ShRNA interference sequence of human iASPP gene as well as construction and application of shRNA interference sequence

Abstract

The invention discloses a shRNA interference sequence of a human iASPP gene and construction and application of the shRNA interference sequence, an effective siRNA target sequence targeting the human iASPP gene is 5 '-AACTCTATCGTGGATT-3', and proliferation of tumor cells is inhibited and apoptosis of the tumor cells is promoted by constructing shRNA which aims at lentivirus mediated expression of the human iASPP gene and efficiently inhibiting expression of the iASPP gene in bladder cancer cells. And a wide prospect is provided for the development of bladder cancer treatment medicines based on the human iASPP gene in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of biological medicines, and in particular to a shRNA interference sequence of a human iASPP gene and a construction and application thereof. Background Art

[0002] Bladder cancer is a common malignant tumor of the urinary system. According to global data in 2018, the incidence of bladder cancer ranked 10th among malignant tumors and 6th among male malignant tumors. In 2014, the incidence of bladder cancer in my country ranked 13th among malignant tumors and 7th among male malignant tumors. Bladder cancer is highly invasive, has a high recurrence rate, is difficult to treat, and treatment research has progressed slowly, with no major breakthroughs.

[0003] Recent studies have shown that some short double-stranded RNA fragments can effectively and specifically block the expression of specific genes in the body and induce cells to show a phenotype of specific gene loss by promoting the degradation of specific gene mRNA, which is called RNA interference. siRNA is such a short double-stranded RNA molecule, which can target mRNA with homologous and complementary sequences and degrade the mRNA of specific genes. The discovery of RNAi is of epoch-making significance. It not only deeply reveals the mechanism of gene silencing in cells, but also is a powerful tool for gene function analysis in the post-genomic era, which greatly promotes the process of human revealing the mysteries of life. Now more and more researchers are beginning to use RNAi to study gene expression in organisms. RNAi technology can be widely used in functional genomics, drug target screening, cell signal transduction pathway analysis and disease gene therapy, etc.

[0004] iASPP gene belongs to the apoptosis stimulating protein of P53 (ASPP) family. Currently, three members of this gene family have been found: ASPP1, ASPP2 and iASPP (inhibitory member of the ASPP family). The names of the three members are derived from their common structures, that is, they all have a large number of ankyrin repeats, SH3 domains and proline-rich domains. After binding to P53 protein, ASPP1 and ASPP2 proteins can activate the apoptosis-promoting function of P53, while iASPP inhibits the tumor suppressor function of P53 after binding to P53. iASPP is abnormally expressed in most tumor tissues and cells, and may play a very important role in the occurrence and development of tumors. iASPP protein is highly expressed in bladder cancer tissues, and the use of RNAi technology provides clues and basis for the development and application of targeted gene molecular therapeutic drugs for bladder cancer. Summary of the invention

[0005] In view of the above problems, the present invention aims to provide a shRNA interference sequence of human iASPP gene and its construction and application.

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

[0007] A shRNA interference sequence for human iASPP gene comprises two inverted repeat sequences targeting human iASPP gene coding DNA, wherein the two inverted repeat sequences are respectively the shRNA target sequence of human iASPP gene and its complementary sequence, separated by a stem-loop sequence in the middle, and the effective siRNA target sequence targeting human iASPP gene is shown in SED ID NO.3.

[0008] Preferably, the shRNA sequence of the human iASPP gene includes a DNA oligo forward strand sequence and a reverse strand sequence;

[0009] Among them: the positive chain sequence is

[0010] 5′-CCGGCC AACTACTCTATCGTGGATT CTCGAG AATCCACGATAGAGTAGTT GGTTTTTG-3′;

[0011] The antisense strand sequence is

[0012] 5′-AATTCAAAAACC AACTACTCTATCGTGGATT CTCGAG AATCCACGATAGAGTAGTT GG-3′.

[0013] A method for constructing a shRNA sequence of a human iASPP gene, the method comprising the steps of

[0014] S101. Design RNAi targets for knocking down expression of human iASPP gene. The obtained effective siRNA target sequence targeting human iASPP gene is: 5′-AACTACTCTATCGTGGATT-3′;

[0015] S102. Using siRNA target sites to synthesize the sense and antisense single strands of single stranded DNA oligo shRNA sequences of human iASPP gene;

[0016] S103. Dissolve the single-stranded DNA oligo interference sequence dry powder of human iASPP gene in annealing buffer to prepare a concentration of 20 μM, incubate in a 90°C water bath for 15 min, and cool naturally to room temperature to obtain double-stranded DNA oligo.

[0017] An application of a shRNA interference sequence of a human iASPP gene in constructing a lentivirus, the specific process of which includes the steps of

[0018] S201. The vector is linearized by enzyme digestion, and the linearized vector GV115 is connected to the double-stranded DNA of the shRNA interference sequence of the human iASPP gene;

[0019] S202. Transform the ligation product into competent E. coli cells, and perform PCR identification on the obtained positive clones;

[0020] S203. Plasmid extraction and virus packaging;

[0021] S204. Using the obtained lentivirus, detect the silencing efficiency of the iASPP gene.

[0022] Preferably, the specific process of connecting the linearized vector GV115 with the double-stranded DNA of the shRNA interference sequence of the human iASPP gene in step S201 includes:

[0023] S2011. Linearized vector preparation

[0024] (1) Prepare 50 μl of restriction enzyme digestion reaction system by adding 41 μl of ddH2O, 5 μl of 10× CutSmart Buffer, 2 μl of purified plasmid DNA, 1 μl of EcoR enzyme, and 1 μl of AgeⅠ enzyme in this order, then gently pipette to mix, centrifuge briefly, and incubate at 37°C for 3 h or overnight to obtain the vector restriction enzyme digestion product;

[0025] (2) Perform agarose gel electrophoresis on the vector digestion product, recover the target band, linearize it, react it at 37°C for 1 h, and then cut the gel to recover the target fragment;

[0026] S2012.Connect

[0027] A 20 μl ligation reaction system was prepared by adding 1 μl of linearized vector DNA 100 ng / ul, 1 μl of DNA oligo 100 ng / ul, 2 μl of 10×T4 phage DNA ligase buffer, 1 μl of T4 phage DNA ligase and 15 μl of ddH2O enzyme in this order, then gently pipette to mix, centrifuge briefly, and place at 16°C for ligation overnight to obtain a ligation product.

[0028] Preferably, the process of obtaining the competent E. coli cells and PCR identification in step S202 includes:

[0029] S2021. Prepare fresh E. coli DH5α competent cells using calcium chloride;

[0030] S2022. Transform E. coli competent cells using the ligation product obtained in step S201;

[0031] S2023. Perform PCR identification on the transformed competent cells for positive clones.

[0032] Preferably, the specific process of performing PCR identification of positive clones on the transformed competent cells in step S2023 includes:

[0033] (1) Construction of RNA interference vector and identification of positive clones

[0034] Take the transformed E. coli competent cells and resuspend them in 10 μl LB solution, mix well and take 1 μl as template; use GV115 universal primers to perform colony PCR identification;

[0035] (2) Prepare 20 μl PCR reaction system

[0036] Comprising: 2 μl 10× buffer, 0.8 μl dNTPs (2.5 mM), 0.4 μl Primer (+), 0.4 μl Primer (-), 0.2 μl Taq polymerase, 1 μl DNA template, 15.2 μl ddH2O; wherein the dNTPs are a mixture of dATP, dCTP, dGTP, and dTTP;

[0037] (3) Then perform PCR amplification

[0038] The reaction conditions of PCR amplification were as follows: first, melt at 94°C for 3 min; then, melt at 94°C for 30 s, anneal at 60°C for 30 s, extend at 72°C for 30 s, and perform 30 cycles of melt-anneal-extend; and finally, extend at 72°C for 6 min.

[0039] (4) After the PCR is completed, 5 μl of the product is taken, and the bands are detected by 1% agarose gel electrophoresis. The iASPP-RNAi (8954-1) detected as positive after amplification is sequenced. The identified positive clone transformants are inoculated into an appropriate amount of LB liquid culture medium containing corresponding antibiotics, cultured at 37°C for 12-16 hours, and an appropriate amount of bacterial liquid is taken for sequencing;

[0040] (5) Sequencing of positive clones was performed using identification primer-F.

[0041] Preferably, the iASPP-RNAi sequencing result obtained by sequencing in step (4) is as shown in SED ID NO.1-2 in the sequence listing.

[0042] Preferably, the human iASPP gene in the lentivirus can be used as a target for bladder cancer cells and applied to the preparation of a drug for treating bladder cancer, wherein the target for bladder cancer cells is an RNA interference target.

[0043] The beneficial effects of the present invention are as follows: the present invention discloses a shRNA interference sequence of human iASPP gene and its construction and application. Compared with the prior art, the improvements of the present invention are:

[0044] The present invention synthesizes a shRNA interference sequence of a human iASPP gene, wherein the effective siRNA target sequence targeting the human iASPP gene is: 5′-AACTACTCTATCGTGGATT-3′, and by constructing a shRNA mediated by a lentivirus for the human iASPP gene and efficiently inhibiting the expression of the iASPP gene in bladder cancer cells, the proliferation of tumor cells is inhibited and the apoptosis of tumor cells is promoted, thereby providing broad prospects for the development of bladder cancer therapeutic drugs based on the human iASPP gene in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a flow chart of the construction process of the shRNA interference sequence of the human iASPP gene in Example 1 of the present invention.

[0046] Figure 2 The sequencing results of the shRNA mediated by lentivirus against the human iASPP gene were constructed in Example 1 of the present invention.

[0047] Figure 3 This is a schematic diagram of the mRNA expression level of iASPP in different bladder cancer cell lines according to Example 2 of the present invention.

[0048] Figure 4 This is a schematic diagram of the protein expression level results of iASPP in different bladder cancer cell lines according to Example 3 of the present invention.

[0049] Figure 5 This is a diagram of the efficiency of iASPP RNAi in bladder cancer cell lines verified by real-time iASPP qPCR in Example 4 of the present invention.

[0050] Figure 6 This is a graph showing the efficiency of iASPP RNAi in bladder cancer cell lines verified by immunoblotting in Example 4 of the present invention.

[0051] Figure 7 This is a schematic diagram of the proliferation ability of each group of human bladder cancer cell lines in Example 4 of the present invention.

[0052] Figure 8 This is the experimental result of bladder cancer cell apoptosis detection in Example 5 of the present invention.

[0053] Fig. 9 This is a cell cycle bar graph of the bladder cancer cell line in Example 6 of the present invention.

[0054] Fig.10This is a schematic diagram of the clone formation of bladder cancer cell lines in Example 7 of the present invention.

[0055] Among them: Figure 2 Middle: The rectangle is the designed shRNA sequence targeting human iASPP gene;

[0056] exist Figure 5 Middle: ** represents P < 0.01, t-test; Figure 6 In the figure, (A) is the result of western blotting to detect the effect of RNAi on the expression of iASPP protein in bladder cancer cells, and (B) is a schematic diagram of the relative quantification of RNAi on the expression of iASPP protein in bladder cancer cells; Figure 7 Middle: (A) is a schematic diagram of the proliferation ability of bladder cancer cell 5637; (B) is a schematic diagram of the proliferation ability of bladder cancer cell T24; (C) is a schematic diagram of the proliferation ability of bladder cancer cell UM-UC-3; Figure 8 Middle: ** represents P < 0.01, t-test; Fig. 9 Middle: (A) is a schematic diagram of the cell cycle of bladder cancer cell 5637; (B) is a schematic diagram of the cell cycle of bladder cancer cell T24; (C) is a schematic diagram of the cell cycle of bladder cancer cell UM-UC-3; Fig.10 In the figure, (A) shows the results of cell clone formation experiment of bladder cancer cells before and after RNAi inhibition of iASPP expression, and (B) shows the schematic diagram of bladder cancer cell clone number. DETAILED DESCRIPTION

[0057] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0058] Embodiment 1: refer to the attached Figure 1-3 The method for constructing a shRNA interference sequence of a human iASPP gene as shown comprises the steps of:

[0059] S1. Design of RNA interference target for human iASPP gene and preparation of double-stranded DNA oligo

[0060] S101. Design and synthesis of effective siRNA targets against human iASPP gene:

[0061] (1) The gene information of human iASPP gene (NM_001142502) was retrieved from Genbank, and RNAi targets were designed to effectively knock down the expression of human iASPP gene;

[0062] (2) The effective siRNA target sequence designed to target the human iASPP gene is:

[0063] 5′-AACTACTCTATCGTGGATT-3′ (as shown in SED ID NO. 3);

[0064] S102. Preparation of single-stranded DNA oligo:

[0065] The human iASPP interference sequence was designed according to the selected target sequence, and appropriate restriction endonuclease sites were added at both ends. The single-stranded DNA oligo was synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.;

[0066] S103. Preparation of double-stranded DNA oligo:

[0067] (1) Dissolve the synthesized single-stranded DNA oligo powder in annealing buffer to prepare a concentration of 20 μM; and heat in a 90°C water bath for 15 min; (2) cool naturally to room temperature to obtain a double-stranded DNA oligo; wherein the double-stranded DNA oligo

[0068] The sense strand sequence is:

[0069] 5′-CCGGCC AACTACTCTATCGTGGATT CTCGAG AATCCACGATAGAGTAGTT GGTTTTTG-3′;

[0070] The antisense strand sequence is:

[0071] 5′-AATTCAAAAACC AACTACTCTATCGTGGATT CTCGAG AATCCACGATAGAGTAGTT GG-3′;

[0072] S2. Using the shRNA interference sequence of the human iASPP gene obtained in step S1 to construct a lentivirus, the process includes the following steps:

[0073] S201. The vector is linearized by enzyme digestion, and the linearized vector is connected to the double-stranded DNA of the shRNA interference sequence of the human iASPP gene, and the specific process is as follows;

[0074] S2011. Linearized vector preparation

[0075] (1) Prepare 50 μl of enzyme digestion reaction system by adding 41 μl of ddH2O, 5 μl of 10× CutSmart Buffer, 2 μl of purified plasmid DNA (1 μg / μL), 1 μl of EcoRⅠ (10 U / μl) enzyme, and 1 μl of AgeⅠ (10 U / μl) enzyme in this order, then gently pipette to mix, centrifuge briefly, and incubate at 37°C for 3 h or overnight to obtain the vector digestion product;

[0076] (2) Perform agarose gel electrophoresis on the vector digestion product, recover the target band, linearize it, react it at 37°C for 1 h, and then cut the gel to recover the target fragment;

[0077] S2012.Connect

[0078] The double-digested linearized vector GV115 and the oligo sequence fragment were ligated in an appropriate buffer by T4 DNA ligase, and the ligated product was subjected to transformation experiment. The ligation reaction system is shown in Table 1:

[0079] Table 1: Components and dosage of general system for ligation reaction

[0080] Reagents Volume (μl) Linearized vector DNA 100ng / μl 1 DNA oligo 100ng / μl 1 10×T4 phage DNA ligase buffer 2 T4 bacteriophage DNA ligase 1 <![CDATA[dd H2O]]> Top up to 20

[0081] The ligation reaction system was ligated overnight at 16° C., thereby ligating the linearized vector GV115 and the double-stranded DNA of the shRNA interference sequence of the human iASPP gene to obtain a ligation product.

[0082] S202. The ligation product is transformed into E. coli competent cells, and the obtained positive clones are subjected to the following specific process:

[0083] S2021. Prepare fresh E. coli competent cells using calcium chloride:

[0084] (1) Pick a single colony from the newly activated E. coli DHSα bacterial plate, inoculate it into 3-5 ml LB liquid culture, and culture it at 37°C with shaking for about 12 h until the logarithmic growth phase; transfer the bacterial suspension to 100 ml LB liquid culture medium at a ratio of 1:100-1:50, and culture it at 37°C with shaking. When the culture solution begins to become turbid, measure the OD600nm every 20-30 min, and stop the culture when the OD600nm is ≤0.5;

[0085] (2) Take three 2 ml aliquots of culture medium from each group and transfer them into 2 ml centrifuge tubes, cool them on ice for 20-30 min, and centrifuge them at 4°C, 4000 rpm for 10 min (from this step onwards, all operations are performed on ice at the fastest and most stable speed possible);

[0086] (3) Pour off the supernatant culture medium, gently suspend the cells with 1 ml of ice-cold 0.1 mol / L CaCl2 solution, and place on ice;

[0087] (4) Centrifugation at 0-4°C, 4000 rpm for 10 min;

[0088] (5) Discard the supernatant, add 500 μl of ice-cold 0.1 mol / L CaCl2 solution, and carefully suspend the cells; centrifuge at 0-4°C, 4000 rpm for 10 min;

[0089] (6) Discard the supernatant, add 100 μl of ice-cold 0.1 mol / L CaCl2 solution, carefully suspend the cells, and place on ice for a while to prepare the competent cell suspension;

[0090] S2022. Transform E. coli competent cells using the ligation product obtained in step S201;

[0091] Add 10 μl of the ligation product to 100 μl of E. coli competent cells and place on ice for 30 min; heat shock at 42°C for 90 sec and place on ice for 2 min; add 500 μl of LB liquid culture medium without antibiotics and shake and culture at 37°C for 1 hr at 200 rpm; take 150 μl of the bacterial solution and spread it evenly on the LB solid culture medium containing Amp and culture it in a 37°C incubator overnight;

[0092] S2023. Perform PCR identification on the transformed competent cells for positive clones. The specific process is as follows:

[0093] (1) Take the transformant (transformed E. coli competent cells) and resuspend it in 10 μl LB solution, mix well and take 1 μl as a template; use GV115 universal primers to perform colony PCR identification experiments. The primer sequences are shown in Table 2:

[0094] Table 2: GV115 universal primer sequences

[0095]

[0096] (2) Prepare 20 μl PCR reaction system, including: 2 μl 10× buffer, 0.8 μl dNTPs (2.5 mM), 0.4 μl Primer (+), 0.4 μl Primer (-), 0.2 μl Taq polymerase, 1 μl DNA template, 15.2 μl ddH2O;

[0097] Wherein: dNTPs is a mixture of dATP, dCTP, dGTP, and dTTP;

[0098] (3) Then, a single colony was picked up with a sterile pipette tip as a template for PCR amplification. The reaction conditions for PCR amplification were as follows: first, 94°C for melting for 3 min; then, 94°C for melting for 30 s, 60°C for annealing for 30 s, and 72°C for extension for 30 s. The melting-annealing-extension cycle was repeated for 30 times; and finally, 72°C for extension for 6 min.

[0099] (4) After the PCR is completed, 5 μl of the product is taken, and the bands are detected by 1% agarose gel electrophoresis. The iASPP-RNAi (8954-1) detected as positive after amplification is sequenced. The identified positive clone transformants are inoculated into an appropriate amount of LB liquid culture medium containing corresponding antibiotics, cultured at 37°C for 12-16 hours, and an appropriate amount of bacterial liquid is taken for sequencing;

[0100] (5) Using the primer (+) in Table 2 as the identification primer-F, the positive clones were sequenced, and the clones whose sequencing results were completely consistent with the target sequence were selected for the next experiment. The sequencing results of iASPP-RNAi (8954-1) are shown in SED ID NO.1-2 in the sequence listing.

[0101] S203. Plasmid extraction and virus packaging, the specific process includes:

[0102] S2031.Plasmid extraction

[0103] (1) The bacterial liquid of the positive clone transformant with correct sequencing was transferred to 150 ml of LB liquid culture medium containing Amp antibiotics and cultured in a shaking incubator at 37°C overnight; (2) Then, the three plasmid DNAs in the lentiviral packaging system, namely GV115, pHelper 1.0, and pHelper 2.0, were extracted using Qiagen's plasmid extraction kit. The plasmid DNA was dissolved in sterile TE and its concentration and purity were determined by ultraviolet absorption to ensure that the A260 / A280 of the extracted plasmid DNA was between 1.8 and 2.0. The plasmids that passed the quality inspection were used for virus packaging;

[0104] S2032. Packaging the extracted Lentivirus

[0105] (1) 24 h before transfection, 293T cells in the logarithmic growth phase were digested with trypsin and the cell density was adjusted to 1.2 x 10 in a medium containing 10% serum. 7cells / 20ml, re-inoculated into a 15cm cell culture dish, and cultured in a 37°C, 5% CO2 incubator; (2) 24h after the cell density reaches 70%-80%, it can be used for transfection, and 2h before transfection, the cell culture medium is replaced with serum-free medium; (3) in a sterile centrifuge tube, add the prepared DNA solution (20μg of GV115 vector, 15μg of pHelper1.0 vector, 10μg of pHelper2.0 vector) and mix evenly with the corresponding volume of Opti-MEM, adjust the total volume to 2.5ml, and incubate at room temperature for 5 minutes; (4) gently shake the Lipofectamine 2000 reagent, take 100μl of Lipofectamine 2000 reagent and mix it with 2.4ml Opti-MEM in another tube, and incubate at room temperature for 5 minutes; (5) mix the diluted DNA with the diluted Lipofectamine 2000, gently invert to mix, and incubate at room temperature for 20 minutes to form a mixture of DNA and Lipofectamine 2000 dilution of the transfection complex; (6) Transfer the mixture of DNA and Lipofectamine 2000 to the culture medium of 293T cells, mix well and culture in a cell culture incubator at 37°C, 5% CO2; (7) After 8 hours of culture, pour out the culture medium containing the transfection mixture, add 20 ml of PBS solution to each bottle of cells, gently shake the culture bottle left and right to wash the remaining transfection mixture, and then pour it out;

[0106] S2033. Harvesting and Concentration of Lentivirus

[0107] (1) Collect the 293T cell supernatant 48 hours after transfection (transfection can be counted from 0 hours); (2) Centrifuge at 4000g for 10 min at 4°C to remove cell debris, and filter the supernatant with a 0.45μm filter into a 40ml ultracentrifuge tube; (3) Add the crude virus extract sample to the filter cup (maximum 19ml) and cover it with a lid; (4) Insert the filter cup into the filtrate collection tube, assemble it, balance it, and place it on the rotor; (5) Centrifuge at 4000×g to the required virus concentration volume. The time usually required is 10-15 minutes; (6) After the centrifugation is completed, remove the centrifuge, separate the filter cup and the filtrate collection cup below, and turn the filter cup upside down on the sample collection cup; (7) The centrifugal force does not exceed 1000g, the time is 2 minutes, and the filter cup is removed from the sample collection cup; the sample collection cup contains the virus concentrate; (8) Remove the virus concentrate, package it and store it in a virus tube, and store it for a long time at -80 degrees; take one of them for virus biological titer determination.

[0108] S3. The obtained lentivirus was used to detect the silencing efficiency of the iASPP gene, which specifically included the following experiments:

[0109] Example 2: S301. Detection of the mRNA expression level of iASPP in bladder cancer cell lines

[0110] 1. Human bladder cancer cell lines 5637, T24 and UM-UC-33 in the logarithmic growth phase were digested with trypsin and prepared into cell suspension (5×10 4 / ml) and inoculated in a 6-well plate, cultured until the cell confluence reached about 30%, added an appropriate amount of lentivirus prepared using the shRNA interference sequence of the human iASPP gene, cultured for 6 hours, replaced the culture medium, and collected the cells after the infection time reached 3 days;

[0111] 2. Cell RNA extraction and cDNA synthesis: ① Collect bladder cancer cell lines 5637, T24 and UM-UC-33 cells (80% cell density in 6-well plates), centrifuge at 2000rpm for 5min, remove the supernatant, add 1mL Trizol to the cell pellet, mix thoroughly and let stand at room temperature for 5min, then transfer to a new 1.5mL EP endof tube; ② Add 200μL chloroform to each tube, turn the EP tube upside down for 15s, and let stand at room temperature for 10min; ③ Centrifuge at 4℃ and 12800rpm for 15min; ④ Aspirate the upper liquid and transfer to a new 1.5mL EP tube, add an equal volume of pre-cooled isopropanol, mix well and let stand at 4℃ for 10min; ⑤ Centrifuge at 4℃ and 12800rpm for 12min and discard the supernatant; ⑥ Add 1mL Wash the precipitate with 75% ethanol freshly prepared with DEPC water; ⑦ Centrifuge at 4°C and 12800rpm for 5min, discard most of the supernatant; ⑧ Centrifuge again at 4°C and 12800rpm for 5min, discard the supernatant, and dry at room temperature; ⑨ When the RNA precipitate is basically transparent, add appropriate amount of RNase-free water to completely dissolve it, and analyze the concentration and quality of the extracted RNA with Nanodrop2000 / 2000C spectrophotometer; ⑩ Reverse transcription to obtain cDNA;

[0112] The process of reverse transcription to obtain cDNA is:

[0113] (1) Add 1 μL Oligo dT (0.5 μg / μL) and 2.0 μg Total RNA to a PCR tube and add RNase-Free H2O to 10 μL;

[0114] (2) After mixing, centrifuge and incubate at 70°C for 10 min. Immediately place in an ice bath in an ice-water mixture to allow Oligo dT and template to anneal.

[0115] (3) In the above mixture, prepare the reaction system according to the ratio in Table 3 (on ice), mix well, and centrifuge briefly;

[0116] Table 3: Ice bath reaction system

[0117]

[0118] (4) The above system was reacted in a 42°C water bath for 1 h, and then in a 70°C water bath for 10 min to inactivate the RT enzyme, and the obtained reverse transcription product cDNA was stored at -20°C for later use;

[0119] Note: dNTPs is a mixture of dATP, dCTP, dGTP, and dTTP at a concentration of 10 mM.

[0120] 3. Design of RT-PCR reaction system

[0121] (1) The RT-PCR primers for human iASPP gene were designed as follows: upstream primer 5'-TGAAGGAGATGAACGACCCG-3'; downstream primer 5'-GGCGCAGTCAGCATAACCC-3', the amplified fragment was 296 bp;

[0122] (2) Using the housekeeping gene GAPDH as the internal reference, the RT-PCR primer sequences are as follows:

[0123] Upstream primer 5'-TGACTTCAACAGCGACACCCA-3',

[0124] The downstream primer was 5′-CACCCTGTTGCTGTAGCCAAA-3′, and the amplified fragment was 121 bp;

[0125] (3) The RT-PCR reaction system was prepared using the primers described in (1) and (2) above as shown in Table 4:

[0126] Table 4: RT-PCR reaction system

[0127]

[0128] 4.RT-PCR detection process

[0129] The program was set as two-step Real-time PCR: pre-denaturation at 95°C for 30s, followed by denaturation at 95°C for 5s; annealing and extension at 60°C for 30s; a total of 45 cycles; absorbance was read each time during the extension phase; after PCR, denaturation was performed at 95°C for 1min, and then cooled to 55°C to allow the DNA double strands to fully bind; from 55°C to 95°C, each step increased by 0.5°C, maintained for 4s, while absorbance was read to create a melting curve; 2- ΔΔCt The analysis method was used to calculate the expression abundance of infected iASPP mRNA;

[0130] The experimental results showed that iASPP mRNA was highly expressed in human bladder cancer cell lines 5637, T24 and UM-UC-33 ( Figure 3 After transfection with lentiviral-iASPP / shRNA, the expression levels of iASPP mRNA in cells were downregulated by 78.4%, 85.9% and 73.2%, respectively, proving that the constructed lentiviral construct expressing iASPP gene shRNA can effectively inhibit the expression of iASPP mRNA ( Figure 4 shown).

[0131] S302. Example 3: Western blotting to detect iASPP protein expression in bladder cancer cell lines:

[0132] 1. Total cell protein extraction:

[0133] (1) Receive the cell sample, wash twice with PBS, take an appropriate amount of RIPA lysis buffer (http: / / www.beyotime.com / ripa-lysis-bufferm.htm), add PMSF within a few minutes before use to make the final concentration of PMSF 1mM; (2) Add an appropriate amount of RIPA lysis buffer and lyse on ice for 10-15min; (3) Use a cell scraper to transfer the cells into a new EP tube, and then ultrasonically disrupt the cells (40W for a total of 20 times, 1s each time, 2s interval); (4) Centrifuge at 4℃ and 12000g for 15min, take the supernatant and determine the protein concentration by BCA method (BCA Protein Assay Kit http: / / www.beyotime.com / p0010s.htm); (5) Add new lysis buffer to adjust the protein concentration of each sample to a consistent level of 2μg / μL; (6) Then add 1 / 5 volume of 6× lodding buffer, mix well, boil at 100℃ for 10min, centrifuge briefly, and store at -80℃ for later use.

[0134] 2. Prepare 10% SDS-PAGE separation gel and 5% concentrated gel. After the gel solidifies, remove the comb, wash the sample well with electrophoresis buffer, load the prepared sample (40 μg of protein is loaded), and perform electrophoresis (electrophoresis conditions are 80 mA for 20 min for concentrated gel and 120 mA for 1 h for separation gel);

[0135] 3. Immunoblotting (wet transfer): after electrophoresis, use a transfer electrophoresis device to transfer the protein to a PVDF membrane at 4°C and 300 mA constant current for 150 min.

[0136] 4. Antibody hybridization

[0137] (1) Blocking: Block the PVDF membrane with blocking solution (TBST solution containing 5% skim milk) at room temperature for 1 h or at 4°C overnight; (2) Primary antibody incubation: Dilute the antibody (rabbit anti-human iASPP monoclonal antibody 1:2000, mouse anti-human β-actin monoclonal antibody 1:5000) with blocking solution, then incubate with the blocked PVDF membrane at room temperature for 2 h or at 4°C overnight, and wash the membrane 4 times with TBST, each time for 8 min; (3) Secondary antibody incubation: Dilute the corresponding secondary antibody (1:10000) with blocking solution, incubate the PVDF membrane at room temperature for 1.5 h, and wash the membrane 4 times with TBST, each time for 8 min;

[0138] 5. X-ray development: (using CST 20X Reagent and 20X Peroxide #7003 Kit)

[0139] (1) Place the PVDF membrane on a flat plastic wrap, mix solution A and solution B in the kit in a 1:1 ratio, invert and mix thoroughly, and let stand for a few minutes before use;

[0140] (2) Wipe the PVDF membrane dry with absorbent paper, lay it flat in a dark box, add an appropriate amount of mixed ECL luminescent liquid, cover with plastic wrap (to avoid bubbles), put the X-ray film on it and prevent it from moving), close the dark box, and expose it for a few seconds to a few minutes (the exposure time needs to be tried several times, and the exposure time should be adjusted appropriately according to whether the fluorescence can be seen by the naked eye and the intensity of the fluorescence);

[0141] (3) Take out the X-ray film, put it into the developer, take it out after the stripes appear, rinse it in clean water for a few seconds, and then put it into the fixer for at least 2 minutes;

[0142] (4) Take out the X-ray film, dry it, and analyze it. The experimental results are as follows: Figure 4 As shown;

[0143] The experimental results showed that iASPP protein was highly expressed in human bladder cancer cell lines 5637, T24 and UM-UC-33. Figure 4 ), after transfection with lentiviral-iASPP / shRNA, iASPP protein in KD group of human bladder cancer cell line 5637, T24 and UM-UC-33 cells was significantly downregulated compared with that in NC group, confirming that the constructed lentiviral construct expressing gene shRNA can effectively inhibit iASPP protein level.

[0144] S303. Example 4: Experiment on the proliferation ability of bladder cancer cells transfected with iASPP-siRNA lentivirus

[0145] 1. Set up blank control group (CON), negative control group (NC) and gene silencing group (KD);

[0146] 2. After digesting the cells in the logarithmic growth phase of each experimental group with trypsin, resuspend them in complete medium into a cell suspension and count them;

[0147] 3. Determine the cell density (2000 cells / well) according to the cell growth rate. Repeat 3 times for each group, plate 5 96-well plates in total, and culture them in a cell culture incubator;

[0148] 4. Starting from the second day after plating, add 20 μL of 5 mg / mL MTT to each well 4 hours before the end of culture. No need to change the medium. After 4 hours, completely aspirate the culture medium. Be careful not to aspirate the formazan particles at the bottom of the well plate. Add 100 μL DMSO to dissolve the formazan particles. Oscillate on an oscillator for 2-5 minutes. Detect the OD value at 490 / 570nm with an enzyme reader. According to the statistical analysis of the data, draw a 5-day cell proliferation curve as shown below: Figure 5 , Figure 6 and Figure 7 As shown;

[0149] The results of MTT test showed that compared with the CON group and NC group, the cell proliferation in the KD group was significantly reduced (P<0.05), indicating that the constructed lentiviral construct expressing iASPP gene shRNA can effectively inhibit the proliferation of tumor cells.

[0150] S304. Example 5: Experiment on detecting apoptosis level of tumor cells transfected with iASPPsiRNA lentivirus

[0151] 1. Set up blank control group (CON), negative control group (NC) and gene silencing group (KD);

[0152] 2. When the cells in each group of 6-well plates grow to a coverage rate of about 70%, trypsinize and resuspend in complete medium to form a cell suspension. Collect the cells in the same 5 mL centrifuge tube with the supernatant. Set up three replicate wells for each group (to ensure that the number of cells is sufficient, the number of cells should be ≥5×10 5 / deal with);

[0153] 3. Centrifuge at 1300 rpm for 5 min, discard the supernatant, wash the cell pellet with 4°C pre-cooled D-Hanks (pH=7.2-7.4); wash the cell pellet once with 1× binding buffer, centrifuge at 1300 rpm for 3 min, and collect the cells;

[0154] 4. Resuspend the cell pellet in 200 μL 1×binding buffer, add 10 μL Annexin V-APC for staining, incubate at room temperature away from light for 10-15 min, add 400-800 μL 1×binding buffer according to the cell volume, and test on the instrument. The test results are as follows: Figure 8 As shown;

[0155] The results showed that when iASPP RNAi lentivirus infected human bladder cancer cell lines 5637, T24 and UM-UC-33 cells, the apoptosis rate in the KD group was significantly increased compared with that in the CON and NC groups (P<0.05), proving that the iASPP gene is related to cell proliferation and its reduced expression can induce apoptosis in bladder cancer cells.

[0156] S305. Example 6: Cell cycle detection experiment

[0157] 1. Set up blank control group (CON), negative control group (NC) and gene silencing group (KD);

[0158] 2. When the cells in each experimental group grow to a coverage rate of about 80% in the 6 cm dish (the cells have not entered the growth plateau phase), trypsinize and resuspend in complete medium into a cell suspension, collect in a 5 mL centrifuge tube, and set up three replicate wells (≥10 6 );

[0159] 3. Centrifuge at 1300 rpm for 5 min, discard the supernatant, wash the cell pellet once with 4°C pre-cooled DPBS (pH = 7.2-7.4); centrifuge at 1300 rpm for 5 min;

[0160] 4. Prepare cell staining solution: 40×PI mother solution (2 mg / mL): 100×RNase mother solution (10 mg / mL): 1×DPBS: 25×Triton-100=25:10:1000:40;

[0161] 5. According to the amount of cells, add a certain volume of liquid (cell staining: according to the amount, add a certain volume of liquid (0.6-1mL) to resuspend, so that the cell throughput rate is 300-800 cells / s when put on the machine;

[0162] 6. Test on the machine and use ModFit software for data analysis. The test results are as follows: Fig. 9 As shown;

[0163] The results showed that when iASPP RNAi lentivirus infected human bladder cancer cell lines 5637, T24 and UM-UC-33 cells, the cell cycle of KD group was mainly arrested in G1 phase, and the number of cells in S phase decreased (P<0.05), which proved that the decreased expression of iASPP gene could induce cell cycle disorder in bladder cancer cells.

[0164] S306. Example 7: Detection of tumor cell clone formation experiment transfected with iASPP-siRNA lentivirus

[0165] 1. Set up blank control group (CON), negative control group (NC) and gene silencing group (KD);

[0166] 2. Digest the cells of each experimental group in the logarithmic growth phase with trypsin, resuspend them in complete medium to make cell suspension, and count them;

[0167] 3. The cells were inoculated in 6-well culture plates. Each experimental group was inoculated with 400-1000 cells / well (determined according to the cell growth conditions), and 3 replicate wells were set up for each experimental group;

[0168] 4. Continue to culture the inoculated cells in the incubator until 14 days or until the number of cells in most single clones is greater than 50. Change the medium every 3 days and observe the cell status. Take pictures of the cell clones under a fluorescence microscope before terminating the experiment and wash the cells once with PBS.

[0169] 5. Add 1 mL of 4% paraformaldehyde to each well and fix the cells for 30-60 min. Wash the cells once with PBS. Add 1000 μL of clean, impurity-free crystal violet stain to each well and stain the cells for 10-20 min. Wash the cells several times with ddH2O, dry them, take pictures with a digital camera, and count the clones. The results are as follows: Fig.10 As shown;

[0170] The results showed that the number of clones in the KD group of human bladder cancer cell lines 5637, T24 and UM-UC-33 cells was significantly reduced compared with the NC group and CON group (P<0.05). The constructed lentiviral construct expressing iASPP gene shRNA could effectively inhibit the proliferation of tumor cells.

[0171] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A shRNA interference sequence of human iASPP gene, Features: It comprises two inverted repeat sequences targeting human iASPP gene coding DNA, wherein the two inverted repeat sequences are respectively the shRNA target sequence of human iASPP gene and its complementary sequence, separated by a stem-loop sequence in the middle, and the effective siRNA target sequence targeting human iASPP gene is shown in SED ID NO.

3.

2. The shRNA interference sequence of the human iASPP gene according to claim 1, Features: The shRNA sequence of the human iASPP gene includes a DNA oligo forward chain sequence and a reverse chain sequence; Among them: the positive chain sequence is 5′-CCGGCC AACTACTCTATCGTGGATT CTCGAG AATCCACGA TAGAGTAGTT GGTTTTTG-3′; The antisense strand sequence is 5′-AATTCAAAAACC AACTACTCTATCGTGGATT CTCGAG AAT CCACGATAGAGTAGTT GG-3′.

3. A method for constructing a shRNA sequence of human iASPP gene, Features: The method comprises the steps of S101. Design RNAi targets for knocking down expression of human iASPP gene. The obtained effective siRNA target sequence targeting human iASPP gene is: 5′-AACTACTCTATCGTGGATT-3′; S102. Using siRNA target sites to synthesize the sense and antisense single strands of single stranded DNA oligo shRNA sequences of human iASPP gene; S103. Dissolve the single-stranded DNA oligo interference sequence dry powder of human iASPP gene in annealing buffer to prepare a concentration of 20 μM, incubate in a 90°C water bath for 15 min, and cool naturally to room temperature to obtain double-stranded DNA oligo.

4. Application of a shRNA interference sequence of human iASPP gene in the construction of lentivirus, Features: The specific process includes the following steps: S201. The vector is linearized by enzyme digestion, and the linearized vector GV115 is connected to the double-stranded DNA of the shRNA interference sequence of the human iASPP gene; S202. Transform the ligation product into competent E. coli cells, and perform PCR identification on the obtained positive clones; S203. Plasmid extraction and virus packaging; S204. Using the obtained lentivirus, detect the silencing efficiency of the iASPP gene.

5. Use of a shRNA interference sequence of a human iASPP gene according to claim 4 in constructing a lentivirus, Features: The specific process of connecting the linearized vector GV115 with the double-stranded DNA of the shRNA interference sequence of the human iASPP gene in step S201 includes: S2011. Linearized vector preparation (1) 41 μl of ddHO 2 Prepare 50 μl of enzyme digestion reaction system by adding 5 μl of 10× CutSmart Buffer, 2 μl of purified plasmid DNA, 1 μl of EcoR enzyme, and 1 μl of AgeⅠ enzyme in this order, then mix by gently pipetting, centrifuge briefly, and react at 37°C for 3 h or overnight to obtain the vector enzyme digestion product; (2) Perform agarose gel electrophoresis on the vector digestion product, recover the target band, linearize it, react it at 37°C for 1 h, and then cut the gel to recover the target fragment; S2012.Connect 1 μl of linearized vector DNA 100 ng / ul, 1 μl of DNA oligo 100 ng / ul, 2 μl of 10× T4 phage DNA ligase buffer, 1 μl of T4 phage DNA ligase and 15 μl of ddHO 2 The addition order of O enzyme was used to prepare 20 μl of ligation reaction system, which was then gently pipetted and mixed, centrifuged briefly, and placed at 16°C for ligation overnight to obtain a ligation product.

6. Use of a shRNA interference sequence of human iASPP gene according to claim 4 in constructing a lentivirus, Features: The process of obtaining competent E. coli cells and PCR identification described in step S202 includes: S2021. Prepare fresh E. coli DH5α competent cells using calcium chloride; S2022. Transform E. coli competent cells using the ligation product obtained in step S201; S2023. Perform PCR identification on the transformed competent cells for positive clones.

7. Use of a shRNA interference sequence of a human iASPP gene according to claim 6 in constructing a lentivirus, Features: The specific process of performing PCR identification of positive clones on the transformed competent cells described in step S2023 includes: (1) Construction of RNA interference vector and identification of positive clones Resuspend the transformed E. coli competent cells in 10 μl LB solution, mix well and take 1 μl as template; use GV115 universal primers to perform colony PCR identification; (2) Prepare 20 μl PCR reaction system Includes: 2μl 10×buffer, 0.8μl dNTPs (2.5mM), 0.4μl Primer(+), 0.4μl Primer(-), 0.2μl Taqpolymerase, 1μl DNA template, 15.2μl ddH 2 O; wherein the above dNTPs are a mixture of dATP, dCTP, dGTP, and dTTP; (3) Then perform PCR amplification The reaction conditions of PCR amplification were as follows: first, melt at 94°C for 3 min; then, melt at 94°C for 30 s, anneal at 60°C for 30 s, extend at 72°C for 30 s, and perform 30 cycles of melt-anneal-extend; and finally, extend at 72°C for 6 min. (4) After the PCR is completed, 5 μl of the product is taken, and the bands are detected by 1% agarose gel electrophoresis. The iASPP-RNAi (8954-1) detected as positive after amplification is sequenced. The identified positive clone transformants are inoculated into an appropriate amount of LB liquid culture medium containing corresponding antibiotics, cultured at 37°C for 12-16 hours, and an appropriate amount of bacterial liquid is taken for sequencing; (5) Sequencing of positive clones was performed using identification primer-F.

8. Use of a shRNA interference sequence of human iASPP gene according to claim 7 in constructing lentivirus, Features: The iASPP-RNAi sequencing results obtained by sequencing in step (4) are shown in SED ID NO.1-2 in the sequence listing.

9. Use of a lentivirus constructed with a shRNA interference sequence of a human iASPP gene according to any one of claims 4 to 8, Features: The human iASPP gene in the lentivirus can be used as a target for bladder cancer cells and applied to the preparation of a drug for treating bladder cancer. The target for bladder cancer cells is an RNA interference target.

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