PGK1 targeted siRNA interference library and its application

Abstract

The present invention discloses a PGK1 targeted siRNA interference library and its application, and belongs to the field of bioengineering technology. Based on the characteristics of abnormal energy metabolism and treatment difficulties of bladder cancer cells, the present invention discloses an application of a PGK1 targeted siRNA interference library in bladder cancer through an anti-bladder cancer gene therapy approach, and provides three groups of siRNA nucleic acid sequences of the interference library. Cytological experiments show that this siRNA interference library has important functions such as inhibiting the protein expression of PGK1 in bladder cancer cells and inhibiting the survival and proliferation of cancer cells. The present invention provides a new drug for the treatment of bladder cancer, and has very important application value for the clinical treatment of bladder cancer and the development of targeted drugs.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and more specifically, relates to a PGK1-targeted siRNA interference library and application thereof. Background Art

[0002] Bladder cancer is the second most common malignancy of the genitourinary system, and nearly 90% of primary bladder cancers arise from the urothelium. 70-80% of urothelial tumors are superficial (Ta, T1), while the rest are muscle-invasive (T2-4) or metastatic.

[0003] During the progression of bladder cancer, the expression of certain genes changes, which are key genes in the occurrence and development of bladder cancer and become potential targets for bladder cancer treatment. Many studies have shown that phosphoglycerate kinase PGK1 is a key enzyme in the glycolysis process, which can catalyze the production of ATP to produce a higher glycolysis rate, leading to abnormal energy metabolism of tumor cells and ultimately promoting tumor growth. These research findings suggest that it has good application prospects as a drug treatment target.

[0004] Abnormal glycolysis metabolism is closely related to tumor metastasis. During the metastasis process, tumors need to transform the internal microenvironment of tissues to promote tumor cells to break through tissue barriers and enter the lymphatic or blood circulation, thereby metastasizing to distant tissues. Abnormal glycolysis metabolism can directly or indirectly provide numerous metabolites to promote tumor metastasis, such as lactate and glutamine. In addition, intermediates produced by abnormal glycolysis, such as acetyl-CoA, can affect the epigenetics of cells and promote epithelial-mesenchymal transition of cells, so that cells change from highly adhesive epithelial morphology to easily metastatic mesenchymal morphology. During the epithelial-mesenchymal transition process, epithelial markers such as E-cadherin on the cell surface are expressed less, while mesenchymal markers such as N-cadherin, β-catenin, Vimentin and related regulatory factors such as Snail, Twist and p-AKT are upregulated. These processes induce morphological changes in cells, thereby promoting tumorigenesis. Therefore, inhibiting the epithelial-mesenchymal transition of tumor cells by intervening in abnormal glycolysis metabolism is an effective strategy for tumor treatment. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a siRNA interference library, which inhibits the proliferation of bladder cancer cells by siRNA interference. Another object of the present invention is to provide an application of the siRNA interference library.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] The PGK1-targeted siRNA interference library consists of three groups of siRNAs, and the specific sequences of the three groups of siRNAs are as follows:

[0008] (1)5′-AGGAAGAAGGGAAGGGAAATT-3′,

[0009] 5′-UUUCCCUUCCCUUCUUCCUTT-3′:

[0010] (2)5′-ACAAACAACCAGAGGAUUATT-3′,

[0011] 5′-UAAUCCUCUGGUUGUUUGUTT-3′;

[0012] (3)5′-ACAGAAGGCUGGUGGGUUUTT-3′,

[0013] 5′-AAACCCACCAGCCUUCUGUTT-3′.

[0014] The application of the PGK1 targeted siRNA interference library in the preparation of anti-bladder cancer drugs.

[0015] Furthermore, in the application, the anti-bladder cancer drug includes a drug that inhibits the proliferation of bladder cancer cells.

[0016] Furthermore, in the application, the anti-bladder cancer drug includes a drug that inhibits glucose uptake by bladder cancer cells.

[0017] Furthermore, in the application, the anti-bladder cancer drug includes a drug that inhibits the activity of lactate dehydrogenase in bladder cancer cells.

[0018] Furthermore, in the application, the anti-bladder cancer drug includes a drug that inhibits the epithelial-mesenchymal transition of bladder cancer cells.

[0019] An anti-bladder cancer drug contains a PGK1-targeted siRNA interference library, wherein the PGK1-targeted siRNA interference library consists of three groups of siRNAs, and the specific sequences are as follows:

[0020] (1)5′-AGGAAGAAGGGAAGGGAAATT-3′,

[0021] 5′-UUUCCCUUCCCUUCUUCCUTT-3′;

[0022] (2)5′-ACAAACAACCAGAGGAUUATT-3′,

[0023] 5′-UAAUCCUCUGGUUGUUUGUTT-3′;

[0024] (3)5′-ACAGAAGGCUGGUGGGUUUTT-3′,

[0025] 5′-AAACCCACCAGCCUUCUGUTT-3′.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention discloses a PGK1-targeted siRNA interference library and its application, and belongs to the field of bioengineering technology. Based on the characteristics of abnormal energy metabolism and treatment difficulties of bladder cancer cells, the present invention discloses the application of a PGK1-targeted siRNA interference library in anti-bladder cancer through an anti-bladder cancer gene therapy approach, and provides three groups of siRNA nucleic acid sequences of the interference library. This siRNA interference library overcomes the problems of off-target effects and unstable interference efficiency of a single PGK1-targeted siRNA, and can achieve more effective and stable interference inhibition of PGK1 in bladder cancer cells. Cytological experiments show that this siRNA interference library has the functions of inhibiting the protein expression of PGK1 in bladder cancer cells, inhibiting cancer cell survival and cancer cell proliferation, and antagonizing the epithelial-mesenchymal transition of cancer cells. The present invention provides a new drug for the treatment of bladder cancer, and has very important application value for the clinical treatment of tumors and the development of targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the result of PGK1 expression in bladder cancer cells after transfection with fragments from the small interference library;

[0029] Figure 2 This is a growth comparison chart of bladder cancer cells transfected with the small interference library and the control group;

[0030] Figure 3 Figure 1 is a graph of the number of cells at different division stages of bladder cancer cells transfected with the small interference library and the control group, wherein Figure A is a flow cytometric graph of bladder cancer cells transfected with the control group, Figure B is a flow cytometric graph of bladder cancer cells transfected with the small interference library, and Figure C is a statistical comparison of the number of cells at different division stages between Figure A and Figure B;

[0031] Figure 4 This is a comparison of glucose content between bladder cancer cells transfected with the small interference library and the control group;

[0032] Figure 5 This is a comparison of lactate concentrations between bladder cancer cells transfected with the small interference library and the control group;

[0033] Figure 6This is a comparison chart of the expression of epithelial-mesenchymal transition markers in bladder cancer cells transfected with the small interference library and the control group. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with specific embodiments.

[0035] Embodiment 1:

[0036] first step:

[0037] The specific sequences of the three siRNAs in the PGK1-targeted siRNA interference library are as follows:

[0038] si-PGK1:

[0039] (1)5′-AGGAAGAAGGGAAGGGAAATT-3′,

[0040] 5′-UUUCCCUUCCCUUCUUCCUTT-3′;

[0041] (2)5′-ACAAACAACCAGAGGAUUATT-3′,

[0042] 5′-UAAUCCUCUGGUUGUUUGUTT-3′;

[0043] (3)5′-ACAGAAGGCUGGUGGGUUUTT-3′,

[0044] 5′-AAACCCACCAGCCUUCUGUTT-3′;

[0045] Control group siRNA sequence (negative control sequence):

[0046] 5′-UUCUCCGAACGUGUCACGUTT-3′,

[0047] 5′-ACGUGACACGUUCGGAGAATT-3′.

[0048] The fragments in the above-mentioned small interference library and the control group siRNA sequences were transfected into the bladder cancer cell line T24, and the medium was changed 12 hours after transfection. On the third day after transfection, the cell samples were collected and the expression of PGK1 was analyzed by immunoblotting. Figure 1 As shown, the PGK1 protein expression level of cells transfected with negative control siRNA sequence in the control group Con-si was significantly higher than that of cells transfected with small interfering library siRNA.

[0049] Step 2: PGK1-targeted siRNA interference library inhibits bladder cancer survival and proliferation

[0050] 1. CCK8 experiments demonstrate the anti-tumor survival effect of PGK1-targeted siRNA interference library

[0051] Bladder cancer cells T24 and UMUC3 were seeded in 96-well culture plates at a density of 2000 cells / well, respectively. The fragments of the PGK1-targeted siRNA interference library and the control group siRNA sequences were transiently transfected into T24 and UMUC3 cells using a transfection reagent. 10μl of CCK-8 solution was added to each well after 0h, 24h, 48h, 72h, and 96h, respectively. After 2 hours, a wavelength of 450nm was selected, and the light absorption value of each well at different time points was measured on an enzyme-linked immunosorbent assay, and the results were recorded. The size of the absorption value reflects the activity of the cells. According to the experimental data, a graph was drawn with time as the horizontal axis and the absorption value as the vertical axis. The CCK-8 results showed that compared with the control group, the small interference library experimental group significantly inhibited the proliferation of T24 and UMUC3 cells, as shown in Figure 2. Figure 2 shown.

[0052] 2. PI staining flow cytometry cycle experiment proves the anti-tumor proliferation effect of PGK1-targeted siRNA interference library

[0053] Bladder cancer cells T24 were seeded in a 6-well culture plate at a density of 20,000 cells / well. The PGK1 small interfering fragment and other control groups were transiently transfected into T24 and UMUC3 cells using a transfection reagent, and the cells were collected after 3 days. The cells were resuspended and washed 3 times with pre-cooled PBS, centrifuged, and the PBS was discarded. The cells were resuspended with pre-cooled 70% ethanol and fixed at -20°C for at least 24 hours. Before detection, the cells were collected by centrifugation, the ethanol was removed, and the cells were washed 3 times with cell PBS, and permeabilized with 200 μL of PBS containing 1% Triton X-100. Subsequently, 300 μL of RNaseA was added and treated at 4°C in the dark. Subsequently, 200 μL of PI dye was added and treated at 4°C in the dark for 20 minutes. Finally, the PI dye was removed by washing with PBS and detected by flow cytometry. The results are shown in Figure 3 As shown: Compared with the control group, T24 cells in the small interference library experimental group were mainly blocked in the G2 phase, indicating that the si-PGK1 interference library can significantly inhibit the cell cycle progression of T24.

[0054] Step 3: PGK1-targeted siRNA interference library inhibits bladder cancer metabolic function

[0055] 1. Glucose concentration detection experiment

[0056] 1) The bladder cancer cell line T24 was cultured and divided into a control siRNA group (Con-si) and a PGK1 siRNA interference library group (si-PGK1). After 3 days, the cells were collected into a centrifuge tube and the supernatant was discarded after centrifugation.

[0057] 2) Collect cells by centrifugation and count them every 10 6 The required lysis solution volume is 0.1 mL, and the mixture is allowed to stand at room temperature for 10 minutes after shaking and lysis.

[0058] 3) The lysates of glucose standard, Con-si group and si-PGK1 group were added to a 96-well plate, and 3 replicates were made for each group. The working solution in the tissue cell glucose oxidase assay kit (Aibixin, abs47047405) was added according to the proportions shown in the following table (8 mL of reagent R1 was mixed with 2 mL of reagent R2 to obtain 10 mL of working solution, which was used on the same day).

[0059]

[0060] 4) The above 96-well plate was reacted at 37°C for 20 minutes.

[0061] 5) Detect the OD value of each well with an ELISA reader at a wavelength of 490nm

[0062] 6) Glucose concentration (mmol / L) = standard concentration * (sample tube OD - blank tube OD) / (standard tube OD - blank tube OD).

[0063] The results are as follows Figure 4 As shown, infection of bladder cancer cell line T24 with siRNA interference library of PGK1 can significantly inhibit the uptake of glucose by cancer cells.

[0064] 2. Lactate dehydrogenase activity detection experiment

[0065] 1) The bladder cancer cell line T24 was cultured and divided into a control siRNA group (Con-si) and a PGK1 siRNA interference library group (si-PGK1). Each group was repeated 3 times or more, and the cell culture supernatant was collected after 3 days;

[0066] 2) Use the recommended ratios in the lactic acid test kit (Nanjing Jiancheng, A019-2-1) to add the corresponding reagents, as shown in the following table:

[0067]

[0068]

[0069] 3) Using standard tubes with different lactic acid concentrations (1mmol / L, 2mmol / L, 3mmol / L, 4mmol / L, 5mmol / L, 6mmol / L) and the measured OD values, draw a lactic acid standard curve and calculate the standard curve equation.

[0070] 4) Use the standard curve to calculate the lactate concentration of the test samples in the Con-si group and the si-PGK1 group.

[0071] The results are as follows Figure 5 As shown, infection of bladder cancer cell line T24 with siRNA interference library of PGK1 can significantly inhibit the activity of lactate dehydrogenase.

[0072] Step 4: PGK1-targeted siRNA interference library inhibits the expression of cell epithelial-mesenchymal transition markers

[0073] 1) The bladder cancer cell line T24 was cultured and transfected with a control siRNA group (Con-si) and a PGK1 siRNA interference library group (si-PGK1), and the cells were collected into a centrifuge tube 3 days after transfection, and the supernatant was discarded after centrifugation;

[0074] 2) The cells were lysed with an appropriate amount of RIPA cell lysis buffer (50 mM Tris-Cl pH 8.0, 150 mM NaCl, 0.5% Sodium Deoxycholate, 0.1% SDS, 1* protease inhibitor), placed on ice for 30 minutes, and then centrifuged at 13000g, 4°C for 15 minutes to collect the supernatant lysate;

[0075] 3) The supernatant lysate was used to quantify the protein concentration in the Con-si and si-PGK1 groups using the Bio-Rad BCA protein quantification method. Methods: Solution A and solution B in the BCA kit were mixed at a ratio of 50:1, added to a 96-well plate at 200 μL / well, and then 10 μL of 2 mg / mL protein standard or 2.5 μL of the supernatant of the Con-si and si-PGK1 groups were added, incubated at 37°C for 30 minutes, and the absorbance at 595 nm in each group was detected in an ELISA instrument. The protein concentration in each group was calculated using linear regression, and the protein sample was diluted to 2 μg / μL using SDS loading buffer and boiled in boiling water for 5 minutes;

[0076] 4) Load the above sample onto 10% SDS-PAGE gel electrophoresis, separate the above protein sample at 100V, and after separation, transfer the sample onto a PVDF membrane using a Bio-Rad protein transfer system;

[0077] 5) The transferred PVDF membrane was blocked with TBST solution (20 mM Tris-C1 pH 7.6, 150 mM NaCl, 0.05% Tween-20) containing 5% skim milk for 1 minute, and then the membrane was incubated with antibodies of various epithelial-mesenchymal markers overnight;

[0078] 6) After the incubation, the PVDF membrane was washed with TBST solution for 3 times, 5 minutes each time, and then the PVDF membrane was incubated with HRP-labeled secondary antibody. After the incubation, the PVDF membrane was washed with TBST solution for 3 times, 5 minutes each time;

[0079] 7) The PVDF membrane is incubated with an enhanced chemiluminescent substrate, and the band signals of each epithelial-mesenchymal marker are developed with film for analysis.

[0080] The results are as follows Figure 6 As shown, compared with the control group, the expression of epithelial marker E-cadherin on the surface of bladder cancer cell line T24 cells infected with PGK1 siRNA interference library was upregulated, while the mesenchymal markers N-cadherin, Vimentin and related regulatory factors such as Twist and p-AKT were downregulated, indicating that the interference library can significantly antagonize the epithelial-mesenchymal transition function of cancer cells. Sequence Listing <110> Nantong University <120> PGK1 targeted siRNA interference library and its application <130> 100 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> RNA <213> si-PGK1-1-F(Artificial) <400> 1 aggaagaagg gaagggaaa 19 <210> 2 <211> 19 <212> RNA <213> si-PGK1-1-R(Artificial) <400> 2 uuucccuucc cuucuuccu 19 <210> 3 <211> 19 <212> RNA <213> si-PGK1-2-F(Artificial) <400> 3 acaaacaacc agaggauua 19 <210> 4 <211> 19 <212> RNA <213> si-PGK1-2-R(Artificial) <400> 4 uaauccucug guuguuugu 19 <210> 5 <211> 19 <212> RNA <213> si-PGK1-3-F(Artificial) <400> 5 acagaaggcu gguggguuu 19 <210> 6 <211> 19 <212> RNA <213> si-PGK1-3-R(Artificial) <400> 6 aaacccacca gccuucugu 19

Claims

1. Application of PGK1-targeted siRNA interference library in the preparation of anti-bladder cancer drugs; the PGK1-targeted siRNA interference library consists of three groups of siRNAs, and the specific sequences of the three groups of siRNAs are as follows: (1)5′-AGGAAGAAGGGAAGGGAAATT-3′, 5′-UUUCCCUUCCCUUCUUCCUTT-3′; (2)5′-ACAAACAACCAGAGGAUUATT-3′, 5′-UAAUCCUCUGGUUGUUUGUTT-3′; (3)5′-ACAGAAGGCUGGUGGGUUUTT-3′, 5′-AAACCCACCAGCCUUCUGUTT-3′; The anti-bladder cancer drugs include drugs that inhibit the proliferation of bladder cancer cells, drugs that inhibit the glucose uptake of bladder cancer cells, drugs that inhibit the activity of lactate dehydrogenase in bladder cancer cells, and drugs that inhibit the epithelial-mesenchymal transition phenomenon of bladder cancer cells.

2. An anti-bladder cancer drug, characterized in that: Contains a PGK1-targeted siRNA interference library, which consists of three groups of siRNAs, and the specific sequences are as follows: (1)5′-AGGAAGAAGGGAAGGGAAATT-3′, 5′-UUUCCCUUCCCUUCUUCCUTT-3′; (2)5′-ACAAACAACCAGAGGAUUATT-3′, 5′-UAAUCCUCUGGUUGUUUGUTT-3′: (3)5′-ACAGAAGGCUGGUGGGUUUTT-3′, 5′-AAACCCACCAGCCUUCUGUTT-3′.

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