Application of KMT2C gene in preparing cells resistant to ALK inhibitors

Abstract

The present invention discloses a nucleotide sequence targeting the KMT2C gene and its application in preparing cells resistant to ALK inhibitors, belonging to the technical field of genetic engineering. The nucleotide sequence targeting the KMT2C gene is shown as SEQ ID NO.1 to SEQ ID NO.6. By reducing the expression of KMT2C, the present invention can obtain a cell model resistant to ALK inhibitors.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a nucleotide sequence targeting the KMT2C gene and its application in preparing cells resistant to ALK inhibitors. Background Art

[0002] Lung cancer is the malignant tumor with the first mortality rate in China and even worldwide. 85% of the pathological types of lung cancer are non-small cell lung cancer (NSCLC); with the discovery of driver genes in NSCLC (especially lung adenocarcinoma) and the successful development of corresponding targeted therapies, the treatment effect of patients has been significantly improved. Anaplastic lymphoma kinase (ALK) is one of the common driver genes in NSCLC, and it is mainly activated by fusing with genes such as echinoderm microtubule associated protein-like 4 (EML4). The successful development of ALK inhibitors (such as tyrosine kinase inhibitors (TKI)) has significantly improved the prognosis of advanced ALK fusion-positive NSCLC patients, but most patients still inevitably develop drug resistance. Although a series of drug resistance mechanisms have been discovered, there are still about 40% of patients with unknown drug resistance mechanisms and cannot receive targeted treatment, resulting in poor prognosis. Therefore, exploring new drug resistance mechanisms of ALK inhibitors and thus developing targeted rescue treatment strategies are expected to further improve the treatment effect of ALK fusion-positive patients, which is of great significance.

[0003] Lysine methyltransferase 2C (KMT2C) is a member of the KMT2 family, which can methylate histone 3 lysine 4 (H3K4) and is one of the key factors in epigenetic regulation. Through the research data of two clinical trials, we found that the mutation frequency of the KMT2C gene increased significantly in the population after drug resistance. The loss of function of KMT2C is involved in the formation of various tumors and can promote distant metastasis of tumors, but its specific role and molecular mechanism in ALK inhibitor resistance are still unclear. In particular, different types of TKIs may cause different types of drug resistance mutations, thus limiting the related applications of KMT2C in the direction of ALK inhibitor resistance in lung cancer cells. In addition, constructing cell lines resistant to ALK inhibitors is of great significance for understanding the drug resistance mechanism, discovering new therapeutic targets, evaluating the efficacy of drug combinations, drug screening, personalized medicine, basic scientific research, and the discovery of prognostic and diagnostic markers. At present, there is no report on constructing drug-resistant cell lines by regulating the expression of the KMT2C gene. Summary of the Invention

[0004] The object of the present invention is to provide the related applications of KMT2C in the direction of ALK inhibitor resistance in lung cancer cells, especially the related applications against alectinib resistance. The applications and methods described in the specification of the present invention all belong to non-disease treatment applications and methods.

[0005] To achieve the above object of the invention, the present invention provides the following technical solutions:

[0006] On the one hand, the present invention provides a small RNA for preparing drug-resistant cells against ALK-TKIs, and the nucleotide sequence of the small RNA is shown as any one of SEQ ID NO.1 to SEQ ID NO.6.

[0007] On the other hand, the present invention provides the application of the above small RNA in preparing drug-resistant cells against ALK-TKIs.

[0008] On the other hand, the present invention provides the application of the above small RNA in screening new compounds to overcome drug resistance.

[0009] On the other hand, the present invention provides the application of the above small RNA in evaluating the efficacy and potential side effects of drugs.

[0010] On the other hand, the present invention provides the application of the above small RNA in analyzing the mechanism of drug action and drug resistance phenomenon.

[0011] On the other hand, the present invention provides a drug-resistant cell model that can be used to screen new anti-tumor drugs, and the cell model is obtained by reducing the expression of KMT2C protein in cells.

[0012] Preferably, the aforementioned cell model is obtained by reducing the expression of KMT2C protein in a lung cancer cell line.

[0013] Preferably, the aforementioned cell model is obtained by reducing the expression of KMT2C protein in a human non-small cell lung cancer cell line.

[0014] Preferably, the aforementioned cell model is obtained by reducing the expression of KMT2C protein in the human non-small cell lung cancer cell line NCI-H3122.

[0015] Preferably, the aforementioned cell model is obtained by using the aforementioned small RNA sequence to reduce the expression of KMT2C protein in cells.

[0016] On the other hand, the present invention provides a method for preparing drug-resistant cells against ALK-TKIs, and the method includes the following steps:

[0017] 1) Culturing cells

[0018] 2) Reducing the expression level of KMT2C protein in the cells described in step 1).

[0019] Preferably, the aforementioned cells are a lung cancer cell line.

[0020] Preferably, the aforementioned cells are a non-small cell lung cancer cell line.

[0021] Preferably, the aforementioned cells are the non-small cell lung cancer cell line NCI-H3122.

[0022] Preferably, step 1) is inoculating 1×107 cells in a 15 cm culture dish for 24 hours.

[0023] Preferably, step 2) can reduce the expression level of KMT2C protein in the cells described in step 1) by gene knockout, gene silencing, and / or gene editing.

[0024] Preferably, step 2) is achieved by using the aforementioned small RNA sequence to reduce the expression of KMT2C protein in cells.

[0025] Preferably, step 3) is to transfect cells with small interfering RNAs (siKMT2C#1: SEQ ID NO.1, siKMT2C#2: SEQ ID NO.2, siKMT2C#3: SEQ ID NO.3) and small hairpin RNAs (shKMT2C#1: SEQ ID NO.4, shKMT2C#2: SEQ ID NO.5, shKMT2C#3: SEQ ID NO.5) of KMT2C using Lipofectamine 3000 transfection reagent for 48 hours.

[0026] On the other hand, the present invention provides the application of the above method in screening novel compounds for overcoming drug resistance.

[0027] On the other hand, the present invention provides a method for screening novel compounds for overcoming drug resistance, the method comprising the following steps:

[0028] 1) Culturing cells

[0029] 2) Reducing the expression level of KMT2C protein in the cells of step 1) to obtain a cell line resistant to alectinib.

[0030] 3) Treating the cell line resistant to alectinib obtained in step 2) with alectinib and a novel compound.

[0031] 4) Determining whether the novel compound can overcome drug resistance by comparing the effects of alectinib and the novel compound on cell treatment.

[0032] Preferably, the aforementioned cells are a lung cancer cell line.

[0033] Preferably, the aforementioned cells are a non-small cell lung cancer cell line.

[0034] Preferably, the aforementioned cells are the non-small cell lung cancer cell line NCI-H3122.

[0035] Preferably, step 1) is to inoculate 1×107 cells in a 15 cm culture dish for 24 hours.

[0036] Preferably, step 2) can reduce the expression level of KMT2C protein in the cells of step 1) by gene knockout, gene silencing, and / or gene editing.

[0037] Preferably, step 2) is achieved by reducing the expression of KMT2C protein in cells using the aforementioned small RNA sequences.

[0038] Preferably, in step 3), Lipofectamine 3000 transfection reagent is used to transfect cells with small interfering RNAs (siKMT2C#1: SEQ ID NO.1, siKMT2C#2: SEQ ID NO.2, siKMT2C#3: SEQ ID NO.3) and small hairpin RNAs (shKMT2C#1: SEQ ID NO.4, shKMT2C#2: SEQ ID NO.5, shKMT2C#3: SEQ ID NO.6) of KMT2C for 48 hours respectively.

[0039] Preferably, in step 4), both alectinib and the novel compound are used to treat cells according to Examples 3-6.

[0040] Preferably, the novel compound can be a compound with the same or similar function after chemical modification or transformation of alectinib, or a chemically acceptable salt.

[0041] Preferably, the ALK-TKIs in any of the foregoing technical solutions are alectinib or a compound with the same or similar function thereto and a chemically acceptable salt.

[0042] Compared with the prior art, the present invention has the following advantages:

[0043] 1. The present invention realizes the decrease in the expression of KMT2C in lung cancer drug-resistant cell lines, and can rapidly prepare a lung cancer cell model resistant to ALK-TKI by silencing the function of KMT2C;

[0044] 3. The silenced cell model can be used to screen novel anti-tumor drugs for reversing drug resistance.

[0045] 4. The present invention optimizes the small RNAs for silencing KMT2C;

[0046] 5. The present invention provides a potential research direction for KMT2C to overcome lung cancer ALK-TKI drug resistance. Description of the Drawings

[0047] The beneficial effects of the present invention will be described in detail below in conjunction with the drawings and specific embodiments.

[0048] Figure 1 For (A): IC50 of alectinib in H3122 and H3122_AR cells; (B): mRNA expression levels of KMT2C in H3122 and H3122_AR cells. **P<0.01.

[0049] Figure 2 For the effect of knocking down KMT2C on the expression of KMT2C in H3122. **P<0.01; ***P<0.001.

[0050] Figure 3 To investigate the effect of knocking down KMT2C on the sensitivity of alectinib in H3122.

[0051] Figure 4 To investigate the effect of knocking down KMT2C on cell proliferation. Figure (A) shows the fluorescence of the EdU cell proliferation assay after knocking down KMT2C in H3122; Figure (B) shows the cell proliferation ratio, **P<0.01; ***P<0.001; ****P<0.0001.

[0052] Figure 5 IncuCyte real-time live cell growth curve after knocking down KMT2C with siKMT2C#1 in H3122, ****P<0.0001.

[0053] Figure 6 IncuCyte real-time live cell growth curve after knocking down KMT2C with shKMT2C#1 in H3122, ****P<0.0001.

[0054] Figure 7 To evaluate the effect of KMT2C knockdown on the formation of ALK-TKI resistant clones, **P<0.01; ***P<0.001; ****P<0.0001. Detailed implementation manners

[0055] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0056] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from regular biochemical reagent stores unless otherwise specified.

[0057] The nucleotide sequences in the embodiments are shown in the following table:

[0058]

[0059] Example 1 Expression of KMT2C in lung cancer parental cells and drug-resistant cells

[0060] The human non-small cell lung cancer cell line NCI-H3122 (Cell Bank of the Chinese Academy of Sciences; CSTR: 19375.09.3101HUMSCSP5028) with EML4-ALK fusion mutation was used. When the cell density of H3122 in the logarithmic growth phase grew to 50-60%, the old culture medium was discarded, and complete medium containing 10 nmol / L alectinib was added. The cells were then cultured in an incubator at 37 °C, 5% CO2, and saturated humidity. The culture medium was changed every 2-3 days. When the cell density grew to 80-90% again, the cells were digested and passaged at a ratio of 1:3. After the cells adhered, they were placed in freshly prepared complete medium containing the original concentration of alectinib and continued to be cultured, and the culture medium was changed every 2-3 days. Again, when the cell density grew to 80-90%, the cells were digested and passaged at a ratio of 1:3. After the cells adhered, they were placed in complete medium with alectinib concentration of 20 nmol / L and continued to be cultured; the above operations were repeated; the concentration of alectinib in the culture medium increased in this order: 10 nmol / L---20 nmol / L---50 nmol / L---100 nmol / L---200 nmol / L---300 nmol / L. After 284 days, lung cancer ALK-TKI resistant cells were gradually established and named H3122-AR, and its IC50 was 181 nmol.

[0061] RNA in the cells was extracted using the TaKaRa MiniBEST Universal RNA Extraction Kit, then reverse transcribed into cDNA, and the expression of KMT2C was detected by qRT-PCR.

[0062] The results were as Figure 1 shown. The expression of KMT2C in lung cancer resistant cells (H3122-AR) was significantly lower than that in parental cells (H3122).

[0063] Example 2 Effect of interfering with KMT2C on the expression of KMT2C in NCI-H3122 cells

[0064] 1×10 7 NCI-H3122 cells were seeded in a 15 cm culture dish for 24 hours, and then transfected with small interfering RNA (siKMT2C#1, siKMT2C#2, siKMT2C#3) and small hairpin RNA (shKMT2C#1, shKMT2C#2, shKMT2C#3) of KMT2C using Lipofectamine 3000 transfection reagent for 48 hours, with non-functional sequence siControl and shControl as controls. The expression of KMT2C was detected by Western blot and qRT-PCR.

[0065] The results are as Figure 2 shown. Compared with the control group, siKMT2C#1, #2, and #3 can respectively reduce the expression of KMT2C in lung cancer drug-resistant cells to 29.89%, 37.59%, and 44.27% of the parent; shKMT2C#1, #2, and #3 can respectively reduce the expression of KMT2C in lung cancer drug-resistant cells to 14.84%, 20.64%, and 56.25% of the parent.

[0066] Example 3 Knockdown of KMT2C can induce resistance of H3122 cells to alectinib

[0067] H3122 cells successfully transfected with KMT2C small interfering RNA and small hairpin RNA were seeded in 96-well plates at a density of 5×10³ cells / well. After 24 hours, gradient-diluted alectinib was added, and after continuous culture for 72 hours, the absorbance at a wavelength of 450 nm was measured by the CCK8 method to calculate the IC50 value.

[0068] As Figure 3 shown, after knocking down the expression of KMT2C in sensitive cells H3122 using small interfering RNA and small hairpin RNA, obvious resistance to alectinib was obtained, and the IC50 increased significantly.

[0069] Example 3 Effect of KMT2C silencing on the proliferation of ALK fusion lung cancer cells

[0070] EdU cell proliferation assay. The specific experimental method is as follows: Cells were seeded in six-well plates at a density of 3×10 5 cells per well. After 24 hours, the cell confluence was observed under the microscope to reach 60%-70%. The EdU solution was diluted with pre-warmed cell culture medium at a ratio of 1:1000. The old culture medium was aspirated, and after washing twice with sterile PBS, it was replaced with freshly prepared fresh medium containing EdU and continued to be cultured for 48 hours. EdU replaces thymidine and incorporates into newly synthesized DNA, and the proliferating cells show bright red fluorescence under a fluorescence microscope through Azide 594 dye labeling. We found that after the expression of KMT2C was knocked down or knocked out, the proliferation of H3122 increased significantly.

[0071] As Figure 4 shown, knocking down KMT2C in the H3122 cell line can significantly increase cell proliferation.

[0072] Example 3 IncuCyte real-time live cell imaging analysis of H3122 cell line after silencing KMT2C

[0073] The specific experimental method is as follows: The H3122 cell line was seeded at 5×10 3Cells were seeded in a 96-well plate at a density of

[0074] As Figures 5 - 6 shown, knockdown of KMT2C increased the proliferation of ALK fusion-positive lung cancer cells under treatment with different concentration gradients of alectinib (p<0.001).

[0075] Example 3 Effect of KMT2C silencing on colony formation of ALK fusion-positive lung cancer cells

[0076] Colony formation assays were used to detect the proliferation and drug resistance of cells with stable low expression of KMT2C. The specific experimental method was as follows: H3122 cells were transfected with short hairpin RNA (shKMT2C) to construct a cell line with stable low expression of KMT2C. Cells were seeded in a six-well plate at a density of 1000 cells / well. Subsequently, the cells were continuously cultured in the absence of alectinib, in the presence of 20 nM alectinib, or in the presence of 100 nM alectinib until 14 days had passed or the number of cells in the vast majority of individual colonies was greater than 50. The culture medium was changed every 3 days during the process, and the cell status was observed. After colony formation, 1 mL of 4% paraformaldehyde was added to each well to fix the cells for 30 - 60 min, and then the cells were washed once with PBS; 1 mL of crystal violet staining solution was added to each well and left at room temperature to stain the cells for 10 - 20 min; subsequently, the crystal violet solution was aspirated, and the cells were washed three times with PBS (or dd water) and left to dry upside down. The colony formation was photographed with a camera and counted using ImageJ software. Finally, the colony formation rate was calculated according to the following formula: Colony formation rate = (number of colonies / number of seeded cells) × 100%.

[0077] As Figure 7 shown, knockdown of KMT2C induced drug resistance of lung cancer cells H3122 to ALK-TKI and increased the colony formation rate of drug-resistant clones.

[0078] The above are only preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or equivalent changes and modifications within the scope of the technical solution of the present invention without departing from the technical content disclosed above. However, any brief modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing ALK-TKIs-resistant cells, characterized in that: The cells are human non-small cell lung cancer cells, and the method comprises the following steps: 1) Cultivate cells; 2) reducing the expression level of KMT2C protein in the cells of step 1) by gene silencing; The ALK-TKIs is Alectinib.

2. The method according to claim 1, characterized in that The step 2) reduces the expression of KMT2C protein in the cell by using small RNA; the nucleotide sequence of the small RNA is shown in any one of SEQ ID NOs: 1-6.

3. Any of the following applications of the method of claim 1: 1) Use in screening compounds that overcome drug resistance, wherein the compounds are compounds for treating human non-small cell lung cancer; 2) Application in evaluating the efficacy and potential side effects of a drug, wherein the efficacy and potential side effects of the drug are for treating human non-small cell lung cancer; 3) Application in analyzing drug action mechanism and drug resistance phenomenon, wherein the drug action mechanism and drug resistance phenomenon are for the treatment of human non-small cell lung cancer.

4. A method for screening a compound that overcomes Alectinib resistance, characterized in that: The method comprises the following steps: 1) culturing cells, wherein the cells are human non-small cell lung cancer cells; 2) reducing the expression level of KMT2C protein in the cells of step 1) by gene silencing to obtain a cell line resistant to Alectinib; 3) treating the alectinib-resistant cell line obtained in step 2) with alectinib and the compound; 4) Determine whether the compound can overcome alectinib resistance by comparing the effects of alectinib and the compound on cell treatment.

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