CN110904104B - Application of human HIST1H2BK gene and related products - Google Patents

Application of human HIST1H2BK gene and related products Download PDF

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CN110904104B
CN110904104B CN201911171162.0A CN201911171162A CN110904104B CN 110904104 B CN110904104 B CN 110904104B CN 201911171162 A CN201911171162 A CN 201911171162A CN 110904104 B CN110904104 B CN 110904104B
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hist1h2bk
gene
liver cancer
shrna
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CN110904104A (en
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陈闯
甘激文
邬国斌
黄山
李科志
何剑波
王宗玉
林栋毅
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Guangxi Medical University Affiliated Tumour Hospital
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Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of a human HIST1H2BK gene serving as a target in preparation of a liver cancer therapeutic drug. The invention is widely and deeply researched, and discovers that the RNAi method is adopted to down regulate the expression of human HIST1H2BK gene, can effectively inhibit proliferation of liver cancer cells, promote apoptosis and can effectively control the growth process of liver cancer. The siRNA or the nucleic acid construct containing the siRNA sequence and the slow virus provided by the invention can specifically inhibit the proliferation rate of liver cancer cells, promote the apoptosis of the liver cancer cells, inhibit the cloning of the liver cancer cells and inhibit the growth of the liver cancer, thereby treating the liver cancer and opening up a new direction for the treatment of the liver cancer.

Description

Application of human HIST1H2BK gene and related products
Technical Field
The invention belongs to the field of biomedical research, and particularly relates to application of a human HIST1H2BK gene and related products.
Background
Histones are the basic nucleoproteins responsible for the chromosomal fibrous nucleosome structure in eukaryotes. Each of the four core histones (H2A, H2B, H and H4) has two molecules forming an octamer surrounded by about 146bp of DNA, and this repeat unit is called the nucleosome. The DNA-linked interactions between the linked histone H1 and nucleosomes play a role in the formation of chromatin into high-level structures. The protein encoded by the HIST1H2BK gene is a replication-dependent histone of one of the members of the histone H2B family, is a core component of nucleosomes, and has activity against bacteria and fungi. Currently, the HIST1H2BK gene contains two transcripts encoding the same protein, is located on chromosome 6p21.33 and is expressed in 25 tissues such as prostate (RPKM 42.3) and testis (RPKM 29.8). HIST1H2BK is involved in meiosis and Rho GTPase signaling pathways. Gene annotation analysis found that the gene was associated with sequence specific DNA binding and protein heterodimer activity. An important homolog of this gene is HIST1H2BI. The HIST1H2BK gene is involved in the activated PKN1 pathway stimulating transcription of the androgen receptor regulated genes KLK2 and KLK 3.
Han J et al found high expression of HIST1H2BK in drug resistant cells of triple negative breast cancer, possibly contributing to the formation of doxorubicin resistant cells by a mechanism of tumor dormancy, and the HDAC inhibitor SAHA increased therapeutic sensitivity of doxorubicin. Investigation and analysis of clinical triple negative breast cancer patients show that: the recurrence-free survival Rate (RFS) of patients with high expression of HIST1H2BK is obviously lower than that of patients with low expression of HIST1H2 BK. Analysis of GEO data by bioinformatic methods for invasive ductal breast cancer, li C, etc., found that HIST1H2BK as a hub gene may be involved in the process of invasive ductal breast cancer.
In addition, HIST1H2BK expression also survived neuroblastoma patients. Sequencing results analysis of serum exosomes from Pancreatic Ductal Adenocarcinoma (PDAC) patients gave: the HIST1H2BK can form a diagnosis marker model with 7 other genes, has higher sensitivity, and can distinguish early-stage (I/II) patients and late-stage (III/IV) patients of pancreatic cancer; can distinguish the PDAC cases with negative CA19-9 from normal persons, and is helpful for accurate diagnosis of pancreatic duct adenocarcinoma.
No document reports the effect of HIST1H2BK in the occurrence and development of liver cancer at present.
Disclosure of Invention
In order to overcome the problems of the prior art, the present invention aims to provide the use of human HIST1H2BK gene and related products.
In order to achieve the above and other related objects, the present invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided the use of the human HIST1H2BK gene as a target in the preparation of a medicament for the treatment of liver cancer.
The human HIST1H2BK gene serving as a target is specifically used for preparing liver cancer therapeutic drugs: the HIST1H2BK gene is taken as an acting object, and medicines or preparations are screened to find medicines capable of inhibiting the expression of the human HIST1H2BK gene to be taken as medicines for treating liver cancer. The small interfering RNA (siRNA) of the HIST1H2BK gene is obtained by taking the human HIST1H2BK gene as an acting object to be screened, and can be used as a medicament with the effect of inhibiting liver cancer cell proliferation. In addition, for example, antibody drugs, small molecule drugs, and the like can also target the HIST1H2BK gene.
The liver cancer therapeutic drug is a molecule capable of specifically inhibiting transcription or translation of the HIST1H2BK gene or specifically inhibiting expression or activity of the HIST1H2BK protein, so that the expression level of the HIST1H2BK gene in liver cancer cells is reduced, and the purposes of inhibiting proliferation, growth, differentiation and/or survival of the liver cancer cells are achieved.
The liver cancer therapeutic drugs prepared by the HIST1H2BK gene include but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins or interfering lentiviruses.
Such nucleic acids include, but are not limited to: antisense oligonucleotides, double-stranded RNAs (dsRNA), ribozymes, small interfering RNAs prepared by endoribonuclease III, or short hairpin RNAs (shRNA).
The liver cancer therapeutic agent is administered in an amount sufficient to reduce transcription or translation of the human HIST1H2BK gene or to reduce expression or activity of the human HIST1H2BK protein. So that the expression of the human HIST1H2BK gene is reduced by at least 50%, 80%, 90%, 95% or 99%.
The method for treating liver cancer by adopting the liver cancer treatment drug mainly achieves the aim of treatment by reducing the expression level of human HIST1H2BK gene and inhibiting proliferation of liver cancer cells. Specifically, a substance effective to reduce the expression level of human HIST1H2BK gene is administered to a patient at the time of treatment.
In one embodiment, the target sequence of the HIST1H2BK gene is set forth in SEQ ID NO: 1. The method comprises the following steps: 5'-GAGAAAGAGTATATAAGCT-3'.
In a second aspect of the invention there is provided the use of an inhibitor of HIST1H2BK in the preparation of a product having at least one of the following effects:
treating liver cancer;
inhibiting proliferation rate of liver cancer cells;
promoting apoptosis of liver cancer cells;
inhibiting liver cancer cell cloning;
inhibit liver cancer growth.
The product necessarily includes a his 1H2BK inhibitor and has the his 1H2BK inhibitor as an active ingredient for the aforementioned efficacy.
In the product, the active ingredient which can play the role can be only an inhibitor of HIST1H2BK, and other molecules which can play the role can also be contained.
That is, the inhibitor of HIST1H2BK is the only active ingredient or one of the active ingredients of the product.
The product can be a single component substance or a multi-component substance.
The form of the product is not particularly limited, and may be solid, liquid, gel, semifluid, aerosol, or the like.
The subject to which the product is primarily directed is a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, etc. The primate is preferably a monkey, ape or human.
Such products include, but are not limited to, pharmaceuticals, nutraceuticals, foods, and the like.
The HIST1H2BK inhibitor can be a nucleic acid molecule, an antibody or a small molecule compound.
As exemplified in the examples of the present invention, the inhibitor of HIST1H2BK may be a nucleic acid molecule that reduces expression of the HIST1H2BK gene in liver cancer cells. Specifically, it may be a double-stranded RNA or an shRNA.
In a third aspect of the present invention, there is provided a method of treating liver cancer by administering to a subject an inhibitor of HIST1H2 BK.
The subject may be a mammal or a liver cancer cell of a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, etc. The primate is preferably a monkey, ape or human. The liver cancer cell may be an ex vivo liver cancer cell.
The subject may be a patient suffering from liver cancer or an individual desiring treatment of liver cancer. Or the subject is an isolated liver cancer cell of a liver cancer patient or an individual desiring to treat liver cancer.
The his 1H2BK inhibitor may be administered to the subject before, during, or after receiving liver cancer therapy.
In a fourth aspect, the invention discloses a nucleic acid molecule for reducing expression of a HIST1H2BK gene in a liver cancer cell, wherein the nucleic acid molecule comprises double-stranded RNA or shRNA.
Wherein the double-stranded RNA contains a nucleotide sequence capable of hybridizing with the HIST1H2BK gene;
the shRNA contains a nucleotide sequence capable of hybridizing with the HIST1H2BK gene.
Further, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary together to form an RNA dimer, and the sequence of the first strand is substantially identical to a target sequence in the HIST1H2BK gene.
The target sequence in the HIST1H2BK gene is a fragment in the HIST1H2BK gene corresponding to an mRNA fragment recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used for specifically silencing the expression of the HIST1H2BK gene.
Further, the target sequence of the double-stranded RNA is shown as SEQ ID NO: 1. The method comprises the following steps: 5'-GAGAAAGAGTATATAAGCT-3'. Further, the sequence of the first strand of the double-stranded RNA is shown in SEQ ID NO: 2. Specifically 5'-GAGAAAGAGUAUAUAAGCU-3'.
Further, the double stranded RNA is small interfering RNA (siRNA).
SEQ ID NO:2 is one strand of small interfering RNA which is designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aims at the human HIST1H2BK gene, the sequence of the other strand, namely the second strand, is complementary with the sequence of the first strand, and the siRNA can play a role in specifically silencing the expression of the endogenous HIST1H2BK gene in liver cancer cells.
The shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, wherein the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is substantially identical to a target sequence in the HIST1H2BK gene.
Further, the target sequence of the sh RNA is shown as SEQ ID NO: 1.
The shRNA can become small interfering RNA (siRNA) after enzyme digestion processing, thereby playing a role in specifically silencing the expression of endogenous HIST1H2BK genes in liver cancer cells.
Further, the sequence of the stem-loop structure of the shRNA may be selected from any one of the following: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU and CCACACC.
Further, the sequence of the shRNA is shown as SEQ ID NO: 3. Specifically 5'-CUGAGAAAGAGUAUAUAAGCUCUCGAGAGCUUAUAUACUCUUUCUCAG-3'.
Further, the HIST1H2BK gene is derived from human.
In a fifth aspect, the invention discloses a HIST1H2BK gene interference nucleic acid construct, which contains a gene fragment encoding shRNA in the nucleic acid molecule and can express the shRNA.
The HIST1H2BK gene interfering nucleic acid construct can be obtained by cloning a gene fragment encoding the human HIST1H2BK gene shRNA into a known vector.
Further, the HIST1H2BK gene interfering nucleic acid construct is a HIST1H2BK gene interfering lentiviral vector.
The HIST1H2BK gene interference slow virus vector disclosed by the invention is obtained by cloning a DNA fragment for encoding the HIST1H2BK gene shRNA into a known vector, wherein most of the known vectors are slow virus vectors, the HIST1H2BK gene interference slow virus vector is packaged into infectious virus particles by viruses, then is used for infecting liver cancer cells, further is transcribed into the shRNA, and finally is used for specifically silencing the expression of the HIST1H2BK gene through the steps of enzyme cutting and the like.
Furthermore, the HIST1H2BK gene interference slow virus vector also contains a promoter sequence and/or a nucleotide sequence for encoding a marker which can be detected in liver cancer cells; preferably, the detectable label is a Green Fluorescent Protein (GFP).
Further, the lentiviral vector may be selected from the group consisting of: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635 any one of pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-lamrishma, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.
The embodiment of the invention specifically enumerates a human HIST1H2BK gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, and is named pGCSIL-GFP-HIST1H2BK-siRNA.
The HIST1H2BK gene siRNA can be used for inhibiting proliferation of liver cancer cells, and further can be used as a medicament or preparation for treating liver cancer. The HIST1H2BK gene interference lentiviral vector can be used for preparing the HIST1H2BK gene siRNA. When used as a medicament or formulation for treating liver cancer, a safe and effective amount of the nucleic acid molecule is administered to a mammal. The particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
In a sixth aspect of the present invention, a HIST1H2BK gene interfering lentivirus is disclosed, wherein the HIST1H2BK gene interfering nucleic acid construct is packaged by virus with the aid of a lentivirus packaging plasmid and a cell line. The slow virus can infect liver cancer cells and generate small interfering RNA aiming at HIST1H2BK genes, thereby inhibiting proliferation of the liver cancer cells. The HIST1H2BK gene interference lentivirus can be used for preparing medicines for preventing or treating liver cancer.
In a seventh aspect of the present invention, there is provided the use of the aforementioned nucleic acid molecule, or the aforementioned HIST1H2BK gene interfering nucleic acid construct, or the aforementioned HIST1H2BK gene interfering lentivirus, as follows: the method is used for preparing medicines for preventing or treating liver cancer or preparing a kit for reducing the expression of HIST1H2BK genes in liver cancer cells.
The application of the medicine for preventing or treating liver cancer provides a method for treating liver cancer, in particular to a method for preventing or treating liver cancer in a subject, which comprises the step of applying an effective dose of the medicine to the subject.
Further, when the medicament is used for preventing or treating liver cancer in a subject, an effective dose of the medicament is required to be administered to the subject. With this method, the growth, proliferation, recurrence and/or metastasis of the liver cancer are inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the portion of the liver cancer that grows, proliferates, recurs and/or metastasizes is inhibited.
The object of the method may be a person.
In an eighth aspect of the present invention, there is provided a composition for preventing or treating liver cancer, comprising:
the nucleic acid molecules as described above; and/or, the aforementioned HIST1H2BK gene interfering nucleic acid construct; and/or, the aforementioned HIST1H2BK gene interferes with lentiviruses, as well as pharmaceutically acceptable carriers, diluents or excipients.
The composition may be a pharmaceutical composition.
When the composition is used for preventing or treating liver cancer in a subject, an effective dose of the composition is required to be administered to the subject. With this method, the growth, proliferation, recurrence and/or metastasis of the liver cancer are inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the portion of the liver cancer that grows, proliferates, recurs and/or metastasizes is inhibited.
The form of the composition is not particularly limited, and may be solid, liquid, gel, semifluid, aerosol, or the like.
The subject to which the composition is primarily directed is a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, etc. The primate is preferably a monkey, ape or human.
In summary, the invention designs an RNAi target sequence aiming at the human HIST1H2BK gene, and constructs a corresponding HIST1H2BK RNAi vector, wherein the RNAi vector pGCSIL-GFP-HIST1H2BK-siRNA can obviously down regulate the expression of the HIST1H2BK gene at mRNA level and protein level. The RNAi sequence aiming at the HIST1H2BK gene can be efficiently introduced into liver cancer SMMC-7721 cells in a targeted manner by using lentiviruses (abbreviated as Lv) as a gene manipulation tool to carry RNAi vectors pGCSIL-GFP-HIST1H2BK-siRNA, so that the expression level of the HIST1H2BK gene is reduced, and the proliferation capacity of the tumor cells is obviously inhibited. Lentivirus mediated HIST1H2BK gene silencing is thus a potential clinical non-surgical treatment modality for malignant tumors.
Compared with the prior art, the invention has the following beneficial effects:
the invention is widely and deeply researched, and discovers that the RNAi method is adopted to down regulate the expression of human HIST1H2BK gene, can effectively inhibit proliferation of liver cancer cells, promote apoptosis and can effectively control the growth process of liver cancer. The siRNA or the nucleic acid construct containing the siRNA sequence and the slow virus provided by the invention can specifically inhibit the proliferation rate of liver cancer cells, promote the apoptosis of the liver cancer cells, inhibit the cloning of the liver cancer cells and inhibit the growth of the liver cancer, thereby treating the liver cancer and opening up a new direction for the treatment of the liver cancer.
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Fig. 1: and detecting the target gene reduction efficiency of the mRNA level of the SMMC-7721 cells by RT-PCR.
Fig. 2: and detecting the SMMC-7721 cell target spot by using a Western Blot to reduce the expression level of the HIST1H2BK gene protein.
Fig. 3: the Celigo cell automatic analysis result reveals the influence on SMMC-7721 liver cancer cell proliferation after infection of HIST1H2BK-shRNA lentivirus. (1, 2,3,4 and 5 days after viral infection, cell numbers were counted respectively)
Fig. 4: the CCK8 method detects the influence of HIST1H2BK-shRNA lentivirus on SMMC-7721 cell proliferation capacity after 5 days of infection.
Fig. 5: the influence of the clone formation experiment on the clone formation ability of SMMC-7721 cells after infection of HIST1H2BK-shRNA lentivirus is shown on the left side of the physical graph, and the average value of three experiments is shown on the right side.
Fig. 6-1: annexin V-APC flow apoptosis detection effects on SMMC-7721 apoptosis after infection with HIST1H2BK-shRNA lentivirus.
Fig. 6-2: annexin V-APC flow apoptosis detection of infection of HIST1H2BK-shRNA lentiviruses on SMMC-7721 apoptosis, columnar results are shown as percentage mean ± standard deviation.
In the drawings of which there are shown,
the bar graph represents the average of three experiments and the error bars represent Standard Deviation (SD).
* shCtrl has P <0.01 compared to the target gene shRNA lentivirus treatment group.
* Compared with the target gene shRNA lentivirus treatment group, the shCtrl has the P of more than or equal to 0.01 and less than or equal to 0.05.
Detailed Description
The invention confirms the function of HIST1H2BK gene in liver cancer occurrence from the aspect of cell functional science. The expression condition of mRNA and protein level target genes in two groups of liver cancer cell lines is detected by constructing target gene shRNA lentivirus and then transfecting liver cancer cells and comparing the target gene shRNA lentivirus with a transfection control lentivirus; and then, cell proliferation, apoptosis and other detection are carried out through a cell functional experiment, and the result shows that the shRNA group is compared with the control group, the liver cancer cell proliferation inhibition degree of the shRNA group is obviously higher than that of the control group, and the apoptosis rate increase degree is higher than that of the control group.
HIST1H2BK inhibitors
Refers to a molecule having an inhibitory effect on HIST1H2 BK. Having inhibitory effects on his 1H2BK includes, but is not limited to: inhibit expression or activity of HIST1H2 BK.
Inhibiting HIST1H2BK activity refers to decreasing HIST1H2BK activity. Preferably, the HIST1H2BK activity is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, most preferably by at least 90% compared to that prior to inhibition.
Inhibition of expression of HIST1H2BK may specifically be inhibition of transcription or translation of the HIST1H2BK gene, and specifically may be: the transcription activity of the gene of HIST1H2BK is reduced, or the translation level of the gene of HIST1H2BK is reduced, or the gene of HIST1H2BK is not translated.
The person skilled in the art can use conventional methods for regulating the gene expression of HIST1H2BK, such as gene knockout, homologous recombination, interfering RNA, etc.
The inhibition of gene expression of HIST1H2BK can be verified by PCR and Western Blot detection.
Preferably, the expression of the HIST1H2BK gene is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, yet more preferably by at least 90% compared to the wild type, and most preferably the HIST1H2BK gene is not expressed at all.
Small molecule compounds
The present invention refers to a compound having a molecular mass of 1000 or less, which is composed of several or several tens of atoms.
Preparation of medicine for preventing or treating liver cancer
Nucleic acid molecules that reduce expression of the HIST1H2BK gene in hepatoma cells can be utilized; and/or, the HIST1H2BK gene interferes with the nucleic acid construct; and/or HIST1H2BK gene interference slow virus is used as an active ingredient to prepare a medicament for preventing or treating liver cancer. Typically, the medicament will include, in addition to the active ingredient, one or more pharmaceutically acceptable carriers or excipients, as required by the different dosage forms.
By "pharmaceutically acceptable" is meant that the molecular entity and composition do not produce adverse, allergic or other untoward reactions when properly administered to an animal or human.
The "pharmaceutically acceptable carrier or adjuvant" should be compatible with the active ingredient, i.e. it can be blended therewith without substantially reducing the efficacy of the drug in the usual manner. Specific examples of some substances which may be pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc. These substances are used as needed to aid stability of the formulation or to aid in enhancing the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.
In the present invention, the pharmaceutical dosage form is not particularly limited unless otherwise specified, and may be formulated into injection, oral liquid, tablet, capsule, dripping pill, spray, etc., and may be prepared by conventional methods. The choice of the pharmaceutical dosage form should be compatible with the mode of administration.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. The test methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the respective manufacturers.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts.
Example 1 preparation of RNAi lentiviruses against human HIST1H2BK Gene
1. Screening effective siRNA target spot aiming at human HIST1H2BK gene
Retrieving HIST1H2BK (NM_ 080593) gene information from Genbank; an effective siRNA target aiming at the HIST1H2BK gene is designed. Table 1-1 shows the effective siRNA target sequences screened against the HIST1H2BK gene.
TABLE 1-1 siRNA target sequences targeting the human HIST1H2BK Gene
SEQ ID NO TargetSeq(5’-3’)
1 GAGAAAGAGTATATAAGCT
2. Preparation of lentiviral vectors
Double-stranded DNA Oligo sequences (tables 1-2) containing the Age I and EcoR I cleavage site sticky ends at both ends are synthesized aiming at siRNA targets (taking SEQ ID NO:1 as an example); the restriction enzymes Age I and EcoR I were used to linearize pGCSIL-GFP vector (available from Shanghai Ji Kai Gene chemical technologies Co., ltd.) and the cut fragments were identified by agarose gel electrophoresis.
TABLE 1-2 double-stranded DNA Oligo containing Age I and EcoR I cleavage sites at both ends
The vector DNA, which was digested with double enzymes and digested with T4 DNA ligase (cleavage system shown in tables 1 to 4, 37 ℃ C., 1h of reaction) was ligated to the purified double-stranded DNA Oligo, and the ligation was performed overnight at 16 ℃ in a suitable buffer system (ligation system shown in tables 1 to 5) to recover the ligation product. The ligation products were transformed into fresh E.coli competent cells prepared from calcium chloride (transformation protocol: see second edition of molecular cloning protocol pages 55-56). Dipping a surface of a clone growing with a transformation product, dissolving in 10 μl of LB culture medium, uniformly mixing, and taking 1 μl as a template; upstream and downstream of the RNAi sequence in the lentiviral vector, universal PCR primers were designed, upstream primer sequences: 5'-CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO: 6); downstream primer sequence: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7) and performing PCR identification experiments (the PCR reaction systems are shown in tables 1-6, and the reaction conditions are shown in tables 1-7). Sequencing and comparing the clones positive to the PCR identification, and comparing the correct clones to obtain the sequence of the sequence shown in SEQ ID NO:1, named pGCSIL-GFP-HIST1H2BK-siRNA.
pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed, the negative control siRNA target sequence was 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). When constructing pGCSIL-GFP-Scr-siRNA negative control plasmid, double-stranded DNA Oligo sequences (tables 1-3) containing Age I and EcoR I restriction enzyme site sticky ends at two ends are synthesized aiming at the Scr siRNA target spot, and other construction methods, identification methods and conditions are the same as those of pGCSIL-GFP-HIST1H2BK-siRNA.
Tables 1-3 double-stranded DNA Oligo containing the sticky ends of the Age I and EcoRI cleavage sites at both ends
Table 1-4 pGCSIL-GFP plasmid cleavage reaction System
Reagent(s) Volume (mul)
pGCSIL-GFP plasmid (1. Mu.g/. Mu.l) 2.0
10×buffer 5.0
100×BSA 0.5
Age I(10U/μl) 1.0
EcoR I(10U/μl) 1.0
dd H 2 O 40.5
Total 50.0
TABLE 1-5 vector DNA and double-stranded DNA Oligo ligation reaction System
Reagent(s) Positive control (μl) Self-connecting control (mul) Connection group (mu l)
Linearized vector DNA (100 ng/. Mu.l) 1.0 1.0 1.0
Annealed double-stranded DNA Oligo (100 ng/. Mu.l) 1.0 - 1.0
10×T4 phage DNA ligase buffer 1.0 1.0 1.0
T4 phage DNA ligase 1.0 1.0 1.0
dd H 2 O 16.0 17.0 16.0
Total 20.0 20.0 20.0
TABLE 1-6 PCR reaction System
Reagent(s) Volume (mul)
10×buffer 2.0
dNTPs(2.5mM) 0.8
Upstream primer 0.4
Downstream primer 0.4
Taq polymerase 0.2
Template 1.0
ddH 2 O 15.2
Total 20.0
TABLE 1-7 Programming of PCR reaction System
3. Packaging HIST1H2BK-shRNA lentiviruses
DNA of RNAi plasmid pGCSIL-GFP-HIST1H2BK-siRNA was extracted with a plasmid extraction kit from Qiagen, and 100 ng/. Mu.l of the stock solution was prepared.
24h before transfection, human embryonic kidney 293T cells in logarithmic growth phase were digested with trypsin and cell density was adjusted to 1.5X10% in DMEM complete medium containing 10% fetal bovine serum 5 Cells/ml, seeded in 6-well plates, 37 ℃,5% CO 2 Culturing in an incubator. And the cell density reaches 70-80% and can be used for transfection. 2h before transfection, the original medium was aspirated and 1.5ml of fresh complete medium was added. 20. Mu.l of Packing Mix (PVM), 12. Mu.l of PEI, 400. Mu.l of serum-free DMEM medium, 20. Mu.l of the extracted plasmid DNA were added to the PVM/PEI/DMEM mixture as described in Sigma-aldrich company MISSION Lentiviral Packaging Mix kit.
Incubating the above transfection mixture at room temperature for 15min, transferring into culture medium of human embryo kidney 293T cells, 37 ℃ and 5% CO 2 Culturing in an incubator for 16h. The medium containing the transfection mixture was discarded, washed with PBS solution, and 2ml of complete medium was added to continue the culture for 48 hours. Cell supernatants were collected, and lentiviruses purified and concentrated by a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) as follows: (1) centrifuging at 4 ℃ for 10min at 4000g to remove cell debris; (2) The supernatant was filtered through a 0.45 μm filter in a 40ml ultracentrifuge tube; (3) Centrifuging at 4000g for 10-15min to obtain the required virus concentration volume; (4) After centrifugation, separating the filter cup from the lower filtrate collecting cup, reversely buckling the filter cup on the sample collecting cup, and centrifuging for 2min until the centrifugal force is not more than 1000g; (5) The centrifuge cup is removed from the sample collection cup and the virus concentrate is present in the sample collection cup. Will be illThe toxic concentrated solution is packaged and stored at-80 ℃. The sequence of the first strand of siRNA contained in the virus concentrate is shown as SEQ ID NO. 2. The packaging process of the control lentivirus is the same as that of the HIST1H2BK-shRNA lentivirus, and only pGCSIL-GFP-Scr-siRNA vector is used for replacing pGCSIL-GFP-HIST1H2BK-siRNA vector.
Example 2 real-time fluorescent quantitative RT-PCR method for detecting silencing efficiency of Gene
Human liver cancer SMMC-7721 cells in logarithmic growth phase are subjected to pancreatin digestion to prepare cell suspension (cell number is about 5×10) 4 Per ml) was inoculated into 6-well plates and cultured until the cell fusion reached about 30%. According to the complex value of infection (MOI, SMMC-7721:10), the lentivirus prepared in example 1 was added in a suitable amount, the medium was changed after 24 hours of culture, and after the infection time reached 5 days, the cells were collected. Total RNA was extracted according to Trizol protocol from Invitrogen. RNA was reverse transcribed to obtain cDNA according to the M-MLV protocol from Promega (reverse transcription reaction system see Table 2-1, 42℃for 1h, followed by inactivation of reverse transcriptase in a water bath at 70℃for 10 min).
Real-time quantitative detection was performed using a Real time PCR instrument model TP800 (TAKARA). Primers for the HIST1H2BK gene were as follows: an upstream primer 5'-TCGTAGTTCGCCTTCAACAT-3' (SEQ ID NO: 11) and a downstream primer 5'-CTTCTTCTGCGCCTTAGTCA-3' (SEQ ID NO: 12). The housekeeping gene GAPDH is taken as an internal reference, and the primer sequences are as follows: an upstream primer 5'-TGACTTCAACAGCGACACCCA-3' (SEQ ID NO: 13) and a downstream primer 5'-CACCCTGTTGCTGTAGCCAAA-3' (SEQ ID NO: 14). The reaction system was prepared in the proportions shown in Table 2-2.
TABLE 2-1 reverse transcription reaction system
Reagent(s) Volume (mul)
5×RT buffer 4.0
10mM dNTPs 2.0
RNasin 0.5
M-MLV-RTase 1.0
RNase-Free 2.6
Total 10.0
TABLE 2-2 Real-time PCR reaction System
Reagent(s) Volume (mul)
SYBR premix ex taq 6.0
Primer MIX (5. Mu.M) 0.3
cDNA 0.6
ddH 2 O 5.1
Total 12.0
The procedure was set as two-step Real-time PCR: pre-denaturation at 95 ℃ for 30s; then each step is denatured at 95 ℃ for 5s; annealing and extending at 60 ℃ for 30s; a total of 40 cycles were performed. The absorbance was read each time during the extension phase. After the PCR was completed, the DNA was denatured at 95℃for 15 seconds, and then cooled to 60℃to allow the DNA double strand to bind sufficiently. Starting from 60 ℃ to 95 ℃, increasing the temperature by 0.5 ℃ in each step, keeping for 4s, and simultaneously reading the light absorption value to prepare a melting curve. By 2 -ΔΔCt The assay calculates the abundance of expression of the mRNA that infects HIST1H2 BK. Cells infected with control virus served as controls. The experimental results are shown in FIG. 1, which shows that the expression level of HIST1H2BK mRNA in human liver cancer SMMC-7721 cells is reduced by 70.6%.
Example 3 Western Blotting detection of silencing efficiency of Gene
1. Extraction of total cell proteins
1) Control viruses and RNAi viruses directed against the HIST1H2BK interference target were determined based on complex values of infection (MOI: 10 The target cells (SMMC-7721 cells) were infected.
2) After 5 days of infection, cell samples were collected and appropriate amounts of RIPA lysate (bi yun day, P0013C) were taken and PMSF was added over the last few minutes to a final concentration of 1mM.
3) Adding proper amount of RIPA lysate, and performing ice lysis for 10-15min. Cells were scraped off and transferred into new EP tubes, and then sonicated (20 times 40W total, 1s each, 2s intervals).
4) Centrifugation was performed at 12000g at 4℃for 15min, and the supernatant was collected by BCA Protein Assay Kit (manufacturer: biyun (a Chinese character) and goods number: P0010S) protein concentration was determined.
5) The addition of fresh lysate adjusts the protein concentration for each sample to be consistent, typically 2. Mu.g/. Mu.L. Then adding 1/5 volume of 6 locking buffer, mixing, decocting in 100 deg.C metal bath for 10min, centrifuging, and preserving at-80deg.C.
2.SDS-PAGE
1) And (3) glue preparation: the glues with different concentrations are prepared according to the molecular weight of the target protein, and the specific systems are shown in tables 3-1, 3-2 and 3-3:
TABLE 3-1 SDS-PAGE separating gel (8 mL system)
Separation gel (8 mL system) 8％ 9％ 10％ 12％ 13％ 15％
H 2 O 3.7 3.4 3.1 2.6 2.3 1.8
30％PAGE 2.1 2.4 2.7 3.2 3.5 4
1.5mol/L Tris(pH 8.8) 2 2 2 2 2 2
10％SDS 0.08 0.08 0.08 0.08 0.08 0.08
10％APS 0.08 0.08 0.08 0.08 0.08 0.08
TEMED 0.005 0.004 0.004 0.004 0.004 0.004
TABLE 3-2 SDS-PAGE separating gel (10 mL system)
Separation gel (10 mL system) 8％ 9％ 10％ 12％ 13％ 15％
H 2 O 4.6 4.3 4 3.3 2.9 2.3
30％PAGE 2.7 3 3.3 4 4.4 5
1.5mol/L Tris(pH 8.8) 2.5 2.5 2.5 2.5 2.5 2.5
10％SDS 0.1 0.1 0.1 0.1 0.1 0.1
10％APS 0.1 0.1 0.1 0.1 0.1 0.1
TEMED 0.006 0.004 0.004 0.004 0.004 0.004
TABLE 3-3 SDS-PAGE gel (different systems)
Concentrated glue (5%) 3mL 4mL 5mL
H 2 O 2.1 2.7 3.4
30％PAGE 0.5 0.67 0.83
1.0mol/L Tris(pH6.8) 0.38 0.5 0.63
10％SDS 0.03 0.04 0.05
10％APS 0.03 0.04 0.05
TEMED 0.003 0.004 0.005
2) Loading: after the glue is solidified, the comb is pulled out, the electrophoresis buffer solution is used for cleaning the sample loading hole, and the prepared sample is loaded.
3) Electrophoresis: concentrating the gel 80mA for 20min; the gel was separated 120mA for 1h.
3. Immunoblot (Wet turn)
After electrophoresis, the protein was transferred to PVDF membrane by using a transfer electrophoresis apparatus and electroblotting at 4℃for 150min under 300mA constant current.
4. Antibody hybridization:
1) Closing: the PVDF membrane was blocked with blocking solution (TBST solution containing 5% skimmed milk) at room temperature for 1h or overnight at 4 ℃.
2) Incubation resistance: histone H2B K primary antibody (abcam) was diluted 1:200 with blocking solution, GAPDH (Santa Cruz) diluted 1:2000, then incubated with blocked PVDF membrane for 2H or overnight at 4℃at room temperature, and washed with TBST 4 times for 8min each.
3) Secondary antibody incubation: the membrane was incubated with blocking solution for 1.5h at room temperature by diluting the rabit IgG and mouse IgG secondary antibodies (SantaCruz from each manufacturer) at 1:2000, and washed with TBST 4 times for 8min each.
X-ray development:
1) Using CST company 20XReagent and 20X Peroxide#7003 kit, mix solution A and solution B in the kit according to 1:1 proportion and mix them upside down, stand for several minutes and then use. />
2) Taking out the film, wiping the water absorbing paper, spreading the film into a magazine, dripping a proper amount of well mixed ECL luminous solution, spreading a preservative film (avoiding generating bubbles), putting an X-ray film (avoiding the movement of the X-ray film), closing the magazine, exposing for 1s to a plurality of minutes (the exposure time needs to be more than tried several times, and properly adjusting the exposure time according to whether the naked eyes can see fluorescence and the intensity of the fluorescence).
3) Taking out the X-ray film, putting into a developing solution, taking out after the strip appears, rinsing in clear water for a few seconds, and putting into a fixing solution for at least 2min.
4) Taking out the X-ray film, airing and analyzing.
As shown in FIG. 2, western Blot experiments show that the target has a knockdown effect on endogenous expression of the HIST1H2BK gene, so that the target is an effective target.
EXAMPLE 4 Celigo assay to examine proliferation potency of tumor cells infected with HIST1H2BK-shRNA lentiviruses
Human liver cancer SMMC-7721 cells in logarithmic growth phase are subjected to pancreatin digestion to prepare cell suspension (cell number is about 5×10) 4 Per ml) was inoculated into 6-well plates and cultured until the cell fusion reached about 30%. According to the complex number of infection (MOI, SMMC-7721:10), a proper amount of virus is added, the culture medium is replaced after 24 hours of culture, and after the infection time reaches 5 days, the cells of each experimental group in the logarithmic growth phase are collected. Complete medium resuspension of the adult cell suspension (2X 10) 4 Per ml), 96-well plates were seeded at a cell density of about 2000 cells per well. Each group had 5 duplicate wells, 100 μl per well. After being paved, the mixture is placed at 37 ℃ and 5 percent of CO 2 Culturing in an incubator. The plates were read once daily with a Celigo instrument (Nexcelom) starting the next day after plating and were continuously tested for 5 days. Accurately calculating the number of cells with green fluorescence in each scanning hole plate by adjusting the input parameters of analysis settings; statistical plots were made on the data to plot 5 day cell proliferation curves.
The results are shown in fig. 3, and the results show that after each tumor in the lentivirus infected group is cultured in vitro for 5 days, the proliferation speed is obviously slowed down, which is far lower than that of the tumor cells in the control group, and the number of viable cells is reduced by 46.4%, which indicates that HIST1H2BK gene silencing leads to inhibition of the proliferation capacity of human liver cancer SMMC-7721 cells.
Example 5 CCK8 assay to examine proliferation potency of tumor cells infected with HIST1H2BK-shRNA lentiviruses
Human liver cancer SMMC-7721 cells in logarithmic growth phase are subjected to pancreatin digestion to prepare cell suspension (cell number is about 5×10) 4 Per ml) was inoculated into 6-well plates and cultured until the cell fusion reached about 30%. According to the complex number of infection (MOI, SMMC-7721:10), a proper amount of virus is added, the culture medium is replaced after 24 hours of culture, and after the infection time reaches 5 days, the cells of each experimental group in the logarithmic growth phase are collected. 96-well plates were seeded at a cell density of about 2500 cells/well. Each group had 3 duplicate wells. After the plates are pavedPlacing at 37deg.C, 5% CO 2 Culturing in an incubator. Beginning the next day after plating, 20. Mu.L of 5mg/mL MTT was added to the wells 4h before termination of the culture without changing the solution. After 4h, the culture was completely aspirated, and the formazan particles at the bottom of the well plate were not aspirated, and 100. Mu. LDMSO was added to dissolve the formazan particles. The oscillator oscillates for 2-5min, and the OD value is detected by an enzyme labeling instrument 490/570 nm. And (5) data statistics and analysis.
The results are shown in fig. 4, and the results show that after each tumor in the lentivirus infected group is cultured in vitro for 5 days, the proliferation speed is obviously slowed down, which is far lower than that of the tumor cells in the control group, and the number of viable cells is reduced by 42.8%, which indicates that HIST1H2BK gene silencing leads to inhibition of the proliferation capacity of human liver cancer SMMC-7721 cells.
Example 6 detection of the clonogenic Capacity of tumor cells infected with HIST1H2BK-shRNA lentivirus
Human liver cancer SMMC-7721 cells are inoculated into 12-hole plates after being digested by pancreatin, and the cell density is 10-15%. The next day was changed to fresh medium containing 5. Mu.g/ml polybrene. The HIST1H2BK-shRNA lentivirus is added into a culture plate according to the infection number of 10, and fresh culture medium is replaced after 12-24 hours of infection. After 72h of infection, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 80%.
After pancreatin digestion of the cells after infection with virus in the logarithmic growth phase, the complete medium is resuspended into a cell suspension; inoculating the cells into a 6-hole plate (800 cells/hole) after counting, continuously culturing the inoculated cells in an incubator for 12 days, and replacing liquid at 3day intervals and observing the cell state; photographing the cell clone under a fluorescence microscope before the experiment is terminated; cells were fixed with paraformaldehyde at the end of the experiment, and after washing the cells with PBS, giemsa was stained and photographed.
As shown in fig. 5, compared with the control interference (shCtrl group), the number of cloning plaques formed by tumor cells is obviously reduced and the volume of cloning plaques is obviously reduced after the expression of the RNA interference reduced gene (shHIST 1H2BK group); it is shown that HIST1H2BK gene silencing results in a decrease in the ability of human liver cancer SMMC-7721 cells to form clones. After the expression of the reduced gene was detected in the plate clone formation assay, the clonality of tumor cells was decreased.
Example 7 detection of apoptosis level of tumor cells infected with HIST1H2BK-shRNA lentivirus
Human liver cancer SMMC-7721 cells are inoculated into 12 pore plates after being digested by pancreatin, and the cell density is 10-15%. The next day was changed to fresh medium containing 5. Mu.g/ml polybrene. The HIST1H2BK-shRNA lentivirus is added into a culture plate according to the infection number of 10, and fresh culture medium is replaced after 12-24 hours of infection. After 72h of infection, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.
After digestion of the cells in the logarithmic growth phase with pancreatin, the complete medium is resuspended into a cell suspension; collecting the supernatant cells in the same 5mL centrifuge tube, and arranging three compound holes (in order to ensure that the number of the cells on the machine is enough, the number of the cells is more than or equal to 5 multiplied by 10) 5 Treatment). Centrifugation at 1300rpm for 5min, supernatant was discarded and cell pellet was washed with PBS pre-chilled at 4 ℃. The cell pellet was washed once with 1×binding buffer (eBioscience, 88-8007-74), centrifuged at 1300rpm for 3min, and the cells were collected. 200 μL of 1 Xbinding buffer resuspended cell pellet. Add 10. Mu.L of Annexin V-APC (eBioscience, 88-8007) for staining, protected from light at room temperature for 10-15min. According to the cell amount, 400-800 mu L of 1×binding buffer is added, and the detection is performed by an up-flow cytometer. The results were analyzed.
The results are shown in FIGS. 6-1 and 6-2, and the Annexin V single-stain method shows the change in the apoptosis ratio of tumor cells after decreasing the expression of the gene. The apoptosis proportion of tumor cells was found to increase after down-regulating gene expression. The number of apoptotic tumor cells is obviously increased after the expression of the RNA interference reduced gene (shHIST 1H2BK group) is interfered with a control (shCtrl group); indicating that gene silencing leads to apoptosis of tumor cells.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
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<213> Artificial sequence (Artificial Sequence)
<400> 12
cttcttctgc gccttagtca 20
<210> 13
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
tgacttcaac agcgacaccc a 21
<210> 14
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
caccctgttg ctgtagccaa a 21

Claims (6)

  1. Use of a hist1h2bk inhibitor in the preparation of a product having at least one of the following effects:
    treating liver cancer;
    inhibiting proliferation rate of liver cancer cells;
    promoting apoptosis of liver cancer cells;
    inhibiting liver cancer cell cloning;
    inhibiting liver cancer growth;
    the HIST1H2BK inhibitor is selected from double-stranded RNA and shRNA, and the shRNA or a target sequence of the double-stranded RNA is shown as SEQ ID NO:1 is shown in the specification;
    the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand being complementary together to form an RNA dimer, the first strand having a sequence as set forth in SEQ ID NO:2 is shown in the figure;
    the nucleotide sequence of the shRNA is shown as SEQ ID NO: 3.
  2. 2. A nucleic acid molecule that reduces expression of a HIST1H2BK gene in a liver cancer cell, the nucleic acid molecule comprising:
    a. a double-stranded RNA comprising a nucleotide sequence capable of hybridizing to the HIST1H2BK gene; or alternatively
    A shRNA, wherein the shRNA contains a nucleotide sequence capable of hybridizing with a HIST1H2BK gene;
    wherein the double-stranded RNA comprises a first strand and a second strand that are complementary together to form an RNA dimer, and the sequence of the first strand is substantially identical to a target sequence in the HIST1H2BK gene; the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, wherein the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is substantially identical to a target sequence in the HIST1H2BK gene;
    the shRNA or double-stranded RNA target sequence is shown as SEQ ID NO: 1.
    1) The double-stranded RNA is siRNA, and the sequence of the first strand of the siRNA is shown as SEQ ID NO:2 is shown in the figure;
    2) The nucleotide sequence of the shRNA is shown as SEQ ID NO: 3.
  3. 3. A his 1H2BK gene interfering nucleic acid construct comprising a gene fragment encoding the shRNA in the nucleic acid molecule of claim 2, capable of expressing the shRNA.
  4. 4. A HIST1H2BK gene interference slow virus, which is formed by the interference nucleic acid construct of claim 3 through virus packaging with the aid of slow virus packaging plasmid and cell line.
  5. 5. The use of the nucleic acid molecule of claim 2, or the HIST1H2BK gene interfering nucleic acid construct of claim 3, or the HIST1H2BK gene interfering lentivirus of claim 4, for: the method is used for preparing medicines for preventing or treating liver cancer or preparing a kit for reducing the expression of HIST1H2BK genes in liver cancer cells.
  6. 6. A composition for preventing or treating liver cancer, comprising:
    the nucleic acid molecule of claim 2; and/or, the HIST1H2BK gene interfering nucleic acid construct of claim 3; and/or the HIST1H2BK gene of claim 4 interferes with lentiviruses, and a pharmaceutically acceptable carrier, diluent or excipient.
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Citations (1)

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CN108367995A (en) * 2015-10-06 2018-08-03 安大略省癌症研究所 Histone approach is targeted to detect and overcome anthracycline resistant

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108367995A (en) * 2015-10-06 2018-08-03 安大略省癌症研究所 Histone approach is targeted to detect and overcome anthracycline resistant

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG ET AL..Mass Spectrometry-Based Proteomic Analysis Reveals the Interacting Partners of Lipin1.IUBMB LIFE.摘要. *

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