CN110117593B - Application of nucleic acid, recombinant vector and recombinant lentivirus for specifically reducing FAM84B gene expression - Google Patents
Application of nucleic acid, recombinant vector and recombinant lentivirus for specifically reducing FAM84B gene expression Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology and genetic engineering, and relates to application of at least one of nucleic acid, a recombinant vector and a recombinant lentivirus for specifically reducing FAM84B gene expression in preparation of a medicament for inhibiting growth and/or proliferation of breast cancer cells or in preparation of a medicament for treating and/or preventing breast cancer.
Description
Technical Field
The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to a nucleic acid for specifically reducing FAM84B gene expression, a recombinant vector and application of a recombinant lentivirus.
Background
Breast cancer is one of common malignant tumors of women worldwide, and the new incidence and mortality of breast cancer are the first of all types of cancer. Although the incidence of breast cancer is relatively low in China, the population is large, the total incidence is large, the incidence rate tends to rise year by year, and the incidence age tends to be younger. Breast cancer presents great complexity and heterogeneity in cell origin, histological morphology, disease grading, clinical presentation, treatment response, metastatic potential and other aspects, and the effectiveness and the universality of the existing breast cancer treatment method are limited. There is still a lack of clinical targets for breast cancer therapy.
The Thioesterase Superfamily Member 6(Thioesterase Superfamily Member 6, FAM84B) protein-encoding gene, also known as C8orf55, is located at 8q24.3 and has 3 exons. FAM84B protein is widely expressed in tissues such as kidney, stomach, lung, etc., and is usually localized in the extracellular, cytoplasmic and nuclear regions. To date, the biological function of FAM84B gene in relation to breast cancer has not been studied.
Disclosure of Invention
The invention aims to provide a nucleic acid for specifically reducing FAM84B gene expression, a recombinant vector and application of a recombinant lentivirus.
In order to achieve the above object, the present invention provides an application of at least one of a nucleic acid, a recombinant vector and a recombinant lentivirus for specifically reducing the expression of FAM84B gene in the preparation of a medicament for inhibiting the growth and/or proliferation of breast cancer cells or for treating and/or preventing breast cancer.
According to a preferred embodiment of the invention, the nucleic acid for specifically reducing the expression of FAM84B gene is siRNA, and the siRNA generally has a nucleotide sequence of 19-27 bp. The present invention focuses on providing a novel breast cancer therapeutic target, and is not limited to a specific siRNA sequence, and siRNA directed to the target can be designed according to various methods conventional in the art.
Specifically, the nucleotide sequence of the siRNA at least comprises the following group of nucleotide sequences:
(1) a first set of nucleotide sequences
The first group of nucleotide sequences are shown as SEQ ID NO 1 and SEQ ID NO 2, the SEQ ID NO 1 is 5'-CAACGAUCUGUACCGCUACAA-3', and the SEQ ID NO 2 is 5'-UUGUAGCGGUACAGAUCGUUG-3';
(2) second set of nucleotide sequences
The second group of nucleotide sequences are shown as SEQ ID NO 3 and SEQ ID NO 4, the SEQ ID NO 3 is 5'-AGUCUAGAGGACCUGAUCAUG-3', and the SEQ ID NO 4 is 5'-CAUGAUCAGGUCCUCUAGACU-3';
(3) a third group of nucleotide sequences
The third group of nucleotide sequences are shown as SEQ ID NO 5 and SEQ ID NO 6, wherein the SEQ ID NO 5 is 5'-GGUGGAAUGCUCCGUGUUCUA-3', and the SEQ ID NO 6 is 5'-UAGAACACGGAGCAUUCCACC-3'.
According to the invention, the nucleic acid for specifically reducing FAM84B gene expression is shRNA corresponding to the siRNA, the shRNA has a single-stranded RNA with a stem-loop structure, and the nucleotide sequence of the shRNA at least comprises the following nucleotide sequences:
(1) nucleotide sequence of the fifth group
The fifth group of nucleotide sequences are shown as SEQ ID NO 9 and SEQ ID NO 10, wherein the SEQ ID NO 9 is 5'-CCGGCAACGAUCUGUACCGCUACAACUCGAGUUGUAGCGGUACAGAUCGUUGUUUUUG-3', and the SEQ ID NO 10 is 5'-AAUUCAAAAACAACGAUCUGUACCGCUACAACUCGAGUUGUAGCGGUACAGAUCGUUG-3';
(2) nucleotide sequence of group VI
The sixth group of nucleotide sequences are shown as SEQ ID NO. 11 and SEQ ID NO. 12, wherein the SEQ ID NO. 11 is 5'-CCGGAGUCUAGAGGACCUGAUCAUGCUCGAGCAUGAUCAGGUCCUCUAGACUUUUUUG-3', and the SEQ ID NO. 12 is 5'-AAUUCAAAAAAGUCUAGAGGACCUGAUCAUGCUCGAGCAUGAUCAGGUCCUCUAGACU-3';
(3) nucleotide sequence of group seven
The seventh group of nucleotide sequences are shown as SEQ ID NO. 13 and SEQ ID NO. 14, wherein the SEQ ID NO. 13 is 5'-CCGGGGUGGAAUGCUCCGUGUUCUACUCGAGUAGAACACGGAGCAUUCCACCUUUUUG-3', and the SEQ ID NO. 14 is 5'-AAUUCAAAAAGGUGGAAUGCUCCGUGUUCUACUCGAGUAGAACACGGAGCAUUCCACC-3'.
According to the invention, the nucleic acid for specifically reducing FAM84B gene expression is DNA encoding the shRNA, and the nucleotide sequence of the DNA encoding the shRNA at least comprises the following group of nucleotide sequences:
(1) nucleotide sequence of the ninth group
The ninth group of nucleotide sequences are shown as SEQ ID NO 17 and SEQ ID NO 18, wherein the SEQ ID NO 17 is 5'-CCGGCAACGATCTGTACCGCTACAACTCGAGTTGTAGCGGTACAGATCGTTGTTTTTG-3', and the SEQ ID NO 18 is 5'-AATTCAAAAACAACGATCTGTACCGCTACAACTCGAGTTGTAGCGGTACAGATCGTTG-3';
(2) tenth group of nucleotide sequences
The tenth group of nucleotide sequences are shown as SEQ ID NO. 19 and SEQ ID NO. 20, wherein the SEQ ID NO. 19 is 5'-CCGGAGTCTAGAGGACCTGATCATGCTCGAGCATGATCAGGTCCTCTAGACTTTTTTG-3', and the SEQ ID NO. 20 is 5'-AATTCAAAAAAGTCTAGAGGACCTGATCATGCTCGAGCATGATCAGGTCCTCTAGACT-3';
(3) nucleotide sequence of the eleventh group
The eleventh group of nucleotide sequences are shown as SEQ ID NO 21 and SEQ ID NO 22, wherein the SEQ ID NO 21 is 5'-CCGGGGTGGAATGCTCCGTGTTCTACTCGAGTAGAACACGGAGCATTCCACCTTTTTG-3', and the SEQ ID NO 22 is 5'-AATTCAAAAAGGTGGAATGCTCCGTGTTCTACTCGAGTAGAACACGGAGCATTCCACC-3'.
According to the present invention, the recombinant vector for specifically reducing the expression of FAM84B gene is preferably a recombinant vector obtained by inserting the DNA encoding the shRNA into the multiple cloning sites AgeI and EcoRI of the GV493 plasmid (available from Kjekay Gene science and technology Co., Ltd., Shanghai).
According to the invention, the recombinant lentivirus specifically reducing the expression of FAM84B gene is preferably obtained by co-transfecting the above recombinant vector with a viral packaging helper plasmid pHelper 1.0 vector and a viral packaging helper plasmid pHelper 2.0 vector into mammalian cells.
The siRNA provided by the invention can specifically reduce the expression of FAM84B gene, so that the siRNA can be applied to the preparation of medicines for inhibiting the growth and/or proliferation of breast cancer cells or medicines for treating and/or preventing breast cancer.
The shRNA provided by the invention can specifically reduce the expression of FAM84B gene, so that the shRNA can be applied to the preparation of medicines for inhibiting the growth and/or proliferation of breast cancer cells or medicines for treating and/or preventing breast cancer.
The DNA for coding shRNA provided by the invention can specifically reduce FAM84B gene expression, so that the DNA for coding shRNA can be applied to preparation of a medicament for inhibiting growth and/or proliferation of breast cancer cells or preparation of a medicament for treating and/or preventing breast cancer.
The recombinant vector provided by the invention can specifically reduce the expression of FAM84B gene, so that the recombinant vector can be applied to the preparation of medicines for inhibiting the growth and/or proliferation of breast cancer cells or medicines for treating and/or preventing breast cancer.
The recombinant lentivirus provided by the invention can specifically reduce the expression of FAM84B gene, so that the recombinant lentivirus can be applied to the preparation of medicines for inhibiting the growth and/or proliferation of breast cancer cells or medicines for treating and/or preventing breast cancer.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
Fig. 1 shows the difference in expression of FAM84B in normal and breast cancer cells. Wherein MCF-10A (abbreviated as 10A) is normal cell, and MCF7, T-47D and BT-474 are breast cancer cells.
Figure 2 shows the effect of knockdown of FAM84B on breast cancer cell proliferation. Wherein shCtrl is a control group, shFAM84B is an experimental group, the upper curve is a control group, and the lower curve is an experimental group.
Figure 3 shows the effect of knockdown of FAM84B on breast cancer growth in tumor-bearing mice. NC is a control group, and shFAM84B is an experimental group.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The human FAM84B referred to in the examples below was numbered as Gene ID 157638.
In the examples below, statistical analysis was performed using GraphPad Prism 6.0 software. All in vitro experiments were repeated three more times. Data are expressed as mean ± Standard Deviation (SD). P <0.05 was considered statistically significant.
Example 1
This example serves to illustrate the high expression of FAM84B in breast cancer cell lines.
RNA extraction and reverse transcription quantitative PCR (RT-qPCR).
1. Total RNA extraction: this example was carried out at low temperature. Culturing cells in 6-well plate, removing culture medium, rinsing with PBS for 3 times, adding 1000 μ l Trizol per well, and shaking for 10 min; collecting into 1.5ml centrifuge tube, adding 200 μ l chloroform into each tube, mixing vigorously for 30sec, standing for 15min, and centrifuging at 4 deg.C 12000rpm for 15 min; gently sucking 400 μ l of the supernatant liquid into another new centrifuge tube, adding isopropanol with the same volume, gently inverting and mixing, and centrifuging at 12000rpm at 4 ℃ for 10 min; discarding the supernatant, adding 1ml of 75% alcohol to wash the precipitate, and centrifuging at 12000rpm at 4 ℃ for 10 min; the supernatant was discarded as much as possible, air-dried at room temperature for 10min, 10. mu.l of RNase-free water was added to each tube, dissolved, and quantified with a spectrophotometer.
2. Reverse transcription, each 25. mu.l reverse transcription system comprises 100pmol random primer, 2. mu.g total RNA, 1. mu.l M-L V reverse transcriptase, 0.625. mu.l RNase inhibitor, 1.25. mu.l dNTPs (10mM), 5. mu.l 5 × M-M L V buffer, and the rest is ddH2The volume of O is filled to 25 μ l. The reaction conditions are as follows: 1h at 37 ℃ and 5min at 95 ℃.
3. Quantitative PCR, each 20. mu.l reaction system contains 2 × PCR Mix (ABI) 10. mu.l, upstream and downstream primers 0.4. mu.l each, cDNA 1. mu.l, ddH2O8.2. mu.l. The reaction conditions are as follows: 94 ℃ for 2min, 94 ℃ for 15s, 60 ℃ for 40s, 40 cycles. The sequences of the primers used in the experiments are shown in Table 1.
TABLE 1 fluorescent quantitative RT-PCR primer sequences
Fig. 1 shows the difference in expression of FAM84B in normal and breast cancer cells. Wherein MCF-10A (abbreviated as 10A) is normal cell, and MCF7, T-47D and BT-474 are breast cancer cells. As shown in fig. 1, the RT-qPCR results show: FAM84B gene expression was significantly elevated in MCF7, T-47D, and BT-474 breast cancer cells relative to control MCF-10A cells (P <0.001, P <0.001, P < 0.001).
Example 2
This example demonstrates that knocking down FAM84B gene expression can inhibit breast cancer proliferation and/or growth.
First, preparation of RNAi lentivirus clone
1. Target design
Aiming at FAM84B gene sequence, 3 RNAi target sequences, nucleotide sequences of 3 siRNAs of FAM84B gene and nucleotide sequence of Negative Control (NC) are designed according to RNAi sequence design principle. The names corresponding to the 3 kinds of siRNAs and the negative control are respectively FAM84B-si-1a, FAM84B-si-1b, FAM84B-si-2a, FAM84B-si-2b, FAM84B-si-3a, FAM84B-si-3b, NC-si-a and NC-si-b which are sequences designed for the negative control group. See table 2 for details.
TABLE 23 nucleotide sequences of RNA interference targets (siRNA) and negative controls
Table 3 shows the nucleotide sequences of 3 kinds of shRNAs used in the examples, which are the nucleotide sequences of FAM84B-sh-1a, FAM84B-sh-1b, FAM84B-sh-2a, FAM84B-sh-2b, FAM84B-sh-3a and FAM84B-sh-3b, and NC-sh-a and NC-sh-b are the nucleotide sequences of a control group. See table 3 for details.
TABLE 33 nucleotide sequences of shRNA and negative controls
The nucleotide sequences of the DNAs used to encode the 3 shRNAs are shown in Table 4. The DNAs encoding shRNA used in the examples were the nucleotide sequences of FAM84B-d-1a, FAM84B-d-1b, FAM84B-d-2a, FAM84B-d-2b, FAM84B-d-3a, and FAM84B-d-3b, and NC-d-a and NC-d-b were the nucleotide sequences of the control group. See table 4 for details.
DNA and nucleotide sequence of negative control of the 43 shRNAs in Table
2. Vector cleavage
A50. mu.l digestion system was prepared according to Table 5. Sequentially adding various reagents according to the sequence of a list, lightly blowing and uniformly mixing by using a pipette, carrying out instantaneous centrifugation, and reacting for 3 hours at 37 ℃. And (4) carrying out agarose gel electrophoresis on the vector enzyme digestion product, and recovering a target band.
TABLE 5 vector cleavage System
3. DNA annealing of shRNA to form double-stranded DNA
And (3) dissolving the DNA dry powder of the synthesized paired shRNA in an annealing buffer solution, carrying out water bath at 90 ℃ for 15min, and naturally cooling to room temperature.
4. Carrier attachment
The double-cut linearized vector and the annealed double-stranded DNA were ligated by T4DNA ligase (T4DNA ligase) for 1-3h at 16 ℃.
TABLE 6 Carrier attachment System
| Reagent | Volume (μ l) |
| Linearized vector (100 ng/. mu.l) | 1 |
| Double-stranded DNA (100 ng/. mu.l) | 1 |
| 10 × T4DNA ligase buffer | 2 |
| T4DNA ligase | 1 |
| Double distilled water (ddH)2O) | Make up to 20 |
5. Transformation of
Adding 10 mu L ligation reaction product into 100 mu L competent cells, flicking the tube wall number, mixing, placing on ice for 30min, performing heat shock at 42 ℃ for 90s, incubating in ice bath for 2min, adding 500 mu L L B culture medium, placing in a shaker at 37 ℃ for shake culture for 1h, taking a proper amount of bacterial liquid, uniformly coating on a plate containing corresponding antibiotics, and performing inverted culture in a constant temperature incubator for 12-16 h.
6. Sequencing identification
Inoculating the identified positive clone transformant into an appropriate amount of L B liquid culture medium containing corresponding antibiotics, culturing at 37 ℃ for 12-16h, and taking an appropriate amount of bacterial liquid for sequencing and identifying.
7. Plasmid transfection and lentivirus harvesting
Viral packaging involves a total of three plasmids: the vector comprises a tool vector plasmid GV493 vector (purchased from Shanghai Jikai gene science and technology Co., Ltd.), a virus packaging Helper plasmid Helper 1.0 vector (purchased from Shanghai Jikai gene science and technology Co., Ltd.) and a virus packaging Helper 2.0 vector (purchased from Shanghai Jikai gene science and technology Co., Ltd.). 293T cells were co-transfected with the above three plasmids.
24h before transfection, 293T cells in the logarithmic growth phase were trypsinized and cell density was adjusted to about 5 × 10 in medium containing 10% serum615ml, reseeding in 10cm cell culture dish, 37 deg.C, 5% CO2Culturing in an incubator; the cell can be used for transfection after 24 hours when the cell density reaches 70-80%; replacing the medium with a serum-free medium 2h before transfection; adding DNA solutions (GV493 plasmid 20 μ g, pHelper 1.0 vector plasmid 15 μ g, pHelper 2.0 vector plasmid 10 μ g) into a sterilized centrifuge tube, mixing with corresponding volume of Gecky transfection reagent, adjusting total volume to 1ml, and incubating at room temperature for 15 min; the mixed solution is slowly dripped into 293T cell culture solution, mixed evenly and treated at 37 ℃ with 5% CO2Culturing in a cell culture box; culturing for 6h, discarding the culture medium containing the transfection mixture, adding 10ml of PBS for cleaning once, gently shaking the culture dish to wash the residual transfection mixture, and then pouring and discarding;
slowly adding 10% serum-containing cell culture medium 20ml, and heating at 37 deg.C with 5% CO2Culturing in the incubator for 48-72 h.
8. Lentivirus concentration and purification and quality control
Collecting 293T cell supernatant 48h after transfection (which can be counted as 0h after transfection) according to cell states; centrifuging at 4000g for 10min at 4 deg.C to remove cell debris; filtering the supernatant with a 0.45 μm filter in a 40ml ultracentrifuge tube; respectively balancing samples, putting the ultracentrifuge tubes with virus supernatant into a Beckman ultracentrifuge one by one, centrifuging for 2h at 4 ℃ and 25000 rpm; after the centrifugation is finished, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding a virus preservation solution, and lightly and repeatedly blowing and resuspending; after full dissolution, centrifuging at high speed of 10000rpm for 5min, and taking the supernatant to subpackage according to the requirement;
the main points of the quality control of lentiviruses comprise physical state detection, sterility detection and virus titer detection.
Second, Lentiviral transfection
In order to ensure the gene interference efficiency, this example designs 3 RNA interference targets (siRNA) for FAM84B gene, and mixes 3 plasmids carrying different targets in equal proportion for lentivirus packaging, thereby ensuring the knockdown efficiency of the target gene after virus infection of cells.
After 12-16 hours of cell subculture into 6-well plates: mixing 0.15ml of virus solution with fresh cell culture solution uniformly, and adding 0.5ml of fresh culture solution and 0.65 μ l of polybrene (final concentration 4ng/ml) into the virus supernatant with the proportion of 0.15 ml; the premixed virus infection liquid is added into a target cell culture dish, and the cell density is not more than 50%. After overnight culture, the culture medium was replaced with fresh medium.
Cells in the logarithmic growth phase were taken 3 days after infection and subjected to cell proliferation assay.
Third, cell proliferation experiment
Pancreatin digestion is carried out on the control group and the cells in the logarithmic growth phase with the expression of the knocked-down FAM84B to prepare cell suspension; the cell suspension (cell number about 3000) was inoculated into a 96-well plate, and the cell number was counted on days 1, 2, 3, 4, and 5, respectively, to draw a growth curve.
Figure 2 shows the effect of knockdown of FAM84B on breast cancer cell proliferation. Wherein shCtrl is a control group, shFAM84B is an experimental group, the upper curve is a control group, and the lower curve is an experimental group. As shown in fig. 2, the cell proliferation experiment result shows that the knocking down of the expression of FAM84B gene significantly inhibits the proliferation of breast cancer cells MCF7 both at day 4 and day 5 (P <0.001 ).
Tumor bearing experiment of nude mouse breast cancer
The control group and the MCF7 breast cancer cell line with the knocked-down FAM84B are respectively prepared into cell suspensions for the implantation of fat pads of nude mice, 6 mice are inoculated with 100 mu l of 5 × 106And (4) cells. After 1.5 months, tumor size and volume were measured and statistically analyzed.
Figure 3 shows the effect of knockdown of FAM84B on breast cancer growth in tumor-bearing mice. NC is a control group, and shFAM84B is an experimental group. As shown in fig. 3, the tumor-bearing experimental results of mice showed that the tumor volume of breast cancer in FAM84B low expression group was significantly lower than that in control group (P < 0.01). It can be seen that reducing the expression of FAM84B gene can significantly inhibit breast cancer cell growth.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Sequence listing
<110> Jiangsu medical profession college
<120> use of nucleic acid, recombinant vector and recombinant lentivirus for specifically reducing FAM84B gene expression
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<213>Artificial Sequence
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<210>26
<211>20
<212>DNA
<213>Artificial Sequence
<400>26
<210>27
<211>20
<212>DNA
<213>Artificial Sequence
<400>27
<210>28
<211>20
<212>DNA
<213>Artificial Sequence
<400>28
Claims (6)
1. The application of the nucleic acid for specifically reducing the expression of the FAM84B gene and the recombinant vector and/or the recombinant lentivirus thereof in preparing the medicament for inhibiting the growth and/or proliferation of the breast cancer cells MCF 7.
2. The use according to claim 1, wherein the nucleic acid that specifically reduces the expression of FAM84B gene is siRNA that specifically reduces the expression of FAM84B gene, and the nucleotide sequence of the siRNA comprises at least one nucleotide sequence selected from the group consisting of:
(1) a first set of nucleotide sequences
The first group of nucleotide sequences are shown as SEQ ID NO 1 and SEQ ID NO 2, the SEQ ID NO 1 is 5'-CAACGAUCUGUACCGCUACAA-3', and the SEQ ID NO 2 is 5'-UUGUAGCGGUACAGAUCGUUG-3';
(2) second set of nucleotide sequences
The second group of nucleotide sequences are shown as SEQ ID NO 3 and SEQ ID NO 4, the SEQ ID NO 3 is 5'-AGUCUAGAGGACCUGAUCAUG-3', and the SEQ ID NO 4 is 5'-CAUGAUCAGGUCCUCUAGACU-3';
(3) a third group of nucleotide sequences
The third group of nucleotide sequences are shown as SEQ ID NO 5 and SEQ ID NO 6, wherein the SEQ ID NO 5 is 5'-GGUGGAAUGCUCCGUGUUCUA-3', and the SEQ ID NO 6 is 5'-UAGAACACGGAGCAUUCCACC-3'.
3. The use according to claim 1, wherein the nucleic acid that specifically reduces the expression of FAM84B gene is shRNA that specifically reduces the expression of FAM84B gene, and the nucleotide sequence of the shRNA comprises at least one of the following nucleotide sequences:
(1) nucleotide sequence of the fifth group
The fifth group of nucleotide sequences are shown as SEQ ID NO 9 and SEQ ID NO 10, wherein the SEQ ID NO 9 is 5'-CCGGCAACGAUCUGUACCGCUACAACUCGAGUUG UAGCGGUACAGAUCGUUGUUUUUG-3', and the SEQ ID NO 10 is 5'-AAUUCAAAAACAACGAUCUGUACCGCUACAACUCGAGUUGUAGCGGUACAGAUCGUUG-3';
(2) nucleotide sequence of group VI
The sixth group of nucleotide sequences are shown as SEQ ID NO. 11 and SEQ ID NO. 12, wherein the SEQ ID NO. 11 is 5'-CCGGAGUCUAGAGGACCUGAUCAUGCUCGAGCAUGAUCAGGUCCUCUAGACUUUUUUG-3', and the SEQ ID NO. 12 is 5'-AAUUCAAAAAAGUCUAGAGGACCUGAUCAUGCUCGAGCAUGAUCAGGUCCUCUAGACU-3';
(3) nucleotide sequence of group seven
The seventh group of nucleotide sequences are shown as SEQ ID NO. 13 and SEQ ID NO. 14, wherein the SEQ ID NO. 13 is 5'-CCGGGGUGGAAUGCUCCGUGUUCUACUCGAGUAGAACACGGAGCAUUCCACCUUUUUG-3', and the SEQ ID NO. 14 is 5'-AAUUCAAAAAGGUGGAAUGCUCCGUGUUCUACUCGAGUAGAACACGGAGCAUUCCACC-3'.
4. The use according to claim 1, wherein the nucleic acid that specifically reduces the expression of FAM84B gene is DNA encoding the shRNA according to claim 3, the nucleotide sequence of the DNA comprising at least one of the following group of nucleotide sequences:
(1) nucleotide sequence of the ninth group
The ninth group of nucleotide sequences are shown as SEQ ID NO 17 and SEQ ID NO 18, wherein the SEQ ID NO 17 is 5'-CCGGCAACGATCTGTACCGCTACAACTCGAGTTGTAGCGGTACAGATCGTTGTTTTTG-3', and the SEQ ID NO 18 is 5'-AATTCAAAAACAACGATCTGTACCGCTACAACTCGAGTTGTAGCGGTACAGATCGTTG-3';
(2) tenth group of nucleotide sequences
The tenth group of nucleotide sequences are shown as SEQ ID NO. 19 and SEQ ID NO. 20, wherein the SEQ ID NO. 19 is 5'-CCGGAGTCTAGAGGACCTGATCATGCTCGAGCATGATCAGGTCCTCTAGACTTTTTTG-3', and the SEQ ID NO. 20 is 5'-AATTCAAAAAAGTCTAGAGGACCTGATCATGCTCGAGCATGATCAGGTCCTCTAGACT-3';
(3) nucleotide sequence of the eleventh group
The eleventh group of nucleotide sequences are shown as SEQ ID NO 21 and SEQ ID NO 22, wherein the SEQ ID NO 21 is 5'-CCGGGGTGGAATGCTCCGTGTTCTACTCGAGTAGAACACGGAGCATTCCACCTTTTTG-3', and the SEQ ID NO 22 is 5'-AATTCAAAAAGGTGGAATGCTCCGTGTTCTACTCGAGTAGAACACGGAGCATTCCACC-3'.
5. The use according to claim 1, wherein the recombinant vector of the nucleic acid specifically reducing the expression of FAM84B gene is a recombinant vector obtained by inserting the DNA encoding shRNA according to claim 4 into the multiple cloning sites AgeI and EcoRI of the GV493 plasmid.
6. The use according to claim 1, wherein the recombinant lentivirus of a nucleic acid that specifically reduces the expression of the FAM84B gene is obtained by co-transfecting mammalian cells with the recombinant vector of claim 5 and the viral packaging helper plasmid phepper 1.0 vector and the viral packaging helper plasmid phepper 2.0 vector.
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