Use of non-coding regulatory RNA in the preparation of drugs for preventing or treating cancer or tissue fibrosis diseases

Abstract

The present invention discloses non-coding RNA that targets and inhibits the expression of mitochondrial-localized base cleavage repair proteins and its use in the preparation of drugs for the prevention or treatment of cancer / fibrotic diseases. The present invention discloses specific long-chain non-coding RNA and small interfering RNA, which can target and knock down the levels of mitochondrial-localized base cleavage repair proteins involved in the repair of mitochondrial DNA oxidative damage, hinder the mitochondrial oxidative phosphorylation function of cells, and then inhibit the M2 polarization of macrophages in the interstitial tissue, inhibit the formation of tissue microenvironments that are conducive to the occurrence and development of cancer and fibrotic diseases, and produce resistance to the occurrence and development of cancer and fibrotic diseases. The present invention discloses for the first time the use of specific lncRNA and siRNA design to prepare drugs for the directional regulation of macrophage polarization. By changing the immunological state of the microenvironment of cancer tissue / fibrotic disease tissue, the effect of preventing and treating diseases is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology. Specifically, the present invention relates to the use of nucleic acid molecules that inhibit the base excision and repair function of mitochondrial DNA and inhibit the M2 polarization of macrophages in the preparation of drugs for preventing or treating cancer and tissue fibrosis diseases. Background Art

[0002] Epidemiological data show that lung cancer is the leading cause of cancer incidence and cancer death in humans. Most lung cancer patients have a poor prognosis, with a five-year survival rate of only 4-17% after diagnosis. Lung cancer is one of the major diseases that seriously threatens human health and life, and its prevention and treatment are also key areas of long-term biomedical research. (1-3) . Pulmonary fibrosis (PF) is a serious diffuse interstitial lung disease caused by a variety of internal and external factors. Clinically, it can be divided into two major categories: (1) Secondary pulmonary fibrosis (SPF), which has a clear cause, such as silicosis and pneumoconiosis caused by environmental pollution, exposure to minerals and industrial dust, pulmonary fibrosis lesions left over from viral infections in the lungs, and pulmonary fibrosis that may be associated with certain autoimmune diseases; (2) Idiopathic pulmonary fibrosis (IPF), which is difficult to determine the cause of the disease. Idiopathic pulmonary fibrosis is an aging-related disease, and the incidence rate increases significantly with age. Pulmonary fibrosis is also a high-mortality disease for which there is no effective treatment in clinical practice. (4-5) .

[0003] The academic community has clearly recognized that the occurrence and progression of lung cancer and pulmonary fibrosis are closely related to the phenotypic differentiation (polarization) of macrophages in the tissue microenvironment: (1) Cancer occurrence and lesion formation depend on two factors: The malignant phenotype of cancer cells is mainly caused by cancer-driving gene mutations within the cell genome; The tissue microenvironment that is conducive to the proliferation and growth of cancer cells is mainly determined by the properties of the immune cells (especially the macrophage population) in the tissue. It is generally believed that the tumor-associated macrophages in the tissue microenvironment that promotes the proliferation and growth of cancer cells are mainly M2-type polarized, showing tumor immunosuppression and promoting tumor development; while the macrophages in the tissue microenvironment that inhibits the proliferation and growth of cancer cells are mainly M1-type polarized, showing anti-tumor effects. (6,7)(2) The occurrence of tissue fibrosis is associated with persistent chronic inflammatory stimulation, which causes the macrophages infiltrating the tissue to be alternatively activated, showing macrophage type 2 (M2) polarization, changing the tissue inflammatory microenvironment and forming a microenvironment that promotes the transformation and proliferation of myofibroblasts, a key signaling pathway that enhances the activity of TGF-β / Smad, leading to the occurrence of tissue fibrosis. (8,9) At present, clinical treatments for lung cancer and pulmonary fibrosis are mainly aimed at killing / inhibiting target cells (cancer cells, myofibroblasts) and inhibiting tissue inflammation. It is rare to see the use of drugs that regulate macrophage polarization in the disease tissue microenvironment. Studies have confirmed that the energy metabolism of M2-polarized macrophages depends on the energy production of mitochondrial oxidative phosphorylation, and mitochondrial oxidative phosphorylation dysfunction will hinder the M2 polarization of macrophages. (10) , and it is difficult to form a tissue microenvironment that is conducive to the development / occurrence of cancer / fibrosis diseases. Given the key role of M2 polarized macrophages in physiological and pathological processes such as tissue inflammation repair, fibrosis, tumor immunosuppression, and allergies to environmental substances, targeted regulation of macrophage M2 polarization has great application prospects in the prevention and treatment of various diseases.

[0004] The average age of onset of lung cancer and idiopathic pulmonary fibrosis (IPF) is over 60 years old. They are aging-related diseases, which are related to the increase of reactive oxygen species (ROS) levels (oxidative stress) and the accumulation of cellular DNA oxidative damage caused by various factors such as the environment. Most patients have no clear family history. The MUTYH gene is one of the important components of the cell base excision repair system. It has glycosylase activity and specifically recognizes and repairs 8-hydroxydeoxyguanosine (8-OHdG), the oxidative damage product of guanine, for DNA damage caused by ROS. 8-OHdG is a common form of guanine oxidation in the DNA chain and is stable in nature. During DNA replication, the ability of 8-OHdG to pair with cytosine is weakened, and it selectively mispairs with adenine (A), resulting in G>T mutations in subsequent DNA replication chains. The MUTYH gene has three transcripts (α-, β- and γ-transcripts). The α-transcript encodes the translation of type 1 MUTYH protein (MUTYH1), which is mainly located in the mitochondria and participates in the repair of oxidative damage to mitochondrial DNA (mtDNA); the β- and γ-transcripts encode the translation of type 2 MUTYH protein (MUTYH2), which is located in the nucleus and participates in the repair of oxidative damage to nuclear genomic DNA (nDNA). (11,12)Germline mutations of the MUTYH gene are associated with a significantly increased risk of intestinal adenomatous polyposis and colorectal cancer.

[0005] The preliminary work of the present invention found that there is a new Alu element (AluYb8) insertion mutation in the human MUTYH gene. Based on the individual's carrying status of this insertion mutation, there are three genotypes of the MUTYH gene in the population: homozygous variant individuals (variant / variant); heterozygous individuals (wild type / variant); homozygous wild type individuals (wild type / wild type). Further analysis found that this Alu element insertion mutation can lead to the suppression of the expression of mitochondrial localized MUTYH type 1 protein, the age-related decrease in the mitochondrial DNA (mtDNA) content of cells, the downregulation of mtDNA encoding gene expression, and the reduction of mitochondrial function.

[0006] References

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[0010] 4.Chanda D,Otoupalova E,Smith,SR,Volckaert T,De Langhe SP,andThannickal V.Developmental pathways in the pathogenesis of lung fibrosis.MolAspects Med,2019;65:56-69.

[0011] 5.Wijsenbeek M,Cottin V.Spectrum of Fibrotic Lung Diseases.N Engl JMed,2020;383(10):958-968.

[0012] 6.Boutilier AJ,Elsawa SF.Macrophage Polarization States in the TumorMicroenvironment.Int J Mol Sci,2021;22(13):6995.

[0013] 7.Sedighzadeh SS,Khoshbin AP,Razi S,Keshavarz-Fathi M,Rezaei N.Anarrative review of tumor-associated macrophages in lung cancer:regulation ofmacrophage polarization and therapeutic implications.Transl Lung Cancer Res,2021;10(4):1889-1916.

[0014] 8.Wojtan P,Mierzejewski M,Osińska I, J.Macrophagepolarization in interstitial lung diseases.Cent Eur J Immunol,2016;41(2):159-164

[0015] 9. Zhang F, Ayaub E, Wang B, Puchulu-Campanella E, Li Y, Hettiarachchi S, et al. Reprogramming of profibrotic macrophages for treatment of bleomycin-induced pulmonary fibrosis[J]. EMBO Mol Med, 2020, 12(8): e12034.

[0016] 10. Van den Bossche J, Baardman J, Otto NA, van der Velden S, Neele AE, van den Berg SM, Luque-Martin R, Chen HJ, Boshuizen MC, Ahmed M, Hoeksema MA, de Vos AF, de Winther MP. Mitochondrial Dysfunction Prevents Repolarization of Inflammatory Macrophages. Cell Rep, 2016; 17(3): 684 - 696.

[0017] 11. Fromme JC, Banerjee A, Huang SJ, Verdine GL. Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase. Nature, 2004, 427: 652 - 656.

[0018] 12. Cheadle JP and Sampson JR. Exposing the MYtH about base excision repair and human inherited disease. Hum Mol Genet, 2003, 12: R159 - 165. Summary of the Invention

[0019] Purpose of the invention: The technical problem to be solved by the present invention is to provide a nucleic acid molecule that targets and inhibits the expression of mitochondrial-localized base excision repair proteins for use in the preparation of drugs for the prevention or treatment of cancer / fibrotic diseases. The nucleic acid molecule is a non-coding regulatory RNA that targets and binds to the α transcript mRNA (α-MUTYH) of the gene MUTYH, an important component of the cellular base excision repair system.

[0020] Technical solution: The present invention provides the use of specifically designed non-coding regulatory RNA in the preparation of drugs for preventing or treating cancer / fibrotic diseases.

[0021] Wherein, the non-coding regulatory RNA is lncRNA and siRNA of specific sequence.

[0022] The drug of the present invention specifically inhibits the expression of the DNA glycosylase type 1 MUTYH (MUTYH1) protein encoded by the gene MUTYH, an important component of the cell base excision repair system, and the protein is localized in mitochondria.

[0023] The drug of the present invention inhibits the repair of oxidative damage to mitochondrial DNA (mtDNA) in cells and suppresses the expression of mtDNA coding genes.

[0024] The drug of the present invention inhibits the oxidative phosphorylation function of cell mitochondria and reduces the production of ATP, a bioenergy molecule of cells.

[0025] The present invention includes the use of non-coding regulatory RNA in the preparation of drugs for preventing or treating cancer or fibrotic diseases, wherein the non-coding regulatory RNA includes long-chain non-coding regulatory RNA or small interfering RNA, wherein the long-chain non-coding regulatory RNA sequence is shown in SEQ ID NO.1, and the small interfering RNA sequence is shown in SEQ ID NO.2 or SEQ ID NO.3 or SEQID NO4.

[0026] Wherein, the non-coding regulatory RNA can target and inhibit the expression function of mitochondrial localized base excision repair protein (subtype).

[0027] Wherein, the non-coding regulatory RNA is used to inhibit the function of mitochondrial oxidative phosphorylation in cells.

[0028] Wherein, the non-coding regulatory RNA is used to inhibit the M2 polarization function of macrophages.

[0029] The cancer includes but is not limited to lung cancer.

[0030] Wherein, the fibrotic disease is pulmonary fibrosis.

[0031] Among them, the non-coding regulatory RNA is used to regulate the immunological state of the lung tissue microenvironment and inhibit the formation of tissue microenvironment that promotes the occurrence and development of tumor / fibrotic lesions.

[0032] The present invention includes the use of an expression vector containing a non-coding regulatory RNA in the preparation of a drug for preventing or treating cancer or fibrotic diseases, wherein the sequence of the non-coding regulatory RNA is shown in SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4.

[0033] Wherein, the expression vector inhibits cell mitochondrial proliferation and mitochondrial function or inhibits M2 polarization of macrophages in the body.

[0034] Functional regulation of cell protein molecules is one of the important paths for the development of modern drugs, including regulation of new variant molecules associated with diseases. So far, many successful examples have emerged. The inventor accidentally discovered that the human base excision repair gene MUTYH has an insertion mutation of an Alu element (AluYb8) during the screening of functional variants in the human genome. Regarding the carrying status of this variant, the MUTYH gene of different individuals in the population has three genotypes: homozygous wild-type (wild-type / wild-type) individuals in which both homologous chromosomes of the MUTYH gene do not carry this variant; homozygous variant (variant / variant) individuals in which both homologous chromosomes carry this variant; heterozygous (wild-type / variant) individuals in which only one chromosome carries this variant of the MUTYH gene, while the other homologous chromosome does not carry this variant. In homozygous variant individuals, the translation expression of the MUTYH1 protein located in the mitochondria of cells is reduced, the transcription of the mtDNA encoding gene is inhibited and the expression is downregulated, and the function of the mitochondrial oxidative respiratory chain (oxidative phosphorylation) is reduced. There is evidence that the M2 differentiation (polarization) of macrophages depends on the oxidative phosphorylation function of cell mitochondria. That is, impaired cellular oxidative phosphorylation function will inhibit the M2 differentiation of macrophages, and the polarization of macrophage M2 is an important basis for building the immunosuppressive microenvironment of cancer tissue and the microenvironment of fibrotic disease tissue. Based on this, the present invention uses a variety of cell and molecular biological experimental techniques to isolate a new transcript (containing AluYb8 sequence) associated with this insertion variation of the MUTYH gene. It was identified that the transcript lacks the function of (encoding) translating protein peptide chains, but has the function of inhibiting the translation of MUTYH1 protein, and is determined to be a long non-coding RNA (lnc-MUTYH) that can inhibit the expression of MUTYH1 protein.

[0035] The present invention also designs a variety of siRNAs. These non-coding regulatory RNAs of specific sequences can specifically bind to the α-transcript base sequence of the MUTYH gene, selectively inhibit the translation and expression of the mitochondrial-localized MUTYH 1 protein, cause dysfunction of the cell mtDNA oxidative damage repair function, and inhibit the expression of the oxidative respiratory chain protein encoded by mtDNA, resulting in impaired mitochondrial oxidative phosphorylation energy metabolism function, which can hinder the M2 polarization of macrophages. The present invention discloses a new path for blocking the M2 polarization of macrophages by blocking the repair of mitochondrial DNA oxidative damage and inhibiting the mitochondrial oxidative phosphorylation function. In view of the fact that the immunosuppressive tissue microenvironment constructed mainly by M2 macrophages has a clear promoting effect in the occurrence and evolution of cancer and fibrotic diseases, the nucleic acid molecules for blocking the M2 polarization of macrophages proposed in the present invention can be used to develop and prepare new drugs for preventing or treating cancer and tissue fibrosis diseases.

[0036] Based on the above findings, the embodiment of the present invention further studied and isolated a new long non-coding regulatory RNA (Long non-coding RNA) associated with the AluYb8 insertion mutation, named lnc-MUTYH (Table 1). This regulatory RNA contains the corresponding sequence of AluYb8, which is specifically complementary to the sequence around the translation start site of the α-transcript of the human MUTYH gene (α-MUTYH). Based on this, the present invention designs and constructs lnc-MUTYH expression plasmids and regulatory small interfering RNAs (Small interfering RNA, siRNA) for the α-transcript of the MUTYH gene (Table 1), and transfects specific genotype cells respectively. Analysis confirmed that lnc-MUTYH selectively inhibits the translation expression of MUTYH 1 protein at the post-transcriptional level of MUTYH, causing mtDNA oxidative damage repair disorders in transfected cells, and mtDNA replication function is inhibited, resulting in impaired mitochondrial oxidative phosphorylation function (cell oxygen consumption rate and cell ATP production are significantly reduced). At the same time, it was confirmed that small interfering RNA (siRNA) transfection targeting the α transcript of the MUTYH gene also significantly reduced the transcription level of α-MUTYH and inhibited the expression of MUTYH1 protein. Further transfection and regulation analysis of macrophages confirmed that lnc-MUTYH-mediated inhibition of MUTYH type 1 protein expression ultimately led to the obstruction of M2 polarization of macrophages. More and more evidence suggests that the inhibition of M2 polarization of macrophages may play a role in anti-cancer and anti-fibrotic diseases. The present invention found through molecular epidemiological case-control analysis and in vitro culture cell model studies that lnc-MUTYH expression associated with MUTYH mutations and MUTYH gene functional defects can significantly reduce the incidence of lung cancer and inhibit the formation of effector cells-myofibroblasts that occur in tissue fibrosis. And using model animal experiments to demonstrate that MUTYH gene defects that damage cell mitochondrial function can alleviate the pathological occurrence of pulmonary fibrosis.

[0037] Beneficial effects: The above research results, which serve as the basis for proposing the present invention, clearly show that the use of specific nucleic acid molecules (lnc-MUTYH, siRNA) can specifically inhibit the expression of mitochondrial-localized base cutting repair protein (MUTYH1), hinder the repair of mtDNA oxidative damage, damage the mitochondrial oxidative phosphorylation function, and achieve the effect of blocking macrophage M2 polarization. In view of the fact that macrophage M2 polarization has a clear promoting effect in the occurrence and evolution of lung cancer and pulmonary fibrosis, nucleic acid molecules that inhibit the expression of base cutting repair proteins for mtDNA oxidative damage repair can be used to develop and prepare new drugs for the prevention and treatment of lung cancer and pulmonary fibrosis. The content of the present invention discloses a new strategic approach to the development of drugs for the prevention and treatment of lung cancer and pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 :Three genotypes formed by the Alu element insertion (AluYb8) mutation of the MUTYH gene in the Chinese population. Primers were designed based on the sequence (NM_001128425) published by the MUTYH gene (Human Genome Project) to implement specific PCR amplification of the base sequence containing the 15th intron of the gene, and the expected PCR product length was 500bp. However, the results of the population sample test unexpectedly showed that the PCR products of some individual samples of the Chinese population showed the expected single 500bp band (second electrophoresis lane) as the homozygous wild type; some individuals showed a single 826bp growth band (third electrophoresis lane) as the homozygous variant, indicating the presence of a 326bp DNA fragment insertion; some individuals showed a 500bp and 826bp heterozygous band (first electrophoresis lane). The sequencing results showed that the inserted fragment was an Alu element, and in-depth analysis showed that it was the subtype AluYb8 in the Alu sequence.

[0039] Figure 2 :The homozygous cells of Alu insertion mutation in MUTYH gene produce new specific transcription sequences. Specific primers were designed to detect the possible new transcription sequences associated with the insertion of Alu elements in MUTYH gene. The homozygous wild-type (Mutation Absent, A / A genotype) cell lines A549 and DU145, the homozygous mutation (Mutation Present, P / P genotype) cell lines 293T and HCT616, and the heterozygous mutation (A / P genotype) cell lines H1299 and SPCA-1 were taken to extract cellular RNA. Reverse transcription PCR (RT-PCR) technology was used for amplification and agarose electrophoresis of PCR products. Results: In the homozygous wild-type cell lines A549 and DU145 (A / A genotype), the amplified product was the expected transcription sequence of the wild-type MUTYH gene; in the homozygous variant cell lines 293T and HCT616 (P / P genotype), only a new unknown sequence was unexpectedly amplified; in the heterozygous variant cell lines H1299 and SPCA-1 (A / P genotype), the bands that appeared in the homozygous wild-type and homozygous variant cells were amplified simultaneously ( Figure 2 A and 2B). We also collected peripheral venous blood from 6 normal subjects (Person 1-Person 6, P1-P6) (including 2 individuals with A / A, A / P and P / P genotypes), extracted RNA, and verified it by reverse transcription PCR (RT-PCR). The results obtained were consistent with those obtained in the three genotype cell lines ( Figure 2 C). The terminal rapid cDNA amplification technology was then used to identify and determine the full-length sequence of the new unknown sequence obtained by accidental amplification, confirming that it was a newly emerged specific transcription sequence associated with the Alu element insertion mutation (lnc-MUTYH, see Table 1).

[0040] Figure 3 :lnc-MUTYH inhibits the expression of MUTYH gene in cells. lnc-MUTYH expression plasmid was constructed. Human lung epithelial cell line A549 cells (MUTYH gene is homozygous wild type A / A, no lnc-MUTYH expression itself) were cultured in vitro, and the expression level of MUTYH protein in cells was high; transfection of recombinant lnc-MUTYH expression plasmid significantly inhibited the expression of MUTYH protein in cells. Control cells were transfected with empty plasmid (Mock).

[0041] Figure 4 :lnc-MUTYH specifically inhibits the translation and expression of MUTYH1 protein. Human epithelial cell line 293T cells (MUTYH gene is homozygous variant P / P, and lnc-MUTYH is expressed by itself) were cultured in vitro. A. The expression level of MUTYH protein in control cells (si-control) was low (inhibited by lnc-MUTYH expressed by the cells themselves); small interfering RNA targeting lnc-MUTYH (si-lnc-MUTYH) was transfected to knock down the expression of lnc-MUTYH in cells, and the expression of MUTYH protein in cells was upregulated. B. The promoter sequence of the α-transcript of the MUTYH gene (α-MUTYH, encoding the mitochondrial localized MUTYH 1 subtype protein) and the green fluorescent protein (Enhanced green fluorescence protein, EGFP) expression plasmid were constructed, and the cells were transfected to observe the regulation of α-MUTYH translation expression by cellular lnc-MUTYH expression. The results confirmed that lnc-MUTYH specifically inhibited the translation and expression of α-MUTYH (MUTYH1 protein).

[0042] Figure 5 :Analysis of the silencing effect of small interfering RNA (siRNA) targeting inhibition of α-MUTYH (encoding MUTYH1 protein). Human lung epithelial cell line A549 cells (MUTYH gene is homozygous wild type) were cultured in vitro. Multiple pairs of small interfering RNA (siRNA) targeting inhibition of α-MUTYH translation activity were designed (three pairs are listed in Table 1). This figure shows the experimental results of transfecting cells with si-α-MUTYH-1. The selected siRNA was transfected into A549 cells at a concentration of 5nM and cultured for 72 hours. The cells were lysed, and the expression levels of three transcripts of the MUTYH gene (α-MUTYH, β-MUTYH and γ-MUTYH) were detected by real-time RT-PCR. The results showed that compared with the control, si-α-MUTYH-1 could stably knock down (inhibit) the expression of α-MUTYH, while there was no visible regulatory effect on the expression of β-MUTYH and γ-MUTYH. ** indicates p<0.01, and ns indicates that there was no significant statistical difference between the groups.

[0043] Figure 6 :lnc-MUTYH expression reduces the mitochondrial DNA (mtDNA) content of cells. Human epithelial cell line 293T cells (MUTYH gene is a homozygous variant, which expresses lnc-MUTYH itself) were cultured in vitro. The mtDNA encoding genes mt-CO1 and mt-tRNA leu The sequence was used as the analysis target, and its relative comparison with the copy number of nuclear genomic DNA (nDNA) was observed to identify the changes in the copy number of cellular mtDNA. As a result, the copy number of mtDNA in the control cells (si-control) was low, indicating that the mitochondrial proliferation function was impaired; after transfection of si-lnc-MUTYH to knock down the lnc-MUTYH expressed by the cells themselves, the copy number of cellular mtDNA increased significantly (* indicates P<0.05). Two mtDNA coding genes (mt-CO1 and mt-tRNA leu ) copies and nDNA copies were consistent with the results, indicating that knocking down lnc-MUTYH expressed in 293T cells (P / P genotype) restored the mitochondrial replication function of 293T cells.

[0044] Figure 7 :lnc-MUTYH expression impairs mitochondrial function of cells. Human monocyte / macrophage cell line THP-1 (MUTYH gene is homozygous wild type A / A, no lnc-MUTYH expression itself) was cultured in vitro. When the control cells were transfected with empty plasmid, the transcriptional expression level of COX2, a gene related to mitochondrial function (mitochondrial DNA encoding gene), was high; when the recombinant plasmid was transfected to express lnc-MUTYH, the transcriptional expression of COX2 gene encoded by mitochondrial DNA of the cells was significantly inhibited (** indicates P<0.01), indicating that mitochondrial function was impaired.

[0045] Figure 8:lnc-MUTYH expression hinders the M2 polarization of macrophages. The human monocyte / macrophage cell line THP-1 (MUTYH gene is homozygous wild type, and there is no lnc-MUTYH expression itself) was cultured in vitro, and IL-4 and IL-13 (IL-4-IL-13) were used to stimulate THP-1 cells to polarize to M2. CD206 and CD163, marker molecules of macrophage M2 polarization, were selected as observation target molecules to analyze and determine the effect of lnc-MUTYH expression status on M2 polarization of THP1 cells. The results showed that when the control cells were transfected with empty plasmid, IL-4-IL-13 induced high levels of M2 polarization marker molecules CD206 and CD163 in THP1 cells, indicating that IL-4-IL-13 induced M2 polarization was good; when the recombinant plasmid was transfected to express lnc-MUTYH, the expression of CD206 and CD163 in THP1 cells was significantly inhibited, indicating that the transfection of lnc-MUTYH significantly inhibited the IL-4-IL-13-induced M2 polarization of macrophages THP-1. (** indicates P<0.01, * indicates P<0.05)

[0046] Fig. 9 :lnc-MUTYH mediates the inhibition of fibroblast transformation into myofibroblast (effector cell of tissue fibrosis) by hindering macrophage M2 polarization. The mononuclear / macrophage cell line THP-1 (MUTYH gene is homozygous wild type, no lnc-MUTYH expression itself) was cultured in vitro, and IL-4-IL13 was used to stimulate THP-1 cells to polarize into M2. THP-1 cells that stimulated M2 polarization were simultaneously transfected with recombinant plasmids expressing lnc-MUTYH (control cells were transfected with empty plasmids). The stimulated and treated THP-1 cells were co-cultured with pre-cultured human lung fibroblasts MRC5 (blank control was MRC5 cells cultured alone). By analyzing the marker characteristics of MRC5 after co-culture, it was determined that lnc-MUTYH mediates the inhibition of fibroblast transformation into myofibroblast by inhibiting macrophage polarization. The results showed that after co-culture with M2-type THP-1 cells transfected with empty plasmid, MRC5 cells showed the characteristics of (already transformed into) myofibroblasts, and the expression of myofibroblast marker molecules Fibronectin and α-SMA protein was significantly increased; lnc-MUTYH treatment significantly inhibited the transformation of MRC5 into myofibroblasts by inhibiting the M2-type polarization of THP-1, and the expression of Fibronectin and α-SMA proteins was significantly reduced. This suggests that lnc-MUTYH can inhibit the transformation and generation of tissue fibrosis effector cells (myofibroblasts) by inhibiting the M2-type polarization of macrophages, thereby achieving the effect of treating or preventing the occurrence / development of pulmonary fibrosis.

[0047] Fig.10:Mutyh gene functional deficiency significantly alleviates the occurrence of bleomycin-induced pulmonary fibrosis in mice. Mutyh gene knockout technology was used to damage the Mutyh function of mice (cells), and a bleomycin (BLM)-induced mouse pulmonary fibrosis model was selected to compare and analyze the effects of Mutyh functional impairment on the occurrence and development of pulmonary fibrosis, in order to determine the potential role of Mutyh functional intervention in the prevention and treatment of pulmonary fibrosis. BLM exposure was administered once by airway instillation (3 mg / kg), and mice were subjected to Micro-CT scanning of the lungs 28 days later to observe the pathological state of pulmonary fibrosis. A double control of wild-type mice with normal saline instillation control and wild-type mice with BLM pulmonary fibrosis modeling was established. The results showed that compared with wild-type mice, the pathological phenotype of BLM-induced pulmonary fibrosis in mice with Mutyh functional deficiency was significantly milder.

[0048] Fig.11 :Mutyh gene function deficiency significantly reduces the deposition of interstitial collagen in the lungs of bleomycin model mice. Mutyh gene knockout technology was used to damage the Mutyh function of mice (cells), and BLM (single airway instillation of 3 mg / kg) was used to induce the construction of a mouse pulmonary fibrosis model, and the effect of Mutyh function impairment on the occurrence of pulmonary fibrosis was compared and analyzed. 28 days after the mouse modeling, the mouse lung tissue was taken, and Masson staining of the lung tissue was performed to compare and analyze the deposition status of collagen, the main component of pulmonary fibrosis. The results showed that after 28 days of BLM exposure, the collagen deposition in the lung tissue of wild-type mice was severe, while the collagen deposition in the lung tissue of BLM-induced modeling mice with Mutyh function deficiency was significantly reduced. DETAILED DESCRIPTION

[0049] Targeting the functional regulation of cellular protein molecules is one of the important paths for the development of modern drugs, including the regulation of new variant molecules associated with diseases, and there are more and more successful examples. Based on the analysis and identification of a new Alu element (AluYb8) insertion mutation in the human base excision repair gene MUTYH, and the discovery / isolation of a new long non-coding RNA (lncRNA) associated with the mutation, the present invention uses systematic biological technology and human population genetics analysis to conduct in-depth research on functional significance, gene expression regulation mechanism and transformation application prospects.

[0050] 1. Identification of DNA fragment insertion mutations in the MUTYH gene

[0051] Peripheral venous blood (0.1 ml each) was randomly collected from 100 normal Chinese people, and genomic DNA of blood cells was extracted. A pair of PCR primers (Forward-TCTTGACCTGGAGACCTTCC; Reverse-AGCTGCTTCCTCCAAACAGC) was designed based on the sequence of the MUTYH gene (NM_001128425) published by the Human Genome Project to specifically amplify the DNA sequence containing the 15th intron. The PCR product is expected to be 500 bp. PCR reaction system (50 μl): 2×Taq Master Mix (Novozymes, Nanjing), 25 μl; Forward primer (10 μM), 1 μl; Reverse primer (10 μM), 1 μl; Genomic DNA (100 ng / μl), 3 μl; ddH 2 O, 20μl. PCR program: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15sec, 56℃ renaturation for 15sec, 72℃ extension for 40sec, 38 cycles; 72℃ extension for 10min, 4℃ storage. Take 10μl PCR product and add 2μl bromophenol blue loading buffer (6×) to mix, and run on 1% agarose gel for electrophoresis. Agarose electrophoresis showed that the amplification products of the population samples unexpectedly presented 3 states, suggesting the existence of 3 genotypes: Homozygous wild type (wild type / wild type, 35 cases) with the expected single electrophoretic band of about 500 bp in length; Unexpected homozygous variant of a single electrophoretic band of more than 800 bp in length (variant / variant, 21 cases); Two electrophoretic bands (500 bp and more than 800 bp) of heterozygous type (wild type / mutant type, 44 cases) ( Figure 1 ). Sequencing analysis showed that there was an Alu element (AluYb8, 326bp) inserted in the variant sequence. Functional analysis suggested that the mitochondrial function of cells in homozygous variant individuals was impaired.

[0052] 2. Isolation and identification of lncRNA associated with variant genes

[0053] After MUTYH genotype analysis of multiple available human cell lines, 6 human cell lines were selected, namely: A549 and DU145 cells (homozygous wild type, two lines); 293T and HCT116 cells (homozygous variant, two lines); H1299 and SPCA-1 cells (heterozygous variant, two lines). All 6 cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai). A pair of reference primers (Forward-CCAGGGACCTGTATGTAGAGAC; Reverse-GGACCTTTTGGAACCCAGCT) (primer1) were designed based on the wild-type sequence of the MUTYH gene (NM_001128425); at the same time, a pair of screening primers (Forward-CACCGCGCCCGGCCCGGTCT; Reverse-GAACCCAGCTGCTTCCTC) (primer2) were designed based on multiple explorations to specifically explore the expression of new RNAs that may be associated with the insertion of the Alu element in the MUTYH gene. Specific operating procedures:

[0054] RNA extraction of cultured cells: Collect cultured cells in 1.5ml centrifuge tubes, add 1.0ml Trizol reagent (Invitgen), shake and place at 4℃ for 10 minutes; After separating the aqueous phase with 0.2ml chloroform (analytical grade), take the upper aqueous phase and mix it with 2 times the volume of isopropanol (analytical grade), invert and mix, and let it stand at -20℃ for 20min to precipitate RNA; After washing with 70% ice-cold ethanol, an appropriate amount of RNase Free ddH 2 The precipitate was dissolved in 1% O and quantified using an Eppendorf Biophotometer plus, and then stored in a deep freezer (-80°C) until use.

[0055] (First chain) cDNA preparation (reverse transcription system: 20 μl): 5× PrimeScript RT Master Mix (Takara), 4 μl; extracted RNA 1 μg; Random hexamers or oligo (dT) 18 primer, 0.5 μl; RNase Free dH 2 O, add to 20μl); reverse transcription program: 37℃, 15min; 85℃, 5sec; 4℃, 5min; the cDNA obtained by reverse transcription was stored in a -80℃ deep low temperature refrigerator for later use.

[0056] PCR amplification was performed using the two designed primer pairs (reaction system: 50 μl): Ex Taq polymerase (5 U / μl; Ex Hot Start Version, TaKaRa), 0.25 μl; 10×Ex Taq Buffer (containing premixed Mg 2+ ), 5 μl; dNTP Mixture (2.5 mM each), 4 μl; upstream primer (10 μM), 1 μl; downstream primer (10 μM), 1 μl; cDNA, 1.0 μl; ddH 2 O, to 50μl; PCR program: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30sec, 60℃ annealing for 30sec, 72℃ extension for 30sec, 35 cycles; after 72℃ extension for 10min, take 10μl PCR product and add 2μl bromophenol blue loading buffer (6×) to mix, and perform 1.5% agarose gel electrophoresis.

[0057] As a result, a newly generated RNA containing the inserted AluYb8 fragment was unexpectedly amplified in homozygous variant cell lines (293T and HCT116); this fragment was not expressed in homozygous wild-type cells (A549 and DU145), and only the expected fragment amplification product was obtained; while in heterozygous variant cell lines (H1299 and SPCA-1), the expression product containing the inserted AluYb8 fragment (new) and the expression product of the wild-type gene ( Figure 2 A, 2B show the test results for three genotype cell lines). This result was confirmed in the analysis of three genotype individual samples of blood cells from an expanded population ( Figure 2 C shows the test results of human peripheral blood samples, 2 individuals of each genotype, a total of 6 cases, Person 1 to Person 6), thus proving that this unexpectedly discovered newly emerged expression fragment is a specific transcribed non-coding lncRNA associated with the Alu insertion mutation of the MUTYH gene, named lnc-MUTYH. The full length and base sequence of this newly transcribed lnc-MUTYH (780bp, see Table 1 for the complete sequence) were determined by sequencing the amplified product using the rapid-amplification of cDNA ends (RACE) technology.

[0058] 3. Identification of lncRNA associated with variant genes that specifically inhibits the expression of MUTYH type 1 protein (MUTYH1)

[0059] (1) The pCDNA3.1 plasmid (purchased from Hanheng Biotechnology, Shanghai) was used to recombine with lnc-MUTYH to construct the lnc-MUTYH expression plasmid pCDNA3.1(+)lnc-MUTYH (pCDNA3.1 plasmid as the backbone, and the recombination insertion restriction site BamHI / XhoI). Specific operation: 500ng pCDNA3.1 plasmid and 500ng synthetic lnc-MUTYH (carrying BamHI and XhoI restriction sites) were double-digested with BamHI and XhoI (NEB; rCutSmart buffer) at 37°C for 15min; the digestion product was purified and recovered, and the pCDNA3.1 vector plasmid and lnc-MUTYH gene were ligated using T4 DNA ligase (TaKaRa) at 16°C for 16h (T4 DNA ligase reaction system: 10×T4 DNA ligase Buffer, 3μl; pCDNA3.1 vector plasmid fragment, 0.03pmol; lnc-MUTYH gene fragment, 0.09pmol; T4 DNA ligase (350U / μl), 1μl; ddH 2 O, to 30 μl); the ligation product was transformed into competent E. coli (DH5α), and the recombinant plasmid was amplified; the recombinant plasmid was extracted and prepared, and Kpn I / Bgl II double restriction digestion and Sanger sequencing were used for verification; the recombinant plasmid (pCDNA3.1(+)lnc-MUTYH) verified by sequencing was quantitatively analyzed for purity using an Eppendorf Biophotometer plus, and stored in a -80°C deep freezer for later use. The A549 cell line (purchased from the cell bank of the Chinese Academy of Sciences, and the MUTYH gene of the cell was homozygous wild type after genotyping, and there was no lnc-MUTYH expression itself) was selected for in vitro culture. The pCDNA3.1(+)lnc-MUTYH expression plasmid was transfected into the cultured A549 cells, and the effect of pCDNA3.1(+)lnc-MUTYH expression plasmid transfection on the expression of MUTYH protein in A549 cells was compared and observed. The specific transfection conditions and process are as follows:

[0060] A549 (adherent cells) were cultured in DMEM medium containing 10% fetal bovine serum. One day before transfection, the cells were trypsinized and detached and the cells were counted. The cell concentration was adjusted to 1.5×10 5 cells / ml, 2 ml of cell suspension was plated in each well for culture, and transfection was performed when the cell growth confluence reached 70-80%;

[0061] Use 125μl OPTI-MEMⅠ medium to dilute 2.0μg of the plasmid to be transfected (pCDNA3.1(+)lnc-MUTYH expression plasmid or control empty plasmid pCDNA3.1) in each well of cells, and add 4μl of P3000 reagent. At the same time, prepare Lipofectamine 3000 liposomes, and use 125μl OPTI-MEMⅠ medium to dilute 3.75μl liposome Lipofectamine 3000 (preparation) in each well of cells. Take 125μl of the prepared liposome Lipofectamine 3000 reagent and 125μl of the prepared plasmid to be transfected (preparation) in equal proportions, mix gently by pipetting, and let stand at room temperature for 15 minutes;

[0062] 250 μl of the mixed plasmid-liposome complex solution was added to the cell culture wells, and the culture plate was gently shaken to mix well. After 6 hours of incubation at 37°C, the culture medium was replaced with 10% fetal bovine serum and continued to be cultured for 48 hours.

[0063] Cells were collected to analyze changes in MUTYH protein expression. The results showed that A549 cells without the AluYb8 insertion mutation in the MUTYH gene had a higher MUTYH protein expression level (mock group, transfected with an empty plasmid); after transfection with the pCDNA3.1(+)lnc-MUTYH expression plasmid, the MUTYH protein expression in A549 cells was significantly reduced ( Figure 3 ). This indicates that lnc-MUTYH has a significant inhibitory effect on the expression of MUTYH.

[0064] (2) Select the homozygous variant 293T cells (which express lnc-MUTYH) and design small interfering siRNA targeting lnc-MUTYH (si-lnc-MUTYH):

[0065] Sequence: sense 5'-ACCUGUAUGUGCUGGGAUUTT-3'

[0066] antisense 5'-AAUCCCAGCACAUACAGGUTT-3'

[0067] Experimental detection of the knockdown (inhibition) effect of the designed and synthesized si-lnc-MUTYH on lnc-MUTYH expressed in homozygous mutant cells (293T cells):

[0068] Adjust the 293T cell concentration to 1.5 × 10 5cells / ml, inoculated into a 24-well culture plate (0.5 ml of cell suspension per well), and cultured for 24 hours until the confluence of the cultured cells reached about 70%.

[0069] si-lnc-MUTYH transfection solution (10 pmol si-lnc-MUTYH in 50 μl culture medium and 1.5 μl liposome Lipofectamine 3000 in 50 μl culture medium per culture well, mixed, and allowed to stand at room temperature for 15 minutes. At the same time, a random sequence microRNA control (si-control) (synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.) was set up.

[0070] Add si-lnc-MUTYH-liposome complex (or si-control-control) to the cell culture wells and gently shake the culture plate.

[0071] After 48 hours of continuous culture, the cells were collected to analyze the expression of the target gene. As a result, the control (si-control) cells were affected by the presence of lnc-MUTYH in the 293T itself, and the expression level of MUTYH protein was very low; after transfection of siRNA (si-lnc-MUTYH) to knock down lnc-MUTYH, the expression of MUTYH protein in the cells was significantly increased ( Figure 4 A).

[0072] (3) To further determine the mechanism by which lnc-MUTYH inhibits MUTYH protein expression, we compared and analyzed the lnc-MUTYH base sequence and the specific promoter sequences of the three transcripts of the MUTYH gene (α-, β-, and γ-transcripts). We predicted that lnc-MUTYH may inhibit the translation of MUTYH type 1 protein (localized in mitochondria) by binding to the α-transcript of the MUTYH gene (α-MUTYH).

[0073] To verify this concept, a recombinant plasmid with the α-transcript promoter of the MUTYH gene was designed and constructed using the green fluorescent protein reporter system (expression plasmid):

[0074] The α-MUTYH 5'UTR sequence was prepared, and primers carrying restriction sites (Forward-AGGAGATCTCAGCCGGAGCCGCGGTGTACAA; Reverse-CCACTGCAGCCACAGACGACTCAGGCGGGA) were designed to amplify the α-MUTYH 5'UTR region (including the α-MUTYH promoter); PCR system (50 μl): Ex Taq polymerase (5 U / μl; TaKaRa), 0.25 μl; 10× Ex Taq Buffer (premixed with MgCl2 2+ ), 5 μl; dNTP Mixture (2.5 mM each), 4 μl; upstream primer (10 μM), 1 μl; downstream primer (10 μM), 1 μl;

[0075] cDNA, 1.0 μl; ddH 2 O, to 50 μl; PCR program: 94°C, 3 min; 94°C, 30 sec, 58°C, 30 sec, 72°C, 30 sec, 32 cycles; 72°C extension for 10 min.

[0076] The pEGFP-N1 empty plasmid (purchased from GeneCare, Shanghai) was selected, and the pEGFP-N1 empty plasmid and the corresponding α-MUTYH 5'UTR region DNA sequence (obtained by amplification) were double-digested with Bgl II and Pst I (NEBuffer TM r3.1, 37°C, 60min; NEB); after purifying the digested product, the cut pEGFP-N1 empty plasmid and α-MUTYH 5'UTR sequence were connected with T4 ligase to form an α-EGFP fusion plasmid;

[0077] 10 μl of the ligation product (fusion plasmid) was transferred into 30 μl of competent (DH5α) E. coli, and then the E. coli was spread on an LB culture plate containing ampicillin resistance;

[0078] After colonies grow on the LB culture plate, single clones are picked and cultured with liquid LB medium, and plasmids are extracted and sequenced. To verify that the proposed fusion plasmid was successfully constructed. The constructed fusion plasmid was transfected into homozygous mutant 293T cells (which express lnc-MUTYH by themselves). In the control cells (transfected with empty plasmids), the promoter function of the α-transcript was significantly inhibited (the expression level of green fluorescent protein was very low), and after transfection of siRNA (si-lnc-MUTYH) to knock down (inhibit) lnc-MUTYH in 293T cells, the function of the promoter sequence of the α-transcript was restored (the expression of green fluorescent protein was significantly increased) ( Figure 4 B). It was demonstrated that lnc-MUTYH specifically inhibited the translation expression of the α-transcript (α-MUTYH) of the MUTYH gene, that is, inhibited the expression of the mitochondrial localized MUTYH type 1 protein.

[0079] 4. Design and functional identification of small interfering RNA (siRNA) targeting MUTYH gene α-transcript

[0080] Based on the base sequence of the α-transcript of the MUTYH gene and the design principles of small interfering RNA, multiple pairs of siRNAs targeting the α-transcript of the MUTYH gene were designed and synthesized (Table 1 lists the three pairs of designed siRNA sequences, namely si-α-MUTYH-1, si-α-MUTYH-2 and si-α-MUTYH-3). In order to identify the effect of its targeted knockdown of the α-transcript of the MUTYH gene, human lung epithelial cell line A549 cells (MUTYH gene is homozygous wild type) were cultured in vitro as experimental cells, and the effect of siRNA transfection on the expression level of the target transcript was analyzed:

[0081] 3×10 5 The cells were evenly seeded in 6-well plates, and transient transfection was performed when the cell confluence reached about 70%;

[0082] Transfer 250 μl Opti-MEM into a 1.5 ml EP tube, add 10 μl Lipofectamine iMax or 7.5 μl Lipofectamine 3000 and 5 μl P3000;

[0083] Add 25 pmol si-α-MUTYH-1 (or si-α-MUTYH-2 or si-α-MUTYH-3) to the above EP tube, mix well, and incubate at room temperature for 15 min;

[0084] Add the mixture into a six-well plate and fill up to 2 ml / well with complete culture medium;

[0085] 37°C, 5% CO 2 After culturing for 48 or 72 hours, the cells were collected, total RNA was extracted, and cDNA was obtained by reverse transcription. Real-time quantitative PCR (RT-PCR) was used to detect the mRNA expression levels of the three transcripts of the MUTYH gene (α-MUTYH, β-MUTYH, and γ-MUTYH) (i. Prepare 10 μL PCR reaction system: Select Master Mix (2×) 5 μl, cDNA sample 4 μl, upstream primers and downstream primers for α-MUTYH (or β-MUTYH or γ-MUTYH) amplification (α-MUTYH amplification primers: 5'-AAACTGCGCCATCGTCACTG-3' and 5'-GAAGGCTTGGCCTGACTGTTG-3'; β-MUTYH amplification primers: 5'-CTCCGTGTTCTGCTGTCTTC-3' and 5'-CTTGGCCTGACTGTTGTTCT-3'; γ-MUTYH amplification primers: 5'-CGCTAATTGCCTATTGGCCTGT-3' and 5'-GAAGGCTTGGCCTGACTGTTG-3') each 0.5 μl. ii. Real-time quantitative PCR reaction conditions: pre-denaturation, 95°C for 30 s; cycle reaction, denaturation at 95°C for 15 s, annealing and extension at 60°C for 30 s (40 cycles); melting curve: 95°C for 15 s, 60°C for 30 s, 95°C for 15 s).

[0086] The results showed that the designed si-α-MUTYH-1 (or si-α-MUTYH-2 or si-α-MUTYH-3) could effectively knock down (inhibit) the expression of α-MUTYH, but did not affect the expression of β-MUTYH and γ-MUTYH ( Figure 5 Figure 3 shows the effect of si-α-MUTYH-1 knocking down the expression of α-MUTYH).

[0087] Table 1. Non-coding regulatory RNA sequences for inhibiting the expression of mitochondrial localized MUTYH1 isoform proteins

[0088]

[0089]

[0090] 5. lncRNA associated with variant genes affects cell mitochondrial proliferation and mitochondrial function

[0091] To further explore the effect of lnc-MUTYH associated with the mutant gene on the proliferation and function of mitochondria, homozygous mutant 293T cells (which express lnc-MUTYH themselves) were cultured, and the function of lnc-MUTYH in cells was inhibited by transfection of siRNA (si-lnc-MUTYH), and the effect on the number and function of mitochondria was observed.

[0092] (1) Select the mitochondrial DNA (mtDNA) encoding mt-CO1 (Accession NC_012920.1) and mt-tRNA leu(Accession NC_012920.1)

[0093] mt-CO1 Primers:

[0094] 5'-TTCGCCGACCGTTGACTATTCTCT-3'(Forward),

[0095] 5'-AAGATTATTACAAATGCATGGGC-3'(Reverse);

[0096] mt-tRNA leu Primers:

[0097] 5'-CACCCAAGAACAGGGTTTGT-3'(Forward),

[0098] 5'-TGGCCATGGGTATGTTGTTA-3'(Reverse).

[0099] At the same time, the single copy gene β-actin (Accession NG_007992.1) in the cell nuclear genome (nDNA) was selected as the nuclear genome DNA reference fragment.

[0100] β-actin primers:

[0101] 5'-TCACCCACACTGTGCCCATCTACGA-3'(Forward), 5'-CAGCGGAACCGCTCATTGCCAATGG-3'(Reverse).

[0102] Extract and test cell DNA, including nuclear genomic DNA (nDNA) and cell mitochondrial DNA (mtDNA), and use it as template DNA for PCR amplification to perform real-time quantitative PCR. According to the PCR amplification amount of mtDNA gene fragments and nDNA gene fragments, calculate the relative ratio of mtDNA / nDNA, evaluate the relative content of mtDNA in individual cells, and predict the changes in the number of mitochondria in cells.

[0103] The real-time quantitative PCR system and data analysis are as follows:

[0104] The real-time quantitative PCR system (10 μl) was as follows: 2×PowerUp SYBR Green Master Mix (Applied Biosystems), 5 μl; upstream primer (10 mM), 0.3 μl; downstream primer (10 mM), 0.3 μl; DNA template (10 ng / μl), 1 μl; ddH2 O, 3.4 μl.

[0105] The PCR program was set as: 50°C, 2 min; 95°C, 2 min; 95°C 3 sec, 60°C 30 sec, 40 cycles.

[0106] The Cp value of the target gene was obtained, and the content of mtDNA was corrected using the nuclear genomic fragments, namely the "2ΔCt" method, to calculate the ratio of mtDNA to nDNA in the cell (ie, the relative content of mtDNA).

[0107] Results: Compared with control cells transfected with (synthetic) random sequence microRNA (si-control, Ruibo Bio, Guangzhou), the content of mtDNA in cultured 293T cells transfected with (si-lnc-MUTYH) was significantly increased (mt-CO1 and mt-tRNA leu The results of the two fragments compared with the β-actin fragment of nDNA are consistent with those of the Figure 6 ), indicating that the expression of lnc-MUTYH in 293T cells itself has a significant inhibitory effect on the replication of their mtDNA (mitochondrial proliferation).

[0108] (2) The expression level of the COX2 gene (encoded by mitochondrial DNA, Accession NC_012920.1) which can reflect the mitochondrial function of cells was further selected. Total RNA of cells was extracted, and the transcriptional expression status of COX2 was detected by reverse transcription (for specific operations, see above) and real-time quantitative PCR technology.

[0109] COX2 real-time quantitative PCR primers:

[0110] Forward: 5′-CCCCACATTAGGCTTAAAAACAGAT-3′ and Reverse: 5′-TATACCCCCGGTCGTGTAGC-3′ (the operation of real-time quantitative PCR is described above).

[0111] The results showed that after si-lnc-MUTYH transfection, the transcription level of the cell mitochondrial DNA encoding gene COX2 was significantly upregulated ( Figure 7 ). It was confirmed that lnc-MUTYH has an inhibitory effect on cell mitochondrial proliferation and mitochondrial function.

[0112] 6. lncRNA associated with variant genes inhibits M2 polarization of macrophages

[0113] Many studies have confirmed that the M2 polarization of macrophages in the body's tissue microenvironment plays an important role in the pathogenesis and development of various diseases (such as cancer, tissue fibrosis), and the M2 polarization of macrophages depends on the oxidative phosphorylation function of cell mitochondria. In order to clarify whether the expression of specific lncRNA associated with the Alu insertion mutation of the MUTYH gene that inhibits mitochondrial function can inhibit the M2 polarization of macrophages, the human monocyte / macrophage cell line THP-1 (purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai) with the homozygous wild type (A / A) of the MUTYH gene was selected for in vitro culture. IL-4 (final concentration of 20ng / ml, P5129, Beyotime) and IL-13 (final concentration of 20ng / ml, P5178, Beyotime) (IL-4-IL-13) confirmed in the field were used to stimulate THP-1 cells to polarize to M2, and the marker molecules CD206 and CD163 were used as observation target molecules to evaluate the M2 polarization state of THP1 cells. The constructed lnc-MUTYH expression plasmid (pCDNA3.1(+)lnc-MUTYH) was used to transfect cultured THP1 cells, and the effect of lnc-MUTYH expression on IL-4-IL-13-induced M2 polarization of macrophages was analyzed and determined.

[0114] Specific operations:

[0115] Count 4×10 5 Cells were seeded in 6-well plates;

[0116] Use 125 μl OPTI-MEMⅠ medium to dilute 2.0 μg of lncMUTYH expression plasmid or control plasmid, and add 4 μl of P3000 reagent;

[0117] Use 125 μl OPTI-MEMⅠ medium to dilute 3.75 μl lipofectamine 3000 reagent;

[0118] The diluted liposome reagent and the diluted plasmid DNA were mixed in equal proportions, and the mixture was gently pipetted and mixed. After standing at room temperature for 15 minutes, the plasmid DNA-liposome complex was added to the cell culture wells and cultured for 24 hours for macrophage polarization induction analysis. The results showed that compared with the control (empty plasmid transfection) cells, lnc-MUTYH transfection expression significantly reduced the expression levels of IL-4-IL-13-induced THP1 cell M2 polarization marker molecules CD206 and CD163, indicating that IL-4-IL-13-induced THP1 cell M2 polarization was blocked ( Figure 8). It was demonstrated that lnc-MUTYH expression associated with MUTYH variant gene could inhibit the M2 polarization of macrophages in variant gene carriers.

[0119] 7. The lncRNA associated with the mutant gene (lnc-MUTYH) inhibits the transformation of fibroblasts into myofibroblasts (effector cells of tissue fibrosis) by inhibiting the polarization of macrophages M2

[0120] More and more data show that M2 polarized macrophages in the tissue microenvironment play an important role in the occurrence of tissue and organ fibrosis. M2 macrophages secrete TGF-β, activate the TGF-β / Smad signaling pathway, and induce fibroblast to myofibroblast transition (FMT). Myofibroblasts are the main effector cells of tissue and organ fibrosis. Their high expression of interstitial molecules such as collagen causes excessive accumulation of tissue interstitial molecules and tissue fibrosis. Fibronectin and α-SMA are the main marker molecules for the differentiation and formation of myofibroblasts. In order to clarify the potential role of lnc-MUTYH in inhibiting macrophage M2 polarization in the prevention and treatment of tissue fibrosis, the macrophage-fibroblast co-culture technique was used to analyze and identify the inhibitory effect of lnc-MUTYH on fibroblast to myofibroblast transition (FMT) by inhibiting macrophage M2 polarization. Human monocyte / macrophage cell line THP-1 and human lung fibroblast MRC5 with homozygous wild type (A / A) MUTYH gene were selected (both cell lines were purchased from Cell Bank of Chinese Academy of Sciences, Shanghai). The lnc-MUTYH expression plasmid (pCDNA3.1(+)lnc-MUTYH) or empty control plasmid (pCDNA3.1) was transferred into THP-1 cells cultured in vitro using lipofectamine 3000 (the transfection process and conditions were the same as described above); the transfected THP-1 cells were cultured in culture medium containing IL-4-IL-13 (the final concentration of both cytokines was 20 ng / ml) (24 hours) to induce their M2 polarization; then the THP-1 cells induced by IL-4-IL-13 polarization were moved to the bottom of the culture inoculated with MRC5 cells and co-cultured for 24 hours; then the MRC5 cells at the bottom (co-culture) were collected, and MRC5 cells cultured alone were set up as blank controls; the cell proteins were extracted, and the expression of myofibroblast marker molecules Fibronectin and α-SMA was detected by Western blot technology. The results showed that lnc-MUTYH inhibited the transformation of fibroblasts to myofibroblasts (FMT) by inhibiting the M2 polarization of macrophages. Given that myofibroblasts are the main effector cells of tissue fibrosis pathology, this result shows the role of lnc-MUTYH in the prevention and treatment of tissue and organ fibrosis diseases ( Fig. 9 ).

[0121] 8. Carriers of MUTYH gene insertion (homozygous) mutations significantly reduce their risk of lung cancer

[0122] It has been revealed that macrophage M2 polarization in the tissue microenvironment has the effect of suppressing tumor immunity and promoting the occurrence and development of cancer. The Alu sequence in the biological genome is unique to primates. The MUTYH gene α-transcript encodes the MUTYH 1 protein located in the mitochondria, which is the characteristic expression form of the human gene. The MUTYH gene Alu insertion mutation is a polymorphic mutation form of the gene unique to the human genome. Therefore, it is difficult to observe the effect of the MUTYH gene Alu insertion mutation lncRNA expression on the occurrence and development of cancer in animal models. In view of this, the molecular epidemiological "case-control analysis" was selected to implement the effect of the MUTYH gene Alu insertion mutation on the incidence of lung cancer. Most lung cancers are sporadic cases in clinic, and there is no traceable family history of lung cancer. With the increase of age, the risk of lung cancer increases. It is an aging-related disease. The median age of lung cancer in Chinese is currently about 62 years old. Considering that the incidence of lung cancer in young patients may be greatly affected by specific factors such as family or self-environment, a total of 438 lung cancer patients in the peak age group (55-69 years old) and the older age group (≥70 years old) around the median age and 1308 normal controls matched by age and gender were selected to analyze the effect of MUTYH gene Alu insertion mutation on the incidence of lung cancer patients at the median age and above in two age groups. The results showed that MUTYH gene Alu insertion mutation has a protective effect on the occurrence of lung cancer in the clinical high-risk age group (Table 2). Given that the expression of lnc-MUTYH associated with the Alu insertion mutation of the MUTYH gene is the (molecular) mechanism of its functional effect, the protective effect of this mutation on the occurrence of lung cancer should be mediated by lnc-MUTYH.

[0123] Table 2. Relationship between the AluYb8 insertion mutation of the MUTYH gene and the risk of lung cancer

[0124]

[0125] Most of the lung cancer patients in Table 2 have no clear family history, which is an aging-related disease, mainly occurring in middle-aged and elderly people. We selected lung cancer patients with an onset age of ≥55 years and healthy individuals of the corresponding age to conduct a case-control analysis of the effects of different genotypes of the MUTYH gene AluYb8 insertion mutation on the occurrence of lung cancer. The results showed that the detection rate of this mutation in the MUTYH gene in middle-aged and elderly lung cancer patients was significantly lower than that in middle-aged and elderly healthy controls, indicating that this mutation is a protective factor for the occurrence of lung cancer in middle-aged and elderly people. This suggests that the functional inhibition of the medicinal mitochondrial-localized MUTYH1 protein has potential intervention and therapeutic effects in the occurrence and development of lung cancer in middle-aged and elderly people.

[0126] 9. Protective effect of MUTYH gene functional deficiency on the occurrence of tissue fibrosis

[0127] Due to the specificity of biological genomes, the study of the expression of lnc-MUTYH associated with the Alu insertion mutation of the human MUTYH gene is difficult to implement in model animals. To further demonstrate that the non-coding regulatory RNA (lnc-MUTYH) associated with the expression of Alu insertion mutation of the human MUTYH gene that can damage the mitochondrial function of cells has a protective effect on the occurrence of tissue fibrosis, the Mutyh gene knockout (Mutyh - / Mutyh - ) mice (Chen J, et al. Free radical biology & medicine. 2019; 143: 482-493). The mouse has been identified to have defective Mutyh gene function, and the mouse mitochondrial DNA oxidative damage repair function is impaired. At the same time, the commonly used bleomycin (Bleomycin, BLM) induced mouse pulmonary fibrosis model was selected, and bleomycin (3 mg / kg) was instilled in the airway once, and normal saline (Normal saline, NS) was instilled in the airway as a control. Comparative observation of Mutyh gene knockout mice (Mutyh - / Mutyh - ) and wild-type mice in terms of the pathological changes in lung fibrosis. 28 days after airway instillation, chest CT scans were performed on the model mice to observe the state of lung fibrosis ( Fig.10 ), and the mice were euthanized and the lung tissues of the model mice were taken for Masson staining to analyze the collagen fiber deposition in the lung tissues. The results showed that the lungs of wild-type mice instilled with bleomycin (BLM) showed extensive fibrotic pathological changes, while the BLM-induced pulmonary fibrosis in Mutyh gene knockout mice was significantly milder ( Fig.11 ). Chest CT scans and Masson staining of lung tissue in model mice confirmed that the functional defect of the Mutyh gene, which can lead to mitochondrial dysfunction, has a protective effect on the pathological occurrence of lung tissue fibrosis.

[0128] The above experimental results, which are the basis for proposing the present invention, clearly show that by designing non-coding regulatory RNA, specifically blocking the repair of mitochondrial DNA oxidative damage and inhibiting the mitochondrial oxidative phosphorylation function, the M2 polarization of macrophages can be effectively blocked. Based on the functional characteristics of the tissue microenvironment mainly constructed by M2 macrophages, the present invention discloses the use of nucleic acid molecules that block the M2 polarization of macrophages in the development and preparation of new drugs for the prevention or treatment of cancer and tissue fibrosis diseases.

Claims

1. Use of non-coding regulatory RNA in the preparation of a drug for preventing or treating cancer or fibrotic diseases, characterized in that: The non-coding regulatory RNA includes a long non-coding regulatory RNA sequence as shown in SEQ ID NO.1 and a small interfering RNA sequence as shown in SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4; The cancer includes lung cancer; The fibrotic disease is pulmonary fibrosis; The non-coding regulatory RNA is used to regulate the immunological state of the lung tissue microenvironment and inhibit the formation of a microenvironment that promotes the occurrence and development of tumors / fibrotic lesions.

2. The use according to claim 1, characterized in that The non-coding regulatory RNA can target and inhibit the expression of mitochondrial-localized base excision repair proteins.

3. The use according to claim 1, characterized in that The non-coding regulatory RNA is used to inhibit the function of mitochondrial oxidative phosphorylation in cells.

4. The use according to any one of claims 1 to 3, characterized in that: The non-coding regulatory RNA is used to inhibit the M2 polarization of macrophages.

5. The use according to claim 4, characterized in that The expression vector inhibits cell mitochondrial proliferation and mitochondrial function or inhibits the M2 polarization of macrophages in the body.

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