Method and pharmaceutical composition for regulating and controlling liver cancer fibrosis and immune microenvironment by SRSF3 variable shear

Abstract

The invention discloses a method and a pharmaceutical composition for regulating and controlling liver cancer fibrosis and immune microenvironment by SRSF3 variable shear. The method comprises the following steps: detecting the expression level of SRSF3 or the variable shear state of SERPINH1 in tumor tissues of a liver cancer patient; for SRSF3 or a short SERPINH1 variant, an effective amount of an inhibitor is administered, said inhibitor being selected from the group consisting of SRSF3-specific shRNA, a SERPINH1 mutant, an antisense oligonucleotide targeting an SRSF3 binding site, or an antibody that blocks the binding of SRSF3 to SERPINH1; by inhibiting the activity of SRSF3 or reducing the generation of short SERPINH1, reducing the fibrosis level of tumor tissues and enhancing immune cell infiltration in a tumor microenvironment, the progress of liver cancer is inhibited, a molecular mechanism of SRSF3 for driving liver cancer fibrosis and immune escape through variable shear SERPINH1 is disclosed, a gene silencing tool of targeted SRSF3 and a SERPINH1 mutant for blocking abnormal shear are developed, and the development of liver cancer is promoted. A pathway inhibitor, an immune checkpoint inhibitor and an anti-fibrosis drug are combined for use, multi-dimensional microenvironment remodeling is achieved, and the SERPINH1 mutant remarkably reduces collagen deposition by retaining exons 4-6.

Description

Technical Field

[0001] The present application relates to the technical field of tumor targeted therapy, and specifically to a method and pharmaceutical composition for regulating liver cancer fibrosis and immune microenvironment through variable splicing of SRSF3. Background Art

[0002] Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide, with an estimated 900,000 deaths annually. Although immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) have made progress in some tumors, the overall response rate for HCC patients is less than 20%, particularly in "cold" tumors lacking immune cell infiltration, which are virtually unresponsive. Studies have shown that fibrosis and immunosuppression within the tumor microenvironment (TME) are key factors in treatment failure.

[0003] Existing treatment strategies have significant limitations. Although anti-fibrotic drugs (such as pirfenidone and nintedanib) can partially inhibit collagen deposition, they cannot reverse immunosuppression; immune checkpoint inhibitors are almost ineffective against "cold" tumors. SRSF3 is abnormally highly expressed in liver cancer and is closely associated with poor patient prognosis (median survival is shortened by 50%). Further studies have shown that SRSF3 generates a short variant (exons 1-3 are retained and exons 4-6 are deleted) through variable splicing of SERPINH1. This variant loses its collagen folding function and instead promotes abnormal collagen deposition and fibroblast activation. At the same time, it inhibits CD8+T cell infiltration by upregulating PD-L1. This discovery reveals the dual role of the SRSF3 / SERPINH1 pathway in liver cancer fibrosis and immune escape, and provides a new direction for the development of treatment strategies targeting this pathway, which is expected to break through the treatment dilemma of "cold" tumors. Summary of the Invention

[0004] The purpose of this application is to provide a method and pharmaceutical composition for regulating liver cancer fibrosis and immune microenvironment through variable splicing of SRSF3, so as to address the significant limitations of existing treatment strategies. Although anti-fibrotic drugs (such as pirfenidone and nintedanib) can partially inhibit collagen deposition, they cannot reverse immunosuppression, and immune checkpoint inhibitors are almost ineffective against "cold" tumors.

[0005] To solve the above technical problems, the present application provides a method for regulating liver cancer fibrosis and immune microenvironment through SRSF3 variable splicing, comprising the following steps:

[0006] a. Detect the expression level of SRSF3 or the alternative splicing state of SERPINH1 in tumor tissues of patients with liver cancer;

[0007] b. administering an effective amount of an inhibitor against SRSF3 or a short SERPINH1 variant, wherein the inhibitor is selected from: SRSF3-specific shRNA, a SERPINH1 mutant, an antisense oligonucleotide targeting the SRSF3 binding site, or an antibody that blocks the binding of SRSF3 to SERPINH1;

[0008] c. By inhibiting SRSF3 activity or reducing the production of short SERPINH1, the level of tumor tissue fibrosis is reduced, and the infiltration of immune cells in the tumor microenvironment is enhanced, thereby inhibiting the progression of liver cancer.

[0009] Preferably, the detection of SRSF3 is achieved by immunohistochemistry.

[0010] Preferably, the detection of the short SERPINH1 is performed by RNA in situ hybridization technique.

[0011] Preferably, the proportion of the short SERPINH1 to the total SERPINH1 is greater than 50%.

[0012] Preferably, the antisense oligonucleotide comprises a sequence complementary to the SRSF3 binding site, is 18-25 nucleotides in length, and is stabilized by phosphorothioate modification.

[0013] A pharmaceutical composition for regulating liver cancer fibrosis and immune microenvironment, comprising a SERPINH1 mutant whose sequence includes retention of exons 4-6 and blocks the splicing of exons 1-3 by SRSF3;

[0014] A pharmaceutically acceptable carrier is selected from liposomes, nanoparticles or viral vectors. The pharmaceutical composition reduces tumor fibrosis and enhances immune cell infiltration by inhibiting SRSF3-mediated SERPINH1 variable shearing.

[0015] Preferably, the SERPINH1 mutant is delivered to tumor tissue via a lentiviral vector and reduces tumor volume by at least 60% in a mouse tumor-bearing model.

[0016] Preferably, the pharmaceutical composition further comprises a detection reagent, wherein the detection reagent includes an antibody that specifically recognizes SRSF3, and is used for real-time monitoring of changes in the SRSF3 / SERPINH1 signaling pathway during treatment.

[0017] Compared with the prior art, the method and pharmaceutical composition provided by this application for regulating liver cancer fibrosis and immune microenvironment through SRSF3 variable splicing have at least the following beneficial effects:

[0018] Reveal the molecular mechanism by which SRSF3 drives liver cancer fibrosis and immune escape through variable splicing of SERPINH1, develop gene silencing tools targeting SRSF3 (such as shRNA) and SERPINH1 mutants that block abnormal splicing, combine pathway inhibitors with immune checkpoint inhibitors and anti-fibrotic drugs to achieve multi-dimensional microenvironment remodeling. SERPINH1 mutants significantly reduce collagen deposition by retaining exons 4-6 (immunofluorescence shows a 55% decrease in collagen area). DETAILED DESCRIPTION

[0019] In order to enable people skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application are clearly and completely described.

[0020] The core of this application is to provide a method and pharmaceutical composition for regulating liver cancer fibrosis and immune microenvironment through variable splicing of SRSF3, which solves the significant limitations of existing treatment strategies. Although anti-fibrotic drugs (such as pirfenidone and nintedanib) can partially inhibit collagen deposition, they cannot reverse immunosuppression, and immune checkpoint inhibitors are almost ineffective against "cold" tumors.

[0021] A method for regulating liver cancer fibrosis and the immune microenvironment through SRSF3 alternative splicing comprises the following steps: detecting SRSF3 expression levels or the alternative splicing status of SERPINH1 in tumor tissues of liver cancer patients; administering an effective amount of an inhibitor targeting SRSF3 or a shortened SERPINH1 variant, selected from the group consisting of SRSF3-specific shRNA, a SERPINH1 mutant, an antisense oligonucleotide targeting the SRSF3 binding site, or an antibody that blocks SRSF3 binding to SERPINH1; inhibiting SRSF3 activity or reducing the production of shortened SERPINH1 reduces tumor tissue fibrosis and enhances immune cell infiltration in the tumor microenvironment, thereby inhibiting liver cancer progression. SRSF3 binding sequences are analyzed using bioinformatics and sequencing technologies, and the SRSF3 action sites are identified based on known exon and intron information. The primary target sites are those that result in splicing of exons 1-3 and loss of exons 4-6, forming shortened SERPINH1. SRSF3 and SERPINH1-flag are then overexpressed to detect different SERPINH1 variants and sizes, and the sizes of the SRSF3 splicing products are verified. SRSF3, WB detection of different variants and sizes of SERPINH1, verification of the size of SRSF3 splicing products and changes in short SERPINH1 in tumors. Establishment of Hep1-6 cell line overexpressing full-length and short SERPINH1 mutations of SERPINH1, mice were subcutaneously loaded with tumors, tumor volume was detected at different times, tumors were collected after the benevolent end point (about 21 days), and tumor size and weight were detected. Analyze the effect of short SERPINH1 on tumor growth. Establishment of Hep1-6 cell line overexpressing full-length and short SERPINH1 mutations of SERPINH1, mice were subcutaneously loaded with tumors, tumors were collected after the benevolent end point (about 21 days), lymphocytes were isolated, and flow cytometry was used to detect the proportion, number and activation level of each lymphocyte subset in tumor tissue. Analyze the effect of short SERPINH1 on lymphocyte infiltration in tumor tissue.

[0022] pMys was used to construct overexpression vectors for SERPINH1 full-length, short SERPINH1 variants, and SERPINH1 mutations. Stable cell lines were screened and established, and 1×106 cells were injected subcutaneously into mice to establish a mouse tumor-bearing model. Tumor volume was measured at different times, 7, 14, and 21 days. Tumors were collected after the benevolent endpoint (approximately 21 days) to measure tumor size and weight. Flow cytometry was used to detect the proportion, number, and activation level of various lymphocyte subsets in tumor tissues, including CD3, CD4, CD8, NK, CD11b, and CD11c positive cells.

[0023] Preferably, the detection of SRSF3 is achieved by immunohistochemistry.

[0024] Preferably, the detection of the short SERPINH1 is performed by RNA in situ hybridization technique.

[0025] Preferably, the proportion of the short SERPINH1 to the total SERPINH1 is greater than 50%.

[0026] Preferably, the antisense oligonucleotide comprises a sequence complementary to the SRSF3 binding site, is 18-25 nucleotides in length, and is stabilized by phosphorothioate modification.

[0027] Preferably, a SERPINH1 mutant is included, the sequence of which contains the retention of exons 4-6 and blocks the splicing of exons 1-3 by SRSF3;

[0028] A pharmaceutically acceptable carrier is selected from liposomes, nanoparticles or viral vectors. The pharmaceutical composition reduces tumor fibrosis and enhances immune cell infiltration by inhibiting SRSF3-mediated SERPINH1 variable shearing.

[0029] Preferably, the SERPINH1 mutant is delivered to tumor tissue via a lentiviral vector and reduces tumor volume by at least 60% in a mouse tumor-bearing model.

[0030] Preferably, the pharmaceutical composition further comprises a detection reagent, wherein the detection reagent includes an antibody that specifically recognizes SRSF3, and is used for real-time monitoring of changes in the SRSF3 / SERPINH1 signaling pathway during treatment.

[0031] When studying the methods of regulating liver cancer fibrosis and immune microenvironment through SRSF3 alternative splicing, it is important to clarify the relationship between SRSF3 and liver cancer:

[0032] Clinical specimens: HCC tissues of varying degrees of malignancy, adjacent tissues, and normal tissues were collected for immunohistochemistry testing of SRSF3 levels. SRSF3 expression in HCC was also assessed using quantitative PCR and Western blotting. The relationship between SRSF3 and HCC progression was analyzed and clarified.

[0033] In vitro cell lines: Quantitative PCR and Western blotting were used to detect SRSF3 levels in various human liver cancer cell lines and mouse cell lines in the laboratory. A knockdown vector, shSRSF3, was constructed using a silencing vector to silence SRSF3 and measure the effect on tumor cell proliferation.

[0034] Mouse Model: Overexpression and silencing vectors were used to establish stable Hep1-6 cell lines with high and low expression levels. Mice were subcutaneously implanted with tumors. Tumor volume was measured at various times. Tumors were harvested after the benevolent endpoint (approximately 21 days) and their size and weight were measured.

[0035] Then we explored the effect of SRSF3 on tumor immune infiltration:

[0036] Hepatocellular carcinomas of varying degrees of malignancy, adjacent tissues, and normal tissues were collected. Immunofluorescence and immunohistochemistry were used to assess the number and type of lymphocyte infiltrates in tumor tissues to clarify the correlation between SRSF3 expression and lymphocyte infiltration. High- and low-expression stable cell lines of the mouse Hep1-6 cell line were established using overexpression and silencing constructs. Mice were subcutaneously implanted with tumors. Tumors were harvested after the benevolent endpoint (approximately 21 days), and tumor tissue cells were isolated. Flow cytometry was used to assess the proportion, number, and activation levels of various lymphocyte subsets in the tumor tissues.

[0037] Further study on SRSF3 targeting SERPINH1:

[0038] Hep1-6 cells were lysed, and the SRSF3 complex was pulled down using RNA immunoprecipitation. RNA sequencing was used to analyze the RNA of genes bound by SRSF3, revealing significant enrichment of SERPINH1. Further analysis of SRSF3-bound sequences was performed. Hep1-6 cells were lysed, and the SRSF3 complex was pulled down using RNA immunoprecipitation. SERPINH1 levels were assessed using PCR and quantitative PCR. In vitro biotin-labeled SERPINH1 RNA sequences were transcribed and subjected to RNA pulldown. SRSF3 levels were then assessed using Western blotting.

[0039] Bioinformatics and sequencing were used to analyze SRSF3 binding sequences. Based on known exon and intron information, SRSF3 interaction sites were identified, primarily leading to splicing of exons 1-3 and loss of exons 4-6, resulting in the formation of short SERPINH1. SRSF3 and SERPINH1-flag were overexpressed to detect different SERPINH1 variants and sizes, verifying the size of SRSF3 splicing products. SRSF3 was silenced, and Western blotting was used to detect different SERPINH1 variants and sizes, verifying changes in the size of SRSF3 splicing products and short SERPINH1 in tumors.

[0040] Exploring the role of short SERPINH1 in promoting tumor growth:

[0041] Methods: Hep1-6 cell lines overexpressing full-length and short SERPINH1 mutants were established. Mice were subcutaneously implanted with tumors. Tumor volume was measured at different times and tumors were harvested after the benevolent endpoint (approximately 21 days). Tumor size and weight were measured. The effect of short SERPINH1 on tumor growth was analyzed. Methods: Hep1-6 cell lines overexpressing full-length and short SERPINH1 mutants were established. Mice were subcutaneously implanted with tumors. Tumors were harvested after the benevolent endpoint (approximately 21 days). Lymphocytes were isolated and flow cytometry was used to analyze the proportion, number, and activation level of various lymphocyte subsets in tumor tissue. The effect of short SERPINH1 on lymphocyte infiltration in tumor tissue was analyzed.

[0042] Exploring the impact of SERPINH1 on tumor tissue fibrosis:

[0043] Hep1-6 cell lines overexpressing full-length and short SERPINH1 mutants were established, and cell morphology and fibrosis gene expression were examined by microscopy and immunofluorescence. Hep1-6 cell lines overexpressing full-length and short SERPINH1 mutants were established, and mice were subcutaneously implanted with tumors. Tumors were harvested after the benevolent endpoint (approximately 21 days) and fibrosis distribution, morphology, and collagen levels were observed by immunofluorescence, HE staining, and two-photon electron microscopy.

[0044] Effects of Inhibiting Short Serpinh1 Production on Tumors:

[0045] A SERPINH1 sequence mutation was established to prevent SRSF3 from recognizing and cleaving SERPINH1, inhibiting the production of short SERPINH1. A stable cell line was then established. Mice were subcutaneously implanted with tumors, and tumor volume was measured at different times. Tumors were harvested after the benevolent endpoint (approximately 21 days) and their size and weight were measured. The effects of short SERPINH1 on tumor growth were analyzed. Immunofluorescence, hematoxylin and eosin staining, and two-photon electron microscopy were used to observe fibrosis distribution, morphology, and collagen levels. Flow cytometry was used to assess the proportion, number, and activation levels of various lymphocyte subsets in tumor tissue.

[0046] Lentiviral-encapsulated shSRSF3 vectors were injected intratumorally. Tumor volume was measured at various times and tumors were harvested after the benevolent endpoint (approximately 21 days) for size and weight. The effects of SERPINH1 short-acting on tumor growth were analyzed. Immunofluorescence staining, hematoxylin and eosin staining, and two-photon electron microscopy were used to observe fibrosis distribution, morphology, and collagen levels. Flow cytometry was used to assess the proportion, number, and activation level of various lymphocyte subsets in tumor tissue.

[0047] Other embodiments of the present invention will readily occur to those skilled in the art after considering the specification and practicing the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of the present invention that follow the general principles of this application and incorporate common knowledge or customary techniques in the art disclosed herein. The description and examples are to be considered merely as exemplary, and the true scope of this application is indicated by the claims.

[0048] It should be understood that the present application is not limited to the precise structure described above, and various modifications and changes can be made without departing from its scope. The above-described embodiments of the present application do not constitute a limitation on the scope of protection of the present application.

Claims

1. A method for regulating liver cancer fibrosis and immune microenvironment through SRSF3 variable splicing, characterized in that: The following steps are involved: a. Detect the expression level of SRSF3 or the alternative splicing state of SERPINH1 in tumor tissues of patients with liver cancer; b. administering an effective amount of an inhibitor against SRSF3 or a short SERPINH1 variant, wherein the inhibitor is selected from: SRSF3-specific shRNA, a SERPINH1 mutant, an antisense oligonucleotide targeting the SRSF3 binding site, or an antibody that blocks the binding of SRSF3 to SERPINH1; c. By inhibiting SRSF3 activity or reducing the production of short SERPINH1, the level of tumor tissue fibrosis is reduced, and the infiltration of immune cells in the tumor microenvironment is enhanced, thereby inhibiting the progression of liver cancer.

2. The method for regulating liver cancer fibrosis and immune microenvironment according to claim 1, characterized in that: The detection of SRSF3 was achieved by immunohistochemistry.

3. The method for regulating liver cancer fibrosis and immune microenvironment according to claim 1, characterized in that: The detection of the short SERPINH1 was accomplished by RNA in situ hybridization technique.

4. The method for regulating liver cancer fibrosis and immune microenvironment according to claim 3, characterized in that: The short SERPINH1 accounts for more than 50% of the total SERPINH1.

5. The method for regulating liver cancer fibrosis and immune microenvironment according to claim 1, characterized in that: The antisense oligonucleotide comprises a sequence complementary to the SRSF3 binding site, is 18-25 nucleotides in length, and is modified with phosphorothioate to enhance stability.

6. A pharmaceutical composition for regulating liver cancer fibrosis and immune microenvironment, characterized in that: These include SERPINH1 mutants whose sequences contain retention of exons 4–6 and block SRSF3 splicing of exons 1–3; A pharmaceutically acceptable carrier is selected from liposomes, nanoparticles or viral vectors. The pharmaceutical composition reduces tumor fibrosis and enhances immune cell infiltration by inhibiting SRSF3-mediated SERPINH1 variable shearing.

7. The pharmaceutical composition according to claim 6, characterized in that The SERPINH1 mutant was delivered to tumor tissue via a lentiviral vector and reduced tumor volume by at least 60% in a mouse tumor-bearing model.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition further comprises a detection reagent, which includes an antibody that specifically recognizes SRSF3 and is used to monitor changes in the SRSF3 / SERPINH1 signaling pathway in real time during treatment.