CN106636200B - A kind of the RNA interference plasmid and its application method of ZNF667 albumen - Google Patents

Abstract

The invention discloses a kind of short hairpin RNA interference plasmid of ZNF667 albumen and its application methods.Short hairpin RNA (ZNF667-shRNA) interference plasmid of ZNF667 albumen is successfully constructed for the first time, is transfected into hepatoma H22 cells, and the cell strain of stable transfection ZNF667-shRNA plasmid is constructed.It was found that the proliferation of HepG2 cell, infiltration, invasion migration can be inhibited by cutting down the ZNF667 albumen in HepG2 cell.Tumor formation in nude mice confirms that the expression for cutting down ZNF667 in HepG2 cell can inhibit tumor formation from body level.Cut down the expression of the ZNF667 albumen in HepG2 cell, it may be possible to by promoting P53, inhibit Bcl-2 albumen to reach the grade malignancy for inhibiting HepG2 cell.The present invention will provide new thread and approach for the treatment of liver cancer, be of great significance.

Description

A kind of RNA interference plasmid of ZNF667 protein and its application method

technical field

The invention belongs to the technical field of tumor molecular biology, and in particular relates to a short hairpin RNA interference plasmid of ZNF667 protein and an application method thereof.

Background technique

ZNF667 is an up-regulated gene found in rat myocardial ischemic preconditioning. Through comparison with the gene bank, it is found that there is no corresponding sequence. It is a brand-new gene, which is named ZNF667 and registered in Genbank The number is AY221750. Through bioinformatics analysis, it was found that the corresponding protein was a KRAB-type zinc finger protein, which contained 14 zinc finger structures and had the function of transcriptional regulation.

The ORF of ZNF667 is 1827bp, encoding 608 amino acids. The current research results on ZNF667-related functions are as follows: (1) ZNF667 is a newly discovered KRAB-type zinc finger protein, which can inhibit the transcriptional activity of SRE and AP-1, and has a transcriptional inhibitory effect; (2) ZNF667 is localized in the nucleus, It is mainly expressed in heart and brain tissue, followed by liver, kidney, skeletal muscle, and testis, and less in other tissues; (3) ZNF667 gene is expressed under the conditions of ischemia, hypoxia, inflammation, and oxidative stress (4) ZNF667 promotes cell growth under stress; (5) ZNF667 has an anti-apoptotic effect on cardiomyocytes, and H ₂ O ₂ treated H ₉ C ₂ cardiomyocytes in the transient transduction ZNF667 can significantly inhibit the expression of Fas and other proteins after transfection, and this anti-apoptotic effect is related to the direct combination with Fas promoter to inhibit its expression; ZNF667 can inhibit the apoptosis caused by TNF-ɑ, and this anti-apoptotic effect Apoptosis is also related to directly binding to the Bid promoter and inhibiting its expression. (6) Overexpression of ZNF667 can inhibit the expression of Bax and P53. Functional research on ZNF667 shows that with the increase of the pathological grade of brain astrocytoma, the content of ZNF667 mRNA and protein is on the rise, especially in highly malignant grade III and IV brain astrocytoma, the content is significantly increased . However, there is no research on the expression of ZNF667 in other cancers, and the use of ZNF667 short hairpin RNA interference plasmids to treat cancer.

The present invention uses the ZNF667 hairpin interfering RNA (shRNA) plasmid to transfect the HepG2 liver cancer cell line to establish a ZNF667 stably reduced liver cancer cell line. Through research, it is found that the HepG2-ZNF667-shRNA cell line exhibits slow cell proliferation and clone formation The rate is reduced, and the ability of infiltration and invasion is reduced. Tumor formation experiments in nude mice confirmed that inhibiting the expression of ZNF667 in HepG2 cells can inhibit the tumorigenesis of HepG2 cells in vivo. The present invention proves that reducing the expression of ZNF667 protein in HepG2 liver cancer cells can inhibit the malignancy of HepG2 cells and play a therapeutic role from the level of cell function and in vivo animal experiments.

Contents of the invention

The purpose of the present invention is to explore the effect of reducing the ZNF667 protein on the biological characteristics of HepG2 cells by constructing a hairpin short hairpin RNA interference plasmid of the ZNF667 protein, and then use this plasmid for the treatment of inhibiting liver cancer.

A short hairpin RNA interference plasmid of the ZNF667 protein is designed according to the sequence of ZNF667 to produce a DNA fragment capable of producing siRNA that interferes with the expression of the ZNF667 protein, and inserted into the short hairpin RNA interference vector to construct.

The above-mentioned plasmid uses the mRNA sequence of human ZNF667 as a template, and the sequence requirements of the vector promoter to initiate the expression of siRNA, and designs and synthesizes a DNA fragment capable of producing siRNA that interferes with the expression of ZNF667 protein.

The target gene ZNF667 sequence of siRNA interference is as follows:

siRNA-1: CGAATATCTCTCACACGACAT;

siRNA-2: CGCCAATCATTTCTTATTGAA;

siRNA-3: CAACCCTTATTCTGCATCTAA.

Preferably siRNA-2: CGCCAATCATTTCTTATTGAA

The original plasmid expression vector selected for the above plasmids is preferably pRNAT-U6.1/Neo.

When the DNA fragment is inserted into the vector, the restriction sites are BamH I and Hind III.

According to the sequence design of ZNF667, a DNA fragment capable of producing siRNA that interferes with ZNF667 protein expression is designed, and the specific sequence is as follows:

ZNF667-shRNA-1a chain sequence:

5'-GATCCCCGAATATCTCTCACACGACATCTCGAGATGTCGTGTGAGAGATATTCGTTTTTGGAT-3'

ZNF667-shRNA-1b chain sequence:

5'-AGCTATCCAAAAACGAATATCTCTCACACGACATCTCGAGATGTCGTGTGAGAG ATATTCG GG-3'

ZNF667-shRNA-2a chain sequence:

5'-GATCCCCGCCAATCATTTCTTATTGAACTCGAGTTCAATAAGAAATGATTGGCG TTTTTGGAT-3'

ZNF667-shRNA-2b chain sequence:

5'-AGCTATCCAAAAAGCCAATCATTTCTTATTGAACTCGAGTTCAATAAGAAATGAT TGGCG GG-3'

ZNF667-shRNA-3a chain sequence:

5'-GATCCCCAACCCTTTATTCTGCATCTAACTCGAGTTAGATGCAGAATAAGGGTTG TTTTTGGAT-3'

ZNF667-shRNA-3b chain sequence:

5'-AGCTATCCAAAAACAACCCTTATTCTGCATCTAACTCGAGTTAGATGCAGAATAA GGGTTGGG-3'.

Control shRNA a-strand sequence:

5'-GATCCCTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA TTTTTGGAT-3'

Control shRNA b-strand sequence:

5'-AGCTATCCAAAAATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAAGG-3'.

The sequences of the three constructed ZNF667 short hairpin interference plasmid vectors are shown in SEQ NO: 17-19. Preferably see SEQ NO:18.

The application method of the short hairpin RNA interference plasmid of the ZNF667 protein is used for preparing a preparation for inhibiting liver cancer.

The invention selects and designs the interference sequence of ZNF667 through online bioinformatics software, and constructs the short hairpin RNA interference plasmid of ZNF667 according to the principle of RNA interference. Construction and screening of plasmids Short hairpin RNA interference plasmids containing ZNF667 siRNA sequences used ampicillin (ampicilin, Amp) resistance gene as a selection marker. HEPG2 cell lines stably transfected with ZNF667-shRNA and its blank control plasmid were constructed by neomycin (G418) selection method. MTT method was used to detect the proliferation of three groups of HepG2 cell lines transfected with interference plasmid ZNF667-shRNA, blank control plasmid, and blank cells, and the colony formation ability of the three groups of cell lines was monitored by clone formation assay. The migration ability of the three groups of cells was detected by the scratch test, and the infiltration ability was detected by the Transwell test. Tumor formation experiments in nude mice were used to examine the malignancy of the three groups of cells in vivo, and the expression of target molecules P53 and Bcl-2 were detected by Western blotting.

The result is that the present invention successfully constructed the ZNF667 hairpin interference plasmid (ZNF667-shRNA), and transfected it into HepG2 cells, and established a cell line stably transfecting the interference plasmid ZNF667-shRNA. MTT experiment data showed that the growth of cell lines transfected with ZNF667-shRNA was significantly slower than that of HepG2 and HepG2-ZNF667E (transfected blank control plasmid) cells; The group weakened; the scratch test showed that: compared with HepG2 blank cells, HepG2-ZNF667E (that is, the blank control plasmid cell line NC group), the migration distance of the cell line transfected with ZNF667-shRNA was significantly reduced at 24 hours and 48 hours The results of Transwell invasion assay showed that the cell invasion ability of the cell lines transfected with ZNF667-shRNA was weaker than that of HepG2 group and HepG2-ZNF667E group. Western blot results showed that the expression of P53 in cells transfected with ZNF667-shRNA was significantly higher than that of HepG2 and HepG2-ZNF667E cell lines, and the expression of Bcl-2 was significantly lower than that of HepG2 and HepG2-ZNF667E cell lines. This provides a new way and idea for the treatment of liver cancer.

Description of drawings

Fig. 1 is the sequencing result of ZNF667-shRNA-1a chain;

Figure 2 shows the sequencing results of the ZNF667-shRNA-2a chain;

Figure 3 shows the sequencing results of the ZNF667-shRNA-3a chain;

Fig. 4 is after HepG2 cell transfection blank control plasmid (NC), ZNF667-shRNA1 (Sh1), ZNF667-shRNA 2 (Sh2), ZNF667-shRNA3 (Sh3) plasmid, and blank cell protein expression detection band;

Fig. 5 is after HepG2 cell transfection blank control plasmid (NC), ZNF667-shRNA1 (Sh1), ZNF667-shRNA 2 (Sh2), ZNF667-shRNA3 (Sh3) plasmid, and blank cell protein expression detection reduction efficiency;

*Compared with blank cells, & compared with blank control plasmid group;

Figure 6 is the result of Western blot detection of ZNF667 protein in HepG2 cells stably transfected with ZNF667-shRNA, NC (blank control plasmid) and Con (blank cells);

Fig. 7 is the statistical analysis of ZNF667 protein expression in the HepG2 cell of stable transfection ZNF667-shRNA, NC (blank control plasmid) and Con (blank cell);

*Compared with blank cells, & compared with blank control plasmid group;

Fig. 8 is after HepG2 cell transfection ZNF667-shRNA plasmid, blank control plasmid (NC) and Con (blank cell) growth curve;

* compared with Con (blank cell), # compared with NC (blank control plasmid) group;

Figure 9 is a diagram of HepG2 cell transfection ZNF667-shRNA plasmid, blank control plasmid (NC) and Con (blank cell) cell clone formation;

Figure 10 is the statistical analysis of HepG2 cell transfection ZNF667-shRNA plasmid, blank control plasmid (NC) and Con (blank cell) cell clone;

Figure 11 is the TransWell experiment result of HepG2 cell transfection ZNF667-shRNA plasmid, blank control plasmid (NC) and Con (blank cell);

Figure 12 is the statistical analysis result of HepG2 cell transfection ZNF667-shRNA plasmid, blank control plasmid (NC) and Con (blank cell) TransWell experiment;

* compared with Con (blank cells), & compared with NC (blank control plasmid) group;

Figure 13 is the cell scratch experiment result of HepG2 cell transfection ZNF667-shRNA plasmid, blank control plasmid (NC) and Con (blank cell);

Figure 14 is the statistical analysis result of the cell scratch experiment of HepG2 cell transfection ZNF667-shRNA plasmid, blank control plasmid (NC) and Con (blank cell);

* compared with Con (blank cell), # compared with NC (blank control plasmid) group;

Figure 15 is Western blot detection of P53 and Bcl2, ZNF667 protein expression in HepG2 cells transfected with ZNF667-shRNA plasmid, blank control plasmid (NC) and Con (blank cells);

Figure 16 is Western blot detection of Bcl2 protein expression analysis results in HepG2 cells transfected with ZNF667-shRNA plasmid, blank control plasmid (NC) and Con (blank cells);

* compared with Con (blank cell), # compared with NC (blank control plasmid) group;

Figure 17 is Western blot detection of P53 protein expression analysis results in HepG2 cells transfected with ZNF667-shRNA plasmid, blank control plasmid (NC) and Con (blank cells);

* compared with Con (blank cell), # compared with NC (blank control plasmid) group;

Figure 18 is a graph showing the actual volume and growth curve of tumor formation in nude mice of HepG2 cells transfected with ZNF667-shRNA plasmid, blank control plasmid (NC) and HepG2 (blank cells);

Figure 19 is a statistical analysis of the tumor volume and weight of the nude mice with HepG2 cells transfected with ZNF667-shRNA plasmid, blank control plasmid (NC) and HepG2 (blank cells);

Figure 20 shows the tumor formation in nude mice of HepG2 cells transfected with ZNF667-shRNA plasmid, blank control plasmid (NC) and HepG2 (blank cells), the content detection and statistical analysis of ZNF667 protein in tumors.

Detailed ways

The following in conjunction with specific embodiments is intended to further illustrate the present invention, rather than limit the present invention.

Materials and methods:

Materials: Human liver cancer cell line HepG2 was purchased from the Cell Molecular Center of Central South University. The normal growth medium was 1640+10% FBS. The cells were long spindle-shaped and adhered to the wall.

Plasmid expression vector pRNAT-U6.1/Neo, full-length 6380bp (see SEQ NO.21 in the sequence listing), including green fluorescent protein (Geenfluorescent protein, GFP) as a detection marker, ampicillin (ampicilin, Amp) resistance gene As a prokaryotic screening marker, it is used to screen that shRNA fragments have been successfully cloned into the pRNAT-U6.1/Neo plasmid, and the neomycin (neomycin, also known as G418) resistance gene is used as a eukaryotic screening marker for screening Cells stably transfected with ZNF667-shRNA plasmid. Cloning restriction sites BamH I and Hind III. Ensure that the orientation of the insert is correct to prevent self-ligation of the vector. This vector was purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.

method:

1. Selection of ZNF667 interference sites and design of oligodeoxynucleotides

Search the complete mRNA sequence of human ZNF667 in the NCBI database, use it as a template, and refer to the design principles of siRNA. According to the sequence requirements of the siRNA expression by the interference vector pRNAT-U6.1/Neo promoter, the online siRNATatgetfinder software (http://genomics.jp/sidirect/) was used to design and synthesize three siRNA DNA fragments targeting the human ZNF667 sequence. The sequence obtained by online input ZNF667mRNA1 sequence (NM_022103.3) was analyzed, and the target gene sequence with GC content between 40% and 55% was selected as a potential optimization, and searched with BLAsT in the GenBank Expressed Sequence Tag (EsT) database, and the selected The identified sequence was compared with the corresponding genome database, and the sequence homologous to other coding sequences was excluded to confirm that it was a specific sequence.

In order to improve the efficiency of correct annealing of the sequence into a double-stranded DNA molecule, the DNA containing the siRNA sequence capable of interfering with the expression of the target gene is designed as a-chain and b-chain oligonucleotide sequences according to the following principles, adding Spacer sequence CTCGAG, to avoid the formation of termination signal, so that it can form a hairpin structure, GATCCC is added to the 5' end of the a-chain template, and GG is added to the 3' end of the b-chain template, which is complementary to the sticky end formed after BamH I cutting , TTTTTGGAT is added to the 3' end of the a chain, and AGCTATCCAAAAA is added to the 5' end of the b chain template, which is complementary to the sticky end formed after Hind III digestion. The a-chain and b-chain of the three siRNAs against ZNF667 were synthesized by Shanghai Jikai Gene Co., Ltd. The a-strand and b-strand of the shRNA sequence (shRNA-NC) of the blank control plasmid as the short hairpin RNA interference plasmid of ZNF667 were synthesized by Shanghai Jikai Gene Chemical Technology Co., Ltd.

2. ZNF667siRNA interference fragment synthesis

The eight synthesized Oligo fragments were first diluted to 100 μM, and 5 μL was mixed in each pair in pairs. After mixing, they were placed in a PCR instrument at 95°C for 5 minutes, and left at room temperature for 20 minutes to form double-stranded Oligo fragments. Then the double-stranded Oligo DNA fragment was diluted to 10 μM for insertion into the vector digestion fragment generated by pRNAT-U6.1/Neo double digestion.

Table 1 The sequence of the interference plasmid constructed

3. Construction of shRNA vector

The pRNAT-U6.1/Neo vector was digested with BamH I and Hind III, and the digested product was placed at 37 °C for 4 hours, separated by 0.8% agarose gel electrophoresis, and the gel recovery kit was used to recover and purify large fragments spare. According to the instructions of Takara Company, the vector and the insert were matched according to a certain ratio, the ligation reaction was set up, and the reaction was carried out overnight at 16°C. The ligation product was transformed into competent Escherichia coli DH5α strain, screened with ampicillin, positive clones were picked, shaken, and plasmids were extracted, and the three ZNF667shRNA eukaryotic vectors constructed were named ZNF667-shRNA1, ZNF667-shRNA2, and ZNF667-shRNA2 respectively. ZNF667-shRNA3, no sense control shRNA eukaryotic vector was named NC. The constructed vector needs to be sequenced and verified.

4. Identification of shRNA vectors

After the plasmid was extracted, it was sent to Shanghai Sangong Company for sequencing.

5. Identification of Reduction Efficiency of Interfering Plasmids

1×10 ⁵ HepG2 cells were seeded into 6-well plates coated with 0.1% gelatin, and transfected 48 hours after plating. Cell transfection was divided into 5 groups: blank cell group, three shRNA transfection groups and blank control shRNA group, cell transfection was operated according to LipofectaminTM2000 instructions, and all cells were placed in a 37°C, 5% CO ₂ constant temperature incubator for culture , Collect the total protein after 48 hours. The reduction efficiency of the three hairpin short hairpin RNA interference plasmids was determined by detecting the expression of ZNF667 protein. Finally, the No. 2 plasmid ZNF667-shRNA2 was determined as the best candidate plasmid.

6. Establishment of HepG2 cell line stably transfected with ZNF667-shRNA2 vector

①Cell preparation Culture HepG2 cells until the bottle is 80-90% full, digest and collect the cells, inoculate two 6-well cell culture plates at a cell density of 1×10 ⁵ cells/mL, inoculate 2 mL of cell suspension in each well, and inoculate each cell line Incubate 1 well in a 37°C, 5% CO ₂ cell incubator for 24 hours until the cells are 80% full and begin transfection.

② On the next day after the plasmids were transfected into the seed plate, take 4 μg of each plasmid (the blank vector is numbered ZNF667-E, and the above-mentioned No. 2 plasmid is numbered ZNF667-AS2) and diluted with 0.25 mL of serum-free medium, and mixed thoroughly for 5 minutes. Take 20 μL (10 μL × 2) of lipo2000 transfection reagent, dilute it with 0.5 mL (0.25 mL × 2) of serum-free medium, and mix well for 5 min. Discard the complete medium for the cells in the 6-well plate, wash twice with serum-free medium, and add 0.5 mL of serum-free medium at the same time. Take 0.25mL of the two plasmid suspensions and add them to the two centrifuge tubes of the transfection reagent, and mix well. Let it stand at room temperature for 20 minutes, and add it to a six-well plate within 30 minutes.

③ Cell selection HepG2 cell G418 pre-screening: culture HepG2 cells to 80-90% full bottle, digest and collect cells, inoculate 24-well plates at a cell density of ¹ ×104 cells/ml, adjust G418 concentration to 0, 100, 200 , 300, 400, 500, 600, 700, 800, 900, 1000g/ml were added to 24 wells, and the cells were observed. Finally, 600 μg/mL was selected as the screening concentration of G418. Stable strain screening: The above HepG2 cells transfected with ZNF667-E and ZNF667-AS2 were labeled as HepG2-ZNF667-E and HepG2-ZNF667-AS2. After 48 hours of transfection, add 600 μg/mL G418 to the six-well plate, observe the cells, and change the medium every two days. After 7 days, the cells in the HepG2 group basically died. Adjust the concentration of G418 to 200 μg/mL as the maintenance concentration, continue to culture and expand Cells: After 10 days of culture, all the cells in the HepG2 group died, and the other groups (ie, the HepG2 cell group transfected with ZNF667-E and ZNF667-AS2) reduced the G418 concentration to 200 μg/mL, and maintained the G418 concentration of 200 μg/mL to expand the cells to full bottle.

7. Detection of cell proliferation ability

①MTT method

Collect the HepG2 stable strain cells in the logarithmic phase of each group respectively, and adjust the concentration of the cell suspension; take three 96-well plates, add 100 μL of the cell suspension to each well, and spread the plates so that the cells to be tested are 2×10 ³ cells/well (for edge wells). Fill with sterile PBS to eliminate edge effect), set 5 duplicate wells for each group; culture in 5% CO ₂ , 37°C incubator, take out a 96-well plate after 24h, 48h, and 72h for detection: add 10 μL to each well for detection MTT solution (5mg/ml, that is, 0.5% MTT) solution, continue to culture in the cell incubator for 4h, stop the culture; add 150ul dimethyl sulfoxide to each well, shake on the shaker at low speed for 10min, so that the crystals are fully dissolved. Use a microplate reader to detect the absorbance of each well at a wavelength of 490 nm.

② Colony formation experiment

The HepG2 cells in each group in the logarithmic growth phase (3 groups in total) were digested with trypsinized solution and made into a single cell suspension with 1640 complete medium, so that the percentage of single cells was above 95%. Count the cells, and use 1640 medium containing 10% FBS to adjust the cell concentration to ¹ ×104 cells/ml for later use; inoculate 35mm cell culture dishes, 200cells/well, and repeat 3 wells for each group of cells to ensure the reliability of experimental data sex. Make up 3ml of complete medium, mix thoroughly and place in 5% CO ₂ , 37°C incubator for culture. Observe frequently, and terminate the culture when colonies visible to the naked eye appear in the dish. Fixation and staining: Discard the supernatant, carefully soak twice with PBS. Add 2 mL of 4% paraformaldehyde to each well to fix the cells for 15 min. Remove the fixative, add 1ml GIMSA staining solution to stain for 20min, rinse slowly with running water to remove excess staining solution, and air dry. Counting clones: Invert the plate and superimpose a transparent film with a grid, and directly count the number of clones with the naked eye. The calculation of the result adopts the following formula: colony formation rate=(number of clones/number of inoculated cells)×100%.

8. Detection of invasion function (Transwell invasion test)

The HepG2 cells in each group were digested and collected, and the cells were resuspended in serum-free medium, and the cell density was adjusted to 1×10 ⁶ cells/ml. Aspirate the culture medium in the invasion chamber that has fully infiltrated the basal layer membrane; add 500 μL of medium containing 10% FBS to the lower chamber (outside the invasion chamber, inside the well of a 24-well plate); add 200 μL Transfer the cell suspension to the invasion chamber; incubate in a 37°C, 5% CO ₂ cell incubator for 24 hours; gently wipe with a cotton swab to remove cells that have not penetrated the basal layer membrane in the invasion chamber, as well as the basal layer (ECMatrix gel), and operate carefully to avoid Pierce the underlying polycarbonate membrane; add 500 μL of staining solution to the wells of a new 24-well plate, and immerse the invasion chambers for 20 minutes; immerse the invasion chambers in a beaker with water for several times to rinse off excess dye, and then in air Dry in medium; randomly take pictures of different fields of view under the microscope; then dissolve the dye on the membrane with 10% acetic acid, 500 μL/well, and then transfer to a 96-well plate (150 μL/well), and detect the OD value of each well at a wavelength of 570 nm.

9. Migration ability test (scratch test)

The HepG2 cells of each group were digested and collected separately, and the cells of each group were prepared into a single cell suspension with the medium containing 10% FBS, and ³ ×105 cells per well were seeded into a 6-well plate; when the cells covered the bottom of the well, the Scratch the surface of the plate vertically with a 20 μL tip, wash twice with serum-free medium, and wash off the detached cells; culture cells with serum-free medium, and replace with 3% FBS medium after 24 hours to continue culturing; Taking the trace time as the starting point, pictures of each group of cells were taken every 24 h.

10. Preparation of xenograft tumor animal model in nude mice

①Nine nude mice were randomly divided into 3 groups: HepG2 cells, ZNF667-NC group, ZNF667-shRNA group;

② Discard the culture medium of cancer cells in the logarithmic growth phase, wash the cells twice with sterile neutral PBS, digest with 0.25% trypsin, elute from the culture flask, pipette into a single-cell suspension, 1000 rpm Centrifuge for 1 min, collect the cells, suspend the cells with 0.9% physiological saline, make up the volume, and adjust the cell density to the injected cell volume of 2×10 ⁶ cells/ml.

③Irradiate the ultra-clean table with ultraviolet light for 30 minutes, wipe the table with 75% alcohol; wipe the culture cage with 75% alcohol,

All other utensils were autoclaved. Before injecting the cell suspension, use 75% alcohol to sterilize the inoculated skin of the nude mice locally. Each nude mouse is inoculated with 0.2ml of the cell suspension, and the nude mice are inoculated on the back of the neck or subcutaneously under the armpit with a sterile disposable syringe.

④Tumor formation and tumor growth were closely observed every 3 days after inoculation. After successful tumorigenesis in nude mice, the tumor volume was measured and recorded every 5 days, and the growth curves of implanted tumors in each group were drawn. At the end of the experiment on the 42nd day, all nude mice were killed by neck dislocation, the tumor tissues were dissected out, the longest diameter and transverse diameter of the tumor were measured, and the mass of the tumor was weighed. Tumor volumes were calculated separately using the formula.

The calculation formula is as follows: tumor volume = tumor long diameter x tumor short diameter ² x 0.5.

result:

1. Identification of ZNF667-shRNA plasmid

Select the positive colony, extract the plasmid, use T7 as the sequencing primer, conduct a sequence test on the constructed vector, and check and analyze the sequencing peak map, confirming that the inserted fragment is a ZNF667shRNA DNA fragment, and there is no base mutation (Figure 1-Figure 3) , indicating that the shRNA vector was constructed successfully, conforming to the expected design, capable of expressing siRNA, and expected to reduce the expression of ZNF667 protein.

2. Inhibitory effect of ZNF667-shRNA on ZNF667 protein expression in HepG2 cells

Western blotting was used to detect the change of ZNF667 protein expression in HepG2 cells after transfection of sh short hairpin RNA interference plasmid. The results showed that: in HepG2 cells, the blank plasmid control group, ZNF667-con group (blank cell group) and ZNF667-shRNA 1 (transfected with No. 1 plasmid), ZNF667-shRNA 2 (transfected with No. 2 plasmid), ZNF667-shRNA 3 (transfected with No. 3 plasmid) for a total of 5 groups of standardized cell total protein were detected, the results showed that ZNF667-shRNA1, ZNF667 -shRNA2, ZNF667-shRNA3 histone expression bands were significantly weaker than the blank cell group (Con) and blank plasmid (NC) group bands (Figure 4), and statistical analysis confirmed that the three groups of plasmids had interference effects on HepG2 cells Significant difference (Figure 5), the interference efficiency of ZNF667-shRNA2 is the best, and it is determined to be an interference plasmid that can be used in subsequent experiments.

3. Establishment and identification of HepG2 cell lines stably transfected with ZNF667-shRNA2 plasmid and control plasmid

According to the above-mentioned method for establishing stable cell lines, the optimal G418 concentration for screening HepG2 stable cell lines is 600ug/ml. After obtaining stable cell lines, maintain the culture concentration at 200ug/ml. The stable transfected cell line finally established was named ZNF667-shRNA (HepG2 cells transfected with ZNF667-shRNA2 plasmid group), NC (blank control plasmid group). The transfected ZNF667-shRNA, NC and blank cell proteins were collected and detected by Western blot, the film was scanned, and the grayscale analysis was performed on the image with the image analysis software IPP6.0 (Fig. 6). The results of statistical analysis showed ( FIG. 7 ), that after the ZNF667-shRNA2 plasmid was stably transferred into HepG2 cells, it could reduce 90% of the protein expression. Transfected with ZNF667-shRNA, there was a statistically significant difference in the expression of ZNF667 between NC and blank cells.

4. The proliferation ability of ZNF667-shRNA cells is weakened

Based on 3-(4,5)-dimethylthiazo(-z-y1)-3,5-di-phenyltetrazoliumromide (chemical name: 3-(4,5-dimethylthiazol-2)-2,5-diphenyl A cell counting kit based on tetrazolium bromide) was used to detect the proliferation ability of the three cell lines. Use a microplate reader to measure the OD value at a wavelength of 490mM. The succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystal formazan and deposit in the cells, while dead cells have no such function. . Measure its light absorption value at a wavelength of 490nm with a microplate reader, and within a certain range of cell numbers, the amount of MTT crystal formation is proportional to the number of cells. Thus indirectly responding to cell proliferation. 0 hour, 24 hours, 48 hours and 72 hours OD values at 490mM wavelength are shown in Table 1. Curve analysis shows that: after HepG2 cells were transfected with the blank control plasmid ZNF667-NC, the growth of stable cells was slightly slower than that of HepG2 cells; After the cells were transfected with the ZNF667-shRNA2 plasmid, the growth of the cell line was significantly slower than that of HepG2-NC and blank cells (Figure 8), and the difference was statistically significant.

Table 1 The MTT data of different time points of HepG2 stable cell lines in each group

5. The clone formation ability of HepG2 cell line stably transfected with ZNF667-shRNA (plasmid No. 2) was reduced

Clonogenic assay is one of the effective methods to measure cell proliferation ability, and the clone formation rate reflects two important traits of cell population dependence and proliferation ability. After the HepG2 cells were transfected with the plasmid ZNF667-shRNA, the clone formation ability of the ZNF667-shRNA group was weaker than that of the HepG2 group, and weaker than that of the blank control plasmid group (Figure 9, Table 2). The difference in colony formation was statistically significant (Figure 10).

Table 2. Cloning formation rate data of HepG2 groups

6. The infiltration and migration ability of HepG2 cell line stably transfected with ZNF667-shRNA (plasmid No. 2) was reduced

Scratch test and transwell invasion test were used to detect whether the expression change of ZNF667 could affect the invasion ability of HepG2 cells. The results of the Transwell invasion test use the 570nm OD value of the acetic acid eluate on the microplate reader to indirectly represent the number of cells passing through the Transwell chamber. Absorbance decreased by 25% at wavelength (FIG. 11, Table 3). The experimental results showed that the cell invasion ability of the ZNF667-shRNA group was weaker than that of the HepG2 group, and weaker than that of the blank control plasmid group NC group, and the difference was statistically significant (Figure 12).

Table 3 Transwell invasion test results of HepG2 three groups of cells

Con NC ZNF667-shRNA OD 1.233±0.026 1.228±0.028 0.814±0.029

Scratch test (Scratching test) is an experimental method used to detect cell movement, which can be used to detect the invasion and metastasis ability of adherent growth tumor cells. After all three groups of cells covered the bottom of the well, use a 10ul pipette tip to scratch vertically on the surface of the plate. Taking the scratching time as the starting point, take pictures of each group of cells every 24 hours, compared with HepG2 cells and NC group of stable cell lines , the migration distance of the HepG2-ZNF667-shRNA cell line was significantly reduced at 24 hours and 48 hours (Table 4, Figure 13), and the difference was statistically significant (Figure 14).

Table 4. Migration distance (mm) of three groups of HepG2 cells in scratch test

7 Expression of P53 and Bcl-2 proteins in three HepG2 cell lines

In order to further explore how reducing the expression of ZNF667 protein causes changes in the growth, proliferation, infiltration and invasion of HepG2 cells, two proteins, Bcl2 and P53, were selected as target molecules for detection. The results showed that, compared with HepG2 cells and NC cell lines, Compared with that, the expressions of P53 and Bcl2 proteins in HepG2-ZNF667-shRNA cell line were significantly changed. The expression of Bcl2 protein was significantly reduced, and the expression of p53 protein was significantly increased (Fig. 15). Fig. 16 and 17 are the analysis results of Bcl2 protein and P53 protein expression; the results show that the reduction of ZNF667 protein in the three HepG2 cell lines is very likely to be By reducing Bcl2 and increasing the expression of p53, the biological characteristics of HepG2 cells can be changed, and the degree of malignancy can be reduced from the level of cells in vitro.

8. Tumor formation experiment in nude mice

HepG2 cells, HepG2-NC cell lines, and HepG2-ZNF667-shRNA cell lines were subjected to tumorigenesis experiments in nude mice. The nude mice were executed on the 42nd day. The tumor volume and growth curve of the mice are shown in Figure 18. The statistical analysis of the tumor volume and weight is shown in Figure 19. The three groups of cells formed tumors in nude mice, and the ZNF667 protein content detection and statistics in the tumor The results of the analysis are shown in Figure 20.

SEQUENCE LISTING

<110> The Second Xiangya Hospital of Central South University

<120> A kind of RNA interference plasmid of ZNF667 protein and its application method

<130> None

<160> 21

<170> PatentIn version 3.3

<210> 1

<211> 21

<212>DNA

<213> siRNA-1

<400> 1

cgaatatctc tcacacgaca t 21

<210> 2

<211> 22

<212>DNA

<213> siRNA-2

<400> 2

cgccaatcat ttcttattga ac 22

<210> 3

<211> 21

<212>DNA

<213> siRNA-3

<400> 3

caacccttat tctgcatcta a 21

<210> 4

<211> 63

<212>DNA

<213> ZNF667-shRNA-1a chain sequence

<400> 4

gatccccgaa tatctctcac acgacatctc gagatgtcgt gtgagagata ttcgtttttg 60

gat 63

<210> 5

<211> 63

<212>DNA

<213> ZNF667-shRNA-1b strand sequence

<400> 5

agctatccaa aaacgaatat ctctcacacg acatctcgag atgtcgtgtg agagatattc 60

ggg 63

<210> 6

<211> 64

<212>DNA

<213> ZNF667-shRNA-2a strand sequence

<400> 6

gatccccgcc aatcatttct tattgaactc gagttcaata agaaatgatt ggcgtttttg 60

gat 63

<210> 7

<211> 62

<212>DNA

<213> ZNF667-shRNA-2b strand sequence

<400> 7

agctatccaa aaagccaatc atttcttatt gaactcgagt tcaataagaa atgattggcg 60

gg 62

<210> 8

<211> 63

<212>DNA

<213> ZNF667-shRNA -3a strand sequence

<400> 8

gatccccaac ccttattctg catctaactc gagttagatg cagaataagg gttgtttttg 60

gat 63

<210> 9

<211> 63

<212>DNA

<213> ZNF667-shRNA -3b strand sequence

<400> 9

agctatccaa aaacaaccct tattctgcat ctaactcgag ttagatgcag aataagggtt 60

ggg 63

<210> 10

<211> 59

<212>DNA

<213> Control shRNA a-strand sequence

<400> 10

gatcccttct ccgaacgtgt cacgtctcga gacgtgacac gttcggagaa tttttggat 59

<210> 11

<211> 59

<212>DNA

<213> Control shRNA b-strand sequence

<400> 11

agctatccaa aaattctccg aacgtgtcac gtctcgagac gtgacacgtt cggagaagg 59

<210> 12

<211> 6

<212>DNA

<213> a-strand and b-strand sequences joined spacer sequence

<400> 12

ctcgag 6

<210> 13

<211> 6

<212>DNA

<213> Sequence added to the 5' end of the a-strand template

<400> 13

gatccc 6

<210> 14

<211> 2

<212>DNA

<213> Sequence added to the 3' end of the b-strand template

<400> 14

gg 2

<210> 15

<211> 9

<212>DNA

<213> Sequence added to 3' end of strand a

<400> 15

tttttggat 9

<210> 16

<211> 13

<212>DNA

<213> Sequence added to the 5' end of the b-strand template

<400> 16

agctatccaa aaa 13

<210> 17

<211> 938

<212>DNA

<213> plasmid shRNA1

<400> 17

tccggatctc ctaagggagg agagcatgaa ttctcgagcg gccgccccct tcaccgaggg 60

cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat 120

tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta 180

ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct 240

taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa 300

caccggcgaa tatctctcac acgacatctc gagatgtcgt gtgagagata ttcgttttta 360

attctcgacc tcgagacaaa tgcagtattc atccacaagc ttaagtttaa accgctgatc 420

agcctcgact gtgccttcta aatagtaatc aattacgggg tcattagttc atagcccata 480

tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 540

cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 600

ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 660

gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 720

ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 780

catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gtagcggtt 840

tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 900

ccaaaatcaa cgggactttc caaaatgtcg taacaact 938

<210> 18

<211> 1008

<212>DNA

<213> plasmid shRNA2

<400> 18

tttccctcac ttaaggggag gagaagcatg aattctcgag cggccgcccc cttcaccgag 60

ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata 120

attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag 180

taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg 240

cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga 300

aacaccggcg ccaatcattt cttattgaac tcgagttcaa taagaaatga ttggcgtttt 360

taattctcga cctcgagaca aatggcagta ttcatccaca agcttaagtt taaaccgctg 420

atcagcctcg actgtgcctt ctaaatagta atcaattacg gggtcattag ttcatagccc 480

atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 540

cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 600

tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 660

agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 720

gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 780

agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcatgggcg tggatagcgg 840

tttgactcac ggggatttcc aagtctccac cccattgacg tcaatggagt ttgttttggc 900

accaaatcac cggactttcc aaaatgtcgt acactccgcc ccattgacgc aaatgggcgg 960

tagccgtgta cggtgggagt tctatataag tccgagctgg ttagtgac 1008

<210> 19

<211> 1017

<212>DNA

<213> plasmid shRNA3

<400> 19

tcccgtcact aaggggaggag agcatgaatt ctcgagcggc cgcccccttc accgagggcc 60

tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg 120

gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat 180

aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta 240

ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca 300

ccggcaaccc ttattctgca tctaactcga gttagatgca gaataagggt tgtttttaat 360

tctcgacctc gagacaaatg gcagtattca tccacaagct taagtttaaa ccgctgatca 420

gcctcgactg tgccttctaa atagtaatca attacggggt cattagttca tagcccatat 480

atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 540

ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 600

cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 660

tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 720

tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 780

atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 840

gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 900

caaaatcaac gggactttcc aaaatgtcgt acaactccgc cccattgacg caatggggcg 960

gtaggcgggg tacggtggga ggtctaatat aaggcagaag ctggtttagt gaaccgt 1017

<210> 20

<211> 1010

<212>DNA

<213> con-shRNA

<400> 20

tacctcacta agggaggaga agcatgaatt ccccagtgga aagacgcgca ggcaaaacgc 60

accacgtgac ggagcgtgac cgcgcgccga gcgcgcgcca aggtcgggca ggaagagggc 120

ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt 180

agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa 240

taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt 300

accgtaactt gaaagtattt cgatttcttg ggtttatata tcttgtggaa aggacgcggg 360

atcccttctc cgaacgtgtc acgtctcgag acgtgacacg ttcggagaat ttttggatag 420

cttaagttta aaccgctgat cagcctcgac tgtgccttct aaatagtaat caattacggg 480

gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 540

gcctggctga ccgcccaacg accccccgccc attgacgtca ataatgacgt atgttcccat 600

agtaacgcca atagggactt tccatgacg tcaatgggtg gagtatttac ggtaaactgc 660

ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 720

cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 780

gcagtacatc tacgtattag tcatcgctat taccatggtg atgcgggttt tggcagtaca 840

tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 900

tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtacaactc 960

cgccccattg acgcaaatgg ggcggtaggc gtgtacggtg ggaggtctat 1010

<210> 21

<211> 6380

<212>DNA

<213> pRNAT-U6.1/neo vector

<400> 21

aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg gtttatatat 60

cttgtggaaa ggacgcggga tccgagctcg gtaccaagct taagtttaaa ccgctgatca 120

gcctcgactg tgccttctaa atagtaatca attacggggt cattagttca tagcccatat 180

atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 240

ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 300

cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 360

tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 420

tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 480

atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 540

gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 600

caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 660

ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc 720

cgctagcgct accggtcgcc accatggcca gcaagggcga ggagctgttc accggcgtgg 780

tgcccatcct ggtggagctg gacggcgatg tgaatggcca caagttcagc gtgagcggcg 840

agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 900

agctgcctgt gccctggccc accctggtga ccaccctgag ctacggcgtg cagtgcttct 960

cacgctaccc cgatcacatg aagcagcacg acttcttcaa gagcgccatg cctgagggct 1020

acatccagga gcgcaccatc ttcttcgagg atgacggcaa ctacaagtcg cgcgccgagg 1080

tgaagttcga gggcgatacc ctggtgaatc gcatcgagct gaccggcacc gatttcaagg 1140

aggatggcaa catcctgggc aataagatgg agtacaacta caacgcccac aatgtgtaca 1200

tcatgaccga caaggccaag aatggcatca aggtgaactt caagatccgc cacaacatcg 1260

aggatggcag cgtgcagctg gccgaccact accagcagaa tacccccatc ggcgatggcc 1320

ctgtgctgct gcccgataac cactacctgt ccaccccagag cgccctgtcc aaggacccca 1380

acgagaagcg cgatcacatg atcctgctgg agttcgtgac cgccgccggc atcacccacg 1440

gcatggacga gctgtacaag tgaggactag ataactgaac ttgtttattg cagcttataa 1500

tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 1560

ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgtg ggaagacaat 1620

agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg 1680

ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 1740

gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 1800

ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcggggggctc 1860

cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt 1920

gatggttcac gtagtgggcc atcgccctga tagacggtttttcgcccttt gacgttggag 1980

tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg 2040

gtctattctt ttgatttata aggattttg ccgatttcgg cctattggtt aaaaaatgag 2100

ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg 2160

gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 2220

caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 2280

tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc 2340

ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttt gcagaggccg 2400

aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag 2460

gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag 2520

gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt 2580

gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg 2640

ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg 2700

gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg 2760

ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg 2820

gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca 2880

tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc 2940

accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc 3000

aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca 3060

aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga 3120

atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg 3180

cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg 3240

aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg 3300

ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga 3360

ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag 3420

gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct 3480

catgctggag ttcttcgccc accccaactt gtttatgca gcttataatg gttacaaata 3540

aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 3600

tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag 3660

cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 3720

acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 3780

actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 3840

gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 3900

cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 3960

tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 4020

gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 4080

ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 4140

aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 4200

tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 4260

ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 4320

gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 4380

tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 4440

caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 4500

ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 4560

cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtttttt 4620

tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 4680

ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 4740

attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 4800

ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 4860

tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 4920

aactacgata cggggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 4980

acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 5040

aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 5100

agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 5160

ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 5220

agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 5280

tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 5340

tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 5400

attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 5460

taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 5520

aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 5580

caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 5640

gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 5700

cctttttcaa tattattgaa gcatttatca gggttatgt ctcatgagcg gatacatatt 5760

tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc aatttcccc gaaaagtgcc 5820

acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc 5880

tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct 5940

gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg 6000

aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg 6060

cgtttaatac gactcactat agggagagag agagaattac cctcactaaa gggaggagaa 6120

gcatgaattc cccagtggaa agacgcgcag gcaaaacgca ccacgtgacg gagcgtgacc 6180

gcgcgccgag cgcgcgccaa ggtcgggcag gaagagggcc tatttcccat gattccttca 6240

tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac 6300

acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca 6360

gttttaaaat tatgttttaa 6380

Claims (7)

1. a kind of application method of the short hairpin RNA interference plasmid of ZNF667 albumen, which is characterized in that be used to prepare and inhibit liver The preparation of cancer, the short hairpin RNA interference plasmid of the ZNF667 albumen be can be generated according to the sequence design of ZNF667 it is dry The DNA fragmentation for disturbing the siRNA of ZNF667 protein expression is inserted into built-up in short hairpin RNA interference carrier.

2. the application method of the short hairpin RNA interference plasmid of ZNF667 albumen according to claim 1, which is characterized in that

Requirement using the mRNA sequence of people ZNF667 as template and Vector promoter starting expression siRNA to sequence, design Synthesis can generate the DNA fragmentation of the siRNA of interference ZNF667 protein expression.

3. the application method of the short hairpin RNA interference plasmid of ZNF667 albumen according to claim 2, which is characterized in that The target gene ZNF667 sequence of siRNA interference is as follows:

SiRNA-1:CGAATATCTCTCACACGACAT；

SiRNA-2:CGCCAATCATTTCTTATTGAA；

SiRNA-3:CAACCCTTATTCTGCATCTAA.

4. the application method of the short hairpin RNA interference plasmid of ZNF667 albumen according to claim 3, which is characterized in that

The target gene ZNF667 sequence of siRNA interference is siRNA-2:CGCCAATCATTTCTTATTGAA.

5. the application method of the short hairpin RNA interference plasmid of ZNF667 albumen according to claim 3, which is characterized in that

The original plasmid expression vector of selection is preferably pRNAT-U6.1/Neo.

6. the application method of the short hairpin RNA interference plasmid of ZNF667 albumen according to claim 5, which is characterized in that Restriction enzyme site when DNA fragmentation is inserted into carrier isBAmH I andHind III。

7. the application method of the short hairpin RNA interference plasmid of ZNF667 albumen according to claim 6, which is characterized in that The DNA fragmentation of the siRNA of interference ZNF667 protein expression can be generated according to the sequence design of ZNF667, particular sequence is as follows:

ZNF667-shRNA-1 a chain-ordering:

5’-

GATCCCCGAATATCTCTCACACGACATCTCGAGATGTCGTGTGAGAGATATTCGTTTTTGGAT -3’

ZNF667-shRNA-1 b chain-ordering:

5’-

AGCTATCCAAAAACGAATATCTCTCACACGACATCTCGAGATGTCGTGTGAGAGATATTCG GG -3’

ZNF667-shRNA-2 a chain-ordering:

5’-

GATCCCCGCCAATCATTTCTTATTGAACTCGAGTTCAATAAGAAATGATTGGCG TTTTTGGAT -3’

ZNF667-shRNA-2 b chain-ordering:

5’-

AGCTATCCAAAAAGCCAATCATTTCTTATTGAACTCGAGTTCAATAAGAAATGATTGGCG GG -3’

ZNF667-shRNA -3a chain-ordering:

5’-

GATCCCCAACCCTTATTCTGCATCTAACTCGAGTTAGATGCAGAATAAGGGTTG TTTTTGGAT -3’

ZNF667-shRNA -3b chain-ordering:

5’-

AGCTATCCAAAAACAACCCTTATTCTGCATCTAACTCGAGTTAGATGCAGAATAAGGGTTGGG-3’。