Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide the application of the human EME1 gene and related products.
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 EME1 gene as a target in the preparation of a gastric cancer treatment drug or a gastric cancer diagnosis drug is provided.
The human EME1 gene as a target for preparing the gastric cancer treatment drug specifically comprises the following steps: the EME1 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 EME1 gene and is used as a candidate medicine for treating the gastric cancer. The EME1 gene small interfering RNA (siRNA) is obtained by screening human EME1 gene serving as an action object and can be used as a medicine for inhibiting gastric cancer cell proliferation. In addition, the EME1 gene can be used as a target of action, for example, for antibody drugs, small molecule drugs, and the like.
The application of the human EME1 gene as a target in preparing gastric cancer diagnosis medicines specifically comprises the following steps: the EME1 gene expression product is used as a gastric cancer diagnosis index to be applied to the preparation of gastric cancer diagnosis medicaments.
The expression level of the EME1 gene in tumor tissue, normal tissue and normal tissue around the tumor was examined by immunohistochemical method. The research finds that: the expression level of EME1 in gastric cancer tissues is significantly higher than that in normal tissues and normal tissues around tumors. It is suggested that the expression level of EME1 gene may be a marker for tumor diagnosis.
The gastric cancer treatment drug is a molecule capable of specifically inhibiting the transcription or translation of an EME1 gene or specifically inhibiting the expression or activity of an EME1 protein, so that the expression level of an EME1 gene in a gastric cancer cell is reduced, and the purpose of inhibiting the proliferation, growth, differentiation and/or survival of the gastric cancer cell is achieved.
The gastric cancer therapeutic drug or gastric cancer diagnostic drug prepared by the EME1 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 gastric cancer therapeutic drug is administered in an amount sufficient to reduce transcription or translation of the human EME1 gene, or to reduce expression or activity of the human EME1 protein. Such that the expression of the human EME1 gene is reduced by at least 50%, 80%, 90%, 95%, or 99%.
The method for treating the gastric cancer by adopting the gastric cancer treatment drug achieves the treatment purpose by mainly reducing the expression level of human EME1 gene and inhibiting the proliferation of gastric cancer cells. In particular, in therapy, a substance effective in reducing the expression level of the human EME1 gene is administered to a patient.
In one embodiment, the EME1 gene has a target sequence as set forth in SEQ ID NO:1 is shown. The method specifically comprises the following steps: 5'-CTGAGAAGACAGGAAAGAA-3' are provided.
In a second aspect of the invention, there is provided the use of an EME1 inhibitor in the manufacture of a product having at least one of the following effects:
treating gastric cancer;
inhibiting the proliferation ability of gastric cancer cells;
inhibiting the growth of gastric cancer.
The product necessarily comprises an EME1 inhibitor and an EME1 inhibitor as an active ingredient for the aforementioned effects.
In the product, the effective component for the above functions can be only an EME1 inhibitor, and can also comprise other molecules for the above functions.
That is, the EME1 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 EME1 inhibitor can be a nucleic acid molecule, an antibody, a small molecule compound.
As exemplified in the examples herein, the EME1 inhibitor can be a nucleic acid molecule that reduces the expression of EME1 gene in gastric cancer cells. Specifically, it may be a double-stranded RNA or shRNA.
In a third aspect of the invention, there is provided a method of treating gastric cancer by administering to a subject an EME1 inhibitor.
The subject may be a mammal or a mammalian gastric 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 gastric cancer cell may be an isolated gastric cancer cell.
The subject may be a patient suffering from gastric cancer or an individual in whom treatment is desired. Or isolated gastric cancer cells of a subject who is a gastric cancer patient or an individual expected to treat gastric cancer.
The EME1 inhibitor can be administered to a subject before, during, or after receiving treatment for gastric cancer.
The fourth aspect of the invention discloses a nucleic acid molecule for reducing the expression of EME1 gene in gastric 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 an EME1 gene;
the shRNA contains a nucleotide sequence capable of hybridizing with an EME1 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 EME1 gene.
The target sequence in the EME1 gene is a fragment in the EME1 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 EME1 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'-CTGAGAAGACAGGAAAGAA-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'-CCCUGAGAAGACAGGAAAGAA-3'.
Further, the double-stranded RNA is small interfering RNA (siRNA).
SEQ ID NO:2 is one strand of small interfering RNA designed by taking the sequence shown in SEQ ID NO. 1 as an RNA interference target sequence and aiming at the human EME1 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 EME1 gene in gastric cancer cells.
The shRNA includes a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, 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 EME1 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 endogenous EME1 gene expression in gastric 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'-CCCUGAGAAGACAGGAAAGAACUCGAGUUCUUUCCUGUCUUCUCAGGG-3'.
Further, the EME1 gene is derived from a human.
In the fifth aspect of the invention, the EME1 gene interference nucleic acid construct contains a gene segment for coding shRNA in the nucleic acid molecule and can express the shRNA.
The EME1 gene interfering nucleic acid construct can be obtained by cloning a gene segment coding the human EME1 gene shRNA into a known vector.
Further, the EME1 gene interfering nucleic acid construct is an EME1 gene interfering lentiviral vector.
The EME1 gene interference lentiviral vector disclosed by the invention is obtained by cloning a DNA fragment for coding the EME1 gene shRNA into a known vector, wherein the known vector is mostly a lentiviral vector, the EME1 gene interference lentiviral vector is packaged into infectious viral particles by viruses, gastric cancer cells are infected, the shRNA is transcribed, and the siRNA is finally obtained through the steps of enzyme digestion processing and the like and is used for specifically silencing the expression of the EME1 gene.
Further, the EME1 gene interference lentiviral vector also contains a promoter sequence and/or a nucleotide sequence encoding a marker which can be detected in gastric cancer 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 EME1 gene interference lentiviral vector constructed by taking pGCSIL-GFP as a vector, which is named as pGCSIL-GFP-EME 1-siRNA.
The EME1 gene siRNA can be used for inhibiting the proliferation of gastric cancer cells, and further can be used as a medicine or a preparation for treating gastric cancer. The EME1 gene interference lentiviral vector can be used for preparing the EME1 gene siRNA. When used as a medicament or formulation for treating gastric 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.
The sixth aspect of the invention discloses an EME1 gene interference lentivirus, which is formed by virus packaging of the EME1 gene interference nucleic acid construct under the assistance of lentivirus packaging plasmids and cell lines. The lentivirus can infect gastric cancer cells and generate small interfering RNA aiming at the EME1 gene, thereby inhibiting the proliferation of the gastric cancer cells. The EME1 gene interference lentivirus can be used for preparing medicines for preventing or treating gastric cancer.
In a seventh aspect of the present invention, there is provided a use of the nucleic acid molecule, or the EME1 gene interfering nucleic acid construct, or the EME1 gene interfering lentivirus, wherein: is used for preparing a medicine for preventing or treating gastric cancer or a kit for reducing the EME1 gene expression in gastric cancer cells.
The application of the medicament for preventing or treating the gastric cancer provides a method for treating the gastric cancer, in particular to a method for preventing or treating the gastric 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 gastric cancer in a subject, an effective dose of the drug needs to be administered to the subject. With this method, the growth, proliferation, recurrence and/or metastasis of the gastric 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 gastric 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 gastric cancer, comprising, as active ingredients:
the aforementioned nucleic acid molecules; and/or, the aforementioned EME1 gene interfering nucleic acid construct; and/or the aforementioned EME1 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 gastric cancer in a subject, an effective dose of the composition needs to be administered to the subject. With this method, the growth, proliferation, recurrence and/or metastasis of the gastric 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 gastric 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 EME1 gene and constructs a corresponding EME1 RNAi vector, wherein the RNAi vector pGCSIL-GFP-EME1-siRNA can obviously reduce the expression of the EME1 gene at the mRNA level and the protein level. An RNAi vector pGCSIL-GFP-EME1-siRNA carried by lentivirus (Lv) serving as a gene manipulation tool can be used for efficiently introducing an RNAi sequence aiming at an EME1 gene into gastric cancer AGS cells in a targeted manner, so that the expression level of the EME1 gene is reduced, and the proliferation capacity of the tumor cells is remarkably inhibited. Lentivirus-mediated EME1 gene silencing 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 gastric cancer cells can be effectively inhibited and the apoptosis can be promoted after the expression of the human EME1 gene is reduced by adopting an RNAi method, and the growth process of the gastric 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 capacity of gastric cancer cells and inhibit the growth of gastric cancer, thereby treating gastric cancer and opening up a new direction for treating gastric cancer.
Detailed Description
The inventors of the present invention have extensively and intensively studied and found that the EME1 gene is significantly highly expressed in gastric cancer cells; the inventor finds that after the expression of the human EME1 gene is down-regulated by an RNAi method, the proliferation of tumor cells can be effectively inhibited, the apoptosis is promoted, the invasion and the transfer capacity of the tumor cells are reduced, and the growth process of the tumor can be effectively controlled, and the research result shows that the EME1 gene is a protooncogene and can be used as a target point for tumor treatment.
EME1 inhibitors
Refers to a molecule having an inhibitory effect on EME 1. Having inhibitory effects on EME1 include, but are not limited to: inhibiting the expression or activity of EME 1.
Inhibiting EME1 activity refers to a decrease in the activity of EME 1. Preferably, the EME1 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% as compared to prior to inhibition.
The inhibition of the expression of the EME1 can specifically be the inhibition of the transcription or translation of the EME1 gene, and specifically can be the inhibition of the expression of the EME 1: the gene of EME1 was not transcribed, or the transcriptional activity of the gene of EME1 was reduced, or the gene of EME1 was not translated, or the translation level of the gene of EME1 was reduced.
The regulation of gene expression of EME1 can be performed by one skilled in the art using conventional methods, such as gene knock-out, homologous recombination, interfering RNA, and the like.
The inhibition of gene expression of EME1 was verified by detecting the expression level by PCR and Western Blot.
Preferably, the expression of the EME1 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 EME1 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 gastric cancer
Nucleic acid molecules that reduce the expression of EME1 gene in gastric cancer cells can be utilized; and/or, an EME1 gene interfering nucleic acid construct; and/or, the EME1 gene interferes with lentivirus to be used as an effective component for preparing a medicament for preventing or treating gastric 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 EME1 Gene
1. Screening of effective siRNA target against human EME1 Gene
Calling EME1 (NM-152463) gene information from Genbank; designing effective siRNA target point aiming at EME1 gene. Table 1-1 lists the effective siRNA target sequences selected against the EME1 gene.
TABLE 1-1 siRNA target sequences targeting the human EME1 gene
SEQ ID NO
|
TargetSeq(5’-3’)
|
1
|
CTGAGAAGACAGGAAAGAA |
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 medicine science and technology Co., Ltd.), so that the vector is linearized, and the enzyme-cut fragment is identified 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-EME 1-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 (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-EME 1-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
TABLE 1-6-1 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 of EME1-siRNA lentivirus
The DNA of RNAi plasmid pGCSIL-GFP-EME1-siRNA was extracted using a plasmid extraction kit from Qiagen corporation to prepare 100 ng/. mu.l of stock solution.
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, transferred to medium of human embryonic kidney 293T cells at 37 ℃ with 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 concentrate is shown in SEQ ID NO. 2. The packaging procedure for the control lentivirus was identical to that of the EME1-siRNA lentivirus except that the pGCSIL-GFP-Scr-siRNA vector was used in place of the pGCSIL-GFP-EME1-siRNA vector.
Example 2 detection of Gene silencing efficiency by real-time fluorescent quantitative RT-PCR
Human gastric cancer AGS cells in logarithmic growth phase are trypsinized to prepare cell suspension (the number of cells is about 5X 10)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, AGS:10), an appropriate amount of the lentivirus prepared in example 1 is added, the medium is changed after 24h 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 EME1 gene were as follows: an upstream primer 5'-TCTGAGGAGTTGCCAACATTTG-3' (SEQ ID NO: 11) and a downstream primer 5'-GGCTTCACAATCTGAGATGTCAA-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
|
DEPC H2O
|
3.5
|
Total
|
11.0 |
TABLE 2-2 Real-time PCR reaction System
Reagent
|
Volume (μ l)
|
SYBR premix ex taq
|
10.0
|
Upstream primer (2.5. mu.M)
|
0.5
|
Downstream primer (2.5. mu.M)
|
0.5
|
cDNA
|
1.0
|
ddH2O
|
8.0
|
Total
|
20.0 |
The program was a two-step Real-time PCR: pre-denaturation at 95 ℃ for 15 s; then, denaturation is carried out at 95 ℃ for 5s in each step; annealing and extending for 30s at 60 ℃; a total of 45 cycles were performed. Each time reading the absorbance value during the extension phase. After the PCR was completed, the DNA was denatured at 95 ℃ for 1min, and then cooled to 55 ℃ to allow the DNA double strands to be sufficiently bound. Melting curves were prepared by increasing the temperature from 55 ℃ to 95 ℃ by 0.5 ℃ for 4 seconds and reading the absorbance. By adopting 2-ΔΔCtThe assay calculated the expression abundance of the EME 1-infected mRNA. Cells infected with the control virus served as controls. The experimental results are shown in fig. 1, and indicate that the expression level of EME1 mRNA in human gastric cancer AGS cells is down-regulated by 50.2%.
Example 3 examination of the proliferative Capacity of tumor cells infected with EME1-siRNA lentivirus
Human gastric cancer AGS cells in logarithmic growth phase are trypsinized to prepare cell suspension (the number of cells is about 5X 10)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, AGS:10), adding a proper amount of virus, culturing for 24h, then replacing the culture medium, and collecting 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 3000 cells 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 readings were performed once a day with Celigo instrument (Thermo Fisher) starting the second day after plating, and were performed continuously for 5 days. By adjusting the input parameters of Celigo, the number of green fluorescent cells in the well plate for each scan was accurately calculated, and the data were statistically plotted to obtain a cell proliferation curve (the result is shown in FIG. 2). The results show that after each tumor of the lentivirus infection group is cultured in vitro for 5 days, the proliferation speed is obviously slowed down and is far lower than that of the tumor cells of the control group, the number of viable cells is reduced by 50.2 percent, and the result shows that the proliferation capacity of AGS cells of human gastric cancer caused by EME1 gene silencing is inhibited.
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.
Sequence listing
<110> Shanghai Jikai Gene medicine science and technology Co., Ltd
Application of <120> human EME1 gene and related product
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<213> Artificial Sequence (Artificial Sequence)
<400> 2
cccugagaag acaggaaaga a 21
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<213> Artificial Sequence (Artificial Sequence)
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cccugagaag acaggaaaga acucgaguuc uuuccugucu ucucaggg 48
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<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccggccctga gaagacagga aagaactcga gttctttcct gtcttctcag ggtttttg 58
<210> 5
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aattcaaaaa ccctgagaag acaggaaaga actcgagttc tttcctgtct tctcaggg 58
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cctatttccc atgattcctt cata 24
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gtaatacggt tatccacgcg 20
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ttctccgaac gtgtcacgt 19
<210> 9
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ccggttctcc gaacgtgtca cgtctcgaga cgtgacacgt tcggagaatt tttg 54
<210> 10
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aattcaaaaa ttctccgaac gtgtcacgtc tcgagacgtg acacgttcgg agaa 54
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tctgaggagt tgccaacatt tg 22
<210> 12
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ggcttcacaa tctgagatgt caa 23
<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