CN104293785A - Application of microRNA302s in preparation of antitumor drugs - Google Patents

Abstract

The invention discloses a nucleotide sequence shown in SEQ ID NO. 1-5, a plasmid and a recombinant lentivirus containing the sequence, and application of the sequence, the plasmid and the lentivirus in preparation of antitumor drugs. The invention also discloses a tumor screening kit which comprises a reagent for detecting the expression level of the cellular microRNA302 a. The microRNA302s can effectively induce tumor cell apoptosis and inhibit tumor growth, and has strong practical application value.

Description

Application of microRNA302s in the preparation of antitumor drugs

technical field

The present invention relates to a new application of microRNA302s. the

Background technique

microRNAs (miRNAs) are a class of non-coding small RNAs newly discovered by humans, which are widely distributed in eukaryotic cells. miRNAs negatively regulate the expression of target genes at the post-transcriptional level by pairing with the complementary sites of target genes, and participate in life processes such as growth and development, cell proliferation, cell apoptosis, and cell differentiation. Mature miRNAs are 17-24bp long, and one miRNA molecule can regulate multiple genes. Therefore, studies have found that the expression of some genes is more dependent on the regulation level of miRNAs than protein-coding mRNA molecules. the

microRNA302s, the miR-302 family of microRNAs, including miR-302a, miR-302b, miR-302c, and miR-302d, clustered on human chromosome 4 and are miRNAs specifically expressed in embryonic stem cells. At present, studies on microRNAs of the miR-302 family have focused on genes related to embryonic development. For example, studies have found that each member of the miR-302 family can simultaneously regulate about 445 genes, and most of the genes they regulate They are all the same. Most of these genes are involved in early embryonic development-related signaling molecules or cytokines that affect cell differentiation. After miR-302s is transferred, it can inhibit the function of these genes, thereby inhibiting the differentiation of tissue cells, and it is possible to regenerate cells. programmed to an embryonic stem cell-like state. the

Contents of the invention

The invention provides a new application of miR-302 family microRNA in the preparation of antitumor drugs. the

First, the present invention provides the nucleotide sequence shown in SEQ ID NO.1-5. the

The nucleotide sequences shown in SEQ ID NO.1～4 are respectively miR-302a, miR-302b, miR-302c, miR-302d, and the nucleotide sequences shown in SEQ ID NO.5 are miR-302a, miR Gene sequence of -302b, miR-302c and miR-302d in series: miR-302s. the

miR-302a:CCACCACUUAAACGUGGAUGUACUUGCUUUGAAACUAAAAGAAGUAAGUGCUUCCAUGUUUUGGUGAUGG;

miR-302b:GCUCCCUUCAACUUUAACAUGGAAGUGCUUUCUGUGACUUUAAAAGUAAGUGCUUCCAUGUUUUUAGUAGGAGU;

miR-302c:CCUUUGCUUUUAACAUGGGGGUACCUGCUGUGUGAAACAAAAGUAAGUGCUUCCAUGUUUCAGUGGAGG;

miR-302d: CCUCUACUUUAACAUGGAGGCACUUGCUGUGACAUGACAAAAAAUAAGUGCUUCCAUGUUUUGAGUGUGG. the

miR-302s(SEQ ID NO.5):

CCACCACTTAAACGTGGATGTACTTGCTTTGAAACTAAAGAAGTAAGTGCTTCCATGTTTTGGTGATGGAGATCTGCTCCCTTCAACTTTAACATGGAAGTGCTTTCTGTGACTTTAAAAGTAAGTGCTTCCATGTTTTAGTAGGAGTCATATGCCTTTGCTTTAACATGGGGGTACCTGCTGTGTGAAACAAAAGTAAGTGCTTCCATGTTTCAGTGGAGGTCTAGACCTCTACTTTAACATGGAGGCACTTGCTGTGACATGACAAAAATAAGTGCTTCCATGTTTGAGTGTGG 

The present invention also provides the use of any one or multiple sequences in the nucleotide sequences shown in SEQ ID NO.1-5 in the preparation of antitumor drugs. The antineoplastic drug is an antineuroma drug. The anti-neuroma drug is an anti-neuroblastoma drug. the

The present invention also provides a recombinant plasmid, which includes the nucleotide sequence shown in any one of SEQ ID NO.1-5. Preferably, the recombinant plasmid is a recombinant GV266 plasmid. the

The invention also provides the use of the recombinant plasmid in the preparation of antitumor drugs. Preferably, the antineoplastic drug is an antineuroma drug. Further preferably, the anti-neuroma drug is an anti-neuroblastoma drug. the

The recombinant lentivirus of the present invention comprises the nucleotide sequence shown in any one of SEQ ID NO.1-5. the

The recombinant lentivirus of the present invention includes the aforementioned recombinant plasmid. the

The present invention also provides the use of the recombinant lentivirus in the preparation of antitumor drugs. Preferably, the antineoplastic drug is an antineuroma drug. Further preferably, the anti-neuroma drug is an anti-neuroblastoma drug. the

The tumor cell screening kit of the present invention includes reagents for detecting the expression level of microRNA302a in cells. the

Preferably, the antineoplastic drug is an antineuroma drug. Further preferably, the anti-neuroma drug is an anti-neuroblastoma drug. the

The nucleotide sequences shown in SEQ ID NO.1-5 provided by the present invention can induce tumor cell apoptosis, inhibit the growth of tumor cells, and can be used to prepare antitumor drugs. The tumor cell screening kit of the present invention can effectively screen Checking whether the cells to be tested are tumor cells has a good clinical application prospect. the

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made. the

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. the

Description of drawings

Figure 1 Comparison of the expression of miR-302a in 293 cells and SK cells

Figure 2 Flowchart for the construction of target gene eukaryotic expression vector

Figure 3 Carrier map

Figure 4 Marker from top to bottom: 5kb, 3kb, 2kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, 100bp, 2#: Enzyme-digested target gene plasmid;

Figure 5 Electropherogram description: 1#: Negative control (ddH2O); 2#: Negative control (empty self-connected control group); 3#: Positive control (GAPDH); 4#: Marker 5kb from top to bottom, 3kb, 2kb, 1.5kb, 1Kb, 750bp, 500bp, 250bp, 100bp; 5#-12#: hsa-mir-302, a, b, c, d transformants 1-8 in series. the

Figure 6 sequence map, Figure 6-1 is the sequence map of the 1st to 295bp; Figure 6-2 is the sequence map of the 296th to 601bp; Figure 6-3 is the sequence map of the 602nd to 807bp; Figure 6-4 is the sequence map of the first The measurement spectrum of 808-1081bp; Figure 6-5 is the sequencing spectrum of 1081-1159bp. the

Figure 7 Virus infection pictures (1E1uL100x b)

Figure 8 Virus infection pictures (1E1uL100x g)

Figure 9 Virus infection pictures (1E1uL100x r)

Figure 10 Virus infection pictures (1E1uL200x b)

Figure 11 Picture of virus infection (1E1uL400x b)

Figure 12 Quantitative PCR experiment results

Figure 13 Real-time PCR detection of miR-302s expression after lentiviral transfection

Figure 14 The expression of miR-302a in SK-purpose virus transfected cells and SK-purpose control cells

Figure 15 The expression of miR-302b in SK-purpose virus transfected cells and SK-purpose control cells

Figure 16 The expression of miR-302c in SK-purpose virus transfected cells and SK-me control cells

Figure 17 The expression of miR-302d in SK-purpose virus transfected cells and SK-me control cells

Figure 18 Hoechst33342 staining experiment results 48h after SK-N-SH cell infection

Figure 19 Hoechst33342 staining detection of apoptosis results of SK-N-SH cells 48h after infection

Figure 20 The cell loss technique detects the changes in the apoptosis rate of SK-N-SH cells at 48 hours after miR-302s overexpression lentivirus transfection

Figure 21 The change of the apoptosis rate of SK-N-SH cells at 72 hours after miR-302s overexpression lentivirus transfection detected by cell loss technique

Figure 22 TUNEL staining experiment results 48h after SK-N-SH cell infection

Figure 23 48h after SK-N-SH cells were infected, the results of TUNEL staining to detect apoptosis

Figure 24 The expression of CASP-3 in SK normal cells and miR-302s transfected cells

Figure 25 The expression of Bax in SK normal cells and miR-302s transfected cells

Figure 26 The expression of MCL-1 in SK normal cells and miR-302s transfected cells

Figure 27 The expression of CCND-1 in SK normal cells and miR-302s transfected cells

Figure 28 Comparison of BMI-1 expression in SK normal cell nuclei miR-302s transfected cells

Figure 29 Comparison of expression of p16Ink4a in miR-302s transfected cells of normal SK nuclei

Figure 30 Comparison of expression of p53 in SK normal cell nuclei miR-302s transfected cells

Figure 31 Western Blotting to detect the expression of BMI-1

Figure 32 Western Blotting detection of p16Ink4a, p53, Bax expression

The body weight of the two groups of nude mice before the interference in Figure 33

Figure 34 Tumor volume of two groups of nude mice before interference

Figure 35 Weighing chart before execution

Figure 36 The tumor volumes of the two groups measured before execution

Figure 37 Comparison chart of live xenograft tumors before execution

Figure 38 Comparison chart of transplanted tumors in vivo before execution

Figure 39 Comparison chart of transplanted tumor weight between two groups obtained after death

Figure 40 Comparison chart of transplanted tumors obtained after death

Figure 41 Growth curve of transplanted tumor

Figure 42 Growth curve of transplanted tumor

Figure 43 Growth multiples of transplanted tumors

Detailed ways

Example 1 The expression of microR-302a in tumor cells

1. Experimental method

(1) Cell recovery, culture, passage (293 cells and SK cells); 293 cells: human renal epithelial cell line transfected with adenovirus E1A gene; SK cells: human neuroblastoma cell line;

(2) Cell seed plate (six-well plate, 3×10 ⁵ per well);

(3) Extraction of total RNA from cell lines (Trizol method);

(4) Reverse transcription reaction (stem-loop reverse transcription stem-loop), the primer is

5'-GTCGTATCCAGTGCTGGGTCCGAGTGATTCGCACTGGATACGACTCACCA-3';

(5) Real-time fluorescent quantitative PCR (Taqman) probe PCR technology to detect the expression level of miR-302a, the normal cell count is 1, and the probe sequence used is:

5'-FAM-TCGTATCCAGTGCGAATCACTC-TAMRA-3'

(6) Data statistics and analysis. the

2. Experimental results

The experimental results are shown in Figure 1 and Table 1:

Table 1 The expression of miR-302a in 293 cells and SK cells

It can be seen from the experimental results that the expression level of miR-302a in tumor cells is significantly lower than that in normal cells. The experiment shows that by detecting the expression of miR-302a, it is possible to screen whether the cells are tumor cells. the

Example 2 Construction of a recombinant lentivirus comprising the nucleotide sequence of the present invention

1. Description of experimental materials

Cell line: 293T, a lentivirus packaging cell, is an anchorage-dependent epithelioid cell, and the growth medium is DMEM (containing 10% FBS). Adherent cells grow and proliferate in culture to form a monolayer of cells. the

Strain: E. coli strain DH5α. For amplification of lentiviral vectors and helper packaging vector plasmids. the

Viral vector: a) pGC-LV recombinant vector; b) pHelper1.0; c) pHelper2.0

Preparation of DNA solution: Extract the three plasmid DNAs in the lentiviral packaging system with Qiagen’s plasmid extraction kit, dissolve the plasmid DNA in sterilized TE, and measure its concentration and purity by ultraviolet light absorption to ensure that the extracted plasmid The A260/A280 of DNA is between 1.8 and 2.0. the

Main reagents:

Main instrument:

2. Plasmid construction

As shown in Figure 2, from the plasmid containing the target gene, the target gene was fished out by PCR method, the target vector was digested, and the digested product was recovered by electrophoresis and exchanged, and the product was transformed into bacterial competent cells. Colony PCR identification was performed on the grown clones first, and then the clones with positive PCR identification were sequenced and compared and analyzed. The correct comparison was the successful construction of the target plasmid. the

1. Gene information

Chemically synthesized target gene sequence microR-302s (miR-302s):

ACCGGT CCACCACTTAAACGTGGATGTACTTGCTTTGAAACTAAAGAAGTAAGTGCTTCCATGTTTTGGTGATGGAGATCTGCTCCCTTCAACTTTAACATGGAAGTGCTTTCTGTGACTTTAAAAGTAAGTGCTTCCATGTTTT AGTAGGAGTCATATGCCTTTGCTTTAACATGGGGGTACCTGCTGTGTGAAACAAAAGTAAGTGCTTCCATGTTTCAGTGGAGGTCTAGACCTCTACTTTAACATGGAGGCACTTGCTGTGACATGACAAAAATAAGTGCTTCCATGTTTGAGTGTGG GCTAGC

Note: The 5' end is underlined for the Age I restriction site, and the 3' end is underlined for the NheI restriction site. the

2. Tool carrier:

Carrier name: GV266

Component order: Ubi-MCS-IRES-puromycin

Cloning site: AgeI/NheI

The carrier map is shown in Figure 3: http://www.genechem.com.cn/Zaiti.aspx? zt=GV266 can download the instruction manual of the carrier. the

3. Construction of recombinant plasmids

3.1 Exchange of PCR products into linearized expression vectors

Reaction conditions:

30 minutes at 25°C → 15 minutes at 42°C

reaction system:

illustrate:

a) The molar ratio of the added linearized carrier DNA to the purified PCR product is between 1:3 and 1:9

b) The purified PCR product added to the positive control is the GAPDH gene (also with an exchange arm)

3.2 Preparation of competent cells:

Configure the following solutions:

1) 0.1M CaCl ₂ solution, sterilized by 0.22μm filter.

2) 250mM KCl solution. the

3) 2M MgCl ₂ solution, autoclaved.

4) SOB: add 100ml LB to 1ml of 250mM KCl solution, adjust the pH value to 7.0 with 5M NaOH, autoclave, add 0.5ml of 2M MgCl ₂ solution before use.

Preparation of fresh E. coli competent cells with calcium chloride

1) Pick a single colony from a fresh plate cultured at 37°C for 16 hours and transfer to a 1L flask containing 100ml LB medium. Incubate vigorously at 37° C. for 3 hours (rotary shaker, 300 rpm). the

2) Aseptically transfer the bacteria to a sterile, single-use, ice-precooled 50ml polypropylene tube, place on ice for 10 minutes to cool the culture to 0°C. the

3) Centrifuge at 4000 rpm for 10 minutes at 4°C to recover the cells. the

4) Pour out the culture medium and invert the tube for 1 minute to drain the last remaining trace of the culture medium. the

5) Resuspend each pellet in 10ml of ice-cooled 0.1mol/L CaCl2 and place on an ice bath. the

6) Centrifuge at 4000 rpm for 10 minutes at 4°C to recover the cells. the

7) Pour out the culture medium and invert the tube for 1 minute to drain the last remaining trace of the culture medium. the

8) Resuspend each cell pellet with 2 ml of ice-cold 0.1 mol/L CaCl ₂ per 50 ml of initial culture.

9) Divide the cells into small portions and freeze them at -70°C. the

(Reference for Competent Cell Preparation: Molecular Cloning Experiment Guide, Second Edition, page 55)

3.3 Conversion:

1) Use a cooled sterile tip to transfer 200 μL of each competent cell suspension to a sterile microcentrifuge tube, add 10 μL of connection solution to each tube, swirl gently to mix the contents, and store in ice Leave for 30 minutes. the

2) Put the tube on the EP tube rack placed in the circulating water bath preheated to 42°C, and place it for exactly 90 seconds without shaking the EP tube rack. the

3) Quickly transfer the tube to an ice bath to cool the cells for 1-2 minutes. the

4) Add 800 μL LB medium to each tube. Warm the medium to 37°C with a water bath, then transfer the tube to a 37°C shaker and incubate for 45 minutes to recover the bacteria. the

5) Transfer 150 μL of transformed competent cells to AMP-resistant (100 ug/ml) LB agar medium. the

6) Leave the plate at room temperature until the liquid is absorbed. the

7) Invert the plate and incubate at 37°C for 16 hours. the

8) Subsequent PCR identification of the grown clones. the

(Reference for transformation operation: Molecular Cloning Experiment Guide, Second Edition, pages 55-56)

3.4 PCR identification of positive clones

vector digestion

Use AgeI/NheI to digest the chemically synthesized plasmid containing the gene of interest:

Enzyme digestion reaction system:

ddH ₂ O 40.5μl 10X buffer 5μl 100×BSA 0.5μl Purified DNA plasmid (1ug/μl) 2μl EcoR I (20U/μl) 1μl NheI (5U/μl) 1μl Total 50μl

The above mixed reactants were placed at 37°C for 2h. the

Digestion fragment size: 296bp, the fragment digestion electrophoresis results are shown in Figure 4. the

3.5 PCR identification of recombinant clones

PCR reaction system:

PCR reaction conditions:

94℃3min

94℃30sec

60℃30sec

72℃30sec

72℃5min

4℃∞

The PCR results are shown in Figure 5, the size of the PCR product of the positive transformant: 503bp, and the size of the PCR product of the negative transformant: 282bp. the

The positive clone sequencing pattern is shown in Figure 6, and the sequencing results are as follows:

AATTCTGGCCGTTTTTGGCTTTTTTGTTAGACGAAGCTTGGGCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGGTCCACCACTTAAACGTGGATGTACTTGCTTTGAAACTAAAGAAGTAAGTGCTTCCATGTTTTGGTGATGGAGATCTGCTCCCTTCAACTTTAACATGGAAGTGCTTTCTGTGACTTTAAAAGTAAGTGCTTCCATGTTTTAGTAGGAGTCATATGCCTTTGCTTTAACATGGGGGTACCTGCTGTGTGAAACAAAAGTAAGTGCTTCCATGTTTCAGTGGAGGTCTAGACCTCTACTTTAACATGGAGGCACTTGCTGTGACATGACAAAAATAAGTGCTTCCATGTTTGAGTGTGGGCTAGCTAAGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGATGATGCCCAGAAAGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTACACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGCCCCCCGAACCACGGGACGTGCTTTCCTTTGAAAAACACGATGATATATGGCACAGTTACATGACCGAGTACAGCCACGGTGCGCCTCGCACCC GCGACGACATCCCAAGGGCGTAGCACCCTCGCGCCGCGTTGCCGACATACCCGCCCAGCCCAACGTCGATCGAACGCCCAATAAGTACCTGACTGCGAATCTTTCACGCTCGCTCGACTCGCAAGTGTTCGACCACGTCACGTGCACTGGTACTCGCAG

The part in bold above is the target gene. The experimental results show that the present invention successfully constructed the recombinant GV266 vector containing the target gene microR-302s, which is a pGC-LV recombinant vector and can be used for lentivirus packaging.

3. Lentiviral packaging

(1), the cultivation of virus packaging cell 293T

live cell count

Dilute the cell suspension to 200-2000 cells/ml with serum-free medium (generally, the dilution factor is 100 times), and add 0.1 ml of 0.4% trypan blue solution to 0.1 ml of the cell suspension. Mix gently, and after a few minutes, count the cells with a hemocytometer. Live cells reject trypan blue, so cells that stain blue are dead cells. the

Cryopreservation of cell lines

1. Take the vigorously growing cells after culture for 2-3 days, and make the cells into 2×10 ⁶ ～2×10 ⁷ /ml with cell culture medium.

2. Add 0.5ml cell suspension, 0.4ml calf serum and 0.1ml dimethyl sulfoxide (or glycerol) to a 1ml cell cryopreservation tube, mix well and seal. Put it at 4°C for 1 hour, and -20°C for 2 hours, then put it directly into liquid nitrogen or put it on the liquid nitrogen vapor overnight and then immerse it in liquid nitrogen. the

Cell recovery

1. Take out the cell cryopreservation tube from the liquid nitrogen tank, and wear protective eyes and gloves. the

2. Quickly put it into a water bath filled with 37°C water, and shake it from time to time to thaw as soon as possible. the

3. After wiping and disinfecting with 70% alcohol, move it to a clean bench, suck out the cell suspension into a culture bottle, add 3ml of DMEM medium containing 10% FBS, and culture in an incubator. the

4. Change the culture medium the next day before continuing the culture. the

cell inheritance

1. Discard the old culture medium, add 5ml sterilized PBS solution, shake gently, wash the cell growth surface, and then discard the PBS solution. the

2. Add 2ml of trypsin digestion solution and digest for 1-2min until the cells are completely digested. the

3. Add 5ml of DMEM medium containing 10% fetal bovine serum and 100U/ml double antibody, and blow it several times with a graduated pipette to wash down the cells on the bottle wall. the

4. After mixing the cells, divide them into two new culture flasks and continue to culture. the

(2) Lentiviral packaging

cell transfection

1) 24 hours before transfection, digest the 293T cells in the logarithmic growth phase with trypsin, adjust the cell density to ^1.2x107 cells/20ml with the medium containing 10% serum, and re-seek in a 15cm cell culture dish, 37°C, 5 Cultured in a %CO ₂ incubator. After 24 hours, when the cell density reaches 70%-80%, it can be used for transfection. Cell state is critical for virus packaging, so good cell state and low passage times need to be guaranteed.

2) The cell culture medium was replaced with serum-free medium 2 hours before transfection. the

3) Add the prepared DNA solutions (recombinant pGC-LV vector (recombinant GV266 vector prepared in step 2) 20 μg, pHelper1.0 vector 15 μg, pHelper2.0 vector 10 μg) to a sterilized centrifuge tube, and the corresponding volume Mix Opti-MEM evenly, adjust the total volume to 2.5ml, and incubate at room temperature for 5 minutes. the

4) Shake the Lipofectamine2000 reagent gently, take 100μl Lipofectamine2000 reagent and mix it with 2.4ml Opti-MEM in another tube, and incubate at room temperature for 5 minutes. the

5) Mix the diluted DNA with the diluted Lipofectamine2000, and gently invert to mix without shaking. Must be mixed within 5 minutes. the

6) After mixing, incubate at room temperature for 20 minutes to form a transfection complex of DNA and Lipofectamine2000 dilution. the

7) Transfer the mixture of DNA and Lipofectamine2000 to the culture medium of 293T cells, mix well, and culture at 37° C., 5% CO ₂ cell incubator.

8) Pour off the medium containing the transfection mixture after culturing for 8 hours, add 20ml of PBS solution to each bottle of cells, 10) gently shake the culture bottle left and right to wash the remaining transfection mixture, and then pour it out. the

9) Add 25 ml of cell culture medium containing 10% serum to each bottle of cells, and continue culturing for 48 hours in a 37° C., 5% CO2 incubator. the

Infection pictures are shown in Figures 7-11. the

(3) Virus harvest and concentration

1. Collect the 293T cell supernatant 48 hours after transfection (transfection can be counted from 0 hours). the

2. Centrifuge at 4000g for 10min at 4°C to remove cell debris. the

3. Filter the supernatant with a 0.45 μm filter into a 40 ml ultracentrifuge tube. the

4. Add the virus crude extract sample to the filter cup (up to 19ml) and cover with the lid. Insert the filter cup into the filtrate collection tube. the

5. After the combination is complete, balance it and place it on the rotor. the

6. Centrifuge at 4000×g to the desired virus concentration volume. Usually takes 10-15 minutes. the

7. After centrifugation, take out the centrifuge device and separate the filter cup from the filtrate collection cup below. the

8. Invert the filter cup onto the sample collection cup. the

9. The centrifugal force does not exceed 1000g, and the time is 2 minutes. Excessively high rotational speeds can result in sample loss. Remove the filter cup from the sample collection cup. The virus concentrate is in the sample collection cup. the

10. Remove the virus concentrate, store in virus tubes after aliquoting, and store at -80 degrees for a long time. One of them was taken for virus biological titer determination. the

(4) Titer determination

Sample Preparation

1. On the day before the test, subculture the 293T cells, add 1× ¹⁰⁵ cells to each 24-well, the volume is 500 μl;

2. The next day, prepare 7-10 sterile Ep tubes, and add 90 μl of medium (DMEM+10% FBS) to each tube;

3. Take 10 μl of the virus stock solution to be tested and add it to the first tube. After mixing, take 10 μl and add it to the second tube. Continue the same operation until the last tube;

4. Select the desired cell wells and suck out 90 μl of medium. Add the diluted virus solution. Place in a 37°C, 5% _CO2 incubator for cultivation;

5. After 24 hours, add 500 μl of fresh medium. Operate carefully, do not blow up the cells, and maintain the normal growth of cells;

After 6.4 days, extract RNA and prepare for RT-qPCR. the

illustrate:

Add 10 μl of virus stock solution to the first Ep tube, recorded as 1E+1 μl;

The first ten-fold dilution was carried out in the second Ep tube, and the obtained virus stock solution was 1/10 of that in the first Ep tube, which was recorded as 1E+0μl;

The second ten-fold dilution was carried out in the third Ep tube, and the obtained virus stock solution was 1/10 of that in the second Ep tube, which was recorded as 1E-1μl;

And so on…

The sixth ten-fold dilution was carried out in the seventh Ep tube, and the obtained virus stock solution was 1/10 of that in the sixth Ep tube, which was recorded as 1E-5 μl;

Total RNA Extraction

Note: According to Invitrogen's TRIZOL operation manual, all operations are RNase-free. the

1. Remove the cell supernatant, add 1ml TRIZOL to each well, pipette, let stand at room temperature for 5min, and then transfer to another new 1.5ml eppendorf tube. the

2. Add 200 μl chloroform to each tube, shake vigorously for 15 seconds, and let stand at room temperature for 15 minutes. the

3.4°C, 12000rpm, centrifuge for 15min. the

4. Pipette the supernatant from each tube into another new 1.5ml eppendorf tube. Add an equal volume of isopropanol pre-cooled at -20°C, mix well and precipitate at -20°C for 10 minutes. the

After centrifugation at 5.4°C and 12000rpm for 10min, the supernatant was removed. the

6. Add at least 1ml of 75% ethanol pre-cooled at 4°C to wash the precipitate and the wall of the centrifuge tube. the

Centrifuge at 10,000 rpm for 5 minutes at 7.4°C, and discard the supernatant. the

8.4°C, centrifuge again at 10,000 rpm for 5 minutes, suck off the residual liquid, and dry at room temperature (complete drying is not required). the

9. Add 20 μl RNase-free water until completely dissolved, and measure the concentration of the extracted RNA by UV analysis. the

RNA reverse transcription to obtain cDNA

M-MLV reverse transcriptase and dNTP were purchased from PROMEGA Company. Oligo dT was purchased from Shanghai Shenggong. RNase-free items were purchased from Axygen. the

Explanation: According to the M-MLV operation manual of Promega Company, all operations are RNase-free. the

Protocol:

1. Add 1 μl Oligo dT (0.5 μg/μl) and 2.0 μg Total RNA to a small PCR tube, and add DEPC-H2O to 9 μl. After mixing, centrifuge and incubate at 70°C for 10 minutes. Immediately thereafter, insert into a 0°C ice-water bath to anneal the Oligo dT and template. the

2. According to the ratio in the table below, calculate the required reagent volume according to the number of reaction tubes. Mix the M-MLV enzyme and the like on ice to obtain the RT reaction solution. the

3. Add 11μl of RT reaction solution to each reaction tube, mix well and centrifuge. the

Add 11μl of RT reaction solution into each reaction tube, mix well and centrifuge. the

Real-time PCR detection

Real-time PCR was completed on TAKARA's TP800. SYBR Master Mixture comes from TAKARA. 

1. Configure the reaction system according to the following ratio:

2. Set the program to two-step Real-Time quantification. Pre-denaturation at 95°C for 15S, followed by each step of denaturation at 95°C for 5S, annealing and extension at 60°C for 30S, for a total of 40 cycles. Read the absorbance each time during the extension phase. the

Cycle1:(1X)

Step1:95.0℃for00:15. 

Cycle2:(40X)

Step1:95.0℃for00:05. 

Step2:60.0℃for00:30. 

Data collection and real-time analysis enabled. 

3. Make a melting curve. After PCR, denature at 95°C for 1 min. Then cool to 55°C to fully bind the DNA double strands. From 55°C to 95°C, increase by 0.5°C in each step, keep for 30S, and read the absorbance value at the same time. the

Cycle3:(1X)

Step1:95.0℃for01:00. 

Cycle4:(1X)

Step1:55.0℃for01:00. 

Cycle5:(81X)

Step1:55.0℃-95.0℃for00:30. 

Increase set point temperature after cycle2by0.5℃

The virus titer detection results are shown in Table 2:

Table 2 Ct value and expression analysis of sample groups after different concentrations of virus infection

In this titer test, there are about 3 differences in Ct values between the 1.00E-04ul group samples and the control group samples, so there are virus particles in the 1.00E-04ul group samples. the

Assuming that the group of samples contains at least 1 virus particle, the titer of the virus is:

1/(1.00E-04)*20=2.00E+5TU/ul=2.00E+8TU/ml. the

The experimental results show that the present invention successfully constructs lentiviral particles, which contain the target gene microR-302s. the

(5) qPCR expression detection of plasmid containing microR-302s

1. Total RNA extraction (according to Invitrogen's Trizol operating instructions)

1) Collect the cells, centrifuge at 1000rpm for 5min, remove the supernatant, add 1ml Trizol to the cell pellet, pipette quickly and let stand at room temperature for 5min, then transfer to a new 1.5ml eppendorf tube

2) Add 200μl chloroform to each tube, turn the eppendorf tube upside down by hand for 15 seconds, and let it stand at room temperature for 10 minutes

3) 4°C, 12000rpm, centrifuge for 15min

4) Transfer the supernatant to a new 1.5ml eppendorf tube, add an equal volume of pre-cooled isopropanol, mix well and precipitate at 4°C for 10min

5) Centrifuge at 12000rpm for 10min at 4°C, remove the supernatant

6) Add at least 1ml of 75% ethanol (freshly prepared with DEPC-treated water) to wash the precipitate

7) Centrifuge at 10,000 rpm for 5 minutes at 4°C, discard most of the supernatant

8) Centrifuge again at 10,000rpm at 4°C for 5min, and suck off the supernatant

9) Dry at room temperature

10) When the RNA is basically transparent, add 20 μl RNase-free water until it is completely dissolved, and measure the concentration of the extracted RNA by UV analysis. the

2. RNA reverse transcription to obtain cDNA

(According to Promega's M-MLV operating instructions) M-MLV reverse transcriptase and dNTPs were purchased from Promega, Oligo dT was purchased from Shanghai Sangong, and RNase-free items were purchased from Axygen. The specific steps are as follows:

1) Add 1μl Oligo dT (0.5μg/μl) and 2.0μg Total RNA to a small PCR tube, add DEPC-H ₂ O to 9ul; mix well, centrifuge, and incubate at 70°C for 10min; immediately place in ice water at 0°C Ice bath in mixture to anneal OligodT and template

2) Prepare the reaction system according to the ratio in the table below (on ice), mix well, and briefly centrifuge

Note: dNTPs is a mixture of dATP, dCTP, dGTP, dTTP, the concentration is 10mM

3) The above system was reacted at 42°C for 1 hour, and then bathed in a water bath at 70°C for 10 minutes to inactivate the RT enzyme

4) Store the obtained RT product--cDNA at -80°C for future use

3. Real-time PCR detection

1) Configure the reaction system according to the following ratio:

2) Set the program as two-step Real-Time PCR: pre-denaturation at 95°C, 15S; subsequent denaturation at 95°C, 5S for each step;

Annealing extension 60 ℃, 30S; a total of 45 cycles. Absorbance readings each time during the extension phase

Cycle1:(1X)

Step1:95.0℃for00:15. 

Cycle2:(45X)

Step1:95.0℃for00:05. 

Step2:60.0℃for00:30. 

Data collection and real-time analysis enabled

3) Make a melting curve: After PCR, denature at 95°C for 1 min, then cool to 55°C to fully bind the DNA double strands. From 55°C to 95°C, increase by 0.5°C in each step, keep for 4S, and read the absorbance value at the same time

Cycle3:(1X)

Step1:95.0℃for01:00. 

Cycle4:(1X)

Step1:55.0℃for01:00. 

Cycle5:(81X)

Step1:55.0℃-95.0℃for00:04. 

Increase set point temperature after cycle2by0.5℃

Quantitative PCR results are shown in Table 3-4 and Figure 12:

Table 3 Quantitative PCR results

Explanation: ΔCt=hsa-miR-302aCt value-U6Ct value

-ΔΔCt=average value of ΔCt in NC group-ΔCt of each sample

2-ΔΔCt reflects the relative expression level of hsa-miR-302a in each sample relative to the CON group samples

"—" means that the instrument has no reading value because the expression abundance of the target gene in the cell is too low

It is normal for duplicate wells to have poor repeatability due to low expression of the target gene (Ct>31)

Table 4 Quantitative PCR statistical results

As can be seen from the quantitative PCR results, compared with the control group, in the cells containing the lentivirus constructed by the present invention, the expression level of the miR-302a gene is more than 23 times that of the former (P<0.05),

As can be seen from the quantitative PCR results, in human 293T cells, relative to the control group, the expression levels of hsa-miR-302a, hsa-miR-302b, hsa-miR-302c, and hsa-miR-302d genes of the recombinant plasmid of the present invention It can reach more than 23 times of NC (P<0.05). the

(6) Expression detection of microR302s in SK-N-SH cells after transfection and overexpression of microR-302s

1. Experimental method

The lentivirus constructed in step (3) was used to infect SK-N-SH cells, and the expression of miR-302a in SK-target virus-transfected cells and SK-me control cells was detected. the

Transfection:

Preliminary experiments indicated that under optimal conditions (in Eni.S Enhanced Infection Solution (pH7.4)), MOI=10 is required to achieve 80% infection efficiency. the

Transfection steps:

1. Ensure that the cells are in a good growth state before the experiment;

2. Inoculate 2-3× ¹⁰⁵ cells/well in a 6-well culture plate the day before the experiment, add a medium volume of 2ml, after 24 hours of cultivation, the cells enter the logarithmic growth phase, adhere to the wall and grow well, and start transfection ;

3. On the day of the experiment, gently wash the cells twice with mild PBS, add transfection enhancement solution (provided by Enhancement Solution Jikai Company), dilute the target virus according to the design requirements of MOI=10, add the transfection solution, and mix gently Uniformly; add empty virus or blank transfection solution to the control group (the fusion rate of the cells during virus infection is about 30-50%, and the density is 20-40%);

4. After 8-12 hours of transfection, discard the virus transfection solution, gently wash the cells twice with mild PBS, and replace with normal culture medium;

5. Culture until 24 hours after transfection, wash the cells again, and replace with fresh normal culture medium;

6. Cultivate until 48h and 72h after transfection, harvest the cells, and carry out downstream experiments. One experiment requires 32 wells (12 control wells, 20 purpose wells, each divided into two infection conditions). The fusion rate of cells during virus infection is about 30-50%, and the density is 20-40%. the

2. Experimental results

The experimental results are shown in Figures 13-17 and Tables 5-8:

Table 5 qPCR detection of microRNA-302a expression 48h after transfection

the SK-N-SH normal control group Lentiviral transfection group containing miR-302s relative expression 1 166.95 the 1 156.3

[0332] the 1 152.73 the the the average 1 158.66 the the the standard deviation 0 7.397925385

Table 6 qPCR detection of microRNA-302b expression 48h after transfection

the SK-N-SH normal control group Lentiviral transfection group containing miR-302s relative expression 1 23.97 the 1 22.62 the 1 26.11 the the the average 1 24.23333333 the the the standard deviation 0 1.759839008

Table 7 qPCR detection of microRNA-302c expression 48h after transfection

the SK-N-SH normal control group Lentiviral transfection group containing miR-302s relative expression 1 36.75 the 1 37.22 the 1 40.78 the the the average 1 38.25 the the the standard deviation 0 2.203610673

Table 8 qPCR detection of microRNA-302d expression 48h after transfection

the SK-N-SH normal control group Lentiviral transfection group containing miR-302s relative expression 1 14.25 the 1 12.22 the 1 15.24 the the the average 1 13.90333333 the the the standard deviation 0 1.539556213

The experimental results show that after transfection of the lentivirus of the present invention, the expression levels of microRNA-302a, microRNA-302b, microRNA-302c, and microRNA-302d in the cells are increased, indicating that the present invention has constructed a recombinant lentivirus containing the target gene microR-302s. the

Example 3 Effect of microR-302s on tumor cells

1. Experimental method

The recombinant lentivirus containing the target gene microR-302s constructed in Example 2 was used to infect SK-N-SH cells (human neuroblastoma cell line)

(1) Transfection

Preliminary experiments indicated that under optimal conditions (in Eni.S Enhanced Infection Solution (pH7.4)), MOI=10 is required to achieve 80% infection efficiency. the

Transfection steps:

1. Ensure that the cells are in a good growth state before the experiment;

2. Inoculate 2-3× ¹⁰⁵ cells/well in a 6-well culture plate the day before the experiment, add a medium volume of 2ml, after 24 hours of cultivation, the cells enter the logarithmic growth phase, adhere to the wall and grow well, and start transfection ;

3. On the day of the experiment, gently wash the cells twice with mild PBS, add transfection enhancement solution (provided by Enhancement Solution Jikai Company), dilute the target virus according to the design requirements of MOI=10, add the transfection solution, and mix gently Uniformly; add empty virus or blank transfection solution to the control group (the fusion rate of the cells during virus infection is about 30-50%, and the density is 20-40%);

4. After 8-12 hours of transfection, discard the virus transfection solution, gently wash the cells twice with mild PBS, and replace with normal culture medium;

5. Culture until 24 hours after transfection, wash the cells again, and replace with fresh normal culture medium;

6. Cultivate until 48h and 72h after transfection, harvest the cells, and carry out downstream experiments. One experiment requires 32 wells (12 control wells, 20 purpose wells, each divided into two infection conditions). The fusion rate of cells during virus infection is about 30-50%, and the density is 20-40%. the

(2) Hoechst33342 staining

Add an appropriate amount of Hoechst33342 staining solution, which must fully cover the sample to be stained;

Incubate at a temperature suitable for cell culture for 20-30 minutes;

Discard the staining solution, wash with PBS or culture medium for 2-3 times to perform fluorescence detection. the

(3) Flow cytometry detection

In this experiment, ANNEXIN V-FITC Apoptosis Kit from BD Company was used, including (ANNEXIN V-FITC and PI10X Binding Buffer). This experiment can distinguish early apoptosis, late apoptosis and dead cells. the

1. After digesting the cells, wash them twice with mild PBS, suspend the cells in 1X binding buffer, and adjust the concentration to 1×10 ⁶ /ml;

2. Take 100ul, 1× ¹⁰⁵ cell number, fully mix the cell suspension and label it in the flow reagent tube for use;

3. Add 5ul Annexin V-FITC and 5ul PI, mix gently;

4. Add 400ul Binding Buffer, incubate at room temperature in the dark for 15 minutes, and immediately test on the machine. the

(4) Tunel staining detection

1. Fix air-dried cell samples with fresh fixative solution for 1h15-25℃;

2. Rinse twice with PBS;

3. Draw a circle with a protein marker and incubate in blocking solution for 10 minutes at 15-25°C;

4. Rinse twice with PBS;

5. Add cell permeabilization solution and incubate for 2 minutes (on ice);

6. Rinse twice with PBS;

7. After the slides are dry, add 50 μl of TUNEL reaction mixture (the negative control group only adds 50 μl of fluorescein-labeled dUTP solution) on the specimens, add a cover glass or a parafilm and react in a dark and humid box at 37°C for 1h. the

8. Rinse with PBS 3 times;

9. Add 1 drop of PBS to count apoptotic cells under a fluorescence microscope (excitation light wavelength is 450-550nm, detection wavelength is 515-565nm.

Apoptotic cells (200-500 cells in total) were observed and photographed with an optical microscope. The morphological characteristics of apoptotic cells can be combined to make a comprehensive judgment (unstained cells become smaller, the cell membrane is complete but foaming occurs, apoptotic bodies appear in the late stage, adherent cells shrink, become round, and fall off; while stained cells show staining chromatin condensation, marginalization, nuclear envelope cleavage, chromatin segmentation into clumps/apoptotic bodies)

(5) Changes of related indicators in the apoptosis process of SK cells after transfection and overexpression of miR-302s

The SK-N-SH cell line was continuously transfected for 24 hours, and the expression of Mcl-1, CCND-1, CASP3, BAX, etc. was detected by qPCR at the time point of 48 hours after transfection. the

The SK-N-SH cell line was continuously transfected for 24 hours. At 48 hours and 72 hours after transfection, the expression of BMI-1, p16Ink4a, p53, etc. was detected by qPCR. the

The SK-N-SH cell line was continuously transfected for 24 hours, and the expression of Mcl-1, Caspase3Cleaved, BMI-1, p16Ink4a, p53, etc. was detected by Western Blotting at 48h and 72h after transfection. the

2. Experimental results

(1) Hoechst33342 staining results

The results of the Hoechst33342 staining experiment are shown in Figure 18, and the apoptosis results are shown in Figure 19 and Table 9:

Table 9 Apoptosis results detected by Hoechst33342 staining 48h after infection of SK-N-SH cells

From the results of Hoechst33342 staining, it can be seen that the apoptosis of SK-N-SH cells in the microR-302s virus group was significantly higher than that in the normal control group, indicating that the transfer of microR-302s into tumor cells can induce tumor cell apoptosis. the

(2) Flow cytometry test results

The test results of flow cytometry are shown in Figures 20-21 and Tables 10-11:

Table 10 The change of apoptosis rate of SK-N-SH cells at 48 hours after miR-302s overexpression lentivirus transfection detected by cell loss technique

Early apoptosis rate of cells 48h after transfection (%) normal control group Empty virome lentiviral transfection group the 5.9 5.6 25.8 the 5.3 6 24.4 the 5.2 6.2 twenty three average 5.466666667 5.933333333 24.4 the 0.37859389 0.305505046 1.4

Table 11 The change of apoptosis rate of SK-N-SH cells at 72 hours after miR-302s overexpression lentivirus transfection detected by cell loss technique

the 2 5.6 30.7 the 2.7 5.9 35.8 the 2.5 6 34.9 average 2.4 5.833333333 33.8 standard deviation 0.360555128 0.2081666 2.722131518

According to the results of flow cytometry, it can be seen that the apoptosis of SK-N-SH cells in the microR-302s virus group was significantly higher than that in the normal control group and the empty vector virus group, indicating that the transfer of microR-302s into tumor cells can induce Tumor cell apoptosis. the

(3) Tunel staining test results

The results of the Tunel staining experiment are shown in Figure 22, and the apoptosis results are shown in Figure 23 and Table 12:

Table 12 Apoptosis results detected by Tunel staining 48h after infection of SK-N-SH cells

It can be seen from the results of Tunel staining that the apoptosis of SK-N-SH cells in the microR-302s virus group was significantly higher than that in the normal control group, indicating that the transfer of microR-302s into tumor cells can induce tumor cell apoptosis. the

(4) Changes of related indicators in the apoptosis process of SK cells after transfection and overexpression of miR-302s

The changes of relevant indicators in the apoptosis process of SK cells after transfection and overexpression of miR-302s are shown in Figures 24-32 and Tables 13-15:

Table 13 Comparison of expression of BMI-1 in SK normal nuclei miR-302s transfected cells

serial number normal group 48h after miR302s group transfection 72h after miR302s transfection 1 1 0.511686946 0.392292049 2 1 0.567752215 0.337587487 3 1 0.466516496 0.370274388 4 1 0.515318552 0.366717975 average the 0.050715472 0.02752514

Table 14 Comparison of expression of p16Ink4a in SK normal nuclei miR-302s transfected cells

serial number normal group 48h after miR302s group transfection 72h after miR302s transfection 1 1 2.143546925 4.924577653 2 1 2.094588246 3.563594873 3 1 2.193649959 4.093495568 4 1 2.143928377 4.193889365 average the 0.049531958 0.686023118

Table 15 Comparison of expression of p53 in SK normal nuclei miR-302s transfected cells

serial number normal group 48h after miR302s group transfection 72h after miR302s transfection 1 1 1.721102874 2.795939868 2 1 1.977028041 2.351095813 3 1 1.844632387 2.60870414 4 1 1.847587767 2.585246607 average the 0.127988177 0.223347824

The experimental results showed that in the SK-N-SH cells containing the microR-302s virus group, the indicators related to cell apoptosis were significantly higher than those in the normal control group, indicating that the transfer of microR-302s into tumor cells can induce tumor cell apoptosis. the

The above various detection results show that the apoptosis of SK-N-SH cells in the virus group containing microR-302s (nucleotide sequence shown in SEQ ID NO.5) is significantly higher than that in the normal control group, indicating that in tumor cells Introducing microR-302s can induce tumor cell apoptosis, and microR-302s can be used to prepare antitumor drugs. the

Example 4 In vivo tumor suppression experiment

1. Materials and methods:

1.1 Experimental animals

Ten Balb/c nude mice, male, of the same age, all 4-6 weeks old, were purchased from Dashuo Biotechnology Co., Ltd., Chengdu, Sichuan. Breeding and Animal Experiment Center of West China Hospital of Sichuan University, rearing in a laminar flow purification room, cages, bedding, water and food are all sterilized, SPF grade feeding for one week, and the body weight before inoculation is between 16 and 20g. the

1.2 Cell lines

The human neuroblastoma line SK-N-MC was purchased from the Medical Cell Center of the Institute of Basic Medical Sciences, Peking Union Medical College, China. It was cultured in MEM with 10% fetal bovine serum and double antibodies at 5% CO2, 37°C, and saturated humidity. in the liquid. When the cells grow to about 80% confluence, they are digested with 0.25% trypsin, and passaged at 1:2 or 1:3. All experimental cells were in logarithmic growth phase. the

2. Main experimental reagents

microRNA320a, microRNA320b, microRNA320c, microRNA320d agomir (double-stranded RNA), Negative Control agomir (Guangzhou Ruibo Biotechnology Co., Ltd.)

miR-302a:CCACCACUUAAACGUGGAUGUACUUGCUUUGAAACUAAAAGAAGUAAGUGCUUCCAUGUUUUGGUGAUGG;

miR-302b:GCUCCCUUCAACUUUAACAUGGAAGUGCUUUCUGUGACUUUAAAAGUAAGUGCUUCCAUGUUUUUAGUAGGAGU;

miR-302c:CCUUUGCUUUUAACAUGGGGGUACCUGCUGUGUGAAACAAAAGUAAGUGCUUCCAUGUUUCAGUGGAGG;

miR-302d: CCUCUACUUUAACAUGGAGGCACUUGCUGUGACAUGACAAAAAAUAAGUGCUUCCAUGUUUUGAGUGUGG. the

Negative Control: the name is cel-miR-239b-5p, miRNA number:

MIMAT0000295, sequence: UUUGUACUACACAAAAGUACUG

HyClone MEM medium (Thermo Scientific, USA)

HyClone Fetal Bovine Serum (Thermo Scientific, USA)

3. Main experimental instruments

3.1. Constant temperature cell incubator (Thermo Scientific, USA)

3.2. Research biological microscope (Olympus, Japan)

3.3 Vernier caliper (Shanghai Shenhan Measuring Tools Co., Ltd.)

3.4. Electronic scale (Germany Sartorius company)

4. Experimental method

4.1 Cell culture

4.11. Liquid for cell culture

(1) 10×PBS buffer (cell _culture , pH7.2～7.4): KCl2.0g+KH2PO42.0g+NaCl80.0g+ _{NaHPO4.12H2O20.8g} , add _ddH2O to 1000ml, fully dissolve, Sterilize at high temperature and store at -20°C. Dilute 10 times when used.

(2) Trypsin cell digestion solution: trypsin 0.5g + 1×PBS buffer 200ml + EDTA 0.02g, dissolve for 1-2 hours, adjust the pH value to 7.5, filter with a 0.22μm filter, aliquot, and store at -20°C. the

(3) Penicillin (PS) solution: 1 million U penicillin (1 g) + 1 million U streptomycin, dissolved in 100 ml ddH ₂ O, filtered through a 0.22 μm filter, aliquoted, and stored at -20°C.

(4) L-glutamine solution: Dissolve 3.0g of L-glutamine in 100ml of ddH2O, filter through a 0.22μm filter, subpackage, and store at -20°C. Dilute 100 times when used. the

(5) NaHCO ₃ solution (5.6%): 5.6g NaHCO ₃ was dissolved in 100ml ddH2O, filtered through a 0.22 μm filter or sterilized at high temperature, aliquoted, and stored at -20°C.

(6) HEPES solution (stock solution 1500-2000mmol/L): 36-50g HEPES dissolved in 100ml ddH=O, filtered through a 0.22μm filter, aliquoted, and stored at -20°C. Dilute 100 times when used (final concentration is 15-20mmol/L). the

(7) MEM culture medium: MEM85ml + fetal bovine serum (FBS) 10ml + L-glutamine 1ml + penicillin streptomycin 1ml, adjust pH to 7.2 with NaHCO ₃ , store at 4°C.

4.12. Cell Recovery

4.13. Cell passage

4.2. Establishment and treatment of subcutaneous xenograft model of human neuroblastoma SK-N-MC in nude mice

4.2.1 Transplanted tumor model

Take the SK-N-MC cells in the logarithmic growth phase, digest them with trypsin, make a PBS single cell suspension, adjust the cell density to 5×10 ⁷ /ml, and inoculate them subcutaneously in the right armpit of nude mice, each time The volume of injection liquid is 0.2ml, and the subcutaneous nodule diameter is about 5mm as the standard for tumor formation, and the drug injection is carried out.

4.2.2 Experimental grouping and treatment intervention

Observe the growth of transplanted tumors. On the 10th day, the average diameter of transplanted tumors is greater than 5mm. Weigh them before administration, number them from small to large, and use the first ten numbers in line 8 of the random number table, 23, 68, 35, 26, 00, 99, 53, 93, 61, 28. Arrange the random numbers from 1 to 10, assign the nude mice corresponding to 1 to 5 to the control group, and assign the nude mice corresponding to 6 to 10 to the experimental group. Then the size of the tumors in each group was measured. Before the interference, the two groups of nude mice grew in good condition, and there was no statistically significant difference in calculated body weight (as shown in Figure 33) and tumor volume (as shown in Figure 34), and all indicators had no statistical significance. The two groups were treated as follows: Control group: intratumoral multi-point injection of negative control agomir, 4nmol/mole, dissolved in 30ul normal saline, once every four days. Experimental group: 1 nmol each of microRNA320a, microRNA320b, microRNA320c, and microRNA320d agomir were injected into the tumor at multiple points, a total of 4 nmol per mouse, dissolved in 30ul normal saline, once every four days. the

4.2.3 General observation and tumor measurement

During the administration period, the mental state, behavior, diet changes and flexibility of the nude mice in each group were observed every day. The growth of the tumor was detected, and the long diameter (L) and short diameter (W) of the subcutaneous tumor were measured with a vernier caliper every three days, and the tumor volume was calculated, and the tumor growth curve was drawn. At the same time, the body weight of the nude mice was weighed every three days. the

Tumor volume (V) = (LW ² )/2

4.2.4 Nude mice were killed

Four days after the last drug injection, the nude mice were weighed, the volume was measured and the growth multiple of the tumor volume was calculated. The two groups of nude mice were killed after anesthesia, and the transplanted tumors were completely removed with surgical instruments. Calculate the tumor inhibition rate. the

Tumor inhibition rate=(1-average tumor weight of experimental group/average tumor weight of control group)×100%

4.4 Statistical analysis

SPSS16.0 software was used for statistical analysis. All data are presented as mean ± standard deviation. The comparison between two samples was performed by t test. Two-sided α=0.05 is the test standard, and P<0.05 is considered statistically significant. the

5. Experimental results

1. Observation on growth of nude mice and observation of toxic and side effects during the intervention process

During the intervention period, the tumor-bearing nude mice in the two groups grew well, and none of them died. No toxic effect was found during the intervention process. They had a normal diet, a good mental state, and a good activity state. the

2. Tumor formation in nude mice

After 10 nude mice were inoculated with cells, subcutaneous nodules formed on the fifth day and grew gradually, with a tumor formation rate of 100%. Drug injections were given on the 10th day. The transplanted tumors were round or oval. Four days after the last drug injection, all the nude mice were killed, and the transplanted tumors were taken out. After dissection, no lymph node metastasis was found, and no abnormality was found in each organ. HE staining of tumor tissue was confirmed as neuroblastoma (NB). the

3. Balance of tumor volume and body weight of two groups of tumor-bearing nude mice before grouping

The tumor volume and body weight of the two groups of tumor-bearing nude mice before intervention (as shown in Figures 33 and 34) had no statistically significant difference by t test (body weight: F=0.502P=0.775; tumor volume: F=0.001P=0.359), The tumor-bearing nude mice in the two groups were in good growth condition. It indicated that the two groups of tumor-bearing nude mice were balanced before the intervention. the

4. Growth status of transplanted tumor in nude mice

During the observation process, it was found that the tumor growth in the experimental group was significantly slower than that in the control group after the intervention. The growth curves of transplanted tumors are shown in Figure 41 and Figure 42. the

The weight before sacrifice was shown in Figure 35 (control group: 23.38±1.06g, experimental group: 23.62±1.28g), and there was no significant difference in average body weight between the two groups (F=1.427P=0.718). the

Comparison of transplanted tumors in vivo before sacrifice is shown in Figure 37 and Figure 38. The tumor growth of the tumor-bearing nude mice in the experimental group was significantly slower than that in the control group. The comparison of tumor volume between the two groups measured before sacrifice is shown in Table 36 (control group: 2520.37±738.94mm3, experimental group: 968.08±708.49mm3), and the difference was statistically significant (F=0.058P=0.009). the

Two groups of transplanted tumors were obtained after being executed, as shown in Figure 40, the tumors of the tumor-bearing nude mice in the experimental group were significantly smaller than those of the control group; the transplanted tumors of the two groups were weighed, and the tumor weights were compared as shown in Figure 39 (the control group 2.03±0.91g, the experimental group group (0.81±0.75g), the difference was statistically significant (F=0.037P=0.048), tumor inhibition rate=1-0.81/2.03=60.10%. the

The growth rate of transplanted tumors is shown in Figure 43, and the experimental group was significantly lower than the control group (P<0.05). the

In this experiment, the human neuroblastoma cell line SK-N-MC cell suspension was inoculated subcutaneously in the right axilla of nude mice, and a nude mouse xenograft tumor model of human neuroblastoma was successfully constructed. With microRNA320a, microRNA320b, microRNA320c and microRNA320d as drug intervention, multi-point intratumoral injection was performed, and Negative Control was used as a negative control. After four times of administration, there was no statistical difference in the average body weight of nude mice between the two groups. However, the growth of transplanted tumors in the experimental group was significantly slower than that in the control group, and even the transplanted tumors shrank. And weighing the weight of the transplanted tumor showed that the weight of the transplanted tumor in the experimental group was significantly smaller than that in the control group, the difference was statistically significant, and the tumor inhibition rate was 60.1%. the

The experimental results show that microRNA320a, microRNA320b, microRNA320c and microRNA320d (nucleotide sequences shown in SEQ ID NO.1-4) have anti-tumor effects, and can be used to prepare anti-tumor drugs and treat tumors such as neuroblastoma. the

Example 5 Screening kit of the present invention

1. Kit of the present invention (50 servings)

(1) Total RNA extraction reagents

components concentration volume Trizol - 100ml Chloroform - 20ml Isopropanol - 50ml ethanol 75% 100ml DEPC water - 50ml

(2) Reverse transcription reagents

components concentration volume Oligo dT 10mmol/L 100ul 5×RT buffer 5X 200ul Rnasin 5U/ul 50ul M-MLV-RTase 5U/ul 50ul DEPC water - 300ul Stem Loop Primer 50mmol/L 50ul random primer 0.05ug/ul 50ul

(3) Real-time PCR detection reagent

components concentration volume PCR Buffer 10X 300ul MgCl ₂ 25mM 300ul dNTP 2.5mM 360ul upstream primer 20uM 50ul Downstream Universal Primer 20uM 50ul TaqMan probes 10uM 100ul Taq enzyme 5U/ul 30ul ddH2O - 2ml

Upstream primer:

MiR302a3PF:5'AAGTGCTTCCATGTTTTCCT3'Tm(JS)56

MiR302b3PF:5'AAGTGCTTCCATGTTTTAGT3'Tm(JS)54

MiR302c3PF:5'AAGTGCTTCCATGTTTCAGT3'Tm(JS)56

MiR302d3PF:5'GTGCTTCCATGTTTGAGTGT3'Tm(JS)58

Downstream universal primer: 5'-CAGTGCTGGGTCCGAGTGA-3'

TaqMan probe: 5'-FAM-TCGTATCCAGTGCGAATCACTC-TAMRA-3'

2. The method of using the kit of the present invention

(1) Total RNA extraction of cell lines (Trizol method);

(2) Reverse transcription reaction (two-step stem-loop reverse transcription stem-loop method: RT1: 70°C for 5 minutes and ice bath immediately; RT2: 16°C for 30 minutes, 42°C for 30 minutes, 85°C for 5 minutes);

(3) Real-time fluorescence quantitative PCR (Taqman) probe PCR technology (94°C 3min pre-denaturation; two-step method: 94°C 20S, 60°C 40S);

(4) Data statistics and analysis. the

Example 6 Screening kit of the present invention

1. Kit of the present invention (50 servings)

(1) Total RNA extraction reagents

components concentration volume Trizol - 100ml Chloroform - 20ml Isopropanol - 50ml ethanol 75% 100ml DEPC water - 50ml

(2) Reverse transcription reagents

components concentration volume Oligo dT 10mmol/L 200ul 5×RT buffer 5X 400ul Rnasin 5U/ul 100ul M-MLV-RTase 5U/ul 100ul DEPC water - 600ul random primer 0.05ug/ul 100ul

(3) Real-time PCR detection reagent

components concentration volume PCR Buffer 10X 250ul MgCl ₂ 25mM 250ul dNTP 2.5mM 300ul BMI-1 upstream primer 20uM 40ul BMI-1 downstream primer 20uM 40ul SYBGREEN dye 10uM 15ul Glycerin - 125ul Taq enzyme 5U/ul 40ul ddH ₂ O - 2ml

BMI-1 upstream primer:

hBMI1F-1:5'GAAATCTAAGGAGGAGGTGA3'456Tm(P5)50.8,Tm(JS)58

BMI-1 downstream primer:

hBMI1R-1:5'catacatgacatcaatctgga3'

586Tm (P5) 50.9, Tm (JS) 56131bp. the

2. The method of using the kit of the present invention

(1) Total RNA extraction of cell lines (Trizol method), the same as the previous RNA extraction kit;

(2) Reverse transcription reaction (ordinary reverse transcription reaction, two-step method: RT1: 70°C for 5 minutes and ice bath immediately; RT2: 20°C for 10 minutes, 40°C for 60 minutes, 70°C for 10 minutes);

(3) Real-time fluorescent quantitative PCR technology (SYBGREEN fluorescent dye method 94°C 3min pre-denaturation; three-step method 40 cycles: 94°C 30S, 62°C 40S, 72°C 1min; 72°C 5min);

(4) Data statistics and analysis. the

In summary, the expression level of microR-302a in tumor cells is significantly lower than that in normal cells, and whether the cells to be tested are tumor cells can be screened by detecting the expression level of microR-302a. At the same time, the five nucleotide sequences of the present invention can effectively induce tumor cell apoptosis, inhibit tumor proliferation and treat tumors. the

Claims (14)

1. Nucleotide sequence shown in 1.SEQ ID NO.1 ~ 5.
2. Any one or any number of sequences in nucleotide sequence shown in 2.SEQ ID NO.1 ~ 5 are preparing the purposes in antitumor drug.
3. 3. purposes according to claim 2, is characterized in that: described antitumor drug is anti-neuroma medicine.
4. 4. purposes according to claim 3, is characterized in that: described anti-neuroma medicine is anti-neuroblast tumor medicine.
5. 5. a recombinant plasmid, is characterized in that: it comprises the nucleotide sequence shown in any one of SEQ ID NO.1 ~ 5.
6. 6. recombinant plasmid according to claim 5, is characterized in that: it is restructuring GV266 plasmid.
7. 7. a recombinant slow virus, is characterized in that: it comprises the nucleotide sequence shown in any one of SEQ ID NO.1 ~ 5.
8. 8. a recombinant slow virus, is characterized in that: it comprises recombinant plasmid described in claim 5 or 6.
9. 9. recombinant plasmid described in claim 5 ~ 8 or recombinant slow virus are preparing the purposes in antitumor drug.
10. 10. purposes according to claim 9, is characterized in that: described antitumor drug is anti-neuroma medicine.
11. 11. purposes according to claim 10, is characterized in that: described anti-neuroma medicine is anti-neuroblast tumor medicine.
12. 12. 1 kinds of tumor screening test kits, is characterized in that: it comprises the reagent detecting cell microRNA302a expression level.
13. 13. test kits according to claim 12, is characterized in that: described tumour is neuroma.
14. 14. test kits according to claim 13, is characterized in that: described neuroma is neuroblastoma.