Application and method of PYCR1 siRNA in enhancing the sensitivity of lung cancer cells to TRAIL

Abstract

The present invention discloses the application and method of PYCR1 siRNA in enhancing the sensitivity of lung cancer cells to TRAIL. The RNAi technology is used to significantly inhibit the expression of PYCR1 gene, and the PYCR1 siRNA1 with the best inhibitory effect is obtained. By transfecting PYCR1 siRNA1 into lung cancer cells NCI-H1299, it is found that the sensitivity of lung cancer cells to TRAIL is reduced from 400ng / mL to 50ng / mL, and the sensitivity is significantly improved. A strategy and method that can significantly enhance TRAIL treatment of lung cancer patients is established. By using PYCR1 siRNA and TRAIL in combination, the efficacy of TRAIL can be improved, the dosage dependence can be reduced, and a good effect can also be achieved for patients who are relatively insensitive to TRAIL, thereby expanding the clinical application of TRAIL treatment in lung cancer patients.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical research, and specifically relates to an application and method of PYCR1 siRNA in enhancing the sensitivity of lung cancer cells to TRAIL. Background Art

[0002] Pyrroline-5-carboxylate reductase 1 (PYCR1) is located on chromosome 17q2.3, contains 319 amino acids, and encodes a 33.4kDa protein. PYCR1 is a key enzyme involved in proline synthesis, which can catalyze the reduction of 5-pyrrolinecarboxylic acid to proline with NAD(P)H as a cofactor. Studies have found that the expression of the PYCR1 gene increases in 79% of cancers, playing the role of an oncogene. The increase in the expression of the PYCR1 gene can promote tumor cell proliferation and inhibit cell apoptosis. Studies have shown that PYCR1 expression is significantly increased in lung adenocarcinoma tissue and is correlated with pathological grading; PYCR1 gene is highly expressed in patients with non-small cell lung cancer, and high expression of PYCR1 gene is associated with poor overall survival and higher TNM stage and mediates cisplatin resistance; PYCR1 siRNA was transfected into lung cancer cell lines A549 and NCI-H1299 to construct lung cancer cell lines with downregulated PYCR1 expression, and it was found that when PYCR1 expression was inhibited, the cell autophagy process may be enhanced (Yang Xingling, Study on the expression and prognostic value molecules and biological functions of PYCR1 in lung cancer, Naval Medical University, 2021).

[0003] Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a member of the TNF family. Studies have found that its receptors are divided into two categories: one is death receptor (DR), such as DR4 and DR5; the other is "attractant" receptors, such as DcR1 and DcR2. Death receptors can induce cell apoptosis, and "attractant" receptors exist in normal cells to protect cells from TRAIL-induced apoptosis. In vitro experiments have shown that TRAIL has a specific killing effect on tumor cells such as Hela cervical cancer cells, U973, A549 lung cancer cells, and human liver cancer cells 7402 and 7721. This killing effect is time- and dose-dependent and does not cause extensive damage to the body. However, TRAIL-induced apoptosis of tumor cells depends on the expression intensity of DR on the surface of tumor cells. Therefore, how to upregulate the expression intensity of DR and increase the sensitivity of tumor cells to TRAIL has become a key issue in the process of tumor treatment. At the same time, there is a very practical drawback in using TRAIL protein as an anti-tumor drug, that is, large doses of TRAIL protein are required to inhibit the proliferation of tumor cells in the body, which makes the treatment cost high. Therefore, enhancing the sensitivity of tumor cells to TRAIL is of great practical significance.

[0004] Chinese patent CN102821778B discloses a composition for enhancing TRAIL sensitivity containing a TIP41 expression or activity inhibitor as a target gene or a TRAIL sensitizer. It is found that after treating TRAIL-resistant liver cancer cells with TIP41 siRNA and TRAIL, there is an effect of inducing cancer cell-specific apoptosis. Chinese patent CN115161394A discloses the application of TP53INP2 in enhancing the sensitivity of acute myeloid leukemia cells to TRAIL. Chinese patent CN105194671B discloses the application of CABYR-a / b in promoting the sensitivity of tumor cells to VP16 and TRAIL. However, no relevant application of whether the PYCR1 gene can enhance the sensitivity of lung cancer cells to TRAIL has been found. Summary of the invention

[0005] The purpose of the present invention is to provide an application and method of PYCR1 siRNA in enhancing the sensitivity of lung cancer cells to TRAIL, so as to provide a reference for preparing drugs for treating lung cancer.

[0006] To achieve the above object, the present invention provides the use of a PYCR1 expression or activity inhibitor in at least one of the following ac:

[0007] a: Preparation of drugs for promoting the sensitivity of cancer cells to TRAIL;

[0008] b: Preparation of drugs for synergistic TRAIL treatment of cancer;

[0009] c: Preparation of drugs for enhancing TRAIL-induced apoptosis of cancer cells.

[0010] Preferably, the PYCR1 expression or activity inhibitor is siRNA, and the sequences of the siRNA are SEQ ID NO:3 and SEQ ID NO:4.

[0011] Preferably, the cancer cell is lung cancer NCI-H1299 cell, and the cancer is lung cancer.

[0012] The present invention also provides a method for enhancing TRAIL sensitivity by using a PYCR1 expression or activity inhibitor, comprising the following steps:

[0013] (1) Design and synthesize siRNA based on the human PYCR1 gene sequence;

[0014] (2) transfecting siRNA into cancer cells and culturing transfected cells;

[0015] (3) The transfected cells were collected, treated with TRAIL, cultured, and the apoptosis and expression of cancer cells were detected.

[0016] Preferably, the sequences of the siRNA in step (1) are SEQ ID NO:3 and SEQ ID NO:4.

[0017] Preferably, the cancer cells in step (2) are lung cancer NCI-H1299 cells, and the cells are cultured to the logarithmic growth phase before transfection, and the cell density during transfection is 60%-70%.

[0018] Preferably, the siRNA concentration in step (2) is 50-100 nM, and the transfection time is 48 h; further preferably, the siRNA concentration is 50 nM.

[0019] Preferably, the treatment concentration of TRAIL in step (3) is 50-200 ng / mL, and the TRAIL treatment time is 48 h; further preferably, the treatment concentration of TRAIL is 50 ng / mL.

[0020] Preferably, the culture conditions in steps (2) and (3) are 5% CO2 and a 37°C humidified incubator.

[0021] The beneficial effect obtained by the present invention is that siRNA1, an inhibitor of PYCR1 expression or activity, is designed and obtained, which can inhibit the expression of PYCR1 mRNA and protein in lung cancer NCI-H1299 cells, the inhibition rate of mRNA can reach 95%, and the activity of NCI-H1299 cells is inhibited, and the proliferation thereof is inhibited. At the same time, the present invention also finds that siRNA1 of PYCR1 can enhance the sensitivity of lung cancer NCI-H1299 cells to TRAIL, and after siRNA1 transfects lung cancer NCI-H1299 cells and then treats them with TRAIL, the cell activity can be significantly reduced at 400-800ng / mL of TRAIL, and the sensitivity is significantly improved; and when siRNA1 and 50ng / mL of TRAIL are used in combination, the apoptosis rate of lung cancer NCI-H1299 cells increases, the expression of apoptosis-related proteins is activated, and the expression levels of death receptors DR4 and DR5 on the cell membrane increase significantly, indicating that after siRNA1 transfection, the sensitivity of lung cancer cells to TRAIL is significantly enhanced. Therefore, siRNA1 can be used to prepare drugs that promote the sensitivity of lung cancer cells to TRAIL or drugs that cooperate with TRAIL to treat lung cancer; in addition, it can also be used to prepare drugs that enhance TRAIL-induced apoptosis of lung cancer cells, which has important guiding significance for the treatment of lung cancer cells. At the same time, PYCR1 has an increased expression in 79% of cancers, so the present invention can also guide the development of other cancer treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a bar graph showing the inhibition of PYCR1 gene mRNA expression in lung cancer NCI-H1299 cells by siRNA in Example 3; * indicates P<0.05, with significant difference; ** indicates P<0.01, with extremely significant difference.

[0023] Figure 2 This is a gel electrophoresis diagram of the siRNA inhibition of PYCR1 protein expression in lung cancer NCI-H1299 cells in Example 4.

[0024] Figure 3 It is a line graph showing the effect of different TRAIL treatment concentrations on cell viability in Example 5, where * indicates P<0.05, with significant difference; ** indicates P<0.01, with extremely significant difference.

[0025] Figure 4The figure is a diagram showing the effects of siRNA1 and TRAIL in promoting apoptosis of lung cancer NCI-H1299 cells in Example 6; wherein A is a scatter plot of cell apoptosis rates in different treatment groups; B is a bar graph of cell apoptosis rates in different treatment groups; wherein points of different colors in A represent cells of different densities in various areas; in B, * indicates P<0.05, and the difference is significant; ** indicates P<0.01, and the difference is extremely significant; "-" indicates that the treatment is not performed, and "+" indicates that the treatment is performed.

[0026] Figure 5 This is a gel electrophoresis diagram of the expression of proteins related to the apoptosis signaling pathway under the treatment of siRNA1 and TRAIL in Example 7; wherein "-" indicates that the treatment is not performed, and "+" indicates that the treatment is performed.

[0027] Figure 6 The bar graph of siRNA1 promoting the expression of death receptors in Example 8; A is DR4, B is DR5; * indicates P<0.05, and the difference is significant.

[0028] Figure 7 This is a gel electrophoresis diagram showing the effect of siRNA1 on promoting the expression of DR4 and DR5 proteins in Example 9; "-" indicates that the treatment is not performed, and "+" indicates that the treatment is performed.

[0029] Figure 8 The figures are the expression of DR4 and DR5 on the cell membrane surface of lung cancer NCI-H1299 in Example 10; wherein A is the expression waveform of DR4 on the cell membrane surface under different treatments; B is the statistical analysis bar graph of DR4 positive molecules on the cell membrane surface; C is the expression waveform of DR5 on the cell membrane surface under different treatments; D is the statistical analysis bar graph of DR5 positive molecules on the cell surface; waves of different colors in the figure represent different treatment conditions; * indicates P<0.05, and the difference is significant. DETAILED DESCRIPTION

[0030] The present invention is further explained below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses, but these embodiments are only preferred embodiments and cannot be understood as limiting the scope of protection of the present invention. It should be understood by those skilled in the art that modifications or replacements to the details and forms of the technical solution of the present invention without departing from the spirit and scope of the present invention all fall within the scope of protection of the present invention.

[0031] Human lung cancer NCI-H1299 cells: purchased from the cell bank of the Chinese Academy of Sciences in Shanghai (catalog: TCHu160), cultured and preserved by the Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy (Three Gorges University);

[0032] RPMI-1640 basic medium, penicillin, streptomycin, and PBS were purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0033] Trypsin digestion solution containing 0.25% trypsin and trypsin-EDTA digestion solution containing 0.25% trypsin and 0.02% EDTA (0.53 mM) were purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0034] RPMI-1640 complete medium: RPMI-1640 basic medium supplemented with 10% fetal bovine serum, 1×10 5 U / L penicillin and 100mg / L streptomycin;

[0035] Fetal bovine serum (Hyclone): purchased from Wuhan Promoter Biotechnology Co., Ltd.;

[0036] TurboFect transfection reagent: purchased from Thermo Fisher Scientific (China) Co., Ltd.;

[0037] RIPA lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0038] BCA kit: purchased from Wuhan Core Biotechnology Co., Ltd.;

[0039] Annexin V-FITC / PI double staining apoptosis detection kit (BestBio): purchased from Wuhan Core Biotechnology Co., Ltd., containing: Annexin V binding solution, Annexin V-FITC staining solution and PI staining solution.

[0040] Anti-target protein antibody (primary antibody): purchased from Wuhan Tri-Eagle Biotechnology Co., Ltd.;

[0041] Horseradish peroxidase-labeled secondary antibodies and 1% goat serum were purchased from Wuhan Saiweier Biotechnology Co., Ltd.;

[0042] HiScript II One Step qRT-PCR SYBR Green Kit: purchased from Nanjing Novozyme Biotechnology Co., Ltd.; contains 2×One Step SYBR Green Mix, One Step SYBR Green Enzyme Mix, 50×ROX Reference Dye 1 and RNase-free ddH2O;

[0043] TRAIL: purchased from Beijing Sino Biological Technology Co., Ltd.;

[0044] Reagents not mentioned in the present invention can be obtained through conventional commercial channels, and all instruments and methods not mentioned are involved in conventional experiments.

[0045] Example 1 Design of siRNA

[0046] The sequence of Negative Control (NC) comes from the control sequence in foreign companies (Ambion / QIAGEN / sigma) and literature (DOI:10.1074 / jbc.M403861200).

[0047] The human PYCR1 gene was obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / ), with gene ID 5831, and transcript information was obtained. The transcript with a short transcript length and the largest number of homologous exons was selected and submitted to the siDESIGN Center website for siRNA design, and siRNA1 and siRNA2 with high knockdown efficiency were obtained. The sequences are shown in Table 1:

[0048] Table 1 Negative Control, siRNA1 and siRNA2 sequences

[0049]

[0050] The above sequences were synthesized by Suzhou Genema Gene Co., Ltd. The artificially synthesized siRNA fragments were dissolved in 125 μL DEPC water and diluted to 20 μmol / L, and stored in a -20°C refrigerator for later use.

[0051] Example 2 Cultivation and transfection of lung cancer cells

[0052] Human lung cancer NCI-H1299 cells were cultured in RPMI-1640 complete medium at 5% CO2 and 37°C humidified incubator until the logarithmic growth phase, and then transfected with the siRNA prepared in Example 1. The specific steps are as follows:

[0053] (1) Inoculate human lung cancer NCI-H1299 cells in a 6-well plate and culture to a density of 60%-70% for later use;

[0054] (2) Wash the cells in each well twice with PBS solution to ensure that the cells are growing well, and then add 1.8 mL of RPMI-1640 complete medium for later use;

[0055] (3) Place a 1.5 mL EP tube on a tube rack, add 200 μL of RPMI-1640 basal medium and 5 μL of slowly dissolved siRNA, mix gently and let stand for 5 min;

[0056] (4) Add 5 μL of TurboFect transfection reagent to the EP tube, mix gently, and place on ice for 15 min. Label and set aside.

[0057] (5) Add the mixture prepared in step (4) to each well of the 6-well plate, shake it in all directions, place it in a cell culture incubator and continue culturing. After 48 hours, collect the cells in each well for subsequent experiments.

[0058] NC group: transfection with Negative Control, working concentration was 50nM;

[0059] siRNA1 group: transfected with siRNA1, working concentration was 50 nM;

[0060] siRNA2 group: transfected with siRNA2, with a working concentration of 50 nM.

[0061] Example 3 Total RNA extraction and expression level detection

[0062] The cells transfected 48 hours after the experiment were taken, RNA was extracted and real-time fluorescence quantitative PCR (qRT-PCR) was performed to detect the expression of PYCR1 mRNA. The specific steps were as follows:

[0063] (1) Based on the human PYCR1 gene sequence obtained from NCBI, qRT-PCR primers were designed on PrimerBank (https: / / pga.mgh.harvard.edu / primerbank / ). The primers were PYCR1 F and PYCR1 R, and the sequences were SEQ ID NO:7 and SEQ ID NO:8. At the same time, the human β-actin gene was selected as the internal reference gene, and primers were designed. The primers were β-actin F and β-actin R, and the sequences were SEQ ID NO:9 and SEQ ID NO:10. The primer series is shown in Table 2.

[0064] Table 2 Primer sequences for PYCR1 and GADPH

[0065]

[0066]

[0067] (2) Collect the cells of the three groups of transfection in Example 2 after 48 hours, and extract the total RNA with Trizol reagent; use Fermentas reverse transcription kit to synthesize the first chain of cDNA: add 1 μL OligodT to the PCR tube with 1 μg RNA template, add DEPC water to 12 μL, place the EP tube at 70°C for 5 minutes, and then immediately place it on ice for 1 minute. Add 4 μL of 5× buffer, 1 μL of 20U / μL ribonuclease inhibitor, 2 μL of 10mM dNTP mix and 1 μL of reverse transcriptase, mix well, and centrifuge instantly. Place the mixture in a PCR instrument to complete the subsequent reaction: 37°C for 5 minutes, 42°C for 60 minutes, and 70°C for 10 minutes.

[0068] (3) The cDNA obtained in step (2) was used as a template to perform qRT-PCR reaction. HiScript II One Step qRT-PCR SYBR Green Kit was used to perform real-time fluorescence PCR reaction, wherein three replicate wells were set for each sample, and the Ct was taken as the average value. The qRT-PCR reaction system is shown in Table 3, and the reaction procedure is shown in Table 4.

[0069] The calculation formula is as follows:

[0070] ΔCT=CT 目的基因 -CT β-actin , ΔΔCT=ΔCT 处理组 -ΔCT 对照组 ;

[0071] Relative difference in target gene expression between different samples = 2 -ΔΔCT .

[0072] Table 3 qRT-PCR reaction system

[0073] Reagents content <![CDATA[RNase-free ddH2O]]> to 20μL 2×One Step SYBR Green Mix 10μL One Step SYBR Green Enzyme Mix 1μL 50×ROX Reference Dye 1 0.4μL Gene Specific Primer Forward(10μM) 0.4μL Gene Specific Primer Reverse(10μM) 0.4μL Template RNA Total RNA: 1pg-1μg

[0074] Table 4 qRT-PCR reaction procedure

[0075]

[0076]

[0077] like Figure 1As shown in the figure, the expression of β-actin did not change significantly between different transfection treatment groups, while the expression of PYCR1 gene changed significantly; compared with the NC group, the expression of PYCR1mRNA in NCI-H1299 cells decreased significantly after siRNA1 and siRNA2 transfection, among which siRNA1 had a very significant inhibitory effect on PYCR1mRNA, with an inhibition rate of about 95%. Therefore, siRNA1 had the best inhibitory effect on PYCR1 mRNA expression, so siRNA1 was selected for transfection of NCI-H1299 cells in subsequent experiments.

[0078] Example 4 Western Blot detection of PYCR1 protein expression level

[0079] The cells transfected 48 hours after the experiment were taken, and the expression level of PYCR1 protein was detected by Western blot. The specific steps were as follows:

[0080] (1) Take the three groups of cells transfected 48 hours after transfection in Example 2;

[0081] (2) Wash the collected cells three times with PBS, then add 100 μL RIPA lysis buffer (containing Cocktail) and lyse on ice for 30 min;

[0082] (3) centrifuging the lysed liquid at 12000 rpm for 15 min to collect the supernatant to obtain a protein solution;

[0083] (4) Use the BCA kit to measure the OD value of the protein solution and calculate the protein content of the sample;

[0084] (5) Load 20 μg / well of the extracted protein sample onto a 10% SDS-PAGE gel and perform electrophoresis at 80 V for 120 min;

[0085] (6) After electrophoresis, the membrane was transferred and blocked at room temperature for 2 h. The membrane was then incubated with an anti-target protein antibody (PYCR1, dilution ratio 1:1000) and a horseradish peroxidase-labeled secondary antibody (dilution ratio 1:8000). β-actin was used as the internal reference protein in the experiment.

[0086] The results show that Figure 2 ), compared with the NC group, the expression level of PYCR1 protein in NCI-H1299 cells transfected with siRNA1 and siRNA2 was significantly decreased, among which the expression level of PYCR1 protein transfected with siRNA1 decreased more significantly, which is consistent with the results in Example 3. Therefore, in subsequent experiments, siRNA1 was used for transfection of NCI-H1299 cells.

[0087] Example 5 MTT assay to detect cell proliferation ability and sensitivity to TRAIL

[0088] After TRAIL binds to the receptor, it induces apoptosis through the death receptor pathway and the mitochondria-dependent apoptosis pathway, resulting in a decrease in cell viability. Therefore, detecting changes in cell viability levels can measure changes in TRAIL sensitivity. Therefore, the MTT experiment was performed using NCI-H1299 cells transfected with 50nM siRNA1 in Example 2 to detect the proliferation ability of the cells and their sensitivity to TRAIL. The specific steps are as follows:

[0089] siRNA1 group: (1) Take the NCI-H1299 cells transfected with 50 nM siRNA1 in Example 2, add 1 ml of trypsin-EDTA digestion solution for 90 s 48 h after transfection, add 1 mL of RPMI-1640 complete medium, and evenly blow to form a cell suspension; centrifuge at 800 rpm for 3 min to collect the cells;

[0090] (2) Add 1 mL of RPMI-1640 complete medium to the cell pellet in (1) to prepare a cell suspension and count the cells. Evenly plate 3,000 cells / well in a 96-well plate and continue culturing for 48 h.

[0091] (3) Discard the liquid in step (2), add fresh RPMI-1640 complete medium with different concentrations of TRAIL for 48 h, then add 20 μL MTT (5 mg / mL) to each well, gently shake the 96-well plate to mix evenly, and continue culturing in the incubator for 48 h; the concentrations of TRAIL are 0 ng / mL, 200 ng / mL, 600 ng / mL, 800 ng / mL and 1000 ng / mL respectively;

[0092] (4) Take out the 96-well plate, discard the supernatant, add 150 μL DMSO to each well, protect from light, and shake at low speed on a shaker at room temperature for 10 minutes to fully dissolve the crystalline formazan (succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function). Then use the enzyme-linked immunosorbent assay to detect the OD 490 By measuring the absorbance of each well, the cell viability of NCI-H1299 cells under different TRAIL concentrations can be detected.

[0093] NC group: The method and steps were the same as those of the siRNA1 group, except that the NCI-H1299 cells transfected with siRNA1 were replaced with the NCI-H1299 cells transfected with 50 nM Negative Control in Example 2.

[0094] The results show that Figure 3), after TRAIL treatment for 48h, with the increase of TRAIL concentration, the viability of NCI-H1299 cells in different treatment groups gradually decreased. Compared with the NC group, the cell viability of the siRNA1 treatment group decreased significantly; when the concentration of TRAIL was 600ng / mL and 800ng / mL, the cell viability of the siRNA1 treatment group was significantly different from that of the NC group; when the concentration of TRAIL was 400ng / mL, the cell viability of the siRNA1 treatment group was significantly different from that of the NC group, indicating that when siRNA1 is used in combination with TRAIL, it can significantly affect the viability of NCI-H1299 cells, that is, siRNA1 can significantly increase the sensitivity of NCI-H1299 cells to TRAIL.

[0095] Example 6 Detection of cell apoptosis by flow cytometry

[0096] After TRAIL binds to receptors on the surface of cancer cells, it can induce cell apoptosis. Therefore, detecting changes in cell apoptosis rate can measure the effect of siRNA1 on cell apoptosis and measure changes in siRNA1 sensitivity to TRAIL. Annexin-V-FITC can bind to phosphatidylserine on the cell surface in the presence of calcium ions, and the externalization of phosphatidylserine is an early event in cell apoptosis. PI is a DNA dye. In the late stage of cell apoptosis, the cell membrane permeability increases, allowing PI to enter the cell nucleus and bind to DNA. Therefore, Annexin-V-FITC / PI double staining can be used to perform flow cytometry to detect cell apoptosis. The specific steps are as follows:

[0097] siRNA1, TRAIL(+): (1) NCI-H1299 cells transfected with 50 nM siRNA1 in Example 2 were taken, and 50 ng / mL TRAIL was added 48 hours after transfection and treated for 48 hours;

[0098] (2) Add 1 mL of EDTA-free trypsin digestion solution (mass fraction 0.25%) for trypsin digestion, centrifuge at 800 rpm for 3 min, and collect the suspended cells and adherent cells;

[0099] (3) The collected cells were washed twice with PBS and resuspended in 400 μL Annexin V binding solution to make the cell density in the cell suspension 1×10 6 / L;

[0100] (4) Add 5 μL of Annexin-V-FITC staining solution to the cell suspension, mix gently, and incubate in an ice box in the dark for 15 min;

[0101] (5) Add 2 μL of PI staining solution, mix gently, and incubate in an ice box away from light for 5 min. Then, use flow cytometry to detect the cell apoptosis rate.

[0102] siRNA1, TRAIL(-): the same as the siRNA1, TRAIL(+) group, except that NCI-H1299 cells were transfected with siRNA1 for 48 h without TRAIL treatment;

[0103] NC, TRAIL(+): Same as the siRNA1, TRAIL(+) group, NCI-H1299 cells were transfected with NegetiveControl and treated with 50 ng / mL TRAIL for 48 h;

[0104] NC, TRAIL(-): Same as siRNA1, TRAIL(+) group, NCI-H1299 cells were transfected with NegetiveControl but not treated with TRAIL.

[0105] Flow cytometry analysis method:

[0106] Q1: upper left quadrant; Annexin-V negative, PI positive; represents the proportion of cells with mechanical damage;

[0107] Q2: upper right quadrant; Annexin-V positive, PI positive; represents the proportion of cells in the late stage of apoptosis;

[0108] Q3: lower right quadrant; Annexin-V positive, PI negative; represents the proportion of cells in the early stage of apoptosis;

[0109] Q4: lower left quadrant; Annexin-V negative, PI negative; represents normal; proportion of living cells;

[0110] The results show that Figure 4 A), compared with the other groups, the ratio of cells in Q2 and Q3 regions in the siRNA1, TRAIL (+) group increased, especially in the Q3 region, indicating that inhibition of PYCR1 gene with siRNA1 can promote TRAIL-induced cell apoptosis.

[0111] The total apoptosis rate was obtained by adding the ratios of early and late apoptotic cells, and statistically found that: Figure 4As shown in Figure B, the apoptosis rate of NCI-H1299 cells was different under different treatment conditions. The apoptosis rate of cells in the NC, TRAIL (-) group was the lowest, which was used as the control group; the difference between the NC, TRAIL (+) group and the control group was not significant, indicating that this group of cells was resistant to TRAIL and could escape drug-induced cell apoptosis. The difference between the siRNA1, TRAIL (-) group and the siRNA1, TRAIL (+) group and the control group was significant, among which the apoptosis rate of NCI-H1299 cells in the siRNA1, TRAIL (+) group was the highest, indicating that when siRNA1 and TRAIL were used in combination to treat lung cancer cells NCI-H1299, the apoptosis of NCI-H1299 cells could be promoted, while when siRNA1 or TRAIL was used alone to treat cancer cells, the apoptosis rate of cells increased, but the effect was not ideal, thus indicating that the use of siRNA1 can enhance the sensitivity of lung cancer cells NCI-H1299 to TRAIL.

[0112] Example 7 Western blot was used to detect the expression level of apoptosis-related proteins.

[0113] Cell apoptosis is induced by the death receptor pathway and the mitochondria-dependent apoptosis pathway, so detecting the changes in the expression levels of related proteins of the death receptor pathway (extrinsic apoptosis pathway) and the mitochondrial pathway (intrinsic apoptosis pathway) can measure the effect of siRNA1 on cell apoptosis and the change in sensitivity to TRAIL. The specific experimental method is the same as that of Example 4, and the experimental cells are taken from the NCI-H1299 cells obtained from the four treatment groups in Example 6; the target proteins to be examined are: PYCR1, caspase-3, caspase-8, Bax and BCL-2, and β-actin is used as the internal reference protein in the experiment; caspase-3 and caspase-8 target proteins are divided into two forms, pro and cleaved (pro-refers to the protein precursor in the inactive state, and cleaved-is the activated form of the former).

[0114] The results are as follows Figure 5As shown in the figure, by detecting the expression of proteins related to the apoptosis signaling pathway, it was found that compared with the control group NC, TRAIL (-), the apoptosis-related proteins in the NC, TRAIL (+) group were not activated, indicating that the cells in this group were resistant to TRAIL and no apoptosis was detected. The activation of related proteins can be detected in both the siRNA, TRAIL (-) group and the siRNA1, TRAIL (+) group, especially the activated protein content in the siRNA1, TRAIL (+) group was higher, indicating that the apoptosis pathway execution protein casepase3 was significantly activated, and the exogenous apoptosis signaling pathway protein casepase8 could be activated at the same time; the upstream protein Bax of the endogenous apoptosis signaling pathway was upregulated, and Bcl-2 was downregulated. Therefore, it is shown that siRNA1 transfection and TRAIL treatment can promote the apoptosis of NCI-H1299 cells.

[0115] Example 8 Total RNA extraction and qRT-PCR analysis of DR4 / DR5 mRNA expression levels

[0116] The binding of TRAIL to the death receptors DR4 and DR5 on the cell membrane is a key step in initiating programmed cell death, so the expression of death receptors directly affects the sensitivity of TRAIL. In order to detect the effect of siRNA1 transfection on the expression of death receptors DR4 and DR5, the total RNA of the transfected cells was extracted to detect the expression levels of DR4 and DR5 mRNA. The specific method is as follows:

[0117] siRNA1+TRAIL group:

[0118] (1) Based on the human DR4 and DR5 gene sequences obtained from NCBI, qRT-PCR primers were designed on PrimerBank (https: / / pga.mgh.harvard.edu / primerbank / ), where the primers for DR4 were DR4 F and DR4R, with sequences of SEQ ID NO: 11 and SEQ ID NO: 12, and the primers for DR5 were DR5 F and DR5 R, with sequences of SEQ ID NO: 13 and SEQ ID NO: 14; see Table 5 for the series.

[0119] At the same time, human β-actin gene was selected as the internal reference gene, and the series is shown in Table 2.

[0120] Table 5 PYCR1 and GADPH primer sequences

[0121]

[0122] (2) Take NCI-H1299 cells transfected with 50 nM siRNA1 in Example 2, add 50 ng / mL TRAIL 48 hours after transfection and treat for 48 hours, collect cells and extract total RNA, and the subsequent qRT-PCR detection is the same as (2) and (3) in Example 3;

[0123] siRNA1 group: NCI-H1299 cells were transfected with siRNA1 for 48 h without TRAIL treatment;

[0124] NC group: NCI-H1299 cells were transfected with Negative Control for 48 h but not treated with TRAIL;

[0125] NC+TRAIL group: NCI-H1299 cells were transfected with Negative Control for 48 hours and then treated with TRAIL for 48 hours.

[0126] The results are as follows Figure 6 As shown in the figure, compared with the NC group, the expression of DR4 and DR5 mRNA in the NC+TRAIL group increased slightly, but not significantly. The expression of DR4 and DR5 mRNA in the siRNA1 group and siRNA1+TRAIL group increased significantly, among which the expression in the siRNA1+TRAIL group was the highest, about 1.99±0.27 times that of the NC group. This shows that siRNA1 transfection can increase the expression level of death receptors DR4 and DR5 and promote the apoptosis of lung cancer cells. At the same time, the sensitivity of death receptors DR4 and DR5 on the surface of lung cancer cell membranes after siRNA1 transfection to TRAIL is significantly enhanced.

[0127] Example 9 Western blot detection of the expression of target proteins DR4 and DR5

[0128] Using β-actin as the internal reference protein, Western Blot was used to detect the expression levels of target proteins DR4 and DR5 in the four treatment groups in Example 7. The specific steps were the same as those in Example 4.

[0129] The results show that Figure 7 ) showed that compared with the NC group, the expression levels of DR4 and DR5 proteins in the NC+TRAIL group, siRNA1 group and siRNA1+TRAIL group were significantly increased, among which the expression level of DR5 in the siRNA1+TRAIL group increased more significantly, indicating that siRNA1 transfection and TRAIL treatment can up-regulate the protein expression levels of DE4 and DR5 and promote the apoptosis of lung cancer cells. At the same time, the sensitivity of death receptors DR4 and RD5 on the membrane surface of lung cancer cells after siRNA1 transfection to TRAIL was significantly enhanced.

[0130] Example 10 Detection of the expression of target proteins DR4 and DR5 by flow cytometry

[0131] Death receptors DR4 / DR5 can be expressed in the cytoplasm and cell membrane at the same time. However, when Triton-X100 is not used to perforate the cells, incubation with membrane protein antibodies combined with immunofluorescence technology can only detect the expression of cell membrane proteins using flow cytometry. Therefore, this technology can be used to detect the expression levels of DR4 and DR5 proteins on the cell membrane. The steps for detecting the expression level of DR4 protein are as follows:

[0132] siRNA1+TRAIL group:

[0133] (1) After transfection of NCI-H1299 cells with siRNA1 at a concentration of 50 nM for 48 h, 50 ng / mL TRAIL was used to treat the cells for another 48 h;

[0134] (2) Add EDTA-free trypsin (mass fraction 0.25%) to digest the cells for 2 min, and then use RPMI-1640 complete medium to perfuse the cells into a single-cell suspension. Count the cells and adjust the cell count to 1×10 4 μL, take 100 μL of the cell suspension, centrifuge at 1000 rpm for 5 min, and wash twice with PBS;

[0135] (3) Add 50 μL of blocking solution containing 1% goat serum to the cell pellet in (2) and incubate for 40 min, then add 50 μL of anti-target protein antibody (DR4, dilution ratio 1:100) and incubate overnight;

[0136] (4) After incubation, wash the cells twice with PBS: add 1 mL of pre-cooled PBS each time, centrifuge at 1000 rpm for 5-10 min, and discard the supernatant;

[0137] (5) Add 50 μL of Cy3-labeled goat anti-rabbit secondary antibody dilution (dilution ratio 1:100) to the remaining material, mix well, incubate in the dark for 1 h, then wash the cells twice with pre-cooled PBS solution and centrifuge at 4°C for 5 min;

[0138] (6) Add 500 μL PBS to resuspend into a single cell suspension and then test on the instrument.

[0139] NC group: NCI-H1299 cells were transfected with Negative Control for 48 h and not treated with TRAIL;

[0140] siRNA1 group: NCI-H1299 cells were transfected with siRNA1 for 48 h without TRAIL treatment;

[0141] NC+TRAIL group: NCI-H1299 cells were transfected with Negative Control for 48 hours and then treated with TRAIL for 48 hours.

[0142] The steps for detecting the expression level of DR5 protein are the same as above, except that in step (3), the DR4 antibody is replaced with an anti-target protein antibody (DR5, dilution ratio 1:100).

[0143] The results are as follows Figure 8 As shown in the figure, flow cytometry showed that compared with the NC group, the expression levels of DR4 and DR5 in the siRNA1 group, NC+TRAIL group and siRNA1+TRAIL group increased, among which the increase in the siRNA1 group and siRNA1+TRAIL group was more significant, and the expression levels of DR4 and DR5 in the siRNA1+TRAIL group were the highest. Among them, the increase in the expression level of DR5 was higher than that of DR4. In summary, siRNA1 transfection can promote the expression of death receptors DR4 and DR5 on NCI-H1299 cells. At the same time, after transfection and treatment with TRAIL, the expression of death receptors increased to a certain extent, indicating that NCI-H1299 cells are sensitive to TRAIL. Transfection of lung cancer cells with siRNA1 can further increase the sensitivity of death receptors DR4 and DR5 to TRAIL.

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Claims

1. Use of a PYCR1 expression or activity inhibitor in at least one of the following: a: Preparation of drugs for promoting the sensitivity of cancer cells to TRAIL; b: Preparation of drugs for synergistic TRAIL treatment of cancer; c: Preparation of drugs for enhancing TRAIL-induced apoptosis of cancer cells; The PYCR1 expression or activity inhibitor is siRNA, and the sequences of siRNA are SEQ ID NO: 3 and SEQ ID NO: 4; The cancer is lung cancer.

2. The use according to claim 1, characterized in that: The cancer cells are lung cancer NCI-H1299 cells.

3. A method for enhancing TRAIL sensitivity by using a PYCR1 expression or activity inhibitor, characterized in that: The steps include: (1) Design and synthesize siRNA based on the human PYCR1 gene sequence; (2) transfecting siRNA into cancer cells and culturing transfected cells; (3) Collect transfected cells, add TRAIL to treat, culture and detect apoptosis and expression of cancer cells; The sequences of the siRNA in step (1) are SEQ ID NO: 3 and SEQ ID NO: 4; The cancer cells described in step (2) are lung cancer NCI-H1299 cells.

4. The method for enhancing TRAIL sensitivity by using a PYCR1 expression or activity inhibitor according to claim 3, characterized in that: The cancer cells described in step (2) are cultured to the logarithmic growth phase before transfection, and the cell density during transfection is 60%-70%.

5. The method for enhancing TRAIL sensitivity by using a PYCR1 expression or activity inhibitor according to claim 3, characterized in that: The siRNA concentration in step (2) is 50 nM and the transfection time is 48 h.

6. The method for enhancing TRAIL sensitivity by using a PYCR1 expression or activity inhibitor according to claim 3, characterized in that: The treatment concentration of TRAIL in step (3) is 50 ng / mL, and the TRAIL treatment time is 48 h.

7. The method for enhancing TRAIL sensitivity by using a PYCR1 expression or activity inhibitor according to claim 3, characterized in that: The culture conditions described in steps (2) and (3) are 5% CO2, 37°C humidified incubator.

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