Application of CLRN3 gene as tumor treatment target
阅读说明：本技术 Clrn3基因作为肿瘤治疗靶点的应用 (Application of CLRN3 gene as tumor treatment target ) 是由 陈新页 张晓静 张琪 龚天宇 于 2021-07-29 设计创作，主要内容包括：本发明涉及生物医学领域,具体而言,涉及一种CLRN3基因作为肿瘤治疗靶点的应用。具体的,涉及特异性抑制CLRN3基因转录或翻译,或能够特异性抑制CLRN3蛋白的表达或活性的分子在制备防治肿瘤的药物中的用途；其中所述肿瘤选自肠癌、肺癌和胃癌。(The invention relates to the field of biomedicine, in particular to application of a CLRN3 gene as a tumor treatment target. In particular to the application of a molecule which can specifically inhibit the transcription or translation of CLRN3 gene or can specifically inhibit the expression or activity of CLRN3 protein in the preparation of a medicament for preventing and treating tumors; wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer.)
1. The application of a molecule which can specifically inhibit the transcription or translation of CLRN3 gene or specifically inhibit the expression or activity of CLRN3 protein in the preparation of a medicament for preventing and treating tumors;
wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer.
2. The use according to claim 1, said molecule being selected from the group consisting of a nucleic acid molecule, an antibody drug and an interfering lentivirus.
3. The use according to claim 2, the nucleic acid molecule being selected from the group consisting of: antisense oligonucleotides, dsRNA, micro RNA, siRNA and shRNA.
4. The use of claim 3, wherein the siRNA comprises a first strand and a second strand, wherein the first and second strands are complementary and form an RNA dimer, and wherein the sequence of the first strand comprises one selected from the group consisting of sequences 1) to 3):
1) SEQ ID NO: 1 to 3 of at least one sequence shown in the specification;
2) sequences which hybridize under conditions of high stringency with the sequences of 1);
3) a sequence complementary to the sequence of 1) or 2).
5. The use according to claim 3, wherein 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 comprises one of the sequences shown in 1) to 3):
1) SEQ ID NO: 1 to 3 of at least one sequence shown in the specification;
2) sequences which hybridize under conditions of high stringency with the sequences of 1);
3) a sequence complementary to the sequence of 1) or 2).
6. The use of the nucleic acid construct in preparing a medicament for preventing and treating tumors;
wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer;
the nucleic acid construct comprises an siRNA as defined in claim 4, or an shRNA as defined in claim 5.
7. The use of claim 6, wherein the nucleic acid construct is a lentiviral vector.
8. The application of the pharmaceutical composition in preparing medicaments for preventing and treating tumors;
wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer;
the pharmaceutical composition comprises at least one of the siRNA as defined in claim 4, the shRNA as defined in claim 5, the nucleic acid construct as defined in claim 6 or 7, and a pharmaceutically acceptable carrier or excipient.
9. The separated CLRN3 gene or CLRN3 protein is used for screening drugs for preventing and treating tumors;
wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer.
10. The use of claim 9, wherein the CLRN3 gene is a partial fragment of a full-length gene and comprises at least one of the sequences 1) to 3):
1) SEQ ID NO: 1 to 3 of at least one sequence shown in the specification;
2) sequences which hybridize under conditions of high stringency with the sequences of 1);
3) a sequence complementary to the sequence of 1) or 2).
Technical Field
The invention relates to the field of biomedicine, in particular to application of a CLRN3 gene as a tumor treatment target.
Background
CLRN3(Clarin 3) is a protein-encoding gene. Previous studies found that CLRN 3-related diseases include Usher syndrome and tubular adenocarcinoma. An important paralogue of the gene is CLRN2, but the research on CLRN3 in the prior art is still very deficient, the function and the relation with other diseases are not well understood, and the relation between the CLRN3 gene and tumors is not reported.
RNA interference (RNAi) refers to a phenomenon of molecular biological double-stranded RNA-induced gene silencing by inhibiting gene expression by blocking transcription or translation of a particular gene. When double-stranded RNA homologous to the endogenous mRNA coding region is introduced into a cell, the mRNA is degraded, resulting in silencing of gene expression. RNA interference is a common laboratory technique for researching gene function and searching disease treatment methods emerging in recent years.
Therefore, the relation between the CLRN3 protein and diseases still needs to be further clarified, and a medicine capable of influencing the expression of the CLRN3 protein is searched on the basis, so that the aim of treating the diseases is fulfilled.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
Through a large number of experiments and repeated researches, the inventor of the invention unexpectedly discovers for the first time that the reduction of CLRN3 gene expression can inhibit the proliferation and/or infection of tumor cells, thereby achieving the purpose of treating tumors; on the basis, related medicaments such as siRNA, shRNA and the like capable of efficiently inhibiting expression of CLRN3 gene are further found, and the invention is completed.
The first purpose of the invention is to provide the application of a molecule which can specifically inhibit the transcription or translation of CLRN3 gene or can specifically inhibit the expression or activity of CLRN3 protein in the preparation of a medicament for preventing and treating tumors;
wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer.
Alternatively, the use as described above, said molecule being selected from the group consisting of a nucleic acid molecule, an antibody drug and an interfering lentivirus.
Alternatively, for use as described above, the nucleic acid molecule is selected from the group consisting of: antisense oligonucleotides, dsRNA, micro RNA, siRNA and shRNA.
Optionally, for use as described above, the siRNA 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 comprises one selected from the group consisting of sequences represented by 1) to 3):
1) SEQ ID NO: 1 to 3 of at least one sequence shown in the specification;
2) sequences which hybridize under conditions of high stringency with the sequences of 1);
3) a sequence complementary to the sequence of 1) or 2).
Optionally, for the above-mentioned use, 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, the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment comprises one of the sequences shown in 1) to 3):
1) SEQ ID NO: 1 to 3 of at least one sequence shown in the specification;
2) sequences which hybridize under conditions of high stringency with the sequences of 1);
3) a sequence complementary to the sequence of 1) or 2).
The second purpose of the invention is to provide the application of the nucleic acid construct in preparing the medicine for preventing and treating the tumor;
wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer;
the nucleic acid construct comprises an siRNA as defined above, or an shRNA as defined above.
Alternatively, the use as described above, wherein the nucleic acid construct is a lentiviral vector.
The third purpose of the invention is to provide the application of the pharmaceutical composition in preparing the medicine for preventing and treating the tumor;
wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer;
the pharmaceutical composition comprises at least one of an siRNA as defined above, an shRNA as defined above, a nucleic acid construct as defined above, and a pharmaceutically acceptable carrier or excipient.
The fourth purpose of the invention is to provide the application of the separated CLRN3 gene or CLRN3 protein in screening of drugs for preventing and treating tumors;
wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer.
Alternatively, the CLRN3 gene is a partial fragment of a full-length gene and comprises at least one of the sequences shown in 1) to 3):
1) SEQ ID NO: 1 to 3 of at least one sequence shown in the specification;
2) sequences which hybridize under conditions of high stringency with the sequences of 1);
3) a sequence complementary to the sequence of 1) or 2).
The invention has the beneficial effects that:
the downregulation of CLRN3 gene expression is found for the first time to be used for treating intestinal cancer, lung cancer and gastric cancer, and a corresponding downregulation means is provided.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a map of a pCMV-GFP-NC empty vector used in one embodiment of the present invention;
FIG. 2 shows that three shRNAs have inhibitory effects on CLRN3mRNA in three tumor cells according to an embodiment of the present invention (A); qRT-PCR detects knock-down efficiency (B) of CLRN3 in cells after human intestinal cancer RKO cells, lung cancer A549 cells and gastric cancer BGC-823 cells infect shCLRN 3-3;
FIG. 3 shows that the CCK-8 reagent detects the proliferation inhibition effect of CLRN3-siRNA lentivirus on human intestinal cancer RKO cells (A), lung cancer A549 cells (B) and gastric cancer BGC-823 cells (C) in one embodiment of the invention;
FIG. 4 is a graph (A) and a statistical graph (B) showing the results of experiments on the clonogenic expression of lentivirus-infected tumor cells according to an embodiment of the present invention;
FIG. 5 is a graph (A) and a statistical graph (B) of the results of a flow-type apoptosis assay in accordance with an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
Unless otherwise defined, all terms (including technical and scientific terms) used in disclosing the invention are to be interpreted as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions serve to better understand the teachings of the present invention by way of further guidance. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
As used herein, the terms "comprising," "including," and "comprising" are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.
The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range, as well as the recited endpoints.
The invention relates to an application of a molecule which specifically inhibits the transcription or translation of a CLRN3 gene or can specifically inhibit the expression or activity of a CLRN3 protein in preparing a medicament for preventing and treating tumors;
wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer.
It is readily understood that according to the teachings of the present invention, inhibition of CLRN3 gene expression, either from the protein level or mRNA level, would be effective. Inhibition may be partial attenuation of expression of CLRN3 gene or silencing of its expression.
The term "preventing or treating a tumor" refers to the prevention, treatment, or co-treatment of a tumor, or the inhibition or reduction of tumor cell growth, proliferation, differentiation, and/or survival.
Such molecules are understood to include, but are not limited to, nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins and interfering lentiviruses, preferably from nucleic acid molecules, antibody drugs or interfering lentiviruses.
In some embodiments, the nucleic acid molecule is selected from the group consisting of: antisense oligonucleotides, dsRNA, micro RNA, siRNA or shRNA.
In the present invention, the siRNA, i.e., small interfering RNA (small interfering RNA), is a double-stranded small RNA molecule, which consists of a first strand and a second strand that are completely complementary, and is processed by Dicer (an enzyme in RNAase iii family that is specific for double-stranded RNA). The first strand and the second strand are complementary to form an RNA dimer, and the sequence of the first strand is identical to a target sequence in CLRN3 gene, or a sequence that hybridizes to the target sequence under high stringency conditions. The length of the first strand and the second strand of the double-stranded RNA are both 10-30 nucleotides; preferably, the length of each nucleotide is 15-27 nucleotides; more preferably 19 to 23 nucleotides, and also 20, 21 or 22 nucleotides can be selected. siRNA is a major member of the siRISC, triggering silencing of the target mRNA to which it is complementary. RNA interference (RNAi) refers to the phenomenon of specific degradation of intracellular mRNA mediated by endogenous or exogenous double-stranded RNA (dsrna), resulting in silencing of expression of a target gene and the corresponding loss of a functional phenotype.
In some embodiments, the siRNA comprises a first strand and a second strand, the first and second strands being complementary to form an RNA dimer, and the sequence of the first strand comprises one selected from the group consisting of sequences set forth in 1) to 3):
1) SEQ ID NO: 1 to 3 of at least one sequence shown in the specification;
2) sequences which hybridize under conditions of high stringency with the sequences of 1);
3) a sequence complementary to the sequence of 1) or 2).
The sirnas of the invention can be screened by the methods disclosed in the examples herein or by methods known in the art.
In the present invention, "hybridization conditions" are classified according to the degree of "stringency" of the conditions used when measuring hybridization. The degree of stringency can be based, for example, on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about Tm-5 ℃ (5 ℃ below the Tm of the probe); "high stringency" occurs at about 5 ℃ to 10 ℃ below Tm; "moderate stringency" occurs at about 10 ℃ to 20 ℃ below the Tm of the probe; "Low stringency" occurs at about 20 ℃ to 5 ℃ below the Tm. Alternatively, or further, hybridization conditions may be based on hybridization salt or ionic strength conditions and/or one or more stringency washes. For example, 6 × SSC is extremely low stringency; 3 × SSC — low to medium stringency; 1 × SSC to medium stringency; high stringency with 0.5 × SSC. Functionally, conditions of maximum stringency can be used to determine nucleic acid sequences that are strictly identical or nearly strictly identical to the hybridization probes; and nucleic acid sequences having about 80% or more sequence identity to the probe are determined using conditions of high stringency.
For applications requiring high selectivity, it is typically desirable to employ relatively stringent conditions to form the hybrid, e.g., relatively low salt and/or high temperature conditions are selected. Hybridization conditions including medium and high stringency are provided by Sambrook et al (Sambrook, J. et al (1989) molecular cloning, A laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).
For ease of illustration, suitable moderately stringent conditions for detecting hybridization of a polynucleotide of the invention to another polynucleotide include: prewashing with 5 XSSC, 0.5% SDS, 1.0mM EDTA (pH8.0) solution; hybridization in 5 XSSC at 50-65 ℃ overnight; followed by two washes with 2X, 0.5X and 0.2 XSSC containing 0.1% SDS at 65 ℃ for 20 minutes each. One skilled in the art will appreciate that hybridization stringency can be readily manipulated, such as by varying the salt content of the hybridization solution and/or the hybridization temperature. For example, in another embodiment, suitable high stringency hybridization conditions include those described above, except that the hybridization temperature is increased, for example, to 60 ℃ to 65 ℃ or 65 ℃ to 70 ℃.
In the present invention, the shRNA, i.e., a small hairpin RNA or a short hairpin RNA (shRNA), is a segment of RNA sequence with tight hairpin loop (shRNA), which comprises a sense strand segment, an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, and is often used for RNA interference silencing target gene expression. Wherein the sequences of the sense strand and the antisense strand are complementary, and the sequence of the sense strand fragment is identical to 10-30 continuous nucleotide sequences in the CLRN3 gene, preferably, the sequence of the sense strand fragment is identical to 15-27 continuous nucleotide sequences in the CLRN3 gene; more preferably, the sense strand fragment is identical to 19 to 23 nucleotides in the CLRN3 gene, and 19, 20 or 21 consecutive nucleotide sequences may be selected to be identical or sequences that hybridize under high stringency conditions to the above sequences. The hairpin structure of the shRNA can be cleaved by cellular machinery into siRNA, which then binds to an RNA-induced silencing complex (RISC) that is capable of binding to and degrading mRNAs of interest.
In some embodiments, 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, the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment comprises one of the sequences set forth in 1) -3):
1) SEQ ID NO: 1 to 3 of at least one sequence shown in the specification;
2) sequences which hybridize under conditions of high stringency with the sequences of 1);
3) a sequence complementary to the sequence of 1) or 2).
Further, the sequence of the stem-loop structure of the shRNA may be routinely selected in the art, for example selected from any of the following: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CUCGAG, AAGCUU, and CCACACC.
According to a further aspect of the invention, the invention also relates to the use of the nucleic acid construct for the preparation of a medicament for the prevention and treatment of tumors;
wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer;
the nucleic acid construct comprises an siRNA as defined above, or an shRNA as defined above.
In the present invention, the nucleic acid construct refers to a sequence comprising a replication system and a coding sequence capable of transcribing and translating the polypeptide in a given target cell.
A nucleic acid construct is also referred to as a vector when it enables expression of a protein encoded by the inserted polynucleotide. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). In some embodiments, regulatory elements commonly used in genetic engineering, such as enhancers, promoters, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.) are included in the vectors of the present invention. Further, the vector also contains a nucleotide sequence encoding a marker detectable in the cell; the detectable marker is, for example, Green Fluorescent Protein (GFP).
In some embodiments, the nucleic acid construct is a lentiviral vector.
In the present invention, the type of lentiviral vector is well known in the art, and is selected, for example, 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.
According to still another aspect of the invention, the invention also relates to the use of the pharmaceutical composition in the preparation of a medicament for preventing and treating tumors;
wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer;
the pharmaceutical composition comprises at least one of an siRNA as defined above, an shRNA as defined above, a nucleic acid construct as defined above, and a pharmaceutically acceptable carrier or excipient.
In preparing these pharmaceutical compositions, the active ingredient is typically mixed with, or diluted with, excipients or enclosed within a carrier which may be in the form of a capsule or sachet. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the composition may be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions and the like. Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The preparation may further comprise a humectant, an emulsifier, a preservative (such as methyl and propyl hydroxybenzoate), a sweetener, etc.
When the pharmaceutical composition is used for preventing or treating tumors in a subject, an effective dose of the pharmaceutical composition needs to be administered to the subject. Using this method, the growth, proliferation, recurrence and/or metastasis of the tumor is inhibited. Further, at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% fraction of the growth, proliferation, recurrence and/or metastasis of the tumor is inhibited.
The subject of administration of the method may be an animal, preferably a mammal, preferably a primate, more preferably a human.
According to a further aspect of the invention, the invention also relates to the use of the isolated CLRN3 gene or CLRN3 protein in screening medicaments for preventing and treating tumors;
wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer.
In some embodiments, the CLRN3 gene is a partial fragment of a full-length gene and comprises at least one of the sequences shown in 1) to 3):
1) SEQ ID NO: 1 to 3 of at least one sequence shown in the specification;
2) sequences which hybridize under conditions of high stringency with the sequences of 1);
3) a sequence complementary to the sequence of 1) or 2).
Embodiments of the present invention will be described in detail with reference to examples.
Example 1 preparation of RNAi lentivirus against human CLRN3 Gene
1) Screening effective shRNA target point aiming at human CLRN3 gene
Calling CLRN3 (NM-152311) gene information from Genbank; designing an effective shRNA target aiming at the CLRN3 gene.
The sequence of the finally designed shRNA is shown in Table 1.
TABLE 1
2) Preparation of Lentiviral vectors
Synthesizing double-stranded DNA Oligo sequences with sticky ends containing Age I and EcoR I enzyme cutting sites at two ends aiming at shRNA targets, linearizing the Oligo sequences, and identifying enzyme cutting fragments by agarose gel electrophoresis, wherein the used system is shown in Table 2.
TABLE 2
Reagent
Amount of the composition used
Vector(1μg/μl)
2ul
CutSmart Buffer
5μl
AgeI(10U/μl)
1μl
EcoRI(10U/μl)
1μl
H2O
Up to 50μl
And (3) connecting the double-enzyme-digested linearized vector DNA with the purified double-stranded DNA Oligo by using T4DNA ligase, and carrying out enzyme digestion system at 37 ℃ for reaction for 1 h. Ligation was performed overnight at 16 ℃ in an appropriate buffer system to recover the ligation product. The ligation product was transformed into fresh E.coli competent cells prepared from calcium chloride. 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; universal PCR primers were designed upstream and downstream of the RNAi sequence in the lentiviral vector for PCR identification experiments. Sequencing and comparing the clones identified to be positive by the PCR, wherein the clones with correct comparison are vectors which are successfully constructed and aim at expressing RNAi, and are named as pCMV-GFP-CLRN 3-siRNA.
An empty-load vector map of a pCMV-GFP-NC-siRNA negative control plasmid is constructed, wherein the target sequence of the negative control siRNA is nonsense sequence TCACCGAGATGAAGATATC, pCMV-GFP-NC vector with the same base composition as the experimental group is shown in figure 1. When constructing pCMV-GFP-NC-siRNA negative control plasmid, synthesizing double-stranded DNA Oligo sequence containing Age I and EcoR I enzyme cutting sites at two ends aiming at ScrsiRNA target spot, and the rest construction method, identification method and conditions are the same as pCMV-GFP-CLRN 3-siRNA.
The linearized vector was digested with T4DNA ligase in the system shown in Table 3 at 37 ℃ for 1 h.
TABLE 3
Reagent
Amount of the composition used
Linearized Vector(100ng/μl)
1μl
Insert(100ng/μl)
1μl
10×T4 DNA ligase Buffer
2μl
T4 DNA ligase
1μl
H2O
Up to 20μl
3) Packaging CLRN3-siRNA lentivirus
The DNA of RNAi plasmid pCMV-GFP-CLRN3-siRNA was extracted using a plasmid extraction kit from Qiagen, 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. Can be used for transfection when the cell density reaches 70-80%. 2h before transfection, the original culture medium is sucked out and added1.5ml of fresh complete medium. 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 packaging process of the control lentivirus was the same as that of CLRN3-siRNA lentivirus, and only pCMV-GFP-NC-siRNA vector was used instead of pCMV-GFP-CLRN3-siRNA vector.
Example 2 detection of silencing efficiency of CLRN3 Gene by real-time fluorescent quantitative RT-PCR
Human intestinal cancer RKO cells, lung cancer A549 cells and gastric cancer BGC-823 cells in logarithmic growth phase are subjected to pancreatin digestion to prepare cell suspension (the number of cells is about 2 multiplied by 10)5/ml) were inoculated in 6-well plates and cultured until the degree of cell confluence reached about 30%. According to the value of the infection complex number (MOI, RKO:10, A549:20, BGC-823: 20), an appropriate amount of the virus prepared in example 1 was added, the medium was changed after 24h of culture, and cells were collected after the infection time reached 3 d. Total RNA was extracted according to the Trizol protocol of Invitrogen corporation. Reverse transcription of RNA to obtain cDNA was performed according to the M-MLV protocol of Promega, as shown in Table 4, at 42 ℃ for 1 hour, and then in a 70 ℃ water bath for 10min to allow reverse transcriptase to proceedAnd (4) inactivating.
TABLE 4
Reagent
Total pipe addition (10 reactions)
5×RT buffer
44μl
10mM dNTPs2
24μl
RNasin
4.4μl
M-MLV
11μl
Nuclease-free water
28.6μl
Real-time quantitative detection was carried out using a TP800 Real time PCR instrument (TAKARA).
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 using 2-ΔΔCtThe assay calculated the abundance of expression of CLRN 3-infected mRNA. Cells infected with a control virus (Lv-NC-shRNA) served as controls.
The results of the experiment are shown in FIG. 2. As can be seen from FIG. 2A, the three shRNAs have strong inhibition effects on CLRN3mRNA in three tumor cells, and shCLRN3-3 has the best effect, so that only shCLRN3-3 is detected in subsequent experiments. FIG. 2B shows that the knockdown efficiency of CLRN3 in human intestinal cancer RKO cells and lung cancer A549 cells detected by qRT-PCR and the knockdown efficiency of shCLRN3-3 infected by stomach cancer BGC-823 cells are all more than 50%.
Example 3 examination of the proliferative Capacity of tumor cells infected with CLRN3-shRNA lentivirus
Human intestinal cancer RKO cells, lung cancer A549 cells and gastric cancer BGC-823 cells in logarithmic growth phase are subjected to pancreatin digestion to prepare cell suspension (the number of cells is about 5 multiplied by 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, RKO:10, A549:20 and BGC-823: 20), a proper amount of virus is added, the culture medium is replaced after 24 hours of culture, and after the infection time reaches 5d, cells of each experimental group in the logarithmic growth phase are collected. 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 had 3-5 duplicate wells, 100. mu.l per well. After the plate is laid, the plate is placed at 37 ℃ and 5% CO2Culturing in an incubator. And adding 10 mu L of CCK-8 reagent into the wells 2-4 h before the culture is terminated from the next day after the plate laying without changing the solution. After 4h, the 96-well plate is placed on an oscillator to oscillate for 2-5min, and an enzyme-labeling instrument detects the OD value at 450 nm.
The results are shown in FIG. 3: the CCK8 test shows that compared with the control group, after shCLRN3-3 is infected, the proliferation of human intestinal cancer RKO cells (figure 3A), lung cancer A549 cells (figure 3B) and gastric cancer BGC-823 cells (figure 3C) is obviously inhibited.
Example 4 examination of the clonogenic Capacity of Lentiviral-infected tumor cells
Human intestinal cancer RKO cells, lung cancer A549 cells and gastric cancer BGC-823 cells are inoculated into a 12-well plate after being digested by pancreatin, and the cell density is 10-15%. The next day, the medium was changed to fresh medium containing 5ug/ml polybrene. CLRN3-shRNA lentivirus was expressed as MOI, RKO:10, A549:20, BGC-823: 20 were added to the plates and the medium was changed fresh 12-24h after infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.
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, 3 multiple wells are arranged in each experimental group), continuously culturing the inoculated cells in an incubator until the number of the cells in 14d or most of single clones is more than 50, changing the liquid every 3d midway and observing the cell state; photographing the cell clone under a fluorescent microscope before the experiment is terminated; when the experiment is ended, the cells are washed 1 time by PBS, the cells are fixed by paraformaldehyde for 30-60 min and washed 1 time by PBS, 500 mu L of clean and impurity-free GIEMSA staining solution is added into each hole to stain the cells for 20min, and ddH is added2And washing the cells for several times until the background on the plate is cleaned, drying, taking a picture of the monoclonal under a microscope, taking a picture of the whole plate by a digital camera, and counting the clones.
The results are shown in fig. 4, and the colony formation experiment shows that the number of cell clones infected with shCLRN3 is significantly reduced compared with the control group.
Example 5 Lentiviral-infected tumor cell apoptosis level assay
Human intestinal cancer RKO cells, lung cancer A549 cells and gastric cancer BGC-823 cells are inoculated in a 12-well plate after being digested by pancreatin, and the cell density is 10-15%. The next day, the medium was changed to fresh medium containing 5ug/ml polybrene. CLRN3-shRNA lentivirus was expressed as MOI, RKO:10, A549:20, BGC-823: 20 were added to the plates and the medium was changed fresh 12-24h after infection. After infection for 72h, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.
Collecting cell culture supernatant of each experimental group after infection in a 5ml centrifuge tube, washing the cells once by D-Hanks, digesting the cells by pancreatin, terminating the culture supernatant, and collecting the cells in the same 5ml centrifuge tube. Each group is provided with three multiple holes; centrifuging at 1500rmp for 5min, and discarding the supernatant; washing the cell precipitate once with PBS, centrifuging for 5min at 1500rmp, and collecting the cell; washing the cell precipitate once by 1 Xbinding buffer, centrifuging for 5min at 1500rmp, and collecting the cell; 1ml (the volume of staining buffer added is determined according to the amount of cell pellet, so that the final density of the cell suspension is 1X 106～1×107cell/ml)1 × stabilizing buffer resuspensionCell precipitation; 100ul (1X 10) of cell suspension was taken5～1×106Cells), adding 5ul annexin V-APC for staining, and keeping away from light for 10-15min at room temperature; transferring the sample to a flow type machine loading pipe, and loading the sample on a machine for detection.
The experimental results are shown in fig. 5, and the flow apoptosis detection results show that the number of apoptosis of shCLRN3 infected cells is obviously increased compared with the control group.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
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