Targeted inhibitor of Mmrn1, inhibition method and application

Abstract

The invention relates to the field of biological medicine, in particular to an Mmrn1 targeted inhibitor, an inhibition method and application. According to the application disclosed by the invention, firstly, the relationship between the expression of the MMRN1 gene in the acute myelogenous leukemia (AML) and the regulation and control of a'siglecan-siglecc 'axis is creatively disclosed. Therefore, the invention firstly provides a small interfering RNA (Ribonucleic Acid) targeting the Mmrn1 in the leukemia stem cell, and an application of the small interfering RNA in preparation of a'sialic acid glycan-siglecc 'axis immune checkpoint inhibitor.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a targeted inhibitor of Mmrn1 and an inhibition method and application thereof. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant clonal disease originating from hematopoietic stem cells, characterized by the uncontrolled growth of primitive or immature myeloid cells in the bone marrow. In recent years, with the application of the "5+3" standard chemotherapy regimen and targeted therapies such as Flt3-ITD in AML, the remission rate of patients has increased to about 70%, but relapsed and refractory AML is still a difficult point in treatment.

[0003] Leukemia stem cells (LSCs) refer to a group of leukemia-initiating cells in the patient's body that are in a relatively static state and have the ability to self-renew and proliferate indefinitely. They can escape the killing of most tumor drugs and are the key factor leading to leukemia relapse and drug resistance. In recent years, scholars have found that LSCs can also undergo immune escape, and their mechanism is unique. Due to the immune escape of LSCs, the therapeutic effect of conventional immunotherapy is not as effective as that of solid tumor treatment.

[0004] At present, the diagnosis of relapsed and refractory acute myeloid leukemia depends on clinical observation of the effect of conventional radiotherapy and chemotherapy or whether relapse occurs again after remission. In this process, a lot of time and money are spent, and patients also suffer physical and mental pain, such as multiple bone punctures. Although more advanced methods have also appeared, such as the patent with application number CN114867479A and invention name "method for treating leukemia and use of leukemia stem cell characteristics for predicting clinical sensitivity to therapy", a method for identifying or predicting the responsiveness of subjects with or suspected of having AML to compound treatment is disclosed, and the method requires measuring the expression level of one or more genes in the subject sample. However, the application of this method in actual clinical practice is relatively limited. On the one hand, genetic testing is usually expensive and ordinary patients may not be able to afford it; on the other hand, genetic testing also puts forward better requirements on the level and hardware conditions of the hospital where the patient seeks medical treatment, which may not be achieved in ordinary hospitals. Therefore, there is an urgent need for a simple, accurate and rapid means of diagnosing relapsed and refractory AML, and adjusting the treatment plan in time to provide patients with personalized and more suitable treatment plans.

[0005] The "sialoglycan-siglec" axis is a new type of sugar immune checkpoint. Existing studies have found that it plays an important role in the immune escape of solid tumor cells. Sialoglycan has been found to be upregulated in a variety of tumors (breast cancer, melanoma, etc.). It can bind to the sialoglycan receptor Siglec on immune cells in the tumor microenvironment, thereby exerting an immunosuppressive effect. However, in AML, it is still unclear what the "sialoglycan-Siglec" expression level is and whether it is involved in the immune escape of leukemia cells.

[0006] In summary, it is necessary to propose new methods and strategies to alleviate the shortcomings of existing technologies. Summary of the invention

[0007] The object of the present invention is to provide a targeted inhibitor of Mmrn1 and an inhibition method and application thereof, so as to partially solve or alleviate the above-mentioned deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0008] A small interfering RNA targeting Mmrn1 in leukemia stem cells, wherein the nucleotide sequence of the small interfering RNA is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0009] Furthermore, the small interfering RNA is used in the preparation of "sialoglycan-siglec" axis immune checkpoint inhibitors.

[0010] The small interfering RNA is used to inhibit the expression of sialoglycan of the "sialoglycan-siglec" axis of leukemia stem cells.

[0011] As an alternative means, the present invention can also provide a use of a small interfering RNA targeting Mmrn1 (gene) in leukemia stem cells in the preparation of a "sialoglycan-siglec" axis immune checkpoint inhibitor.

[0012] A use of small interfering RNA in the preparation of an immunomodulatory agent for relapsed and refractory acute myeloid leukemia, wherein the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0013] Furthermore, the small interfering RNA is used to assist or assist or indirectly enhance the activity and / or proliferation capacity of T cells.

[0014] A method for inhibiting sialic acid glycan in leukemia stem cells in vitro: co-transfecting leukemia stem cells with a plasmid vector containing shRNA; the nucleotide sequence of the shRNA is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0015] Further, the method comprises the following steps: S01: preparing a plasmid vector containing the shRNA; S02: transfecting a plasmid vector containing the shRNA into leukemia stem cells; S03: Flow cytometry was used to detect the expression level of "sialoglycan-siglec" axis marker molecules on the surface of leukemia stem cells transfected with the shRNA plasmid vector to detect the inhibitory effect.

[0016] Furthermore, the "sialoglycan-siglec" axis marker molecules include MALL-II and / or SNA; when a decrease in the expression level of MALL-II and / or SNA is detected, it is determined that sialoglycan in the leukemia stem cells is inhibited.

[0017] In some preferred embodiments, the marker molecules are MALL-II and SNA.

[0018] A method for screening drugs that potentially target the Mmrn1 gene in leukemia stem cells comprises the following steps: S01: Provide bone marrow fluid or blood samples from AML subjects; S02: selecting leukemia stem cells from the blood sample of the AML subject; S03: co-culturing or co-transfecting the drug to be tested or a vector containing the drug to be tested with the leukemia stem cells as an experimental group; the drug to be tested includes small interfering RNA, small molecule chemical drugs and / or antibody drugs; S04: After the co-culture or co-transfection, the expression levels of "sialoglycan-siglec" axis marker molecules MALL-II and / or SNA on the surface of leukemia stem cells in the experimental group were detected by flow cytometry, and the expression levels of MALL-II and / or SNA on the surface of leukemia stem cells without treatment were used as the control group; In some preferred embodiments, the expression levels of MALL-II and SNA in the cell surface fluid of leukemia stem cells are detected simultaneously.

[0019] S05: When it is detected that the expression level of MALL-II and / or SNA on the surface of leukemia stem cells in the experimental group is lower than that of MALL-II and / or SNA on the surface of leukemia stem cells in the control group, it is determined that the drug to be tested may be effective in targeting the Mmrn1 gene in leukemia stem cells.

[0020] Furthermore, the vector containing the drug to be tested includes a plasmid vector and / or other pharmaceutically acceptable carriers or adjuvants.

[0021] Furthermore, the expression level of MALL-II on the surface of leukemia stem cells in the experimental group was decreased by no less than 15% compared with the expression level of MALL-II on the surface of leukemia stem cells in the control group, indicating that the test drug may be effective in targeting the Mmrn1 gene in leukemia stem cells.

[0022] Furthermore, the SNA expression level on the surface of leukemia stem cells in the experimental group was decreased by no less than 20% compared with the SNA expression level on the surface of leukemia stem cells in the control group, indicating that the test drug may be effective in targeting the Mmrn1 gene in leukemia stem cells.

[0023] Beneficial technical effects: The present invention first innovatively reveals the relationship between the expression of the MMRN1 gene in acute myeloid leukemia (AML) and the regulation of the "sialoglycan-siglec" axis.

[0024] Furthermore, the present invention provides a small interfering RNA that can effectively knock out the MMRN1 gene in leukemia cells without affecting normal hematopoietic stem cells. Animal experiments have confirmed that the survival of AML mice treated with this small interfering RNA is prolonged.

[0025] Furthermore, the present invention also confirms that after knocking out the MMRN1 gene with this small interfering RNA, the level of NPL can be upregulated, thereby inhibiting the mediated sialic acid glycan expression. Therefore, this small interfering RNA can also be used as an alternative means to prepare "sialic acid glycan-siglec" axis immune checkpoint inhibitors.

[0026] Finally, the present invention also provides a method for screening potential drugs targeting the Mmrn1 gene in leukemia stem cells. Since the present invention verifies that the Mmrn1 gene can regulate the "sialic acid polysaccharide-siglec" axis, the method provided by the present invention does not need to perform Mmrn1 gene detection, but only needs to detect the expression of marker molecules in the "sialic acid polysaccharide-siglec" axis, and reversely deduce whether there is a high expression of the Mmrn1 gene in the sample to be tested, which greatly saves and reduces the expensive cost of gene testing and the requirements for the hardware conditions of scientific research and medical systems. In addition, the drug screening method provided by the present invention also greatly saves the time of the traditional method that relies on clinical observation of the effects of conventional radiotherapy and chemotherapy on patients or whether relapse occurs after remission, and avoids the cost of trial and error of patients to a certain extent. Based on this, the method provided by the present invention has a high practical value for preclinical screening of potential drugs targeting the Mmrn1 gene in leukemia stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0028] Figure 1 It was shown that Mmrn1 is highly expressed in leukemia stem cells; Figure 2 It was shown that in the MLL-AF9-induced AML mouse model, Mmrn1 was highly expressed in mouse leukemic stem cells; Figure 3 showed that low expression of Mmrn1 was associated with a better prognosis; Figure 4 The results of knockout of Mmrn1 in Mmrn1-iKO mice are shown; Figure 5 showed that Mmrn1 knockdown delayed the onset of acute myeloid leukemia; Figure 6 showed that Mmrn1 knockdown slowed acute myeloid leukemia progression; Figure 7 The detection of MMRN1 expression levels in multiple leukemia cells was shown; Figure 8 It was shown that knockdown of Mmrn1 did not affect the normal hematopoietic system function of mice; Fig. 9 It was shown that Mmrn1 upregulates NPL-mediated sialoglycan expression; Fig.10 Mmrn1 was shown to inhibit CD8 + T cell proliferation and its immune function; Fig.11 The sialic acid analog 3Fax-Neu5Ac was shown to block the activation of Mmrn1 to CD8 + The inhibitory effect of T cells; Fig.12 showed that Mmrn1 knockdown could alleviate the progression of relapsed and refractory AML. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0031] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0032] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0033] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0034] Detailed description with drawings Figure 1 :Bone marrow samples of AML patients were collected and CD34 + CD38 - , CD34 + CD38 + , CD34 - CD38 - , CD34 - CD38 + Cells were cultured and RNA was extracted, and the expression of MMRN1 was detected by q-PCR.

[0035] Figure 2 : Isolation of GFP from an MLL-AF9-induced AML mouse model +Leukemia cells and LSCs were isolated from wild-type C57BL / 6 mice, and normal hematopoietic stem cells were sorted. RNA was extracted and the relative expression of Mmrn1 was detected by q-PCR.

[0036] Figure 3 : TCGA database analysis of the relationship between Mmrn1 expression and prognosis of AML patients.

[0037] Figure 4 :Successful construction of Mmrn1 knockout mice. (A) Schematic diagram of the construction of Mmrn1 knockout mice, using the Cre-Loxp system to knock out MMRN1 exon 3 to achieve the effect of Mmrn1 knockout; (B) Semi-quantitative PCR to detect the knockout efficiency of Mmrn1; (C) q-PCR to detect the knockout efficiency of Mmrn1.

[0038] Figure 5 :Mmrn1 regulates the occurrence of AML. (A) Construction process of MLL-AF9-induced AML model; (B) Survival analysis of mice; (C) Flow cytometry to detect the proportion of GFP+ leukemia cells in mouse peripheral blood; (D) HE to detect the infiltration of leukemia cells in the liver and spleen of mice; (E) Flow cytometry to detect the proportion of leukemia stem cells in the mouse bone marrow.

[0039] Figure 6 :Mmrn1 regulates the development of AML. (A) Construction process of MLL-AF9-induced AML development model; (B) MLL-AF9-Mmrn1 fl / fl -Mx1-Cre mice survived longer.

[0040] Figure 7 :Western Blotting and ELISA were used to detect the expression level of MMRN1 in leukemia cells.

[0041] Figure 8 :Mmrn1 has no significant effect on the normal hematopoietic system. (A) Flow cytometry (FCM) was used to analyze the expression of Mmrn1 fl / fl (B) Flow cytometry was used to detect the changes in the proportions of mature cells in the peripheral blood, bone marrow, thymus and spleen of Mmrn1 knockout mice, mainly including myeloid, B cells, T cells and erythrocytes; (C) HSC differentiation was detected by cloning; (D-E) Ki67+DAPI (D) and BrdU+DAPI (E) flow cytometry was used to analyze the cycle and proliferation of hematopoietic stem and progenitor cells (HSC, LSK, HPC) in Mmrn1 knockout mice.

[0042] Fig. 9:Knockout of MMRN1 in human leukemia cells upregulates the expression of NPL and inhibits the expression of sialic acid glycans. (A) Western blotting to detect the knockout efficiency of MMRN1; (B) q-PCR to detect the effect of MMRN1 knockout on NPL; (C-D) Flow cytometry analysis of the effect of MMRN1 knockout on sialic acid glycans.

[0043] Fig.10 MMRN1 in LSCs inhibits CD8 + T cell immune function. (A) Co-culture model of LSCs and CD8+T cells; (B) CFSE labeled CD8 + T, CD8 + T cell proliferation; (C) Flow cytometry analysis was used to detect the expression levels of CD25 and Granzyme B in CD8+ T cells.

[0044] Fig.11 :3Fax-Neu5Ac can block MMRN1 from regulating CD8 + T cell inhibitory effect. (A) Co-culture model; (B) CFSE labeled CD8 + T, CD8 + T cell proliferation (left), CD8 T cells were detected by flow cytometry + Expression levels of CD25 (middle) and Granzyme B (right) on T cells.

[0045] Fig.12 : In a humanized AML mouse model, targeting MMRN1 can inhibit progression and improve survival. (A) Construction process of a humanized AML mouse model; (BD) Flow cytometry was used to detect the proportion of peripheral blood leukemia cells (left) and to count the survival of mice (right).

[0046] Definition of noun: The "immune checkpoint inhibitor" of the present invention refers to a drug or preparation for alleviating or treating cancer, which mainly activates the immune system by blocking immune checkpoint molecules. There are many immune checkpoint inhibitors that have been approved for marketing, such as inhibitors targeting CTLA-4, such as ipilimumab; and inhibitors targeting the PD-1 / PD-L1 pathway, such as nivolumab.

[0047] The "immunomodulatory agent" described in the present invention refers to an agent that enhances or assists in enhancing the function or proliferation capacity of immune cells and is used in in vitro experiments.

[0048] Embodiment 1 This example verifies that Mmrn1 is highly expressed in leukemia stem cells (LSCs) and is involved in the occurrence and development of acute myeloid leukemia (AML).

[0049] Bone marrow samples were collected from AML patients and CD34 + CD38 - (representing LSCs cells), CD34 + CD38 + (representing non-stem leukemia cells), CD34 - CD38 - (represents non-stem leukemia cells), CD34 - CD38 + (represents non-stem leukemia cells) cells, and the expression of Mmrn1 in the four groups of cells was detected. Figure 1 As shown, Mmrn1 is expressed in CD34 + CD38 - The expression level was the highest in the cell population, and there was a significant difference compared with the other three cell populations, indicating that Mmrn1 was highly expressed in LSCs.

[0050] GFP was isolated from the MLL-AF9-induced AML mouse model. + Cells (leukemia cells) and LSCs, normal hematopoietic stem cells were sorted from wild-type C57BL / 6 mice, RNA was extracted, and the expression level of Mmrn1 was detected by qRT-PCR and ELISA. Figure 2 As shown, in the MLL-AF9-induced AML mouse model, Mmrn1 was highly expressed in mouse LSCs, demonstrating that the Mmrn1 gene was highly expressed in acute myeloid leukemia stem cells.

[0051] Furthermore, the TCGA database was used to analyze the relationship between Mmrn1 expression and the prognosis of AML patients. Figure 3 As shown in the figure, the expression of Mmrn1 is negatively correlated with the patient's prognosis, that is, the lower the expression of Mmrn1, the higher the patient's overall survival rate, indicating that low expression of Mmrn1 is associated with a better prognosis. Furthermore, the chromosome site-specific recombinase system Cre-loxp was used to knock out Mmrn1, and a Loxp sequence was placed at each end of exon 3 of Mmrn1 to obtain Floxed mice, referred to as Mmrn1 fl / + Mouse, Mmrn1 fl / + Mmrn1 was obtained by crossing mice with Mx1-Cre mice. fl / fl Mice and Mmrn1 fl / fl -Mx1-Cre mice, under the condition of pIpC-induced Mmrn1fl / fl -Mx1-Cre mice can achieve Mmrn1 knockout. For simplicity, Mmrn1 after pIpC treatment fl / fl Mmrn1 fl / fl Mouse, Mmrn1 after pIpC treatment fl / fl -Mx1-Cre mice became Mmrn1-iKO mice ( Figure 4 A). Semi-quantitative PCR was used to Figure 4 B) and qRT-PCR ( Figure 4 C) Means for detecting Mmrn1 knockout efficiency. Figure 4 The results showed that the expression level of Mmrn1 in Mmrn1-iKO mice was significantly lower than that in non-knockout mice. fl / fl Mice, indicating that conditional Mmrn1 knockout mice (i.e., mice in which Mmrn1 is knocked out only in leukemia cells) were successfully constructed.

[0052] Mmrn1 was treated with 5-fluorouracil (5-Fu) fl / fl After the mice and Mmrn1-iKO mice were isolated, the bone marrow cells were removed. 5-Fu can kill the cells in the proliferation phase, so the remaining cells are stem cells. The mice were further infected with MLL-AF9 virus and then irradiated with 5 Gy (to reduce immune rejection) to construct an MLL-AF9-induced AML mouse model ( Figure 5 A). Observe the survival of AML mice and draw a survival curve; use FCM to detect peripheral blood GFP + The liver and spleen were removed, weighed and photographed, and liver and spleen tissue sections were prepared for H&E staining and microscopic examination to determine the leukemia infiltration of mice. Figure 5 As shown, the survival time of MLL-AF9-induced Mmrn1-iKO mice was shorter than that of Mmrn1 fl / fl Mouse length ( Figure 5 B) GFP in peripheral blood + The proportion of cells is also smaller ( Figure 5 C), the results of liver and spleen tissue staining showed that Mmrn1 knockout inhibited the hepatic and splenic infiltration of leukemia cells (Figure 5D). In addition, Mmrn1 knockdown also reduced the proportion of leukemia stem cells (LSC) in the bone marrow ( Figure 5 E). The above results indicate that Mmrn1 regulates the occurrence of AML.

[0053] Furthermore, 5-Fu was used to treat Mmrn1 fl / fl Mice and Mmrn1 fl / fl-Mx1-Cre mice were then generated, bone marrow cells were removed and infected with MLL-AF9 virus to construct an MLL-AF9-induced AML mouse model. The successfully constructed AML mice were treated with pIpC three times to induce Mmrn1 knockout. The overall survival of the mice was observed and a survival curve was drawn. Figure 6 As shown, MLL-AF9-Mmrn1 fl / fl -Mx1-Cre mice survived longer ( Figure 6 B), indicating that Mmrn1 knockout can slow down the progression of AML. Overall, it shows that Mmrn1 regulates the development of AML.

[0054] However, not all leukemia cells have high expression of Mmrn1. This experiment further used Western Blotting and ELISA to detect the expression level of MMRN1 in other leukemia cells. Figure 7 The results showed that Mmrn1 was highly expressed in KG-1a and KG-1 cells with stronger stemness, but was less expressed in Kasumi1 and HL60 cells.

[0055] The above results suggest that conditional knockout of Mmrn1 in a model with low Mmrn1 expression may not be effective in slowing down the progression of AML.

[0056] Embodiment 2 This example verifies the effect of Mmrn1 on the function of the hematopoietic system.

[0057] Next, we adopt Mmrn1 fl / fl The effects of Mmrn1 knockout on the normal hematopoietic system were analyzed by using mice and Mmrn1-iKO, and it was found that Mmrn1 knockout did not affect the number of stem and progenitor cells ( Figure 8 A), does not affect the number of mature cells ( Figure 8 B) does not affect the differentiation of stem cells ( Figure 8 C) does not affect the cell cycle and proliferation of stem cells ( Figure 8 D- Figure 8 E). The above results show that Mmrn1 knockout has no significant effect on the normal hematopoietic system.

[0058] Embodiment 3 This example verifies that Mmrn1 promotes LSC immune escape by promoting NPL (sialic acid-binding immunoglobulin-like lectin)-mediated expression of sialic acid glycans.

[0059] Knockdown of LSC-like cell line KG-1a cells and human primary CD34 by shRNA technology + MMRN1 in leukemia cells (a group of cells with strong stemness) Fig. 9 A). MMRN1 knockout upregulated the level of NPL ( Fig. 9 B), inhibited the level of sialoglycan ( Fig. 9 C- Fig. 9 D). The specific operations were as follows: (1) synthesizing plasmids containing the shRNA in Table 3; (2) using Lipofectamine3000 (Invitrogen, USA) to express the shRNA in LSC cells, KG-1a cells and human primary CD34 + The plasmid was transfected into leukemia cells. Fig. 9 B results show that in KG-1a and CD34 + In the -#1 cell line, the relative NPL expression level was the highest after shMMRN1#1 knockout. Fig. 9 C and Fig. 9 D. Flow cytometry was used to detect the expression of marker molecules MALL-II and SNA on the "sialoglycan-siglec" axis. In all cell lines tested, it was found that the expression of marker molecules MALL-II and SNA on the "sialoglycan-siglec" axis decreased after knocking out MMRN1.

[0060] Table 1 SNA expression results Table 2 MALL-II expression results The above results show that Mmrn1 inhibits NPL expression, and knocking out MMRN1 can upregulate the level of NPL, thereby inhibiting the mediated sialic acid glycan expression. By knocking out MMRN1, the expression of the marker molecules MALL-II and SNA on the "sialic acid glycan-siglec" axis can be significantly reduced.

[0061] Furthermore, LSCs were isolated from the bone marrow of AML patients and CD8 + T cells, LSCs knocked out of Mmrn1 were combined with CD8 + T cell co-culture, detection of CD8 after 5 days + The proliferation of T cells, as well as the expression levels of CD25 (T cell activation indicator) and Granzyme B (CD8+ T cell killing function indicator). Fig.10 As shown, CD8 + The various indicators of the T+LSC-shMMRN1 experimental group were higher than those of the CD8 + T+LSC experimental group and CD8 + The T+LSC-shNC experimental group was even close to CD8 +This indicates that leukemia stem cells inhibit the proliferation and function of T cells, while the inhibitory effect of leukemia stem cells on T cells after MMRN1 knockout disappears, that is, Mmrn1 inhibits CD8 + T cell proliferation and immune function.

[0062] Furthermore, when the above two cells were co-cultured, 100 μmol / L of sialic acid fully acetylated analog 3Fax-Neu5Ac (MCE, Catalog No.: HY-110288) was added to the culture medium, and CD8 + T cell proliferation, and CD25 and Granzyme B expression levels. Fig.11 As shown, CD8 + The proliferation of T cells and the expression levels of CD25 and Granzyme B were similar to those in the experimental group, indicating that the sialic acid acetylation analog 3Fax-Neu5Ac could inhibit the activation of Mmrn1 on CD8 + T cell inhibitory effect.

[0063] These results suggest that MMRN1 inhibits CD8 by regulating NPL-mediated sialic acid glycans. + Immune function of T cells.

[0064] The shRNA sequences involved in this embodiment are as follows: Table 3 Embodiment 4 This example verifies that targeting Mmrn1 can alleviate the progression of relapsed and refractory AML.

[0065] Normal hematopoietic stem cells were collected and injected into 32 6-week-old NSG mice via the tail vein to reconstruct the human immune system. Leukemia cells were collected from the bone marrow of clinically relapsed and refractory AML patients and labeled with GFP lentivirus, and then MMRN1 was knocked down. On the 19th day after the successful reconstruction of the immune system, they were transplanted into NSG mice via the tail vein (5 shNC and 5 shMMRN1#1). The proportion of peripheral blood leukemia cells and the survival of mice were detected every week. The results are shown in Fig.12 As shown, GFP in peripheral blood of mice after knockdown of Mmrn1 + The reduction in the proportion of cells indicates that the progression of AML is slowed down, and the overall survival time is relatively longer, from 40 days to more than 60 days, indicating that the method of targeting Mmrn1 provided by the present invention can alleviate the progression of relapsed and refractory AML and significantly prolong the survival of mice.

[0066] Embodiment 5 This embodiment provides an example of a method for screening drugs that potentially target the Mmrn1 gene in leukemia stem cells.

[0067] S01: Provide a bone marrow fluid or blood sample from an AML subject; for example, the sample may come from a blood test or puncture sample from the hematology / oncology department of a hospital.

[0068] S02: sorting and collecting leukemia stem cells from the bone marrow fluid or blood sample of the AML subject.

[0069] S03: Cultivate cells to the desired number.

[0070] S04: co-culturing or co-transfecting the drug to be tested or a vector containing the drug to be tested with the leukemia stem cells.

[0071] As an implementation method, the drug to be tested is a small interfering RNA drug. The plasmid containing the small interfering RNA is co-transfected with the cultured leukemia stem cells according to conventional operation techniques in the art.

[0072] As an implementation method, the drug to be tested is a small molecule chemical drug. The small molecule chemical drug to be tested is co-cultured with the cultured leukemia stem cells according to conventional operation techniques in the art.

[0073] S05: After the co-culture or co-transfection is completed, the expression levels of "sialoglycan-siglec" axis marker molecules MALL-II and / or SNA on the surface of leukemia stem cells are detected by flow cytometry, and the expression levels of MALL-II and / or SNA on the surface of untreated leukemia stem cells are used as a control.

[0074] As an implementation method, the expression levels of MALL-II and SNA on the surface of leukemia stem cells are detected simultaneously.

[0075] S06: When it is detected that the expression level of MALL-II on the surface of leukemia stem cells in the experimental group is significantly decreased compared with the expression level of MALL-II on the surface of leukemia stem cells in the control group; and / or it is detected that the expression level of SNA on the surface of leukemia stem cells in the experimental group is significantly decreased compared with the expression level of SNA on the surface of leukemia stem cells in the control group, it is determined that the drug to be tested may be effective in targeting the Mmrn1 gene in leukemia stem cells, and the next clinical trial is carried out as a potential drug targeting the MMRN1 gene.

[0076] As an implementation means, the expression level of MALL-II on the surface of leukemia stem cells in the experimental group is decreased by not less than (at least) 15% compared with the expression level of MALL-II on the surface of leukemia stem cells in the control group, or the expression level of SNA on the surface of leukemia stem cells in the experimental group is decreased by not less than (at least) 20% compared with the expression level of SNA on the surface of leukemia stem cells in the control group, which is considered to be a significant decrease.

[0077] In some cases, if only one of the expression levels of MALL-II and SNA reaches the "significant decrease" level, the drug to be tested will be temporarily listed as a drug that needs to be retested or tested by other means.

[0078] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0079] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A small interfering RNA targeting Mmrn1 in leukemia stem cells, characterized in that: The nucleotide sequence of the small interfering RNA is shown as SEQ ID NO.1 or SEQ ID NO.

2.

2. Use of the small interfering RNA according to claim 1 in the preparation of "sialoglycan-siglec" axis immune checkpoint inhibitors.

3. Use of small interfering RNA in the preparation of an immunomodulatory agent for relapsed and refractory acute myeloid leukemia, characterized in that: The nucleotide sequence of the small interfering RNA is shown as SEQ ID NO.1 or SEQ ID NO.

2.

4. A method for inhibiting sialic acid glycans in leukemia stem cells in vitro, characterized in that: The leukemia stem cells are co-transfected with a plasmid vector containing shRNA; the nucleotide sequence of the shRNA is shown in SEQ ID NO.1 or SEQ ID NO.

2.

5. The method according to claim 4, characterized in that The steps include: S01: preparing a plasmid vector containing the shRNA; S02: transfecting a plasmid vector containing the shRNA into leukemia stem cells; S03: Flow cytometry was used to detect the expression level of "sialoglycan-siglec" axis marker molecules on the surface of leukemia stem cells transfected with the shRNA plasmid vector to detect the inhibitory effect.

6. The method according to claim 5, characterized in that The "sialoglycan-siglec" axis marker molecules include MALL-II and / or SNA; when a decrease in the expression level of MALL-II and / or SNA is detected, it is determined that the sialoglycan in the leukemia stem cells is inhibited.

7. A method for screening drugs that potentially target the Mmrn1 gene in leukemia stem cells, characterized in that: The following steps are involved: S01: Provide bone marrow fluid or blood samples from AML subjects; S02: selecting leukemia stem cells from the bone marrow fluid or blood sample of the AML subject; S03: co-culturing or co-transfecting the drug to be tested or a vector containing the drug to be tested with the leukemia stem cells as an experimental group; the drug to be tested includes small interfering RNA, small molecule chemical drugs and / or antibody drugs; S04: After the co-culture or co-transfection, the expression of "sialoglycan-siglec" axis marker molecules MALL-II and / or SNA on the surface of leukemia stem cells in the experimental group was detected by flow cytometry, and the expression of MALL-II and / or SNA on the surface of leukemia stem cells without treatment was used as the control group; S05: When it is detected that the expression level of MALL-II and / or SNA on the surface of leukemia stem cells in the experimental group is lower than that of MALL-II and / or SNA on the surface of leukemia stem cells in the control group, it is determined that the drug to be tested may be effective in targeting the Mmrn1 gene in leukemia stem cells.

8. The method according to claim 7, characterized in that The vector containing the drug to be tested includes a plasmid vector and / or other pharmaceutically acceptable carriers or adjuvants.

9. The method according to claim 7, characterized in that The expression level of MALL-II on the surface of leukemia stem cells in the experimental group was decreased by no less than 15% compared with the expression level of MALL-II on the surface of leukemia stem cells in the control group, indicating that the drug to be tested may be effective in targeting the Mmrn1 gene in leukemia stem cells.

10. The method according to claim 7, characterized in that The SNA expression level on the surface of leukemia stem cells in the experimental group decreased by no less than 20% compared with the SNA expression level on the surface of leukemia stem cells in the control group, indicating that the drug to be tested may be effective in targeting the Mmrn1 gene in leukemia stem cells.

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