CN110904104A - Application of human HIST1H2BK gene and related product - Google Patents
Application of human HIST1H2BK gene and related product Download PDFInfo
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- CN110904104A CN110904104A CN201911171162.0A CN201911171162A CN110904104A CN 110904104 A CN110904104 A CN 110904104A CN 201911171162 A CN201911171162 A CN 201911171162A CN 110904104 A CN110904104 A CN 110904104A
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- hist1h2bk
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- liver cancer
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Abstract
The invention belongs to the field of biomedical research, and particularly relates to application of a human HIST1H2BK gene as a target in preparation of a liver cancer treatment drug. The invention discovers that the proliferation of liver cancer cells can be effectively inhibited and the apoptosis can be promoted after the expression of the human HIST1H2BK gene is reduced by adopting an RNAi method, and the growth process of liver cancer can be effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus 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 cells, thereby treating the liver cancer and opening up a new direction for treating the liver cancer.
Description
Technical Field
The invention belongs to the field of biomedical research, and particularly relates to application of a human HIST1H2BK gene and a related product.
Background
Histones are the basic nucleoproteins responsible for the structure of chromosomal fibronucleosomes in eukaryotes. Each of the four core histones (H2A, H2B, H3, and H4) has two molecules forming an octamer surrounded by approximately 146bp of DNA, and this repeat unit is called a nucleosome. The connecting DNA between the connecting histone H1 and nucleosomes interacts and plays a role in the formation of high-level structures in chromatin. The protein encoded by the HIST1H2BK gene is a replication-dependent histone from one of the histone H2B family members, is a core component of the nucleosome, and has activity against bacteria and fungi. Currently, the HIST1H2BK gene comprises two transcripts encoding the same protein, is located on chromosome 6p21.33, and is expressed in 25 tissues such as prostate (RPKM42.3) and testis (RPKM 29.8). HIST1H2BK is involved in meiosis and the Rho GTPase signaling pathway. Gene annotation analysis found that the gene was associated with sequence specific DNA binding, protein heterodimer activity. An important homologue of this gene is HIST1H2 BI. The involvement of the HIST1H2BK gene in the activated PKN1 pathway stimulates transcription of the androgen receptor regulatory genes KLK2 and KLK 3.
Han J et al found that HIST1H2BK is highly expressed in drug-resistant cells of triple negative breast cancer, and may promote the formation of adriamycin-resistant cells through a tumor dormancy mechanism, and that the HDAC inhibitor SAHA can increase the therapeutic sensitivity of adriamycin. Survey analysis of clinical triple negative breast cancer patients found: the recurrence-free survival Rate (RFS) of patients with high expression of HIST1H2BK was significantly lower than that of patients with low expression of HIST1H2 BK. As a result of analyzing GEO data invasive ductal carcinoma of breast, Li C and the like by a bioinformatic method, HIST1H2BK as a hub gene was found to be possibly involved in the development process of invasive ductal carcinoma of breast.
Furthermore, HIST1H2BK expression was also associated with neuroblastoma patient survival. Sequencing result analysis of serum exosomes of Pancreatic Ductal Adenocarcinoma (PDAC) patients gave: HIST1H2BK can form a diagnostic marker model with other 7 genes, has higher sensitivity, and can distinguish patients with early stage (I/II) and late stage (III/IV) pancreatic cancer; can distinguish the CA19-9 negative PDAC case from normal people, and is favorable for accurate diagnosis of pancreatic ductal adenocarcinoma.
At present, no document reports the role of HIST1H2BK in the development of liver cancer.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide the application of the human HIST1H2BK gene and a related product.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:
in a first aspect of the invention, the use of the human HIST1H2BK gene as a target in the preparation of a liver cancer treatment drug is provided.
The human HIST1H2BK gene as a target for preparing the liver cancer treatment drug specifically comprises the following steps: the HIST1H2BK gene is used as an action object, and the medicine or the preparation is screened to find the medicine which can inhibit the expression of the human HIST1H2BK gene and is used as a candidate medicine for treating the liver cancer. The HIST1H2BK gene small interfering RNA (siRNA) is obtained by screening human HIST1H2BK gene serving as an action object and can be used as a medicament with the effect of inhibiting the proliferation of liver cancer cells. In addition, the HIST1H2BK gene can be used as an active ingredient, such as an antibody drug, a small molecule drug, or the like.
The liver cancer treatment drug is a molecule which can specifically inhibit the transcription or translation of the HIST1H2BK gene, or can specifically inhibit the expression or activity of the HIST1H2BK protein, thereby reducing the expression level of the HIST1H2BK gene in liver cancer cells and achieving the purpose of inhibiting the proliferation, growth, differentiation and/or survival of the liver cancer cells.
The liver cancer therapeutic drug prepared by the HIST1H2BK gene includes but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins, or interfering lentiviruses.
Such nucleic acids include, but are not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA produced by endoribonuclease III or short hairpin RNA (shRNA).
The amount of the liver cancer therapeutic drug to be administered is an amount sufficient to reduce transcription or translation of the human HIST1H2BK gene, or to reduce expression or activity of the human HIST1H2BK protein. Such 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 medicine mainly achieves the purpose of treatment by reducing the expression level of human HIST1H2BK gene and inhibiting the proliferation of liver cancer cells. Specifically, in treatment, a substance effective in reducing the expression level of human HIST1H2BK gene is administered to a patient.
In one embodiment, the hit 1H2BK gene has a target sequence as set forth in SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-GAGAAAGAGTATATAAGCT-3' are provided.
In a second aspect of the invention, there is provided the use of an inhibitor of HIST1H2BK in the manufacture of a product having at least one of the following effects:
treating liver cancer;
inhibiting the proliferation rate of hepatoma cells;
promoting the apoptosis of the liver cancer cells;
inhibiting the cloning of liver cancer cells;
inhibiting liver cancer growth.
The product necessarily comprises the HIST1H2BK inhibitor and takes the HIST1H2BK inhibitor as an effective component of the above effects.
In the product, the effective component for the above functions can be only the HIST1H2BK inhibitor, and can also comprise other molecules for the above functions.
That is, the HIST1H2BK inhibitor is the only active ingredient or one of the active ingredients of the product.
The product may be a single component material or a multi-component material.
The form of the product is not particularly limited, and can be various substance forms such as solid, liquid, gel, semifluid, aerosol and the like.
The product is primarily directed to mammals. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. 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 nucleic acid molecule, antibody, small molecule compound.
As exemplified in the examples herein, the HIST1H2BK inhibitor can be a nucleic acid molecule that reduces the expression of the HIST1H2BK gene in liver cancer cells. Specifically, it may be a double-stranded RNA or shRNA.
In a third aspect of the invention, a method of treating liver cancer is provided by administering to a subject an inhibitor of HIST1H2 BK.
The subject may be a mammal or a mammalian liver cancer cell. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human. The liver cancer cell can be an isolated liver cancer cell.
The subject may be a patient suffering from liver cancer or an individual in whom treatment is desired for liver cancer. Or the subject is an isolated liver cancer cell of a liver cancer patient or an individual expected to treat liver cancer.
The HIST1H2BK inhibitor can be administered to a subject before, during, or after treatment for liver cancer.
The fourth aspect of the invention discloses a nucleic acid molecule for reducing the expression of HIST1H2BK gene in liver cancer cells, 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 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 which is 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 is shown. The method specifically comprises the following steps: 5'-GAGAAAGAGTATATAAGCT-3' are provided. Further, the sequence of the first strand of the double-stranded RNA is shown as SEQ ID NO:2, respectively. Specifically 5'-GAGAAAGAGUAUAUAAGCU-3'.
Further, the double-stranded RNA is small interfering RNA (siRNA).
SEQ ID NO:2 is designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aiming at one strand of small interfering RNA of the human HIST1H2BK gene, and 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 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 basically identical to a target sequence in the HIST1H2BK gene.
Further, the target sequence of the sh RNA is shown as SEQ ID NO:1 is shown.
The shRNA can become small interfering RNA (siRNA) after enzyme digestion and processing, and further plays a role in specifically silencing the expression of endogenous HIST1H2BK gene in liver cancer cells.
Further, the sequence of the stem-loop structure of the shRNA can be selected from any one of the following sequences: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, and CCACACC.
Further, the sequence of the shRNA is shown as SEQ ID NO: 3, respectively. Specifically 5'-CUGAGAAAGAGUAUAUAAGCUCUCGAGAGCUUAUAUACUCUUUCUCAG-3'.
Further, the HIST1H2BK gene is derived from human.
In the fifth aspect of the invention, the HIST1H2BK gene interfering nucleic acid construct contains a gene segment for coding shRNA in the nucleic acid molecule and can express the shRNA.
The HIST1H2BK gene interference nucleic acid construct can be obtained by cloning a gene segment for coding the shRNA of the human HIST1H2BK gene into a known vector.
Further, the HIST1H2BK gene interference nucleic acid construct is a HIST1H2BK gene interference lentiviral vector.
The HIST1H2BK gene interference lentiviral vector disclosed by the invention is obtained by cloning a DNA fragment for coding the HIST1H2BK gene shRNA into a known vector, wherein most of the known vectors are lentiviral vectors, the HIST1H2BK gene interference lentiviral vector is packaged into infectious viral particles by virus, and then infects liver cancer cells to further transcribe the shRNA, and the siRNA is finally obtained by the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the HIST1H2BK gene.
Further, the HIST1H2BK gene interference lentiviral vector also contains a promoter sequence and/or a nucleotide sequence for encoding a marker which can be detected in the hepatoma cells; preferably, the detectable label is 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-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-laminsham, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti 6.2/N-Lumio/V5-GW/lacZ.
The embodiment of the invention specifically discloses a human HIST1H2BK gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, which is named as pGCSIL-GFP-HIST1H2 BK-siRNA.
The HIST1H2BK gene siRNA can be used for inhibiting the 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 will 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 the sixth aspect of the invention, the HIST1H2BK gene interference lentivirus is formed by virus packaging of the HIST1H2BK gene interference nucleic acid construct under the assistance of lentivirus packaging plasmids and cell lines. The lentivirus can infect liver cancer cells and generate small interfering RNA aiming at HIST1H2BK gene, thereby inhibiting the 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 a use of the nucleic acid molecule, or the HIST1H2BK gene-interfering nucleic acid construct, or the HIST1H2BK gene-interfering lentivirus, wherein: is used for preparing a medicine for preventing or treating liver cancer or a kit for reducing the expression of HIST1H2BK gene in liver cancer cells.
The application of the medicament 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 administering an effective dose of the medicament to the subject.
Further, when the drug is used for preventing or treating liver cancer in a subject, an effective dose of the drug needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and/or metastasis of the liver cancer is inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the growth, proliferation, recurrence and/or metastasis of the liver cancer is inhibited.
The subject of the method may be a human.
In an eighth aspect of the present invention, there is provided a composition for preventing or treating liver cancer, comprising the following effective ingredients:
the aforementioned nucleic acid molecules; and/or, the foregoing HIST1H2BK gene interfering nucleic acid construct; and/or, the aforementioned HIST1H2BK gene interfering lentivirus, and a pharmaceutically acceptable carrier, diluent or excipient.
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 needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and/or metastasis of the liver cancer is inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the growth, proliferation, recurrence and/or metastasis of the liver cancer is inhibited.
The form of the composition is not particularly limited, and may be in the form of various substances such as solid, liquid, gel, semifluid, aerosol, etc.
The subject to which the composition is primarily directed is a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human.
In conclusion, 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 reduce the expression of the HIST1H2BK gene at the mRNA level and the protein level. The slow virus (Lv) is used as a gene operation tool to carry an RNAi vector pGCSIL-GFP-HIST1H2BK-siRNA, so that the RNAi sequence aiming at the HIST1H2BK gene can be efficiently introduced into the liver cancer SMMC-7721 cells in a targeted mode, the expression level of the HIST1H2BK gene is reduced, and the proliferation capacity of the tumor cells is remarkably inhibited. Lentivirus-mediated silencing of the HIST1H2BK gene is therefore a potential clinical non-surgical treatment modality for malignancies.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers that the proliferation of liver cancer cells can be effectively inhibited and the apoptosis can be promoted after the expression of the human HIST1H2BK gene is reduced by adopting an RNAi method, and the growth process of liver cancer can be effectively controlled. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus 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 cells, thereby treating the liver cancer and opening up a new direction for treating the liver cancer.
Drawings
FIG. 1: RT-PCR detects the target gene reduction efficiency of SMMC-7721 cell mRNA level.
FIG. 2: western Blot detection of SMMC-7721 cell target to reduce the protein level expression of HIST1H2BK gene.
FIG. 3: the result of Celigo cell automatic analysis reveals the effect of infecting HIST1H2BK-shRNA lentivirus on the proliferation of SMMC-7721 liver cancer cells. (cell count was made 1, 2, 3, 4 and 5 days after viral infection)
FIG. 4: the CCK8 method is used for detecting the influence of infecting HIST1H2BK-shRNA lentivirus on the proliferation capacity of SMMC-7721 cells after 5 days.
FIG. 5: clone formation experiment the influence on the clone formation ability of SMMC-7721 cells after infecting HIST1H2BK-shRNA lentivirus is shown in the left side of a real object graph and in the right side of the graph, the average value of three experiments is shown.
FIG. 6-1: annexin V-APC flow apoptosis test shows the effect of infecting HIST1H2BK-shRNA lentivirus on SMMC-7721 apoptosis.
FIG. 6-2: the Annexin V-APC flow apoptosis test shows the influence of the infected HIST1H2BK-shRNA lentivirus on the apoptosis of SMMC-7721 cells, and the column results are shown as the average value of cell percentage +/-standard deviation.
In the drawings, there is shown in the drawings,
bar graphs represent the mean of three experiments and error bars represent Standard Deviation (SD).
P <0.01 for shCtrl compared to target gene shRNA lentivirus treatment group.
And compared with the target gene shRNA lentivirus treatment group, the shCtrl is not less than 0.01 and P is less than 0.05.
Detailed Description
The invention proves the function of the HIST1H2BK gene in liver cancer generation from the perspective of cell function. Transfecting liver cancer cells after constructing a target gene shRNA lentivirus, and comparing with a transfection control lentivirus to detect the expression conditions of target genes at the mRNA and protein levels in two groups of liver cancer cell lines; and then cell proliferation, apoptosis and other detection are carried out through cytofunctional experiments, and the results show that the liver cancer cell proliferation inhibition degree of the shRNA group is obviously higher than that of the control group and the increase degree of the cell apoptosis rate of the shRNA group is higher than that of the control group compared with the control group.
HIST1H2BK inhibitors
Refers to a molecule having an inhibitory effect on HIST1H2 BK. Having inhibitory effects on HIST1H2BK include, but are not limited to: inhibiting the expression or activity of HIST1H2 BK.
Inhibition of HIST1H2BK activity refers to a reduction in HIST1H2BK activity. Preferably, 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%, and most preferably by at least 90% compared to prior to inhibition.
Specifically, the inhibition of the expression of HIST1H2BK can be the inhibition of the transcription or translation of the HIST1H2BK gene, and specifically can be the inhibition of the expression of HIST1H2 BK: the method comprises the steps of not transcribing the gene of HIST1H2BK, reducing the transcriptional activity of the gene of HIST1H2BK, not translating the gene of HIST1H2BK, or reducing the translation level of the gene of HIST1H2 BK.
One skilled in the art can use conventional methods to modulate the expression of HIST1H2BK gene, such as gene knock-out, homologous recombination, interfering RNA, etc.
Inhibition of gene expression of HIST1H2BK was confirmed by PCR and Western Blot detection of the expression level.
Preferably, the expression of the HIST1H2BK gene is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, still more preferably by at least 90%, most preferably the HIST1H2BK gene is not expressed at all, compared to the wild type.
Small molecule compounds
The invention refers to a compound which is composed of several or dozens of atoms and has the molecular mass of less than 1000.
Preparation of medicine for preventing or treating liver cancer
Nucleic acid molecules that reduce expression of the HIST1H2BK gene in liver cancer cells can be used; and/or, a HIST1H2BK gene interfering nucleic acid construct; and/or, the HIST1H2BK gene interferes with lentivirus, and is used as an effective component for preparing a medicament for preventing or treating liver cancer. Generally, the medicament can comprise one or more pharmaceutically acceptable carriers or auxiliary materials besides the effective components according to the requirements of different dosage forms.
By "pharmaceutically acceptable" is meant that the molecular entities and compositions 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., capable of being blended therewith without substantially diminishing the effectiveness of the drug under ordinary circumstances. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or adjuvants are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; powdered gum tragacanth; 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; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. These materials are used as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration.
In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and may be prepared into injection, oral liquid, tablet, capsule, dripping pill, spray, etc., and may be prepared by a conventional method. The choice of the pharmaceutical dosage form should be matched to the mode of administration.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.
Example 1 preparation of RNAi lentivirus against human HIST1H2BK Gene
1. Screening of effective siRNA target against human HIST1H2BK Gene
Retrieval of HIST1H2BK (NM-080593) gene information from Genbank; effective siRNA targets were designed against the HIST1H2BK gene. Table 1-1 lists the effective siRNA target sequences selected 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
Synthesizing double-stranded DNA Oligo sequences (Table 1-2) containing Age I and EcoR I enzyme cutting sites at two ends aiming at siRNA targets (taking SEQ ID NO:1 as an example); the restriction enzymes Age I and EcoR I act on pGCSIL-GFP vector (provided by Shanghai Jikai Gene chemistry Co., Ltd.), linearize it, and identify the enzyme-cleaved fragments by agarose gel electrophoresis.
TABLE 1-2 double-stranded DNA Oligo with Age I and EcoR I cleavage sites at both ends
The vector DNA linearized by double digestion (digestion system shown in tables 1-4, 37 ℃ C., reaction 1h) and the purified double-stranded DNA Oligo were ligated by T4 DNA ligase at 16 ℃ C. overnight in an appropriate buffer system (ligation system shown in tables 1-5), and the ligation product was recovered. The ligation product was transformed into calcium chloride prepared fresh E.coli competent cells (transformation protocol reference: molecular cloning protocols second edition, pages 55-56). Dipping the surface of the clone of the strain growing out of the connected transformation product, dissolving the surface in 10 mul LB culture medium, uniformly mixing and taking 1 mul as a template; designing universal PCR primers at the upstream and downstream of RNAi sequence in the lentiviral vector, wherein the upstream primer sequence: 5'-CCTATTTCCCATGATTCCTTCATA-3' (SEQ ID NO: 6); the sequence of the downstream primer is as follows: 5'-GTAATACGGTTATCCACGCG-3' (SEQ ID NO: 7), and PCR identification experiments were performed (PCR reaction system shown in tables 1-6, reaction conditions shown in tables 1-7). Sequencing and comparing the clones which are identified to be positive by the PCR, wherein the correctly compared clones are the clones which are successfully constructed and are directed at the nucleotide sequence shown in SEQ ID NO:1, named pGCSIL-GFP-HIST1H2 BK-siRNA.
pGCSIL-GFP-Scr-siRNA negative control plasmid was constructed with negative control siRNA target sequence 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 8). When pGCSIL-GFP-Scr-siRNA negative control plasmids are constructed, double-stranded DNA Oligo sequences (shown in tables 1-3) containing adhesive ends of Age I and EcoR I enzyme cutting sites at two ends are synthesized aiming at Scr siRNA targets, and the rest construction methods, identification methods and conditions are the same as pGCSIL-GFP-HIST1H2 BK-siRNA.
TABLE 1-3 double-stranded DNA Oligo with Age I and EcoR I cleavage sites at both ends
TABLE 1-4 pGCSIL-GFP plasmid digestion reaction System
| Reagent | Volume (μ l) |
| 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 H2O | 40.5 |
| Total | 50.0 |
TABLE 1-5 ligation reaction System of vector DNA and double-stranded DNA Oligo
| Reagent | Positive control (μ l) | Self-contained control (μ l) | Connecting 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 |
1.0 | 1.0 | 1.0 |
| T4 phage DNA ligase | 1.0 | 1.0 | 1.0 |
| dd H2O | 16.0 | 17.0 | 16.0 |
| Total | 20.0 | 20.0 | 20.0 |
TABLE 1-6 PCR reaction System
| Reagent | Volume (μ l) |
| 10×buffer | 2.0 |
| dNTPs(2.5mM) | 0.8 |
| Upstream primer | 0.4 |
| Downstream primer | 0.4 |
| Taq polymerase | 0.2 |
| Form panel | 1.0 |
| ddH2O | 15.2 |
| Total | 20.0 |
TABLE 1-7 PCR reaction System Programming
3. Packaging HIST1H2BK-shRNA lentivirus
The DNA of RNAi plasmid pGCSIL-GFP-HIST1H2BK-siRNA was extracted using a plasmid extraction kit from Qiagen, Inc., and 100 ng/. mu.l of stock solution was prepared.
24h before transfection, human embryonic kidney cell 293T cells in logarithmic growth phase were trypsinized and cell density was adjusted to 1.5X 10 in DMEM complete medium containing 10% fetal bovine serum5Cells/ml, seeded in 6-well plates at 37 ℃ with 5% CO2Culturing in an incubator. The cell density can reach 70-80% to be used for transfection. 2h before transfection, the original medium was aspirated and 1.5ml of fresh complete medium was added. Mu.l of Packing Mix (PVM), 12. mu.l of PEI, and 400. mu.l of serum-free DMEM medium were added to a sterilized centrifuge tube according to the instructions of the MISSION Lentiviral Packing Mix kit from Sigma-aldrich, and 20. mu.l of the above-mentioned extracted plasmid DNA was added to the above-mentioned PVM/PEI/DMEM mixture.
The transfection mixture was incubated at room temperature for 15min and then transferredTransferred to the medium of 293T cells of human embryonic kidney cells at 37 ℃ and 5% CO2Culturing for 16h in an incubator. The medium containing the transfection mixture was discarded, washed with PBS solution, 2ml of complete medium was added and incubation continued for 48 h. The cell supernatant was collected, and the lentivirus was purified and concentrated by a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) according to the following steps: (1) centrifuging at 4 deg.C and 4000g for 10min to remove cell debris; (2) filtering the supernatant with 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 the centrifugation is finished, 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 1000 g; (5) the centrifuge cup is removed from the sample collection cup, and the virus concentrate is obtained. Subpackaging the virus concentrated solution and storing at-80 ℃. The sequence of the first strand of siRNA contained in the virus concentrated solution is shown in SEQ ID NO. 2. The packaging procedure for the control lentivirus was identical to that of the HIST1H2BK-shRNA lentivirus, except that pGCSIL-GFP-Scr-siRNA vector was used instead of pGCSIL-GFP-HIST1H2BK-siRNA vector.
Example 2 detection of Gene silencing efficiency by real-time fluorescent quantitative RT-PCR
Human liver cancer SMMC-7721 cells in logarithmic growth phase were trypsinized to prepare a cell suspension (about 5X 10 cells in number)4/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the complex infection value (MOI, SMMC-7721: 10), an appropriate amount of the lentivirus prepared in example 1 is added, the culture medium is replaced after 24 hours of culture, and cells are collected after the infection time reaches 5 days. Total RNA was extracted according to the Trizol protocol of Invitrogen corporation. The RNA was reverse-transcribed to obtain cDNA according to the M-MLV protocol of Promega (reverse transcription reaction system shown in Table 2-1, reaction at 42 ℃ for 1 hour, and then reverse transcriptase was inactivated by water bath for 10min at 70 ℃ in a water bath).
Real-time quantitative detection was carried out using a TP800 Real time PCR instrument (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 used 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 | Volume (μ l) |
| 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 | Volume (μ l) |
| SYBR premix ex taq | 6.0 |
| Primer MIX (5. mu.M) | 0.3 |
| cDNA | 0.6 |
| ddH2O | 5.1 |
| Total | 12.0 |
The program was a two-step Real-time PCR: pre-denaturation at 95 ℃ for 30 s; then, denaturation is carried out at 95 ℃ for 5s in each step; annealing and extending for 30s at 60 ℃; a total of 40 cycles were performed. Each time reading the absorbance value during the extension phase. After completion of PCR, the DNA double strand was sufficiently bound by denaturation at 95 ℃ for 15 seconds and then cooling to 60 ℃. Melting curves were prepared by increasing the temperature from 60 ℃ to 95 ℃ by 0.5 ℃ for 4 seconds and reading the absorbance. By using 2-ΔΔCtThe assay calculated the abundance of expression of the HIST1H2 BK-infected mRNA. Cells infected with the control virus served as controls. The results are shown in FIG. 1, which shows that the expression level of HIST1H2BK mRNA in human liver cancer SMMC-7721 cells is down-regulated by 70.6%.
Example 3 detection of Gene silencing efficiency by Western Blotting method
1. Extraction of Total cellular proteins
1) Control viruses and RNAi viruses against HIST1H2BK interfering targets were evaluated based on complex infection values (MOI: 10) infecting the target cells (SMMC-7721 cells).
2) 5 days after infection, cell samples were collected, an appropriate amount of RIPA lysate (Biyun day, P0013C) was taken, and PMSF was added to a final concentration of 1mM PMSF within minutes prior to use.
3) Adding appropriate amount of RIPA lysate, and lysing on ice for 10-15 min. Cells were scraped off and transferred to a new EP tube, and then cells were sonicated (20 times at 40W, 1s each, 2s apart).
4) After centrifugation at 12000g for 15min at 4 ℃, the supernatant was removed and purified using BCA Protein Assay Kit (manufacturer: biyuntian, goods number: P0010S) to determine the protein concentration.
5) The protein concentration of each sample was adjusted to be consistent by adding fresh lysate, typically 2. mu.g/. mu.L. Then adding 1/5 volumes of 6Xlodding buffer, mixing, boiling in 100 degree metal bath for 10min, centrifuging for a short time, and storing at-80 ℃ for later use.
2.SDS-PAGE
1) Preparing glue: according to the molecular weight of the target protein, glue with different concentrations is prepared, and the specific system is shown in tables 3-1, 3-2 and 3-3:
TABLE 3-1 SDS-PAGE gels (8mL system)
| Separating glue (8mL system) | 8％ | 9％ | 10％ | 12％ | 13％ | 15％ |
| H2O | 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 gels (10mL system)
| Separating glue (10mL system) | 8％ | 9％ | 10％ | 12％ | 13％ | 15％ |
| H2O | 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 SDS-PAGE gels (different systems)
| Concentrated gum (5%) | 3mL | 4mL | 5mL |
| H2O | 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 gel 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 at 80mA for 20 min; the separation gel was 120mA, 1 h.
3. Immunoblotting (Wet transfer)
After the electrophoresis is finished, the protein is transferred to the PVDF membrane by using a transfer electrophoresis device and electrotransfer for 150min under the constant current condition of 300mA at 4 ℃.
4. Antibody hybridization:
1) and (3) sealing: PVDF membrane was blocked with blocking solution (TBST solution containing 5% skim milk) at room temperature for 1h or overnight at 4 ℃.
2) Primary antibody incubation: histone H2B K primary antibody (abcam) was diluted 1:200 with blocking solution, GAPDH (Santa Cruz) was diluted 1:2000, and then incubated with the blocked PVDF membrane at room temperature for 2H or overnight at 4 ℃ and washed 4 times with TBST for 8min each.
3) And (3) secondary antibody incubation: the rabbitIgG and mouse IgG secondary antibodies (both Santa Cruz from the manufacturer) were diluted with blocking solution at a rate of 1:2000, respectively, and the PVDF membrane was incubated at room temperature for 1.5h and washed with TBST for 8min each time for 4 times.
X-ray development:
1) using CST corporation 20X
Reagent and 20X Peroxide #7003 kit, mixing solution A and solution B in the kit according to the proportion of 1:1, reversing and mixing evenly, and standing for a plurality of minutes for use.
2) Taking out the film, wiping the absorbent paper dry, spreading into a cassette, dripping a proper amount of uniformly mixed ECL luminous liquid, spreading a preservative film (avoiding generating bubbles), putting an X-ray film (avoiding the movement of the X-ray film), closing the cassette, and exposing for 1s to a plurality of minutes (the exposure time needs to be tried for a plurality of times, and the exposure time is properly adjusted according to whether the naked eye can see fluorescence and the strength of the fluorescence.
3) Taking out the X-ray film, placing in developing solution, taking out after banding occurs, rinsing in clear water for several seconds, and placing in fixing solution for at least 2 min.
4) Taking out the X-ray film, drying and analyzing.
As shown in FIG. 2, Western Blot experiments show that the target has a knockdown effect on the endogenous expression of HIST1H2BK gene, and therefore, the target is an effective target.
Example 4 Celigo assay to examine the proliferation potency of tumor cells infected with HIST1H2BK-shRNA lentivirus
Human liver cancer SMMC-7721 cells in logarithmic growth phase were trypsinized to prepare a cell suspension (about 5X 10 cells in number)4/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the infection complex number (MOI, SMMC-7721: 10), adding a proper amount of virus, culturing for 24h, then replacing the culture medium, and collecting the cells of each experimental group in the logarithmic growth phase after the infection time reaches 5 days. Complete medium resuspension into cell suspension (2X 10)4Per ml) at a cell density of about 2000 per well, 96-well plates were seeded. Each set of 5 duplicate wells, 100. mu.l per well. After the plate is laid, the plate is placed at 37 ℃ and 5% CO2Culturing in an incubator. The plate reading was performed once a day with Celigo instrument (Nexcelom) starting the next day after plating, and the plate reading was performed continuously for 5 days. Accurately calculating the number of cells with green fluorescence in each scanning pore plate by adjusting input parameters of analysis settings; the data were statistically plotted and cell proliferation curves were plotted for 5 days.
The results are shown in fig. 3, and the results show that after each tumor in the lentivirus infection group is cultured in vitro for 5 days, the proliferation speed is remarkably reduced, which is far lower than that of the tumor cells in the control group, the number of viable cells is reduced by 46.4%, and the HIST1H2BK gene silencing causes the inhibition of the proliferation capacity of the human liver cancer SMMC-7721 cells.
Example 5 CCK8 experiment to examine the proliferation potency of tumor cells infected with HIST1H2BK-shRNA lentivirus
Human liver cancer SMMC-7721 cells in logarithmic growth phase were trypsinized to prepare a cell suspension (about 5X 10 cells in number)4/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the infection complex number (MOI, SMMC-7721: 10), adding a proper amount of virus, culturing for 24h, then replacing the culture medium, and collecting the cells of each experimental group in the logarithmic growth phase after the infection time reaches 5 days. 96-well plates were seeded at a cell density of about 2500 cells/well. Each group of 3 multiple wells. After the plate is laid, the plate is placed at 37 ℃ and 5% CO2Culturing in an incubator. Starting the day after plating, 20. mu.L of 5mg/mL MTT was added to the wells 4h before termination of the culture without changing the medium. After 4h, the culture was completely aspirated, and the formazan particles were dissolved by adding 100. mu.L of LDMSO, taking care not to aspirate the formazan particles at the bottom of the well plate. Oscillating for 2-5min with oscillator, and detecting OD value with enzyme labeling instrument 490/570 nm. And (6) carrying out data statistical analysis.
The results are shown in fig. 4, and the results show that after each tumor in the lentivirus infection group is cultured in vitro for 5 days, the proliferation speed is remarkably reduced, which is far lower than that of the tumor cells in the control group, the number of viable cells is reduced by 42.8%, and the HIST1H2BK gene silencing causes the inhibition of the proliferation capacity of the human liver cancer SMMC-7721 cells.
Example 6 examination of the clonogenic Capacity of tumor cells infected with HIST1H2BK-shRNA lentivirus
Human liver cancer SMMC-7721 cells are trypsinized and inoculated into a 12-well plate, and the cell density is 10-15%. The next day was changed to fresh medium containing 5. mu.g/ml polybrene. HIST1H2BK-shRNA lentivirus was added to the plates at a multiplicity of infection of 10, and the medium was changed to fresh after 12-24H infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 80%.
After the cells infected with the virus in the logarithmic growth phase are digested by pancreatin, the complete culture medium is re-suspended into cell suspension; after counting the cells, inoculating the cells into a 6-well plate (800 cells/well), continuously culturing the inoculated cells in an incubator for 12 days, changing the liquid every 3day in the middle, and observing the cell state; photographing the cell clone under a fluorescent microscope before the experiment is terminated; at the end of the experiment, cells were fixed with paraformaldehyde, washed with PBS, Giemsa stained, and photographed.
The results are shown in fig. 5, and compared with the control interference (shCtrl group), the expression of the gene is reduced by RNA interference (shHIST1H2BK group), the number of the clone spots formed by the tumor cells is obviously reduced, and the volume of the clone spots is obviously reduced; it was shown that silencing of HIST1H2BK gene resulted in a decrease in the ability of human liver cancer SMMC-7721 cells to form clones. The plate cloning test detects that after the expression of the gene is reduced, the cloning capacity of the tumor cells is reduced.
Example 7 detection of apoptosis levels in tumor cells infected with HIST1H2BK-shRNA lentivirus
Human liver cancer SMMC-7721 cells are inoculated in a 12-well plate 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. HIST1H2BK-shRNA lentivirus was added to the plates at a multiplicity of infection of 10, and the medium was changed to fresh after 12-24H infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.
After trypsinizing the cells in logarithmic growth phase, resuspending the complete medium into a cell suspension; collecting the supernatant in the same 5mL centrifuge tube, each group having three multiple holes (the number of cells is not less than 5 × 10 to ensure enough cells on the machine5Treatment). Centrifuge at 1300rpm for 5min, discard the supernatant, wash the cell pellet with 4 ℃ pre-cooled PBS. The cell pellet was washed once with 1 Xbinding buffer (eBioscience, 88-8007-74), centrifuged at 1300rpm for 3min, and the cells were collected. 200 μ L of 1 × binding buffer resuspended cell pellets. Add 10. mu.L Annexin V-APC (eBioscience, 88-8007) for staining, protected from light at room temperature for 10-15 min. According to the cell amount, 400-800. mu.L of 1 × binding buffer is added, and detection is carried out by an up-flow cytometer. The results were analyzed.
As a result, as shown in FIGS. 6-1 and 6-2, the Annexin V single staining method detected the change in the apoptosis ratio of tumor cells after decreasing the expression of the gene. It was found that the apoptosis rate of tumor cells increases after down-regulating gene expression. After the control interference (shCtrl group) and the RNA interference reduce the expression of the gene (shHIST1H2BK group), the number of apoptotic tumor cells is increased; indicating that gene silencing leads to apoptosis of tumor cells.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
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Claims (10)
1. The use of human HIST1H2BK gene as target in preparing medicine for treating liver cancer.
Use of an inhibitor of HIST1H2BK in the manufacture of a product having at least one of the following effects:
treating liver cancer;
inhibiting the proliferation rate of hepatoma cells;
promoting the apoptosis of the liver cancer cells;
inhibiting the cloning of liver cancer cells;
inhibiting liver cancer growth.
3. Use according to claim 2, further comprising one or more of the following features:
1) the HIST1H2BK inhibitor is a molecule having an inhibitory effect on HIST1H2 BK;
2) the HIST1H2BK inhibitor is the only effective component or one of the effective components of the product;
3) the HIST1H2BK inhibitor is selected from double-stranded RNA, shRNA, antibody or small molecule compound.
4. Use according to claim 3, further comprising one or more of the following features:
1) the shRNA or double-stranded RNA target sequence is shown as SEQ ID NO:1 is shown in the specification;
2) the double-stranded RNA comprises a first strand and a second strand, wherein the first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is shown as SEQ ID NO:2 is shown in the specification;
3) the nucleotide sequence of the shRNA is shown as SEQ ID NO: 3, respectively.
5. A nucleic acid molecule that reduces expression of HIST1H2BK gene in a liver cancer cell, the nucleic acid molecule comprising:
a. a double-stranded RNA comprising a nucleotide sequence capable of hybridizing with the HIST1H2BK gene; or
shRNA containing a nucleotide sequence capable of hybridizing with the HIST1H2BK gene;
wherein the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary 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 basically identical to a target sequence in the HIST1H2BK gene.
6. The nucleic acid molecule of claim 5, further comprising one or more of the following characteristics:
1) the shRNA or double-stranded RNA target sequence is shown as SEQ ID NO:1 is shown in the specification;
2) 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 specification;
3) the nucleotide sequence of the shRNA is shown as SEQ ID NO: 3, respectively.
7. A HIST1H2BK gene interfering nucleic acid construct containing a gene segment encoding the shRNA in the nucleic acid molecule according to any one of claims 5 to 6, capable of expressing the shRNA.
8. A HIST1H2BK gene interference lentivirus, which is prepared by virus packaging the interference nucleic acid construct of claim 7 with the help of lentivirus packaging plasmid and cell line.
9. The nucleic acid molecule of any one of claims 5 to 6, or the HIST1H2BK gene-interfering nucleic acid construct of claim 7, or the use of the HIST1H2BK gene-interfering lentivirus of claim 8, wherein said nucleic acid molecule is selected from the group consisting of: is used for preparing a medicine for preventing or treating liver cancer or a kit for reducing the expression of HIST1H2BK gene in liver cancer cells.
10. A composition for preventing or treating liver cancer, which comprises the following effective components:
the nucleic acid molecule of any one of claims 5-6; and/or, the HIST1H2BK gene interfering nucleic acid construct of claim 7; and/or the HIST1H2BK gene interfering lentivirus of claim 8, in association with a pharmaceutically acceptable carrier, diluent or excipient.
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| CN115927322A (en) * | 2022-08-31 | 2023-04-07 | 首都医科大学附属北京友谊医院 | Application of inhibitor of Thbs2 gene or THBS2 protein in preparation of anti-hepatic fibrosis medicine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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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| 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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| WANG ET AL.: "Mass Spectrometry-Based Proteomic Analysis Reveals the Interacting Partners of Lipin1", IUBMB LIFE * |
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| CN115927322A (en) * | 2022-08-31 | 2023-04-07 | 首都医科大学附属北京友谊医院 | Application of inhibitor of Thbs2 gene or THBS2 protein in preparation of anti-hepatic fibrosis medicine |
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