging at 8000 rpm for 3 minutes each time. Resuspend the RNA pellet in 50-100 µL of ddH2O.

[0092] (2) RNA concentration was detected using NanoDrop 2000. Reverse transcription was performed using the TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit. qPCR was performed using the PerfectStart Green qPCR SuperMix kit. After the reaction was completed, the mRNA expression levels of IL6, CCL5, ISG15, and OAS1 were calculated based on the CT values ​​of each reaction well, with GAPDH as an internal control. The primer pair sequences for detecting the expression levels of each validation factor are as follows: IL6: The upstream sequence of the primer pair is shown in SEQ ID NO: 5 (5'-ACTCACCTCTTCAGAACGAATTG-3'), and the downstream sequence is shown in SEQ ID NO: 6 (5'-CCATCTTTGGAAGGTTCAGGTTG-3'); CCL5: The upstream sequence of the primer pair is shown in SEQ ID NO: 7 (5'-CCAGCAGTCGTCTTTGTCAC-3'), and the downstream sequence is shown in SEQ ID NO: 8 (5'-CTCTGGGTTGGCACACACTT-3'); ISG15: The upstream sequence of the primer pair is shown in SEQ ID NO: 9 (5'-CGCAGATCACCCAGAAGATCG-3'), and the downstream sequence is shown in SEQ ID NO: 9 (5'-CGCAGATCACCCAGAAGATCG-3'). NO: 10 is shown (5'-TTCGTCGCATTTGTCCACCA-3'); OAS1: the upstream sequence of the primer pair is shown as SEQ ID NO: 11 (5'-TGTCCAAGGTGGTAAAGGGTG-3'), and the downstream sequence is shown as SEQ ID NO: 12 (5'-CCGGCGATTTAACTGATCCTG-3').

[0093] The results were consistent with the GSEA analysis above, indicating that inhibiting intracellular WBSCR22 expression significantly increased the levels of related inflammatory factors, with IL6, CCL5, ISG15, and OAS1 expression levels all significantly upregulated (see [link to GSEA analysis]). Figure 9This suggests that leakage of mitochondrial DNA (mtDNA) may lead to excessive mtROS production, which peroxidizes mitochondrial membrane lipids, thereby activating the cGAS-STING pathway. To verify this conclusion, Western blotting experiments were performed using HCC cells according to the aforementioned method. The results showed that the phosphorylation levels of STING, TBK1, and IRF3 were significantly upregulated when WBSCR22 was inhibited, while almost no changes were observed after the mitochondrial-targeted antioxidant mitoquinone (MitoQ) was applied (see [link to study]). Figure 10 This indicates that suppressing WBSCR22 can effectively activate the cGAS-STING signal.

[0094] Subsequently, tumor tissue samples (n=90) from liver cancer patients at the First Affiliated Hospital of Sun Yat-sen University were obtained and subjected to immunohistochemical (IHC) analysis according to the aforementioned experimental methods. The experimental results are as follows: Figure 11-12 As shown in the figure. The results showed a significant negative correlation between WBSCR22 protein expression level and STING S366 phosphorylation level, indicating that WBSCR22-mediated 18S rRNA m7G modification can weaken the body's anti-tumor immune response by inhibiting STING pathway activation (S366 phosphorylation), thereby promoting liver cancer progression, further supporting the above conclusions. In summary, these data suggest that targeting WBSCR22-mediated 18S rRNA m7G modification can trigger cGAS-STING activated inflammatory responses in HCC, and based on the tumor immune surveillance function of the cGAS-STING signaling pathway, it may have a synergistic effect with immune checkpoint blockade (ICB) therapy.

[0095] Example 3

[0096] During tumor immunotherapy, CD8 + T cells promote ferroptosis in cancer cells by secreting IFNγ and remodeling fatty acid metabolism. Furthermore, recent studies have shown that abnormal mtDNA released into the cytoplasm due to mitochondrial damage can strongly activate the cGAS-STING signaling pathway and type I interferon responses. A cohort analysis of patients receiving immune checkpoint blockade (ICB) therapy revealed that WBSCR22 expression was significantly higher in the exhausted non-responder group than in the responder group (see [link to study]). Figure 13 Based on this, it can be inferred that targeting WBSCR22-mediated 18S rRNA m7G modification may synergistically enhance the antitumor activity of ICB therapy.

[0097] To verify the above conclusions, a PD-1 blockade experiment was conducted using a mouse model of liver cancer. The specific steps are as follows:

[0098] (1) Four-week-old C57BL / 6J mice were used to construct a liver-specific knockout of WBSCR22 by introducing AAV 2 / 8-hTBG-iCre virus (WBSCR22). fl / wt +AAV2 / 8-hTBG-iCre (cKO) C57BL / 6 mouse model, while constructing a control group (WBSCR22) wt / wt +AAV2 / 8-hTBG-iCre (Control) model. The cKO group mice were randomly divided into two groups, designated as group 1 and group 2, and the Control group mice were randomly divided into two groups, designated as group 3 and group 4, with 5 mice in each group.

[0099] (2) On the 21st day after tumor formation, mice in each group were treated. Groups 1 and 3 were treated with anti-PD-1 antibody (Selleck, A2122), and groups 2 and 4 were treated with an equal volume of anti-IgG antibody. The treatment was carried out once every 3 days for a total of 4 times.

[0100] (3) On day 35, the mice were sacrificed, the tumors and livers were removed and their weight was measured.

[0101] Experimental results are as follows Figure 12-13 As shown in the figure. The results showed that, compared with the control group, inhibiting WBSCR22 expression alone or administering anti-PD-1 antibody alone could effectively inhibit the progression of liver cancer; while knocking out WBSCR22 and combining it with anti-PD-1 antibody therapy could more significantly inhibit liver cancer growth, indicating that targeting WBSCR22-mediated 18S rRNA m7G modification can synergistically enhance the therapeutic activity of anti-PD-1 antibody against liver cancer (see...). Figure 14-15 ).

[0102] Subsequently, mouse tumor tissue was collected for HE staining and immunohistochemical analysis. The results are as follows: Figure 16-18 As shown in the figure. The results showed that the tumor lesions and diameter were significantly reduced in the WBSCR22 knockout combined with anti-PD-1 antibody treatment group. Immunohistochemical analysis showed that the expression level of Ki67 in the liver cancer tissue of the WBSCR22 knockout combined with anti-PD-1 antibody treatment group was the lowest, while the expression level of 4-HNE was the highest, indicating that the anti-tumor activity of WBSCR22 knockout combined with anti-PD-1 antibody was significantly better than that of single therapy.

[0103] To analyze the effect of WBSCR22 knockout on the recruitment of cytotoxic immune cells, flow cytometry was used to detect the interaction between tumor-infiltrating NK cells that directly target cancer cells and CD8+ cells. +T cells. Results showed that, compared to inhibiting WBSCR22 expression alone or administering anti-PD-1 antibody alone, the combination therapy of WBSCR22 knockout and anti-PD-1 antibody not only increased NK cells and CD8+, but also... + T cell infiltration also leads to CD8 + The significant increase in TNFα and IFNγ expression in T cells further activated CD8. + Effector functions of T cells (see...) Figure 19 In summary, it is clear that targeting WBSCR22-mediated 18S rRNA m7G modification can enhance ICB-mediated anti-tumor immunity by regulating tumor ferroptosis.

[0104] This invention, through extensive research, has clarified that WBSCR22-mediated 18S rRNA m7G modification is a factor highly relevant to hepatocellular carcinoma (HCC) immunotherapy, and WBSCR22 is significantly upregulated in most common cancers, including HCC. Survival analysis results show that HCC patients with high WBSCR22 expression have a poorer prognosis, and DepMap analysis results show that most tumor cell lines, including HCC, are significantly associated with WBSCR22. In vitro experiments show that the level of m7G-modified 18S rRNA is significantly increased in HCC cells. Tumor tissue analysis of HCC patients revealed that patients with high WBSCR22 expression were significantly associated with advanced pathological grade and advanced cancer stage (T3-T4 classification, stage III-IV disease), and had a lower overall survival rate than patients with low expression, indicating that the level of 18S rRNA m7G modification plays a key role in the occurrence and development of HCC. Regarding the regulation of immunotherapy, gene enrichment analysis (GSEA) was used to study its transcriptome information, and the results showed that WBSCR22 knockdown significantly upregulated the levels of genes related to inflammation response in HCC. Inhibiting the expression level of WBSCR22 in liver cancer cells significantly increased the levels of related inflammatory factors, with IL6, CCL5, ISG15, and OAS1 showing significant upregulation. This is speculated to be due to leakage of mitochondrial DNA (mtDNA), leading to excessive mtROS production and peroxidation of mitochondrial membrane lipids, thereby activating the cGAS-STING pathway. Western blot analysis of HCC cells validated these conclusions, showing that inhibition of WBSCR22 significantly upregulated the phosphorylation levels of STING, TBK1, and IRF3, while MitoQ treatment showed almost no change, indicating that inhibition of WBSCR22 effectively activates the cGAS-STING signaling pathway. Immunohistochemical analysis of clinical liver cancer tissue samples revealed a significant negative correlation between WBSCR22 protein expression levels and STING S366 phosphorylation levels. This indicates that WBSCR22-mediated 18S rRNA m7G modification can inhibit STING pathway activation (S366 phosphorylation), thereby weakening the body's anti-tumor immune response and promoting liver cancer progression, further supporting the above conclusions. In summary, these data suggest that targeting WBSCR22-mediated 18S rRNA m7G modification can trigger cGAS-STING activated inflammatory responses in HCC. Based on the tumor immune surveillance function of the cGAS-STING signaling pathway, it may synergize with immune checkpoint blockade (ICB) therapy.

[0105] To clarify whether targeting WBSCR22-mediated 18S rRNA m7G modification can synergistically enhance the anti-tumor activity of ICB therapy, a mouse model of hepatocellular carcinoma was constructed to conduct a PD-1 blockade experiment. The results showed that, compared to the control group, inhibiting WBSCR22 expression alone or administering anti-PD-1 antibody alone effectively inhibited hepatocellular carcinoma progression. Furthermore, combining WBSCR22 knockout with anti-PD-1 antibody therapy significantly inhibited hepatocellular carcinoma growth, indicating that targeting WBSCR22-mediated 18S rRNA m7G modification can synergistically enhance the therapeutic activity of anti-PD-1 antibodies against hepatocellular carcinoma. HE staining and immunohistochemical analysis of mouse tumor tissues revealed that the tumor lesions and diameter were significantly reduced in the WBSCR22 knockout combined with anti-PD-1 antibody treatment group. The hepatocellular carcinoma tissue in the WBSCR22 knockout combined with anti-PD-1 antibody treatment group showed the lowest Ki67 expression level and the highest 4-HNE expression level, indicating that the anti-tumor activity of WBSCR22 knockout combined with anti-PD-1 antibody therapy was significantly superior to single therapy. To analyze the effect of WBSCR22 knockout on the recruitment of cytotoxic immune cells, flow cytometry was used to detect the interaction between tumor-infiltrating NK cells that directly target cancer cells and CD8+ cells. + T cells. Results showed that, compared to inhibiting WBSCR22 expression alone or administering anti-PD-1 antibody alone, the combination therapy of WBSCR22 knockout and anti-PD-1 antibody not only increased NK cells and CD8+, but also... + T cell infiltration also leads to CD8 + The significant increase in TNFα and IFNγ expression in T cells further activated CD8. + The effector function of T cells can be clearly seen in the above. Targeting WBSCR22-mediated 18S rRNA m7G modification can enhance ICB-mediated anti-tumor immunity by regulating tumor ferroptosis.

[0106] In summary, this invention, through extensive in vitro and in vivo experiments, reveals that HCC exhibits a tumor-promoting effect dependent on WBSCR22-mediated 18S rRNA m7G modification, deepening our understanding of the interaction between rRNA epigenetic control of mRNA translation and ferroptosis defense. WBSCR22-mediated 18S rRNA m7G modification, as a tumor-promoting factor in liver cancer, is closely associated with tumor metastasis and poor prognosis due to its high expression and modification levels. Mechanistically, WBSCR22 weakens the anti-tumor immune response by mediating 18S rRNA m7G modification. Targeting WBSCR22-mediated 18S rRNA m7G modification not only inhibits tumor migration but also reshapes the immune microenvironment, promotes mtROS generation, and increases the expression levels of inflammatory factors including IL6, CCL5, ISG15, and OAS1, thereby activating the type I interferon response induced by the cGAS-STING signaling pathway and significantly enhancing the efficacy of anti-PD-1 therapy. This invention reveals the association between WBSCR22-mediated 18S rRNA m7G modification and the development and progression of liver cancer, as well as anti-PD-1 antibody therapy. This finding has significant practical implications for addressing the challenges of inter-individual variability in clinical efficacy and the lack of prognostic assessment, thereby enabling more precise treatment. It provides a novel drug target for conquering liver cancer, offering a new direction for subsequent drug development and clinical treatment, and possesses immense social value and market application prospects.

[0107] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. 18S rRNA m7G modification inhibitors in the preparation of drugs that improve the sensitivity of liver cancer to immunotherapy.

2. The application according to claim 1, characterized in that, The 18S rRNA m7G modification inhibitor is selected from shRNAs that can specifically reduce the level of m7G-modified 18S rRNA.

3. The application according to claim 1, characterized in that, The immunotherapy mentioned is anti-PD-1 antibody therapy. 4.18S rRNA m7G inhibitors and anti-PD-1 antibodies in the preparation of drugs for treating liver cancer.

5. The application according to claim 4, characterized in that, The 18S rRNA m7G modification inhibitor is selected from shRNAs that can specifically reduce the level of m7G-modified 18S rRNA.

6. The application according to claim 4, characterized in that, The immunotherapy mentioned is anti-PD-1 antibody therapy.

7. A pharmaceutical composition for treating liver cancer, characterized in that, Including 18S rRNA m7G modification inhibitors and anti-PD-1 antibodies.

8. The pharmaceutical composition according to claim 7, characterized in that, The 18S rRNA m7G modification inhibitor is selected from shRNAs that can specifically reduce the level of m7G-modified 18S rRNA.

9. Application of reagents for detecting 18S rRNA m7G modification levels in the preparation of products for predicting the efficacy and / or prognostic assessment of immunotherapy for liver cancer.

10. The application according to claim 9, characterized in that, The reagents for detecting 18S rRNA m7G modification levels include primers for detecting 18S rRNA levels and / or reagents for detecting m7G levels.

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