SiRNA for inhibiting expression of corneal epithelium-derived ALYREF-UHRF1 signal axis gene and application thereof

Abstract

The invention provides siRNA for inhibiting expression of a corneal epithelium-derived ALYREF-UHRF1 signal axis gene and application of the siRNA, and belongs to the technical field of gene therapy. The siRNA for inhibiting the expression of the ALYREF-UHRF1 signal axis gene comprises a siRNA for inhibiting the UHRF1, a siRNA for inhibiting the ALYREF, and a siRNA group containing the siRNA for inhibiting the UHRF1 and the siRNA for inhibiting the ALYREF. According to the present invention, the expression of the ALYREF-UHRF1 signal axis gene can be inhibited through the siRNA or the siRNA group, such that the proliferation and the growth of the epithelium-derived tumor can be inhibited so as to achieve the effect of treating the epithelium-derived tumor.

Description

Technical Field

[0001] The present invention relates to the technical field of gene therapy, and in particular to siRNA for inhibiting the expression of ALYREF-UHRF1 signal axis gene derived from corneal epithelium and application thereof. Background Art

[0002] ALYREF (Aly / REF export factor) is an RNA binding protein that plays an important role in RNA metabolism, especially in the nuclear export of mRNA. ALYREF acts as a reader of RNA m5C modification, recognizing and binding to 5-methylcytosine (m5C) modification on RNA molecules. m5C is a common RNA modification that plays an important role in the stability, export and translation of mRNA. ALYREF participates in regulating RNA metabolism by recognizing m5C modification, including promoting the transport of mRNA from the nucleus to the cytoplasm.

[0003] UHRF1 (Ubiquitin-like with PHD and Ring Finger domains 1) is a multifunctional nuclear protein. UHRF1 includes ubiquitin-like domain, PHD domain (planthomeodomain finger), SRA domain and RING finger domain. UHRF1 recognizes and binds to methylated DNA and histones through the above domains, thereby participating in cell life activities such as DNA replication, cell cycle regulation, cell proliferation and apoptosis, and DNA damage response.

[0004] The signal axis is a relatively specific concept, which usually refers to a complex system composed of a series of proteins or other biological molecules that can interact and transmit signals. These molecules form one or more signal transmission pathways in the body through specific interaction mechanisms, thereby regulating cell function, tissue development, and physiological and pathological processes. In medical research, the concept of signal axis is widely used to explain a variety of physiological and pathological phenomena.

[0005] At present, it has been found that both ALYREF and UHRF1 have the ability to promote cell proliferation, differentiation and migration, but the regulatory relationship between ALYREF and UHRF1 is unknown. As for how to regulate ALYREF and / or UHRF1 to achieve the application of treating epithelial tumors, it is also an urgent problem to be solved. Summary of the invention

[0006] The present invention proposes siRNA for inhibiting the expression of ALYREF-UHRF1 signaling axis genes derived from corneal epithelium and its application, which can inhibit the expression of ALYREF-UHRF1 signaling axis genes by siRNA inhibiting UHRF1 and / or siRNA inhibiting ALYREF, thereby inhibiting the proliferation of epithelial tumors and achieving the effect of treating epithelial tumors.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an siRNA for inhibiting the expression of ALYREF-UHRF1 signal axis gene derived from corneal epithelium, wherein the siRNA sequence is an siRNA for inhibiting UHRF1;

[0009] Sense strand of siRNA: 5′-GGACGAAGTCTTCAAGATT-3′;

[0010] Antisense strand of siRNA: 3′-CCUGCUUCAGAAGUUCUAA-5′.

[0011] In a second aspect, the present invention further provides a siRNA for inhibiting the expression of the ALYREF-UHRF1 signal axis gene derived from corneal epithelium, wherein the siRNA sequence is an siRNA for inhibiting ALYREF;

[0012] Sense strand of siRNA: 5′-GCACGATCTTTTCGACAGT-3′;

[0013] Antisense strand of siRNA: 3'-CGUGCUAGAAAAGCUGUCA-5'.

[0014] In an implementation of the first and second aspects, the 3' ends of the sense strand and antisense strand of the UHRF1-inhibiting siRNA of the first aspect are dangled with dTdT, and the 3' ends of the sense strand and antisense strand of the ALYREF-inhibiting siRNA of the second aspect are dangled with dTdT.

[0015] In a third aspect, the present invention also provides a siRNA group for inhibiting the expression of ALYREF-UHRF1 signaling axis genes derived from corneal epithelium, including the siRNA for inhibiting UHRF1 of the first aspect and the siRNA for inhibiting ALYREF of the second aspect, or, the siRNA for inhibiting UHRF1 and the siRNA for inhibiting ALYREF in the above-mentioned implementation method.

[0016] In a fourth aspect, the present invention further provides a siRNA for inhibiting the expression of ALYREF-UHRF1 signal axis gene derived from corneal epithelium, wherein the siRNA sequence is an siRNA for inhibiting UHRF1;

[0017] The sense strand of the siRNA: 5'-CGAGATCTTTCCGGCAACA-3';

[0018] The antisense strand of the siRNA: 3'-GCUCUAGAAAGGCCGUUGU-5'.

[0019] In a fifth aspect, the present invention also provides siRNA for inhibiting the expression of ALYREF-UHRF1 signal axis gene derived from corneal epithelium, wherein the siRNA sequence is siRNA for inhibiting ALYREF;

[0020] The sense strand of the siRNA: 5'-GCGAGAATGGACACCAGTT-3';

[0021] The antisense strand of the siRNA: 3'-CGCUCUUACCUGUGGUCAA-5'.

[0022] In an implementation of the fourth and fifth aspects, the 3' ends of the sense and antisense strands of the UHRF1-inhibiting siRNA of the fourth aspect are dangled with dTdT, and the 3' ends of the sense and antisense strands of the ALYREF-inhibiting siRNA of the fifth aspect are dangled with dTdT.

[0023] In a sixth aspect, the present invention further provides a siRNA group for inhibiting the expression of ALYREF-UHRF1 signaling axis genes derived from corneal epithelium, including the siRNA for inhibiting UHRF1 of the fourth aspect and the siRNA for inhibiting ALYREF of the fifth aspect, or the siRNA for inhibiting UHRF1 and the siRNA for inhibiting ALYREF in the above-mentioned implementation methods.

[0024] In a seventh aspect, the present invention further provides an application of siRNA that inhibits UHRF1 or siRNA that inhibits ALYREF in the preparation of a drug for treating epithelial tumors, based on the siRNA provided in any one of the first, second, third, fourth, fifth and sixth aspects above.

[0025] In one implementation of the seventh aspect, the epithelial tumor is at least one of bladder urothelial carcinoma, head and neck squamous cell carcinoma, breast invasive cancer, cervical squamous cell carcinoma and adenocarcinoma, bile duct cancer, colon cancer, rectal adenocarcinoma, esophageal cancer, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, prostate cancer, skin melanoma, gastric cancer, thyroid cancer, thymic carcinoma and endometrial cancer.

[0026] In an eighth aspect, the present invention provides an anti-tumor drug prepared by the siRNA or siRNA group that inhibits the expression of the ALYREF-UHRF1 signaling axis gene derived from the corneal epithelium according to any one of the first, second, third, fourth, fifth and sixth aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is an analysis diagram of the inhibition of corneal epithelial damage repair by in vivo / in vitro knockdown of UHRF1 provided in the examples of the present application;

[0028] Figure 2 It is a schematic diagram showing that the UHRF1 inhibitor Hinokitiol provided in the examples of the present application can significantly inhibit the proliferation and migration ability of HCEC;

[0029] Figure 3 is the RNA m provided in the examples of this application 5 Schematic diagram of the C reader ALYREF binding to UHRF1 mRNA and regulating the expression of UHRF1;

[0030] Figure 4 is the RNA m provided in the examples of this application 5 Schematic diagram of the C reader ALYREF recognizing UHRF1 mRNA;

[0031] Figure 5 It is a schematic diagram of the correlation analysis of ALYREF and UHRF1 expression in 21 epithelial tumors provided in the examples of the present application;

[0032] Figure 6 It is a schematic diagram for verifying the existence of the ALYREF / UHRF1 signal axis provided in an embodiment of the present application;

[0033] Figure 7 This is a functional schematic diagram of verifying the ALYREF / UHRF1 signaling axis on the breast cancer cell line provided in the examples of the present application;

[0034] Figure 8 It is a schematic diagram of the analysis of the results of the human breast cancer cell nude mouse tumor formation experiment provided in the examples of the present application. DETAILED DESCRIPTION

[0035] The “and / or” in the embodiments of the present application indicates the relationship between objects. For example, A and / or B may indicate the following three situations: A exists alone, B exists alone, and A and B exist at the same time.

[0036] In the embodiments of the present application, words such as "exemplary" or "for example" are used to indicate examples, illustrations or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of the present application should not be interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0037] The method provided in the embodiment of the present application relates to siRNA that inhibits the expression of the ALYREF-UHRF1 signaling axis gene derived from the corneal epithelium and its application. Epithelial tumors can be treated by inhibiting the gene expression of the ALYREF-UHRF1 signaling axis derived from the corneal epithelium. Specifically, the gene expression of the ALYREF-UHRF1 signaling axis derived from the corneal epithelium can be inhibited by siRNA that inhibits UHRF1 and siRNA that inhibits ALYREF, thereby further inhibiting the proliferation, differentiation and migration of epithelial tumors, thereby achieving the effect of treating epithelial tumors.

[0038] The following is an introduction to the relevant concepts involved in the embodiments of the present application.

[0039] UHRF1 (Ubiquitin-like with PHD and Ring Finger domains 1) is an important epigenetic integrator (a protein or protein complex that can integrate multiple epigenetic mechanisms or factors to jointly regulate gene expression or cell fate during the epigenetic regulation of organisms. UHRF1 is a nuclear protein) that can recognize methylated DNA and methylated histones and is specifically highly expressed in a variety of tumors.

[0040] Among them, epigenetic regulation mainly includes DNA methylation, histone modification and non-coding RNA. These biological codes characterized by flexibility and adaptability have the ability to interpret the DNA genome carried by cells.

[0041] The above DNA methylation refers to the chemical modification process in which the active methyl group is transferred to a specific base (mainly the C-5 position of cytosine) in the DNA chain under the catalysis of DNA methyltransferase (DNMT) using S-adenosylmethionine (SAM) as a methyl donor. This modification can occur at multiple locations in the genome, especially on the cytosine of CpG dinucleotides, forming 5-methylcytosine (m5C or m 5 C).

[0042] The above-mentioned histone modification is a complex and important field in epigenetics, which refers to the process in which histone undergoes a series of chemical changes under the action of related enzymes, including methylation, acetylation, phosphorylation, adenylation, ubiquitination, ADP ribosylation, etc. These modifications do not change the nucleotide sequence of DNA, but can regulate the accessibility of DNA to the transcriptional machinery, thereby affecting gene expression.

[0043] The above-mentioned non-coding RNAs (ncRNAs) are a type of RNA that does not encode proteins. They are widely present in cells. Although they are not translated into proteins, they can perform their respective biological functions at the RNA level.

[0044] ALYREF (Aly / REF export factor) is an RNA binding protein that is also highly expressed in a variety of tumors. It acts as a "reader" to recognize and bind to 5-methylcytosine (m5C) modifications on RNA molecules, and participates in regulating RNA metabolism, including promoting the transport of mRNA from the nucleus to the cytoplasm.

[0045] ALYREF is the first RNA m5C reader discovered in the nucleus. It specifically binds to the m5C modification site at lysine 171, promotes mRNA nuclear export, and then exerts its biological function. High expression of ALYREF has been found to promote cell proliferation, differentiation and migration in a variety of epithelial tumors. Further preliminary experiments found that knocking down ALYREF in the mouse corneal epithelium did block the corneal epithelial wound healing (CEWH) process; multi-omics joint analysis identified UHRF1, a potential downstream core target of ALYREF; Luciferase detection preliminarily showed that ALYREF can recognize downstream m5C-UHRF1. UHRF1, as a hub of DNA methylation and histone modification, is an important epigenetic regulator that plays a key role in regulating cell proliferation, differentiation and migration; in particular, high expression of UHRF1 in CEWH significantly promotes CEWH, and UHRF1 mRNA has abundant RNA m5C modification sites. These data suggest that ALYREF may regulate CEWH by recognizing and binding to RNA m5C-UHRF1.

[0046] Among them, 5-methylcytosine (m5C) modification is a type of RNA modification that has attracted widespread attention. This modification is species-conservative, tissue-specific, and developmentally dynamic, and is significantly enriched downstream of the translation start codon and in the CG-rich region. In the early stage, Yang Yungui's team at the Beijing Institute of Genomics, Chinese Academy of Sciences, established RNA m5C sequencing technology, mapped the mRNA m5C modification map of mammalian cells and tissues, and identified NSUN2 as an mRNA-specific m5C methylase whose catalytic activity depends on two cysteine ​​sites, C271 and C321. It was further discovered that m5C regulates mRNA nuclear export through its intranuclear reader ALYREF. Based on the previously discovered RNA m5C modification regulation, more and more studies have reported that m5C plays an important regulatory role in cell metabolism, growth and development, and physiological and pathological changes.

[0047] In order to solve the problem that the regulatory relationship between ALYREF and UHRF1 in the background technology is unknown. As for how to achieve the application of treating epithelial tumors by regulating ALYREF and / or UHRF1, it is also a problem that needs to be solved urgently. The embodiments of the present application provide siRNA and its application for inhibiting the gene expression of ALYREF-UHRF1 signal axis, and find the signal transduction pathway of ALYREF regulating the translation of UHRF1, and obtain the ALYREF-UHRF1 signal axis, and then inhibit the gene expression of ALYREF-UHRF1 signal axis by inhibiting UHRF1 siRNA and / or ALYREF siRNA, inhibit the proliferation, differentiation and migration of epithelial tumors, so as to achieve the effect of treating epithelial tumors.

[0048] The following example of the present application provides a detailed introduction to the process of determining the ALYREF-UHRF1 signal axis derived from the corneal epithelium.

[0049] The present application example found through the study of corneal epithelial damage repair that the expression of UHRF1 is regulated by ALYREF, that is, there is such an apparent signal axis: ALYREF-UHRF1, and this signal axis not only regulates the corneal epithelial damage repair.

[0050] Specifically, the cornea can be divided into five layers from a structural perspective: epithelial cell layer, Bowman layer, stroma layer, Descemet's membrane, and endothelial cell layer. Among them, the corneal epithelium, which is about 50 μm thick and exposed to the surface of the eyeball, originates from limbal stem cells (LSCs), is in direct contact with the external environment, and has a strong "damage repair" ability. In recent years, the mechanism of epigenetic regulation in corneal epithelial wound healing (CEWH) has also attracted more and more attention from the academic community.

[0051] In view of the relatively small number of studies on epigenetics in CEWH, the present application embodiment "pieces together" the epigenetic regulation mode of CEWH:

[0052] 1) Epigenetic Regulators: The NanoString technology system was used to construct the epigenetic regulator map of CEWH;

[0053] 2) DNA methylation level: MethylFlash was used to quantify the dynamic changes of DNA methylation in CEWH and to analyze the detailed mechanism of DNA methylation regulating CEWH;

[0054] 3) Histone modification level: The histone modification spectrum of CEWH was established based on LC-MS / MS, and the molecular mechanism of H3K9 methylase SUV39H1 regulating CEWH was elucidated;

[0055] 4) Non-coding RNA level: The miRNA expression profile of CEWH was constructed using the NanoString technology system, revealing the important role of miR-204 and miR-184, which are highly abundant in the corneal epithelium, in CEWH.

[0056] In summary, CEWH is a typical epigenetic regulation process. On this basis, the discovery and application of ALYREF / UHRF1 are elaborated in detail as follows.

[0057] 1. Silencing UHRF1 in vivo and in vitro significantly inhibits corneal epithelial damage and repair

[0058] The knockdown ability of siUHRF1 was verified by western blot experiments. Figure 1 As can be seen from Figures A and B, after transfection with siUHRF1, the protein expression level of UHRF1 decreased significantly. Figure 1 Figure B in Figure 1 is a statistical graph of protein expression, which shows significant differences. To characterize the function of UHRF1 in CEWH, we first injected siUHRF1 into the anterior corneal stroma. Figure 1 As shown in Figure C, the expression level of UHRF1 protein was significantly reduced during CEWH. Compared with the cornea injected with NC (negetive control, purchased from Thermo Fisher Scientific), the unhealed area of ​​the cornea injected with siUHRF1 was larger. Figure 1 Figure D in the figure is a statistical graph of corneal healing, which shows a significant difference. These data indicate that UHRF1 silencing delays CEWH, suggesting that UHRF1 plays an important role in regulating CEWH.

[0059] Next, the present application example uses human corneal epithelial cells (hereinafter referred to as HCECs) to study how UHRF1 silencing affects wound healing function. Proliferation experiments were performed ( Figure 1 Figure E) and cell migration assay ( Figure 1 Figure G), Figure 1 Figures F and H in the figure are statistical graphs of proliferation and migration experiments, respectively, with significant differences. The results show that the proliferation and migration of HCECs transfected with siUHRF1 were significantly inhibited compared with those transfected with NC. Overall, these results indicate that UHRF1 is essential for regulating CEWH.

[0060] 2. Inhibition of UHRF1 and overexpression of UHRF1 have opposite effects on cell proliferation and migration

[0061] To further confirm the functional relevance of UHRF1 in CEWH, we examined the corresponding effects of silencing or overexpressing UHRF1 on the migration and proliferation of HCECs. Figure 2 Figure A in the figure is a cell proliferation experiment using the UHRF1 inhibitor Hinokitiol group and the control group (CT). From the statistical chart, it can be seen that the UHRF1 inhibitor Hinokitiol inhibited cell proliferation; Figure 2 Figure B in the figure is a cell migration experiment using the UHRF1 inhibitor Hinokitiol group and the control group (CT). Through statistics, it was found that the UHRF1 inhibitor Hinokitiol inhibited the migration ability of cells. Then we used lentivirus to overexpress UHRF1, namely LV-UHRF1. LV-NC means that the lentivirus carries the same fragment size but meaningless sequence into the cell. Cell proliferation and migration experiments were also performed, such as Figure 2 Figures C and D in the figure show that after overexpression of UHRF1, the proliferation and migration abilities of cells also increased. Through the above experiments, it was concluded that the UHRF1 inhibitor Hinokitiol delayed scratch healing and inhibited the proliferation of HCECs. On the contrary, the upregulation of UHRF1 significantly promoted the proliferation and migration of HCECs. In general, these in vitro experiments show that UHRF1 promotes CEWH.

[0062] 3. RNA m5C reader ALYREF regulates the expression of UHRF1

[0063] Previous studies have shown that m5C-methylated mRNA can be directly recognized by ALYREF and YBX1 ( Figure 3Figure A), YBX1 binding has been reported to promote mRNA degradation, however, we found that NSUN2 regulates the translation of UHRF1. More importantly, during CEWH, ALYREF protein levels increased significantly, while YBX1 showed no significant changes. Therefore, we investigated whether ALYREF is involved in the regulation of UHRF1 expression. Figure 3 The RIP-qPCR in Figure B shows that ALYREF-bound UHRF1 mRNA is more enriched than IgG (antibody to immunoglobulin G)-bound UHRF1 mRNA. In addition, RT-qPCR experiments ( Figure 3 Figure C) and western blot experiment ( Figure 3 Figures D and E in Figure 2 showed that silencing ALYREF barely changed the expression level of UHRF1 mRNA ( Figure 3 C), although it reduced the expression level of UHRF1 protein ( Figure 3 D and E), suggesting that ALYREF regulates the translation of UHRF1.

[0064] It should be noted that the above RIP-qPCR was performed according to the instructions provided by Magna RIP Kit (Merk Millipore, Burlington, MA, USA) to detect the enrichment of ALYREF on UHRF1 mRNA. Briefly, HCECs were lysed using complete RIP lysis buffer. Next, magnetic beads protein A / G coupled with ALYREF antibody or IgG antibody were prepared and mixed with RIP lysis buffer. The pulled-down RNA was digested with proteinase K buffer and purified with phenol, chloroform, and isoamyl alcohol. The retrieved RNA was analyzed by RT-qPCR, and the IgG RIP experiment was performed as NC.

[0065] 4. ALYREF regulates UHRF1 expression in an RNA m5C-dependent manner

[0066] The above studies suggest that RNA m5C-modified UHRF1 may regulate CEWH through ALYREF, but it is still unclear how ALYREF recognizes UHRF1. To address this issue, we constructed UHRF1 mutants for luciferase reporter gene assays (corresponding to Figure 4 Figure A), compared with UHRF1-WT (wild-type UHRF1, i.e., the non-mutated UHRF1 gene), UHRF1-Mut (the mutant form of the UHRF1 gene) was not affected by knockdown of wild-type ALYREF (corresponding to Figure 4 This suggests that RNA m5C modification is key to ALYREF regulation.

[0067] It can be seen from this that the wild-type UHRF1 is affected by ALYREF knockdown, indicating the existence of an ALYREF-UHRF1 signaling axis; UHRF1 after the m5C modification site is mutated is not affected by ALYREF knockdown, indicating that the two recognize and bind through this m5C site, thus proving that RNA m5C modification is the key to ALYREF regulation.

[0068] 5. Correlation analysis of the ALYREF-UHRF1 signaling axis in 21 epithelial tumors

[0069] Since 90% of human cancers originate from epithelial cells, the above experiments have demonstrated the existence of an ALYREF-UHRF1 signaling axis in which ALYREF regulates UHRF1 expression in human corneal epithelial cells.

[0070] In order to verify the hypothesis that the ALYREF-UHRF1 signaling axis plays a key role in epithelial tumors, the present application example uses the GEPIA website to perform a correlation analysis between ALYREF and UHRF1 in tumors. As shown in the figure above, the correlation analysis found that the ALYREF-UHRF1 signaling axis is positively correlated in 21 different epithelial tumors (corresponding to Figure 5 BLCA bladder urothelial carcinoma, HNSC head and neck squamous cell carcinoma, BRCA invasive breast carcinoma, CESC cervical squamous cell carcinoma and adenocarcinoma, CHOL bile duct carcinoma, COAD colon cancer, READ rectal adenocarcinoma, ESCA esophageal cancer, KICH chromophobe renal cell carcinoma, KIRC renal clear cell carcinoma, KIRP renal papillary cell carcinoma, LIHC hepatocellular carcinoma, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, PAAD pancreatic cancer, PRAD prostate cancer, SKCM skin melanoma, STAD gastric cancer, THCA thyroid cancer, THYM thymic carcinoma and UCEC endometrial cancer (correlation analysis).

[0071] The above positive correlation refers to the positive correlation between ALYREF and UHRF1. Specifically, Figure 5 In these 21 epithelial tumors, when ALYREF increased, the expression level of UHRF1 also increased; when ALYREF expression decreased, the expression level of UHRF1 also decreased.

[0072] 6. Validation of the ALYREF-UHRF1 signaling axis in breast cancer cells

[0073] Breast cancer is a representative epithelial tumor. The present invention also verifies this epigenetic signal axis in breast cancer cell lines. Figure 6As shown in Figure A, ALYREF-UHRF1 are significantly elevated in breast cancer. We then knocked down ALYREF in two breast cancer cell lines (MDA-MB-231 and BT-549) and examined the changes in UHRF1 protein levels by western blot. Figure 6 Figures B and C in the figure show that knocking down ALYREF in breast cancer cell lines significantly reduced the expression of UHRF1, indicating that this signaling axis also exists in breast cancer. These results provide additional suggestive evidence that this new signaling axis exists in representative epithelial tumors.

[0074] 7. Silencing the ALYREF-UHRF1 signaling axis inhibits the proliferation of breast cancer cells

[0075] Breast cancer cell lines were used as subjects to study the inhibitory effect of silencing the ALYREF-UHRF1 signaling axis on the proliferation of breast cancer cells. In the present application, two independent triple-negative breast cancer cell lines (MDA-MB-231 and BT-549) were subjected to ALYREF knockdown, and it was found that in all the tested cell lines, the cell proliferation rate was significantly reduced ( Figure 7 We then performed an independent experiment, the colony formation assay, and the results confirmed that the number of colonies in cells with reduced ALYREF expression was significantly reduced ( Figure 7 We also further investigated the effect of ALYREF expression changes on cell proliferation by EdU experiments and found that in both cell types, ALYREF knockdown significantly reduced the intensity of EdU fluorescence ( Figure 7 Figure C in Figure 2). These results strongly support the important role of ALYREF in breast cancer cell proliferation. To further explore the role of the ALYREF-UHRF1 signaling axis in breast cancer cell proliferation, we performed a rescue experiment. The results showed that after knocking down ALYREF, overexpression of UHRF1 could rescue the proliferation inhibition phenotype of MDA-MB-231 and BT-549 cells induced by ALYREF knockdown ( Figure 7 This result indicates that silencing the ALYREF-UHRF1 signaling axis can inhibit the proliferation of breast cancer cells.

[0076] As the above contents confirm that the ALYREF-UHRF1 signaling axis exists in epithelial tumors and silencing the ALYREF-UHRF1 signaling axis can inhibit the proliferation of epithelial tumor cells, the embodiments of the present application are directed to the ALYREF-UHRF1 signaling axis, and siRNA that inhibits UHRF1 and siRNA that inhibits ALYREF are obtained through design and selection.

[0077] In one implementation, for siRNA that inhibits UHRF1, the sense strand of the siRNA is: 5'-GGACGAAGTCTTCAAGATT-3'; and the antisense strand of the siRNA is: 3'-CCUGCUUCAGAAGUUCUAA-5'.

[0078] For siRNA inhibiting ALYREF, the sense strand of siRNA is: 5'-GCACGATCTTTTCGACAGT-3'; the antisense strand of siRNA is: 3'-CGUGCUAGAAAAGCUGUCA-5'.

[0079] In the art, dTdT is often selected to be hanging at the 3' end of siRNA, which can be synthesized by the above siRNA for subsequent experiments and treatments.

[0080] In the case of dTdT hanging, for siRNA that inhibits UHRF1, the sense strand of siRNA is: 5'-GGACGAAGTCTTCAAGATTdTdT-3'; and the antisense strand of siRNA is: 3'-TdTdCCUGCUUCAGAAGUUCUAA-5'.

[0081] For siRNA inhibiting ALYREF, the sense strand of siRNA is: 5'-GCACGATCTTTTCGACAGTdTdT-3'; the antisense strand of siRNA is: 3'-TdTdCGUGCUAGAAAAGCUGUCA-5'.

[0082] Based on the above-mentioned siRNA that inhibits UHRF1 and the above-mentioned siRNA that inhibits UHRF1, the embodiments of the present application provide a group of siRNA groups that inhibit the expression of ALYREF-UHRF1 signaling axis genes, including the above-mentioned siRNA that inhibits UHRF1 and the above-mentioned siRNA that inhibits ALYREF, or, siRNA that inhibits UHRF1 with dTdT hanging at the 3' end and siRNA that inhibits ALYREF with dTdT hanging at the 3' end.

[0083] In another implementation, for siRNA that inhibits UHRF1, the sense strand of siRNA is: 5'-CGAGATCTTTCCGGCAACA-3'; and the antisense strand of siRNA is: 3'-GCUCUAGAAAGGCCGUUGU-5'.

[0084] For siRNA inhibiting ALYREF, the sense strand of siRNA is: 5'-GCGAGAATGGACACCAGTT-3'; the antisense strand of siRNA is: 3'-CGCUCUUACCUGUGGUCAA-5'.

[0085] In the art, dTdT is often selected to be hanging at the 3' end of siRNA, which can be synthesized by the above siRNA for subsequent experiments and treatments.

[0086] In the case of dTdT hanging, for siRNA inhibiting UHRF1, the sense strand of siRNA is: 5'-CGAGATCTTTCCGGCAACAdTdT-3'; the antisense strand of siRNA is: 3'-TdTdGCUCUAGAAAGGCCGUUGU-5'.

[0087] For siRNA inhibiting ALYREF, the sense strand of siRNA is: 5'-GCGAGAATGGACACCAGTTdTdT-3'; the antisense strand of siRNA is: 3'-TdTdCGCUCUUACCUGUGGUCAA-5'.

[0088] Based on the above-mentioned siRNA that inhibits UHRF1 and the above-mentioned siRNA that inhibits UHRF1, the embodiments of the present application provide a group of siRNA groups that inhibit the expression of ALYREF-UHRF1 signaling axis genes, including the above-mentioned siRNA that inhibits UHRF1 and the above-mentioned siRNA that inhibits ALYREF, or, siRNA that inhibits UHRF1 with dTdT hanging at the 3' end and siRNA that inhibits ALYREF with dTdT hanging at the 3' end.

[0089] 8. Silencing the ALYREF-UHRF1 signaling axis in vivo inhibits breast cancer growth

[0090] After confirming that inhibiting the ALYREF-UHRF1 signaling axis reduces the proliferation of breast cancer cells in vitro, we then constructed a nude mouse tumorigenesis experiment with human breast cancer cells. After the tumors of the mice were stable, the mice were divided into four groups and injected with NC, siALYREF, siUHRF1, and siALYREF+siUHRF1, respectively. The tumor volumes of the mice were measured every week. The results showed that the siALYREF+siUHRF1 group significantly reduced the tumor volume ( Figure 8 (A); (B) After 35 days, tumor tissues were obtained and weighed. The results showed that the siALYREF+siUHRF1 group significantly reduced tumor weight ( Figure 8 Targeting the ALYREF-UHRF1 signaling axis dramatically inhibited breast cancer growth in mice.

[0091] Based on the above content, the present application also provides the use of siRNA that inhibits UHRF1 or siRNA that inhibits ALYREF in the preparation of drugs for treating epithelial tumors. According to the specificity and sensitivity of the tumor to the ALYREF / UHRF1 signaling axis, the siRNA drug is used in the following three ways: ① single siALYREF drug; ② single siUHRF1 drug; ③ two siRNA combination drugs: siALYREF and siUHRF1.

[0092] It should be noted that the embodiment of the present application adopts the strategy of siRNA targeted inhibition of gene expression, and designs tumor drugs based on RNA interference (gene silencing mechanism caused by double-stranded RNA, Nobel Prize in Physiology or Medicine in 2006). The specific design principle of tumor drugs is: first, Primer3 plus is used to design small interfering RNA (Smallinterfering RNA; siRNA) targeting ALYREF or UHRF1; then the synthesized siRNA is transfected into cells and combined with the RNA-induced silencing complex (RISC) in the cytoplasm, and unwound into a single strand. The positive strand will be degraded, and the remaining antisense strand will guide RISC to bind to the corresponding complementary ALYREF mRNA or UHRF1 mRNA, causing RISC to cut off this mRNA and degrade it, thereby inhibiting the expression of ALYREF or UHRF1 mRNAs, so that it cannot translate into protein. Specifically, the embodiment of the present application adopts the process of designing tumor drugs based on siRNA, which is common in the field of this technology, to design tumor drugs, which will not be repeated here.

[0093] Among them, the epithelial tumor is at least one of bladder urothelial carcinoma, head and neck squamous cell carcinoma, breast invasive cancer, cervical squamous cell carcinoma and adenocarcinoma, bile duct cancer, colon cancer, rectal adenocarcinoma, esophageal cancer, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, prostate cancer, skin melanoma, gastric cancer, thyroid cancer, thymic carcinoma and endometrial cancer.

[0094] The embodiments of the present application also provide an anti-tumor drug, which is prepared from the aforementioned siRNA that inhibits the expression of ALYREF-UHRF1 signaling axis genes, or the aforementioned siRNA group that inhibits the expression of ALYREF-UHRF1 signaling axis genes.

[0095] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An siRNA for inhibiting the expression of ALYREF-UHRF1 signal axis gene derived from corneal epithelium, characterized in that: The siRNA sequence is an siRNA that inhibits UHRF1; The sense strand of the siRNA: 5'-GGACGAAGTCTTCAAGATT-3'; The antisense strand of the siRNA: 3'-CCUGCUUCAGAAGUUCUAA-5'.

2. An siRNA for inhibiting the expression of ALYREF-UHRF1 signaling axis gene derived from corneal epithelium, characterized in that: The siRNA sequence is an siRNA that inhibits ALYREF; The sense strand of the siRNA: 5'-GCACGATCTTTTCGACAGT-3'; The antisense strand of the siRNA: 3'-CGUGCUAGAAAAGCUGUCA-5'.

3. siRNA for inhibiting the expression of ALYREF-UHRF1 signal axis gene derived from corneal epithelium, characterized in that: The siRNA sequence is an siRNA that inhibits UHRF1; The sense strand of the siRNA: 5'-CGAGATCTTTCCGGCAACA-3'; The antisense strand of the siRNA: 3'-GCUCUAGAAAGGCCGUUGU-5'.

4. An siRNA for inhibiting the expression of ALYREF-UHRF1 signal axis gene derived from corneal epithelium, characterized in that: The siRNA sequence is an siRNA that inhibits ALYREF; The sense strand of the siRNA: 5'-GCGAGAATGGACACCAGTT-3'; The antisense strand of the siRNA: 3'-CGCUCUUACCUGUGGUCAA-5'.

5. The siRNA for inhibiting the expression of the ALYREF-UHRF1 signaling axis gene derived from corneal epithelium according to claim 1 or 3, characterized in that: The 3' ends of the sense strand and the antisense strand of the UHRF1-inhibiting siRNA are dangled with dTdT.

6. The siRNA for inhibiting the expression of the ALYREF-UHRF1 signaling axis gene derived from corneal epithelium according to claim 2 or 4, characterized in that: The 3' ends of the sense strand and the antisense strand of the siRNA for inhibiting ALYREF are dangled with dTdT.

7. A siRNA group for inhibiting the expression of ALYREF-UHRF1 signal axis gene derived from corneal epithelium, characterized in that: The method comprises the siRNA for inhibiting UHRF1 according to claim 1 and the siRNA for inhibiting ALYREF according to claim 2.

8. A siRNA group for inhibiting the expression of ALYREF-UHRF1 signal axis gene derived from corneal epithelium, characterized in that: The method comprises the siRNA for inhibiting UHRF1 as claimed in claim 3 and the siRNA for inhibiting ALYREF as claimed in claim 4. 9 . Use of the siRNA for inhibiting UHRF1 or the siRNA for inhibiting ALYREF according to any one of claims 1 to 8 in the preparation of a drug for treating epithelial tumors.

10. The use according to claim 9, characterized in that The epithelial tumor is at least one of bladder urothelial carcinoma, head and neck squamous cell carcinoma, breast invasive cancer, cervical squamous cell carcinoma and adenocarcinoma, bile duct cancer, colon cancer, rectal adenocarcinoma, esophageal cancer, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, prostate cancer, skin melanoma, gastric cancer, thyroid cancer, thymic carcinoma and endometrial cancer.

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