Application of KMT2C gene in preparation of ALK inhibitor drug-resistant cells

Abstract

The invention discloses a nucleotide sequence of a targeted KMT2C gene and application of the targeted KMT2C gene in preparation of ALK inhibitor drug-resistant cells, and belongs to the technical field of gene engineering. The nucleotide sequences of the targeted KMT2C gene are as shown in SEQ ID NO. 1 to SEQ ID NO. 6. According to the invention, the ALK inhibitor drug-resistant cell model can be obtained by reducing the expression of KMT2C.

Description

Technical Field

[0001] The present invention belongs to the field of gene engineering technology, and specifically relates to a nucleotide sequence targeting a KMT2C gene and an application thereof in preparing cells resistant to ALK inhibitors. Background Art

[0002] Lung cancer is the malignant tumor with the highest mortality rate in my country and even in the world. 85% of lung cancers are of non-small cell lung cancer (NSCLC) pathological type; with the discovery of NSCLC (especially lung adenocarcinoma) driver genes 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, which is mainly activated by fusion 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 patients with advanced ALK fusion-positive NSCLC, but most patients still inevitably develop drug resistance. Although a series of drug resistance mechanisms have been discovered, about 40% of patients still have unknown drug resistance mechanisms, cannot receive targeted treatment, and have a poor prognosis. Therefore, it is of great significance to explore new resistance mechanisms of ALK inhibitors and develop targeted rescue treatment strategies, which are expected to further improve the treatment effect of ALK fusion-positive patients.

[0003] Lysine methyltransferase 2C (KMT2C) is a member of the KMT2 family. It 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 functional loss 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 TKI may cause different types of resistance mutations, thus limiting the relevant application of KMT2C in the direction of ALK inhibitor resistance in lung cancer cells. In addition, the construction of ALK inhibitor-resistant cell lines is of great significance for understanding the resistance mechanism, discovering new therapeutic targets, evaluating the effect of drug combinations, drug screening, personalized medicine, basic scientific research, and the discovery of prognostic and diagnostic markers. At present, there are no reports on the construction of resistant cell lines by regulating the expression of KMT2C gene. Summary of the invention

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

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0007] In another aspect, the present invention provides use of the above-mentioned small RNA in preparing drug-resistant cells against ALK-TKIs.

[0008] On the other hand, the present invention provides the use of the above-mentioned small RNA in screening new compounds that overcome drug resistance.

[0009] In another aspect, the present invention provides the use of the above-mentioned small RNA in evaluating the efficacy and potential side effects of a drug.

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

[0011] On the other hand, the present invention provides a drug-resistant cell model that can be used to screen new anti-tumor drugs, wherein 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 cells of lung cancer cell lines.

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

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

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

[0016] In another aspect, the present invention provides a method for preparing ALK-TKIs-resistant cells, the method comprising the following steps:

[0017] 1) Cultivate cells

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

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

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

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

[0022] Preferably, step 1) is to seed 1×10 7 cells in a 15 cm culture dish for 24 hours.

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

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

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

[0026] In another aspect, the present invention provides application of the above method in screening novel compounds that overcome drug resistance.

[0027] In another aspect, the present invention provides a method for screening novel compounds that overcome drug resistance, the method comprising the following steps:

[0028] 1) Cultivate 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 alectinib-resistant cell line obtained in step 2) with alectinib and the novel compound.

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

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

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

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

[0035] Preferably, step 1) is to seed 1×10 7 cells in a 15 cm culture dish for 24 hours.

[0036] Preferably, in step 2), the expression level of the KMT2C protein in the cells of step 1) can be reduced 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 sequence.

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

[0039] Preferably, in step 4), cells are treated with alectinib and the novel compound according to Examples 3 to 6.

[0040] Preferably, the novel compound may be a compound having the same or similar function after chemical modification or transformation of Alectinib, or a chemically acceptable salt thereof.

[0041] Preferably, the ALK-TKIs in any of the aforementioned technical solutions is Alectinib or a chemically acceptable salt thereof having the same or similar function.

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

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

[0044] 3. The silenced cell model can be used to screen new anti-tumor drugs that reverse drug resistance.

[0045] 4. The present invention optimizes the small RNA that silences KMT2C;

[0046] 5. The present invention provides a potential research direction for KMT2C to overcome ALK-TKI resistance in lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

[0053] Figure 6 This is the IncuCyte real-time cell growth curve after knocking down KMT2C in H3122 using shKMT2C#1, ****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 DESCRIPTION

[0055] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only 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 creative work are within the scope of protection of the present invention.

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

[0057] The nucleotide sequences in the examples 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 with EML4-ALK fusion mutation (Cell Bank of the Chinese Academy of Sciences; CSTR: 19375.09.3101HUMSCSP5028) was used. When the density of H3122 cells in the logarithmic growth phase grew to 50-60%, the old culture medium was discarded, and complete culture medium containing 10nmol / L alectinib was added, and the cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity; the culture medium was replaced every 2-3 days, and when the cell density grew to 80-90% again, the cells were digested and passaged at a ratio of 1:3, and after the cells adhered to the wall, they were placed in a newly configured complete culture medium containing the original concentration of alectinib for continued culture, and the culture medium was replaced every 2-3 days. Again, when the cell density grows to 80-90%, digest the cells, subculture them at a ratio of 1:3, and after the cells adhere to the wall, continue to culture them in complete culture medium with an alectinib concentration of 20 nmol / L; repeat the above operation; the alectinib concentration in the culture medium increases in the following order: 10 nmol / L---20 nmol / L---50 nmol / L---100 nmol / L---200 nmol / L---300 nmol / L, and after 284 days, lung cancer ALK-TKI-resistant cells are gradually established, named H3122-AR, with an IC50 of 181 nmol.

[0061] TaKaRa MiniBEST Universal RNA Extraction Kit was used to extract RNA from cells, which was then reverse transcribed into cDNA. The expression of KMT2C was detected by qRT-PCR.

[0062] The results are as follows Figure 1 As 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 KMT2C Interference on KMT2C Expression in NCI-H3122 Cells

[0064] 1×10 7 NCI-H3122 cells were seeded in 15 cm culture dishes for 24 hours, and then transfected with KMT2C small interfering RNA (siKMT2C#1, siKMT2C#2, siKMT2C#3) and small hairpin RNA (shKMT2C#1, shKMT2C#2, shKMT2C#3) using Lipofectamine3000 transfection reagent for 48 hours, with non-functional sequences siControl and shControl as controls. Western blot and qRT PCR were used to detect the expression of KMT2C.

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

[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 a 96-well plate at a density of 5×103 cells / well. Serial dilutions of alectinib were added after 24 hours. 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] like Figure 3 As shown, after knocking down KMT2C expression in sensitive cells H3122 using small interfering RNA and small hairpin RNA, significant resistance to alectinib was acquired and the IC50 was significantly increased.

[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: 3×10 5 The cells were seeded in a six-well plate at a density of 10 cells per well. After 24 hours, the cell confluence reached 60%-70% under the microscope. EdU solution was diluted with preheated cell culture medium at a ratio of 1:1000, the old culture medium was discarded, and the cells were washed twice with sterile PBS, and then replaced with a newly prepared fresh culture medium containing EdU and continued to be cultured for 48 hours. EdU replaces thymidine and is incorporated into newly synthesized DNA. The proliferating cells are labeled with Azide 594 dye, which makes them show bright red fluorescence under a fluorescence microscope. We found that after the expression of KMT2C was knocked down or knocked out, the proliferation of H3122 increased significantly.

[0071] like Figure 4 As shown, knockdown of KMT2C in 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: H3122 cell line was cultured at 5×10 3The cells were seeded in a 96-well plate at a density of 10 cells / well. After the cells adhered to the plate, different concentrations of alectinib were added, and five replicates were set up for each group. The cells in different groups were photographed every two hours by IncuCyte real-time living cell imaging, and the confluence was calculated to draw a cell growth curve.

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

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

[0076] The clone formation experiment was used to detect the proliferation and drug resistance of KMT2C stable low-expressing cells. The specific experimental method is as follows: H3122 cells were transfected with short hairpin RNA (shKMT2C) to construct a stable KMT2C low-expressing cell line. The cells were seeded in a six-well plate at a density of 1000 cells / well. Subsequently, they were cultured continuously in the culture environment without alectinib treatment, 20nM alectinib and 100nM alectinib, respectively, until 14 days or the number of cells in most single clones was greater than 50. The medium was changed every 3 days and the cell state was observed. After the clones were formed, 1mL of 4% paraformaldehyde was added to each well for fixation for 30-60min, and PBS was washed once; 1ml of crystal violet stain was added to each well, placed at room temperature, and the cells were stained for 10-20min; then, the crystal violet solution was aspirated, the cells were washed three times with PBS (or dd water), and inverted to dry. The clone formation was photographed by a camera and counted by ImageJ software. Finally, the clone formation rate was calculated according to the following formula: clone formation rate = (clone number / inoculated cell number) × 100%

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

[0078] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. 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 are still within the scope of the technical solution of the present invention.

Claims

1. A small RNA for preparing ALK-TKIs-resistant cells, characterized in that: The nucleotide sequence of the small RNA is shown in any one of SEQ ID NO.1 to SEQ ID NO.

6.

2. A drug-resistant cell line against ALK-TKIs, characterized in that: The cells are obtained by reducing the expression of KMT2C protein in the cells.

3. A method for preparing ALK-TKIs-resistant cells, characterized in that: The method comprises the following steps: 1) culturing cells; 2) reducing the expression level of KMT2C protein in the cells of step 1).

4. The method according to claim 3, characterized in that Step 1) The cells are human non-small cell lung cancer cell lines.

5. The method according to claim 3, characterized in that Step 2) reducing the expression level of the KMT2C protein in the cells of step 1) by gene knockout, gene silencing and / or gene editing.

6. Any of the following uses of the small RNA according to claim 1: 1) Application in the preparation of ALK-TKIs-resistant cells; 2) Application in the screening of new compounds that overcome drug resistance; 3) Application in the evaluation of drug efficacy and potential side effects; 4) Application in the analysis of drug action mechanisms and drug resistance phenomena.

7. Any of the following uses of the drug-resistant cells according to claim 2: 1) Application in screening new compounds that overcome drug resistance; 2) Application in evaluating the efficacy and potential side effects of drugs; 3) Application in analyzing the mechanism of drug action and drug resistance.

8. Any of the following applications of the method of claim 3: 1) Application in screening new compounds that overcome drug resistance; 2) Application in evaluating the efficacy and potential side effects of drugs; 3) Application in analyzing the mechanism of drug action and drug resistance.

9. A method for screening novel compounds that overcome drug resistance, characterized in that: The method comprises the following steps: 1) Cultivating cells; 2) Reducing the expression of KMT2C protein in the cells of step 1) to obtain a cell line resistant to alectinib; 3) Treating the cell line resistant to alectinib obtained in step 2) with alectinib and a novel compound; 4) Determining whether the novel compound can overcome the drug resistance by comparing the effects of alectinib and the novel compound on the cells.

10. The method according to claim 3, characterized in that Step 1) The cells are human non-small cell lung cancer cell lines.

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