CN113521287B - Application of CLRN3 gene as tumor treatment target - Google Patents

Abstract

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 molecules which can specifically inhibit the transcription or translation of CLRN3 genes or can specifically inhibit the expression or activity of CLRN3 proteins in preparing medicaments for preventing and treating tumors; wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer.

Description

CLRN3Application of gene as tumor treatment target

Technical Field

The invention relates to the biomedical field, in particular to a biological preparation method of a biological preparationCLRN3The gene is used as a tumor treatment target.

Background

CLRN3 (Clarin 3) is a protein-encoding gene. Previous studies have found diseases associated with CLRN3 including Usher syndrome and gastric tubular adenocarcinoma. An important paralog of this gene is CLRN2, but the prior art has been very deficient in the study of CLRN3, and its function and relationship to other diseases is poorly understood, andCLRN3the relationship between genes and tumors has not been reported yet.

RNA interference (RNAi) refers to a phenomenon of gene silencing induced by double-stranded RNA in molecular biology by inhibiting gene expression by blocking transcription or translation of a specific gene. When double stranded RNA homologous to the coding region of endogenous mRNA is introduced into a cell, the mRNA is degraded resulting in silencing of gene expression. RNA interference is a commonly used laboratory technique in recent years to study gene function and find methods for disease treatment.

Therefore, there is still a need in the art to further define the relationship between CLRN3 protein and disease, and on this basis, search for drugs that can affect CLRN3 protein expression, so as to achieve the goal of treating disease.

In view of this, the present invention has been made.

Disclosure of Invention

The inventors of the present invention have unexpectedly found down-regulation for the first time through a large number of experiments and repeated studiesCLRN3The gene expression can inhibit proliferation and/or infection of tumor cells, thereby achieving the purpose of treating tumors; and further found that the inhibition can be effectively realized on the basisCLRN3The invention is completed by the fact that the gene expression of siRNA, shRNA and other related drugs.

A first object of the present invention is to provide a specific inhibitionCLRN3Use of a gene transcription or translation, or a molecule capable of specifically inhibiting expression or activity of CLRN3 protein, in the preparation of a medicament for controlling a tumor;

wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer.

Alternatively, the use as described above, the molecule is selected from the group consisting of a nucleic acid molecule, an antibody drug and an interfering lentivirus.

Alternatively, the nucleic acid molecule is selected from the group consisting of: antisense oligonucleotides, dsrnas, micro RNAs, sirnas, and shrnas.

Alternatively, for use as described above, the siRNA comprises a first strand and a second strand that are complementary together to form an RNA dimer, and the sequence of the first strand comprises one selected from the sequences shown in 1) to 3):

1) SEQ ID NO: 1-3 at least one sequence shown;

2) A sequence which hybridizes to the sequence of 1) under higher stringency conditions;

3) A sequence complementary to the sequence of 1) or 2).

Alternatively, for use as described above, the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure linking the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment being complementary, and the sequence of the sense strand segment comprising one of the sequences shown in 1) to 3):

1) SEQ ID NO: 1-3 at least one sequence shown;

2) A sequence which hybridizes to the sequence of 1) under higher stringency conditions;

3) A sequence complementary to the sequence of 1) or 2).

A second object of the present invention is to provide a use of a nucleic acid construct in the preparation of a medicament for controlling a tumor;

wherein the tumor is selected from the group consisting of intestinal cancer, lung cancer and gastric cancer;

the nucleic acid construct comprises an siRNA as defined above, or a shRNA as defined above.

Alternatively, the nucleic acid construct is a lentiviral vector for use as described above.

The third object of the invention is to provide the application of the pharmaceutical composition in preparing medicines for preventing and treating tumors;

wherein the tumor is selected from the group consisting of intestinal cancer, lung cancer and gastric cancer;

the pharmaceutical composition contains 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.

A fourth object of the present invention is to provide a separationCLRN3The application of the gene or the CLRN3 protein in screening medicaments for preventing and treating tumors;

wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer.

Optionally, the use as described above, saidCLRN3The gene is a partial fragment of a full-length gene and at least comprises one of the sequences shown in 1) to 3):

1) SEQ ID NO: 1-3 at least one sequence shown;

2) A sequence which hybridizes to the sequence of 1) under higher stringency conditions;

3) A sequence complementary to the sequence of 1) or 2).

The beneficial effects of the invention are as follows:

down-regulation was first foundCLRN3The gene expression is used for treating intestinal cancer, lung cancer and gastric cancer, and provides a corresponding down-regulating means.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

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 in one embodiment of the invention have inhibitory effects on CLRN3 mRNA in three tumor cells (A); qRT-PCR (quantitative reverse transcription-polymerase chain reaction) is used for detecting the knockdown efficiency (B) of CLRN3 in human intestinal cancer RKO cells, lung cancer A549 cells and cells after the gastric cancer BGC-823 cells are infected with shCLRN 3-3;

FIG. 3 shows the inhibition of proliferation of human intestinal cancer RKO cells (A), lung cancer A549 cells (B) and gastric cancer BGC-823 cells by CCK-8 reagent according to one embodiment of the present invention;

FIG. 4 is a graph (A) and a statistical graph (B) of experimental results of cloning of lentivirus-infected tumor cells according to one embodiment of the present invention;

fig. 5 is a graph (a) and a statistical graph (B) of results of flow apoptosis detection according to an embodiment of the present invention.

Description of the embodiments

Reference now will 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. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.

The present invention relates to specific inhibitionCLRN3Use of a gene transcription or translation, or a molecule capable of specifically inhibiting expression or activity of CLRN3 protein, in the preparation of a medicament for controlling a tumor;

wherein the tumor is selected from intestinal cancer, lung cancer and gastric cancer.

It will be readily appreciated that the protein level or mRNA level pairs according to the teachings of the present inventionCLRN3Inhibition of gene expression will be effective. Inhibition may be partial attenuationCLRN3Expression of the gene may also be such that its expression is silenced.

The term "preventing or treating a tumor" refers to preventing, treating or aiding in the treatment of a tumor, or inhibiting or reducing the growth, proliferation, differentiation and/or survival of tumor cells.

Such molecules should be 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, dsrnas, micro RNAs, sirnas or shrnas.

In the present invention, the siRNA, i.e. small interference RNA (small interfering RNA), is a double-stranded small RNA molecule consisting of a first strand and a second strand that are perfectly complementary, 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 together to form an RNA dimer, and the sequence of the first strand is complementary to that of the second strandCLRN3The target sequence in the gene is the same or a sequence that hybridizes to the target sequence under high stringency conditions. The lengths of the first strand and the second strand of the double-stranded RNA are 10-30 nucleotides; preferably, the length is 15-27 nucleotides; better19-23 nucleotides may be selected, or 20, 21 or 22 nucleotides may be selected. siRNA is a major member of sirrisc, triggering silencing of target mRNA complementary thereto. RNA interference (RNAinterference, RNAi) refers to the phenomenon that endogenous or exogenous double-stranded RNA (dsRNA) mediates specific degradation of intracellular mRNA, thereby leading to silencing of target gene expression and corresponding loss of functional phenotype.

In some embodiments, the siRNA comprises a first strand and a second strand that are complementary together to form an RNA dimer, and the sequence of the first strand comprises one selected from the sequences shown in 1) to 3):

1) SEQ ID NO: 1-3 at least one sequence shown;

2) A sequence which hybridizes to the sequence of 1) under higher stringency conditions;

3) A sequence complementary to the sequence of 1) or 2).

The siRNA of the invention can be screened by 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 in measuring hybridization. The degree of stringency can be based on, for example, 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 about 5 ℃ to 10 ℃ below Tm; "moderate stringency" occurs about 10 ℃ to 20 ℃ below the probe Tm; "Low stringency" occurs about 20℃to 5℃below Tm. Alternatively, or in addition, hybridization conditions may be based on salt or ionic strength conditions of hybridization and/or one or more stringent washes. For example, 6 x SSC = very low stringency; 3x SSC = low to medium stringency; 1x SSC = medium stringency; 0.5 x SSC = higher stringency. Functionally, maximum stringency conditions can be used to determine nucleic acid sequences that are identical or nearly identical to the hybridization probes; while higher stringency conditions are used to determine nucleic acid sequences that have about 80% or more sequence identity to the probe.

For applications requiring high selectivity, it is typically desirable to employ relatively stringent conditions to form hybrids, e.g., to select relatively low salt and/or high temperature conditions. Sambrook et al (Sambrook, J. Et al (1989) molecular cloning, A laboratory Manual, cold Spring Harbor Press, planview, N.Y.), provide hybridization conditions including medium and high stringency.

For ease of illustration, suitable moderately stringent conditions for detecting hybridization of a polynucleotide of the invention with other polynucleotides include: pre-washed with 5 XSSC, 0.5% SDS, 1.0mM EDTA (pH 8.0) solution; hybridization in 5 XSSC at 50-65℃overnight; followed by washing twice with 2×, 0.5× and 0.2×ssc each containing 0.1% sds at 65 ℃ for 20 minutes. It will be appreciated by those skilled in the art that hybridization stringency can be readily manipulated, such as by altering 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 raised, for example, to 60℃to 65℃or 65℃to 70 ℃.

In the present invention, the shRNA, i.e., small hairpin or short hairpin RNA (a small hairpin RNA or shorthairpin RNA, shRNA), is an RNA sequence with a tight hairpin loop (tight hairpin turn) comprising a sense strand segment, an antisense strand segment, and a stem loop structure connecting the sense strand segment and the antisense strand segment, is often used for RNA interference silencing expression of a target gene. Wherein the sequences of the sense strand and the antisense strand are complementary and the sequence of the sense strand fragment is complementary toCLRN310 to 30 consecutive nucleotide sequences in the gene are identical, preferably the sense strand fragment is identical toCLRN315-27 continuous nucleotide sequences in the gene are identical; more preferably, the sense strand fragment is linked toCLRN3The gene may have 19 to 23 nucleotides identical, or 19, 20 or 21 consecutive nucleotides identical, or may be hybridized with the above sequences under high stringency conditions. The hairpin structure of shRNA can be cleaved by cellular mechanisms into siRNA, which then binds to RNA-induced silencing complexes (RNA-inducedsilencing complex, RISC) that are capable of binding to and degrading the desired mRNAs.

In some embodiments, the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure linking the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment being complementary, and the sequence of the sense strand segment comprising one of the sequences shown in 1) to 3):

1) SEQ ID NO: 1-3 at least one sequence shown;

2) A sequence which hybridizes to the sequence of 1) under higher stringency conditions;

3) A sequence complementary to the sequence of 1) or 2).

Further, the sequence of the stem-loop structure of the shRNA may be a routine choice in the art, e.g., selected from any one of the following sequences: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CUCGAG, AAGCUU and CCACACC.

According to a further aspect of the invention, it also relates to the use of a nucleic acid construct for the preparation of a medicament for the prevention and treatment of tumors;

wherein the tumor is selected from the group consisting of intestinal cancer, lung cancer and gastric cancer;

the nucleic acid construct comprises an siRNA as defined above, or a shRNA as defined above.

In the present invention, the nucleic acid construct is meant to include a replication system and sequences capable of transcribing and translating a polypeptide coding sequence in a given target cell.

When a nucleic acid construct enables expression of a protein encoded by an inserted polynucleotide, it is also referred to as a vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that 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 Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). In some embodiments, the vectors of the invention comprise 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.). Further, the vector also contains a nucleotide sequence encoding a marker that can be detected in the cell; the detectable label is, for example, green Fluorescent Protein (GFP).

In some embodiments, the nucleic acid construct is a lentiviral vector.

In the present invention, the lentiviral vector is of a type well known in the art, for example selected from: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635 pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-lamisnna, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.

According to a further aspect of the invention, it also relates to the use of a pharmaceutical composition for the preparation of a medicament for the prevention and treatment of tumors;

wherein the tumor is selected from the group consisting of intestinal cancer, lung cancer and gastric cancer;

the pharmaceutical composition contains 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 admixed with or diluted with an excipient or enclosed in a carrier which may be in the form of a capsule or sachet. When the excipient acts as a diluent, it may be a solid, semi-solid, or liquid material 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, starches, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The formulation may further comprise wetting agents, emulsifying agents, preserving agents (e.g., methyl and propyl hydroxybenzoates), sweetening agents, and the like.

For use in preventing or treating a tumor in a subject, an effective amount of the pharmaceutical composition is administered to the subject. With this method, the growth, proliferation, recurrence and/or metastasis of the tumor is inhibited. Further, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the portion of the tumor that grows, proliferates, recurs and/or metastasizes is inhibited.

The subject to which the method is administered may be an animal, preferably a mammal, preferably a primate, more preferably a human.

According to a further aspect of the invention, it also relates to a separationCLRN3The application of the gene or the 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, theCLRN3The gene is a partial fragment of a full-length gene and at least comprises one of the sequences shown in 1) to 3):

1) SEQ ID NO: 1-3 at least one sequence shown;

2) A sequence which hybridizes to the sequence of 1) under higher stringency conditions;

3) A sequence complementary to the sequence of 1) or 2).

Embodiments of the present invention will be described in detail below with reference to examples.

Example 1 directed to humansCLRN3Preparation of Gene RNAi lentiviruses

1) Screening for humansCLRN3Efficient shRNA target of gene

Clan 3 (nm_ 152311) gene information is called from Genbank; design is aimed atCLRN3Efficient shRNA targets of genes.

The sequence of the finally designed shRNA is shown in table 1.

TABLE 1

2) Preparation of lentiviral vectors

Double-stranded DNA Oligo sequences containing Age I and EcoR I restriction enzyme cutting sites at two ends and sticky ends are synthesized aiming at shRNA targets, linearization is carried out on the double-stranded DNA Oligo sequences, the restriction enzyme cutting fragments are identified by agarose gel electrophoresis, and the system is shown in Table 2.

TABLE 2

The vector DNA which is tangentially treated by double enzymes and the purified double-stranded DNA Oligo are connected by T4DNA ligase, and the enzyme digestion system is reacted for 1h at 37 ℃. Ligation was performed overnight at 16℃in an appropriate buffer system and the ligation product was recovered. Fresh E.coli competent cells prepared by transforming the ligation product with calcium chloride. Dipping a surface of a clone growing with a transformation product, dissolving in 10 μl of LB culture medium, uniformly mixing, and taking 1 μl as a template; a PCR identification experiment was performed by designing universal PCR primers upstream and downstream of RNAi sequences in lentiviral vectors. And (3) sequencing and comparing the clones positive to the PCR identification, wherein the clones with correct comparison are successfully constructed vectors aiming at expressing RNAi, and are named as pCMV-GFP-CLRN3-shRNA.

Constructing a pCMV-GFP-NC-shRNA negative control plasmid, wherein the negative control shRNA target sequence is a nonsensical sequence TCACCGAGATGAAGATATC, pCMV-GFP-NC vector with the same base composition as the experimental group, and the empty vector map of the negative control shRNA target sequence is shown in figure 1. When constructing a pCMV-GFP-NC-shRNA negative control plasmid, synthesizing a double-stranded DNA Oligo sequence with Age I and EcoR I restriction enzyme cutting sites at two ends aiming at a ScrshRNA target spot, and the rest construction methods, the identification methods and the conditions are the same as those of the pCMV-GFP-CLRN3-shRNA.

The vector was digested with T4DNA ligase and digested with the same two enzymes at 37℃for 1 hour.

TABLE 3 Table 3

3) Packaging CLRN3-shRNA lentiviruses

The DNA of RNAi plasmid pCMV-GFP-CLRN3-shRNA was extracted with a plasmid extraction kit from Qiagen, and 100 ng/. Mu.l of the stock solution was prepared.

24h before transfection, human embryonic kidney 293T cells in logarithmic growth phase were digested with trypsin and cell density was adjusted to 1.5X10% in DMEM complete medium containing 10% fetal bovine serum ⁵ Cells/ml, seeded in 6-well plates, 37 ℃,5% CO ₂ Culturing in an incubator. And (5) when the cell density reaches 70% -80%, the cell can be used for transfection. 2h before transfection, the original medium was aspirated and 1.5 ml of fresh complete medium was added. 20. Mu.l of Packing Mix (PVM), 12. Mu.l of PEI, 400. Mu.l of serum-free DMEM medium, 20. Mu.l of the extracted plasmid DNA were added to the PVM/PEI/DMEM mixture as described in Sigma-aldrich company MISSION Lentiviral Packaging Mix kit.

Incubating the above transfection mixture at room temperature for 15min, transferring into culture medium of human embryo kidney 293T cells, 37 ℃ and 5% CO ₂ Culturing in an incubator for 16h. The medium containing the transfection mixture was discarded, washed with PBS solution, and 2ml of complete medium was added to continue the culture for 48 hours. Cell supernatants were collected, and lentiviruses purified and concentrated by a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) as follows: (1) centrifuging at 4 ℃ for 10min at 4000g to remove cell debris; (2) The supernatant was filtered through a 0.45 μm filter in a 40ml ultracentrifuge tube; (3) Centrifuging at 4000g for 10-15min to obtain the required virus concentration volume; (4) After centrifugation, separating the filter cup from the lower filtrate collecting cup, reversely buckling the filter cup on the sample collecting cup, and centrifuging for 2min until the centrifugal force is not more than 1000g; (5) The centrifuge cup is removed from the sample collection cup and the virus concentrate is present in the sample collection cup. Packaging the virus concentrate, and storing at-80deg.C. The packaging process of the control lentivirus is the same as that of the CLRN3-shRNA lentivirus, and only the pCMV-GFP-CLRN3-shRNA vector is replaced by the pCMV-GFP-NC-shRNA vector.

Example 2 real-time fluorescent quantitative RT-PCR detectionCLRN3Gene of geneSilencing efficiency

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 (cell number is about 2×10) ⁵ Per ml) was inoculated into 6-well plates and cultured until the cell fusion reached about 30%. According to the values of complex infection (MOI, RKO:10, A549:20, BGC-823: 20), the virus prepared in example 1 was added in a proper amount, the culture medium was changed after culturing for 24 hours, and after the infection time reached 3d, the cells were collected. Total RNA was extracted according to Trizol protocol from Invitrogen. RNA was reverse transcribed to obtain cDNA according to the M-MLV protocol from Promega, table 4, and the reverse transcription reaction system was reacted at 42℃for 1 hour, followed by inactivation of the reverse transcriptase in a water bath at 70℃for 10 minutes.

TABLE 4 Table 4

Real-time quantitative detection was performed using a Real time PCR instrument model TP800 (TAKARA).

The procedure was set as two-step Real-time PCR: pre-denaturation at 95 ℃,15s; then each step is denatured at 95 ℃ for 5s; annealing and extending at 60 ℃ for 30s; a total of 45 cycles were performed. The absorbance was read each time during the extension phase. After the PCR was completed, the DNA was denatured at 95℃for 1min, and then cooled to 55℃to allow the DNA double strand to bind sufficiently. Starting from 55 ℃ to 95 ℃, increasing the temperature by 0.5 ℃ in each step, keeping for 4s, and simultaneously reading the absorbance value to prepare a melting curve. By 2 ^-ΔΔCt Analysis calculated the abundance of expression of CLRN3 mRNA. Cells infected with control virus (Lv-NC-shRNA) served as controls.

The experimental results are shown in FIG. 2. As can be seen from fig. 2A, the three shRNA have strong inhibition effect on CLRN3 mRNA in the three tumor cells, and the effect of shCLRN3-3 is optimal, so that only shCLRN3-3 is detected in the subsequent experiments. FIG. 2B shows that the knockdown efficiency of CLRN3 in cells after qRT-PCR detection of human intestinal cancer RKO cells, lung cancer A549 cells and gastric cancer BGC-823 cells infected with shCLRN3-3 is more than 50%.

Example 3 detection of proliferation 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 (cell number is about 5×10) ⁴ Per ml) was inoculated into 6-well plates and cultured until the cell fusion reached about 30%. According to the complex number of infection (MOI, RKO:10, A549:20, BGC-823: 20), a proper amount of virus was added, the culture medium was changed after culturing for 24 hours, and after the infection time reached 5 days, the cells of each experimental group in the logarithmic growth phase were collected. Complete medium resuspension of the adult cell suspension (2X 10) ⁴ Per ml), 96-well plates were seeded at a cell density of about 2000 cells per well. 3-5 wells per group, 100 μl per well. After being paved, the mixture is placed at 37 ℃ and 5 percent of CO ₂ Culturing in an incubator. Starting from the next day after plating, 10. Mu.L of CCK-8 reagent is added to the wells 2-4 hours before the end of the culture without changing the liquid. After 4h, the 96-well plate is placed on an oscillator to oscillate for 2-5 min, and an enzyme-labeled instrument 450 nm detects the OD value.

The results are shown in FIG. 3: CCK8 detection shows that compared with a control group, after shCLRN3-3 is infected, proliferation of human intestinal cancer RKO cells (figure 3A) and lung cancer A549 cells (figure 3B) and gastric cancer BGC-823 cells (figure 3C) is obviously inhibited.

Example 4 detection of the clonogenic Capacity of lentivirus-infected tumor cells

Human intestinal cancer RKO cells, lung cancer A549 cells and gastric cancer BGC-823 cells are inoculated into a 12-hole plate after being digested by pancreatin, and the cell density is 10-15%. The next day was changed to fresh medium containing 5ug/ml polybrene. CLRN3-shRNA lentivirus was tested according to MOI, RKO:10, a549:20, bgc-823:20 are added into the culture plate, and fresh culture medium is replaced after 12 to 24 hours of infection. After 72h of infection, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.

After pancreatin digestion of the cells after infection with virus in the logarithmic growth phase, the complete medium is resuspended into a cell suspension; inoculating the cells into a 6-hole plate (800 cells/hole, 3 compound holes are arranged in each experimental group), continuously culturing the inoculated cells in an incubator until the number of the cells in 14d or most single clones is more than 50, and replacing the liquid in 3d and observing the cell state; cell alignment under fluorescence microscope before termination of experimentCloning and photographing; washing cells 1 time with PBS at the end of the experiment, fixing the cells for 30-60 min with paraformaldehyde, washing the cells 1 time with PBS, adding 500 mu L of clean and impurity-free GIEMSA dye solution into each hole, dying the cells for 20min, and ddH ₂ O washing the cells for several times until the cells are washed with the background, airing, photographing the monoclone under a microscope, photographing the whole plate by a digital camera, and counting the clones.

The results are shown in fig. 4, which shows that the clone numbers of cells infected with shCLRN3 are significantly reduced compared to the control group.

Example 5 detection of apoptosis level in lentivirus-infected tumor cells

Human intestinal cancer RKO cells, lung cancer A549 cells, gastric cancer BGC-823 cells and pancreatin are digested and inoculated into 12-hole plates, and the cell density is 10-15%. The next day was changed to fresh medium containing 5ug/ml polybrene. CLRN3-shRNA lentivirus was tested according to MOI, RKO:10, a549:20, bgc-823:20 are added into the culture plate, and fresh culture medium is replaced after 12 to 24 hours of infection. After 72h of infection, fluorescence is observed under a fluorescence microscope, and the infection efficiency reaches 90%.

Cell culture supernatants from each experimental group after infection were collected in 5ml centrifuge tubes, the cells were washed once with D-Hanks, the cells were pancreatin digested, the culture supernatants were terminated, and the cells were collected in the same 5ml centrifuge tubes. Each group is provided with three complex holes; centrifuging at 1500rmp for 5min, and discarding the supernatant; washing the cell pellet once with PBS, centrifuging at 1500rmp for 5min, and collecting the cells; washing the cell pellet once by 1×binding buffer, centrifuging at 1500rmp for 5min, and collecting the cells; 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 10) ⁶ ~1×10 ⁷ cell/ml) 1×starting buffer resuspended cell pellet; 100ul (1X 10) of cell suspension was taken ⁵ ~1×10 ⁶ Cells), adding 5ul of annexin V-APC for dyeing, and keeping out of light at room temperature for 10-15 min; transferring to a flow type on-machine pipe, and detecting on-machine.

The experimental results are shown in fig. 5, and the flow apoptosis detection results show that the number of apoptosis infected with shCLRN3 is obviously increased compared with the control group.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

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Claims (6)

1. Specific inhibitionCLRN3Use of a nucleic acid molecule capable of gene transcription or translation, or specifically inhibiting expression or activity of CLRN3 protein, in the manufacture of a medicament for treating a tumor, said nucleic acid molecule being shRNA;

the shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure linking the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment being complementary, and the sequence of the sense strand being selected from the group consisting of SEQ ID NOs: 1-3 at least one sequence shown;

wherein the tumor is selected from intestinal cancer, lung cancer or gastric cancer.

2. Specific inhibitionCLRN3Use of a gene transcription or translation, or a nucleic acid construct capable of specifically inhibiting expression or activity of CLRN3 protein, in the manufacture of a medicament for treating a tumor;

wherein the tumor is selected from intestinal cancer, lung cancer or gastric cancer;

the nucleic acid construct comprises the shRNA of claim 1.

3. The use of claim 2, wherein the nucleic acid construct is a lentiviral vector.

4. Specific inhibitionCLRN3Use of a gene transcription or translation, or a pharmaceutical composition capable of specifically inhibiting expression or activity of CLRN3 protein, in the preparation of a medicament for treating a tumor;

wherein the tumor is selected from intestinal cancer, lung cancer or gastric cancer;

the pharmaceutical composition comprises the shRNA of claim 1, and a pharmaceutically acceptable carrier or excipient.

5. Specific inhibitionCLRN3Use of a gene transcription or translation, or a pharmaceutical composition capable of specifically inhibiting expression or activity of CLRN3 protein, in the preparation of a medicament for treating a tumor;

wherein the tumor is selected from intestinal cancer, lung cancer or gastric cancer;

the pharmaceutical composition contains the nucleic acid construct of claim 2, and a pharmaceutically acceptable carrier or excipient.

6. Specific inhibitionCLRN3Use of a gene transcription or translation, or a pharmaceutical composition capable of specifically inhibiting expression or activity of CLRN3 protein, in the preparation of a medicament for treating a tumor;

wherein the tumor is selected from intestinal cancer, lung cancer or gastric cancer;

the pharmaceutical composition comprises the lentiviral vector of claim 3, and a pharmaceutically acceptable carrier or excipient.

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