CN116515833A - Use of ACVR1C inhibitors in the treatment of colorectal cancer - Google Patents
Use of ACVR1C inhibitors in the treatment of colorectal cancer Download PDFInfo
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- CN116515833A CN116515833A CN202310460378.9A CN202310460378A CN116515833A CN 116515833 A CN116515833 A CN 116515833A CN 202310460378 A CN202310460378 A CN 202310460378A CN 116515833 A CN116515833 A CN 116515833A
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- colorectal cancer
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Abstract
The invention discloses an application of an ACVR1C inhibitor in treating colorectal cancer, and relates to the technical field of biomedicine. The invention suppresses proliferation of colorectal cancer cells and weakens invasion capacity of the cells by suppressing protein activity and/or expression level of ACVR1C, so that colorectal cancer tumor growth is suppressed, a new target is provided for colorectal cancer treatment, and the invention has clinical application prospect.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to an application of an ACVR1C inhibitor in treating colorectal cancer.
Background
Colorectal cancer has its morbidity and mortality in tumors worldwide, located at 3 rd and 2 nd, respectively. Colorectal cancer occurs and evolves as a result of a multi-step, complex interaction involving activation, mutation, inactivation, and deletion of numerous tumor regulatory factors. Because of the characteristics of high heterogeneity, hidden disease, rapid development and the like, many intestinal cancer patients are in middle and late stages when diagnosis is confirmed, and the selection of treatment means is relatively limited.
With the continued depth of research into colorectal cancer, one starts to search for indicators of tumorigenesis, development and prognosis at the gene level, and some colorectal cancer related mutant genes and signaling pathways have been discovered, and these findings promote clinical transformation of molecular targeted therapeutic drugs. For example, EGFR monoclonal antibodies-cetuximab, targeting KRAS-WT in colorectal cancer patients, demonstrate superior anti-cancer effects, highlighting the importance of molecular precise therapies.
Further screening of key genes capable of colorectal cancer onset and/or targeted drugs for colorectal cancer targeted therapy is one of the problems to be solved in the present day.
In view of this, the present invention has been made.
Disclosure of Invention
The object of the present invention is to provide the use of an ACVR1C inhibitor in the treatment of colorectal cancer.
The invention is realized in the following way:
in a first aspect, embodiments of the present invention provide an shRNA molecule comprising any one of shRNA35 and shRNA 37: the target sequence of the shRNA35 is shown as SEQ ID NO. 1, and the target sequence of the shRNA37 is shown as SEQ ID NO. 2;
optionally, the nucleotide sequence of the sense strand of the shRNA35 is shown as SEQ ID NO. 3, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 4;
the nucleotide sequence of the sense strand of the shRNA37 is shown as SEQ ID NO. 5, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 6.
In a second aspect, embodiments of the present invention provide a lentiviral vector comprising an shRNA molecule as described in the previous embodiments.
In a third aspect, embodiments of the present invention provide a recombinant lentivirus obtained by transfecting a cell with the recombinant lentivirus vector of the previous embodiments.
In a fourth aspect, embodiments of the present invention provide a medicament, the active ingredients of which include: the shRNA molecule of the preceding embodiment or the lentiviral vector of the preceding embodiment or the recombinant lentiviral of the preceding embodiment.
In a fifth aspect, embodiments of the present invention provide a colorectal cancer cell model that is a colorectal cancer cell line infected with a recombinant lentivirus comprising the foregoing embodiments.
In a sixth aspect, an embodiment of the present invention provides a method for constructing a colorectal cancer cell model, including: colorectal cancer cell lines were infected with the recombinant lentiviruses described in the previous examples.
In a seventh aspect, an embodiment of the present invention provides an ACVR1C inhibitor or a colorectal cancer cell model according to the previous embodiment or an application of the colorectal cancer cell model constructed by the construction method according to the previous embodiment in preparing a medicament for treating or assisting in treating colorectal cancer.
In an eighth aspect, embodiments of the present invention provide use of an ACVR1C inhibitor as described in the previous embodiments or a colorectal cancer cell model constructed by the method of construction as described in the previous embodiments in the preparation of an agent that inhibits at least one of proliferation, migration and invasion of colorectal cancer cells.
In a ninth aspect, embodiments of the present invention provide the use of an ACVR1C inhibitor as described in the preceding embodiments or a colorectal cancer cell model constructed by a method of constructing the preceding embodiments for inhibiting at least one of proliferation, migration and invasion of colorectal cancer cells, which is not directly for the diagnosis or treatment of a disease.
The invention has the following beneficial effects:
the invention suppresses proliferation of colorectal cancer cells and weakens invasion capacity of the cells by suppressing protein activity and/or expression level of ACVR1C, so that colorectal cancer tumor growth is suppressed, a new target is provided for colorectal cancer treatment, and the invention has clinical application prospect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is the effect of 2 AVCR1C shRNAs on human colorectal cancer cell HCT116 AVCR1 CmRNAs; wherein P <0.0001, compared to NC group, student's t-test was used;
FIG. 2 is an effect of ACVR1C shRNA on human colorectal cancer cell HCT116 ACVR1C protein expression;
FIG. 3 is the effect of ACVR1C shRNA on tumor growth in nude mice;
FIG. 4 is the effect of ACVR1C shRNA on lung metastasis of colorectal cancer cells in nude mice.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Short hairpin RNAs (shrnas), which comprise two short inverted repeats, separated by a stem-loop (loop) sequence, constitute a hairpin structure, controlled by the pol iii promoter, followed by 5-6 additional ts as transcription terminators for RNA polymerase iii, have many different biological uses. At present, shRNA is known to be mainly involved in RNA interference (RNAi), and double-stranded RNA with the same sequence as an endogenous target gene is artificially introduced to induce the degradation of mRNA of the endogenous target gene, so that the aim of weakening gene expression is fulfilled. shRNA can be introduced into cells via a variety of different transfection techniques and produce specific knockdown effects on specific genes. At present, two methods for introducing shRNA into a body exist, namely, directly injecting naked shRNA or shRNA subjected to chemical modification, and expressing shRNA by using a viral vector (such as lentivirus, adenovirus or adeno-associated virus). The shRNA has high specificity, high interference efficiency and simple operation, so that the complementarity of the shRNA which is properly cut can be utilized to mark the gene with a known sequence, and the phenomenon makes the shRNA an important tool for researching the gene function and the drug target.
ACVR1C is a type I receptor of TGF- β series signaling molecules that phosphorylates cytoplasmic SMAD transcription factors after binding to ligands, and then transfers into the nucleus, either directly interacting with DNA or complexing with other transcription factors of Nodal, activin a/B and GDF3, primarily for expression in the central nervous system, retinoblastoma, colon and pancreas. In the present invention, it is disclosed that inhibiting ACVR1C signaling is effective in inhibiting the metastatic phenotype of colorectal cancer cells, both in vitro and in vivo.
The inventors found and verified that ACVR1C-shRNA can inhibit proliferation of human colorectal cancer cells, and after ACVR1C-shRNA interferes with ACVR1C, the invasive capacity of the cells is weakened, and colorectal cancer tumor growth is inhibited. The RNAi technology or the gene editing technology is used for inhibiting the expression of ACVR1C genes in cancer cells, constructing a colorectal cancer cell model with low ACVR1C expression, researching the effect of ACVR1C in colorectal cancer cell generation and development, and helping to elucidate the mechanism of ACVR1C expression in colorectal cancer cells from the molecular level, thereby providing a useful clue for uncovering the pathogenesis and treatment of cancers and having important application value.
Specific technical scheme
In one aspect, embodiments of the present invention provide an shRNA molecule comprising any one of shRNA35 and shRNA 37: the target sequence of the shRNA35 is shown as SEQ ID NO. 1, and the target sequence of the shRNA37 is shown as SEQ ID NO. 2.
Optionally, the nucleotide sequence of the sense strand of the shRNA35 is shown as SEQ ID NO. 3 (5'-CcggcgGAGGAATTGTTGAGGAGTACTCGAGTACTCCTCAACAATT CCTCCGTTTTTg-3'), and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 4 (5'-aatt caaaaacgGAGGAATTGTTGAGGAGTACTCGAG TACTCCTCAACAATTCC TCCG-3');
the nucleotide sequence of the sense strand of the shRNA37 is shown as SEQ ID NO. 5 (5'-Ccgg gcAACACCTCAACTCATCTTTCTCGAGAAAGATGAGTTGAGGTGTTGC TTTTTg-3'), and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 6 (5'-aattcaaaaagcA ACACCTCAACTCATCTTTCTCGAGAAAGATGAGTTGAGGTGTTGC-3').
Remarks: a represents the sense strand and b represents the antisense strand.
Based on the present invention discloses the nucleotide sequence of shRNA molecule, one skilled in the art will easily think of preparing shRNA molecule by genetic engineering technique or other technique (chemical synthesis and recombinant expression). This is readily accomplished by those skilled in the art, and based on this, it is within the scope of the present invention to prepare shRNA molecules of the present invention regardless of the technique used.
shRNA35 and shRNA37 are effective in inhibiting/interfering with ACVR1C expression relative to other shRNA. "expression of ACVR 1C" includes mRNA expression levels and/or protein expression levels.
The ACVR1C amino acid sequence is as follows (SEQ ID NO: 7): MTRALCSALRQALLLLAAAAELSPGLKCVCLLCDSSNFTCQTEGACWASVMLTNGKEQVIKSCVSLPELNAQVFCHSSNNVTKTECCFTDFCNNITLHLPTASPNAPKLGPMELAIIITVPVCLLSIAAMLTVWACQGRQCSYRKKKRPNVEEPLSECNLVNAGKTLKDLIYDVTASGSGSGLPLLVQRTIARTIVLQEIVGKGRFGEVWHGRWCGEDVAVKIFSSRDERSWFREAEIYQTVMLRHENILGFIAADNKDNGTWTQLWLVSEYHEQGSLYDYLNRNIVTVAGMIKLALSIASGLAHLHMEIVGTQGKPAIAHRDIKSKNILVKKCETCAIADLGLAVKHDSILNTIDIPQNPKVGTKRYMAPEMLDDTMNVNIFESFKRADIYSVGLVYWEIARRCSVGGIVEEYQLPYYDMVPSDPSIEEMRKVVCDQKFRPSIPNQWQSCEALRVMGRIMRECWYANGAARLTALRIKKTISQLCVKEDCKA.
In another aspect, embodiments of the present invention provide a lentiviral vector comprising an shRNA molecule as described in the previous embodiments.
The term "lentiviral vector" refers to a vector that can efficiently integrate a foreign gene or foreign shRNA into a host chromosome, thereby achieving the effect of permanently expressing a sequence of interest. In the aspect of infection capability, the lentiviral vector can effectively infect various types of cells such as neuron cells, liver cells, cardiac muscle cells, tumor cells, endothelial cells, stem cells and the like, thereby achieving good gene therapy effect. For some cells which are difficult to transfect, such as primary cells, stem cells, undifferentiated cells and the like, the lentiviral vector is used, so that the transduction efficiency of the target gene or the target shRNA can be greatly improved, the probability of integrating the target gene or the target shRNA into the host cell genome is greatly increased, and the long-term and stable expression of the target gene or the target shRNA can be conveniently and rapidly realized. In view of this, lentiviral vectors have been widely used in scientific experiments and CAR-T cell therapies in research in vitro and in vivo, and their biosafety has been demonstrated, and the "pseudoviruses" thus prepared are non-infectious and pathogenic.
In the present invention, lentiviral vectors that can be used are those conventionally used in the art.
In some embodiments, the lentiviral vector comprises a vector backbone and a shRNA molecule.
In some embodiments, the carrier scaffold comprises: hU6-MCS-CBh-gcGFP-IRES-puromycin.
In some embodiments, the method of constructing a lentiviral vector comprises: synthesizing a sense strand and an antisense strand of the shRNA molecule, mixing the sense strand and the antisense strand, and annealing to obtain a DNA double strand;
and mixing and connecting the fragments (linearization vectors) of the vector skeleton after double enzyme digestion with double-stranded shRNA molecules to obtain the lentiviral vector inserted with the shRNA molecules.
In another aspect, embodiments of the present invention provide a recombinant lentivirus obtained by transfecting a cell with the recombinant lentivirus vector of any of the previous embodiments.
In some embodiments, the cells may be mammalian cells and cell lines. "mammalian cells" include cells derived from any member of a mammal, such as human cells, mouse cells, rat cells, monkey cells, hamster cells, and the like. In some specific embodiments, the cell may be a mouse cell, a human cell, a Chinese Hamster Ovary (CHO) cell, a CHOK1 cell, a CHO-DXB11 cell, a CHO-DG44 cell, a CHOK1SV cell (including all variants (e.g., lough, slough, UK)) or a CHOK1SV GS-KO (glutamine synthetase knockout) cell (including all variants (e.g., XCEEDTM, slalong, UK)) exemplary human cells include Human Embryonic Kidney (HEK) cells, such as HEK-293, hela cells, or HT1080 cells.
In some embodiments, the cell is: HEK-293T cells.
In another aspect, the embodiment of the present invention provides a medicament, including: the shRNA molecule of the preceding embodiment or the lentiviral vector of the preceding embodiment or the recombinant lentiviral of the preceding embodiment.
In some embodiments, the medicament may be formulated as a suspension in an aqueous, non-aqueous or mixed medium.
In some embodiments, the medicament further comprises: pharmaceutically acceptable auxiliary materials. Such adjuvants include, but are not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulates, excipients, fillers, binders, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, and adsorption carriers.
In some embodiments, the medicament is for use in the treatment or co-treatment of colorectal cancer.
The term "treating" includes preventing or alleviating a condition, reducing the rate at which a condition is raised or developed, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination thereof.
For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cell, or some combination of the foregoing. For tumors, "treatment" includes clearing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or slowing tumor progression, or some combination thereof.
In another aspect, embodiments of the invention provide a colorectal cancer cell model that is a colorectal cancer cell line infected with a recombinant lentivirus comprising any of the foregoing embodiments.
In some embodiments, the colorectal cancer cell line comprises human colorectal cancer cell HCT116.
In some embodiments, the colorectal cancer cell model underexpresses ACVR1C.
In another aspect, an embodiment of the present invention provides a method for constructing a colorectal cancer cell model, including: colorectal cancer cell lines were infected with the recombinant lentiviruses described in any of the previous examples.
In some embodiments, the colorectal cancer cell line comprises human colorectal cancer cell HCT116.
In some embodiments, the colorectal cancer cell model underexpresses ACVR1C.
In some embodiments, the method of constructing further comprises, after infecting the colorectal cancer cell line: colorectal cancer cell lines that underexpress ACVR1C were screened as a final colorectal cancer cell model. The screening conditions can be specifically as follows: screening was performed using 0.5 puromycin.
On the other hand, the embodiment of the invention provides an application of the ACVR1C inhibitor or the colorectal cancer cell model in any embodiment or the colorectal cancer cell model constructed by the construction method in any embodiment in preparation of medicines for treating or assisting in treating colorectal cancer.
In some embodiments, the ACVR1C inhibitor comprises: an agent that inhibits ACVR1C protein activity and/or expression level.
In some embodiments, the ACVR1C inhibitor comprises: at least one of an ACVR1C antibody, a gene editing agent that inhibits ACVR1C protein activity and/or expression level, and a gene interfering agent that inhibits ACVR1C protein activity and/or expression level.
In some embodiments, the gene interfering agent comprises: any one of siRNA, shRNA, lentiviral vector and recombinant lentivirus.
In some embodiments, the shRNA is a shRNA molecule as described in any of the foregoing examples.
In some embodiments, the lentiviral vector is a lentiviral vector as described in any of the preceding examples.
In some embodiments, the recombinant lentivirus is a recombinant lentivirus as described in any of the preceding examples.
In another aspect, embodiments of the present invention provide the use of an ACVR1C inhibitor as described in any of the preceding embodiments, or a colorectal cancer cell model constructed by a method as described in any of the preceding embodiments, in the preparation of an agent that inhibits at least one of proliferation, migration, and invasion of colorectal cancer cells.
In some embodiments, the colorectal cancer cell comprises: human colorectal cancer cell HCT116.
In addition, the embodiments of the present invention further provide the use of the ACVR1C inhibitor described in any of the preceding embodiments or the colorectal cancer cell model constructed by the method of constructing any of the preceding embodiments for inhibiting at least one of proliferation, migration and invasion of colorectal cancer cells, which is not directly aimed at diagnosis or treatment of a disease.
In some embodiments, the colorectal cancer cell comprises: human colorectal cancer cell HCT116.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1 design and Synthesis of shRNA targeting mRNA of ACVR1C Gene (abbreviated as ACVR1C shRNA)
The inventor designs ACVR1C shRNA according to human ACVR1C sequence in GenBank, and the design basic principle is as follows: 1. shRNA cloned into shRNA expression vectors comprises two short inverted repeats, separated by a stem-loop (loop) sequence in the middle, constituting a hairpin structure, controlled by the pol iii promoter. Then 5-6T's are ligated as transcription terminators for RNA polymerase III. 2. Two complementary oligonucleotides must bear restriction sites at both ends. StrataGENE found that 29 oligonucleotides were more effective in inhibiting the gene of interest than the 23 oligonucleotides originally recommended. 4. A C is placed immediately below the cleavage site downstream of the promoter, allowing a certain spacing between the insert and the promoter to ensure transcription. The first base of the shrna sequence of interest must be G to ensure RNA polymerase transcription. If the selected sequence of interest does not begin with G, a G must be added immediately upstream of the sense strand. The stem loop in the shrna insert should be near the center of the oligonucleotide. Stem loops of different sizes and nucleotide sequences have been successfully used. Wherein the stem loop comprising a unique restriction site facilitates detection of clones harboring the shRNA insert. The 5'tcaagag3' sequence is most effective (AMBION use) when compared to numerous stem loops of different lengths and sequences. 7.5-6T must be placed at the tail of the shRNA insert to ensure that RNA polymerase III terminates transcription (stop). 8. No consecutive 3 or more T's can occur on the sense and antisense strand sequences. This may lead to premature termination of shRNA transcription. 9. Starting from the AUG start code of the transcript (mRNA), the "AA or NA" diad sequence was found and the 19 base sequence at its 3' end was noted as a potential siRNA target site. Both sense and antisense strands were designed with these 19 bases (excluding AA or NA repeats).
According to the above principle, lentiviruses of ACVR1C shRNA sequences and 2 ACVR1C shRNAs packaged by Shanghai Ji Kai company were designated ACVR1C shRNA35 and ACVR1CshRNA37, respectively, and the nucleotide sequences are shown in Table 1.
TABLE 1 target sequences for ACVR1C shRNA and NC shRNA
Example 2 detection of ACVR1C interfering Effect of 2 ACVR1C shRNAs in human colorectal cancer cell HCT116
Human colorectal cancer cell HCT116 is a product of the american ATCC cell bank.
1. Infection with
(1) Human colorectal cancer cells HCT116 were inoculated into a culture dish (6 cm in diameter) containing a DMEM medium containing 10% (v/v) of newborn calf serum (the cell density at the time of transfection is preferably 30-40%) and cultured for 24 hours in a conventional manner.
(2) After the step (1) is completed, shRNA (NC shRNA, ACVR1C shRNA35 and ACVR1C shRNA 37) is added into a culture medium, and after 48 hours of infection, puromycin with the concentration of 1 mug/ml is used for screening, and after one week of screening, a stable transgenic cell strain expressing ACVR1CshRNA is obtained from human colorectal cancer cells HCT116.
2. cDNA acquisition
Total RNA of shRNA transfected human colorectal cancer cell HCT116 was extracted with Trizol (Invitrogen corporation, USA), followed by reverse transcription to obtain cDNA of shRNA transfected human colorectal cancer cell HCT116.
The reverse transcription steps are as follows:
(1) The preparation system comprises: the system was 15.9. Mu.l composed of total RNA of 2. Mu.g shRNA transfected human colorectal cancer cells HCT116, 1. Mu.l of 1. Mu.g/. Mu.l of random primer solution and DEPC water.
(2) The system was taken, incubated at 70℃for 5min and cooled on ice.
(3) After completion of step (2), 5. Mu. l M-MLV 5 Xbuffer, 2.5. Mu.l of 10mM dNTP and 1.0. Mu. l M-MLV reverse transcriptase (concentration: 200U/. Mu.l) were added, mixed, incubated at 42℃for 60min, and terminated at 95℃for 5min to obtain cDNA of shRNA transfected human colorectal cancer cell HCT116.
The random primer is synthesized by Beijing Saighur company. M-MLV 5 Xbuffer and M-MLV reverse transcriptase were purchased from Promega, USA.
3. Identification of interference effect of ACVR1C shRNA on ACVR1C Gene by qRT-PCR
And (2) respectively using the cDNA of the shRNA transfected human colorectal cancer cell HCT116 obtained in the step (2) as a template, and detecting the relative expression quantity of the ACVR1C gene (using the beta-actin gene as an internal reference gene) in the shRNA transfected human colorectal cancer cell HCT116 by qRT-PCR. The mRNA expression level of ACVR1C was calculated using the 2-DeltaCT method.
The primers for identifying the ACVR1C gene were: 5'-TGAACAGGGCTCCTTATATGACT-3' and 5'-GTGTGCCAGACCACTAGCAA-3'.
Primers identifying beta-actin were 5'-CATGTACGTTGCTATCCAGGC-3' and 5'-CTCCTTAATGTCACGCACGAT-3'.
The detection results are shown in FIG. 1A. The results show that after the ACVR1C shRNA35 and the ACVR1CshRNA37 are respectively transfected into human colorectal cancer cells HCT116, the expression level of ACVR1C mRNA is obviously reduced.
4. Western blot identification of ACVR1C shRNA inhibition effect on ACVR1C protein
(1) SDS-PAGE electrophoresis
Respectively extracting total protein of the shRNA transfected human colorectal cancer cell HCT116 obtained in the step 1, and carrying out SDS-PAGE electrophoresis after denaturation; the voltage is 120V for about 1.5h.
(2) Transfer film
After step (1) is completed, transferring the protein to a nitrocellulose membrane; the voltage was 16V and the transfer was about 1.5h.
(3) Closure
After completion of step (2), the nitrocellulose membrane was blocked with 5% nonfat dry milk (formulated with TBST solution containing TriS2.42g, naCl 8g, tween-20 10ml, pH7.6 per 1L of water) at room temperature for 1h and at 4℃overnight.
(4) Primary antibody binding
After the step (3) is completed, adding the primary antibody diluted by 5% of skimmed milk powder according to a certain proportion on the nitrocellulose membrane, and gently shaking for 1h at room temperature, and washing the membrane with TBST solution for 3 times, each time for 5min.
The primary antibody is a rabbit anti-human ACVR1C antibody (Sieimer, inc.) or a horseradish peroxidase-conjugated beta-actin antibody (ProteinTech, inc., U.S.).
(5) Secondary antibody binding
After the step (4) is completed, horseradish peroxidase-conjugated IgG (goat anti-rabbit IgG antibody, U.S. Santacruz Biotech) diluted with 5% skimmed milk powder in a certain proportion is added to the nitrocellulose membrane, and the membrane is gently shaken at room temperature for 1h, and washed with TBST solution for 3 times, 5min each time.
(6) Development process
After the step (5) is completed, the color is developed for 8min by a chemiluminescence method, and the film is pressed and developed.
The detection results are shown in fig. 2, and the results show that compared with the negative control, the expression level of the ACVR1C protein in human colorectal cancer cells HCT116 transfected with the ACVR1C shRNA35 and the ACVR1C shRNA37 is obviously reduced, and the reduction effect of the ACVR1C shRNA37 is more obvious than that of the ACVR1C shRNA 35.
The results indicated that ACVR1C shRNA35 and ACVR1C shRNA37 have good interfering effects on ACVR1C expression in human colorectal cancer cells HCT116.
Example 3 effect of ACVR1C knockdown on growth of colorectal cancer tumor in nude mice
To investigate the effect of ACVR1C knockdown on tumor growth in nude mice, the ACVR1C knockdown human colorectal cancer cell HCT116 stable cell lines constructed in example 2 (group: HCT116-NC, HCT116-ACVR1C-KD35 (constructed with shRNA 35), HCT116-ACVR1C-KD37 (constructed with shRNA 37)) and control cell lines were each made into cell suspensions, and nude mouse fat pad plantation was performed. Each group of 6 mice was inoculated with 100. Mu.L of the cell suspension (containing 5X 10) 6 Individual cells). After 45 days, tumor size and volume were measured and statistically analyzed.
The results of the analysis are shown in fig. 3, which shows that the ACVR1C shRNA colorectal cancer tumor volume is significantly reduced (P < 0.01) compared to NC shRNA control group. Thus, it can be seen that decreasing the expression of the ACVR1C gene can significantly inhibit colorectal cancer tumor growth.
Example 4 effect of ACVR1C knockdown on pulmonary metastasis of nude mouse colorectal cancer cells
To investigate the effect of ACVR1C knockdown on lung metastasis of nude mouse colorectal cancer cells, the construction of example 2 was performedHuman colorectal cancer cell HCT116 stable cell lines (groups: HCT116-NC, HCT116-ACVR1C-KD35 and HCT116-ACVR1C-KD 37) with knockdown ACVR1C and control cell lines were made into cell suspensions, respectively, and mice (4-6 week old BALB/C or C57 BL/6) were subjected to pulmonary metastasis model construction by tail intravenous injection. Each group of 6 mice was inoculated with 200. Mu.l of cell suspension (containing 1-5X 10) 6 Individual cells). After 2-3 weeks of the tail vein injection transfer period, lung transfer was observed by in vivo animal imaging on days 0,5, 10, 21, respectively. Statistical analysis was performed subsequently.
The results of the analysis are shown in fig. 4, which shows that ACVR1C shRNA colorectal cancer cell lung metastasis is significantly reduced (P < 0.01) compared to NC shRNA control group. Thus, it can be seen that decreasing the expression of the ACVR1C gene can significantly inhibit colorectal cell lung metastasis.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An shRNA molecule comprising any one of shRNA35 and shRNA 37:
the target sequence of the shRNA35 is shown as SEQ ID NO. 1, and the target sequence of the shRNA37 is shown as SEQ ID NO. 2;
optionally, the nucleotide sequence of the sense strand of the shRNA35 is shown as SEQ ID NO. 3, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 4;
the nucleotide sequence of the sense strand of the shRNA37 is shown as SEQ ID NO. 5, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 6.
2. A lentiviral vector comprising the shRNA molecule of claim 1;
optionally, the lentiviral vector further comprises a vector backbone;
optionally, the carrier scaffold comprises: hU6-MCS-CBh-gcGFP-IRES-puromycin.
3. A recombinant lentivirus obtained by transfecting a cell with the recombinant lentivirus vector of claim 2;
optionally, the cell comprises: HEK-293T cells.
4. A medicament, characterized in that the active ingredients thereof comprise: the shRNA molecule of claim 1 or the lentiviral vector of claim 2 or the recombinant lentiviral vector of claim 3;
optionally, the medicament further comprises: pharmaceutically acceptable auxiliary materials;
optionally, the medicament is for use in the treatment or co-treatment of colorectal cancer.
5. A colorectal cancer cell model, characterized in that it is a colorectal cancer cell line infected with a recombinant lentivirus of claim 3;
optionally, the colorectal cancer cell line comprises human colorectal cancer cell HCT116;
alternatively, the colorectal cancer cell model underexpresses ACVR1C.
6. A method of constructing a colorectal cancer cell model, comprising: infecting a colorectal cancer cell line with the recombinant lentivirus of claim 3;
optionally, the colorectal cancer cell line comprises human colorectal cancer cell HCT116;
alternatively, the colorectal cancer cell model underexpresses ACVR1C.
Use of an acvr1c inhibitor or a colorectal cancer cell model according to claim 5 or a colorectal cancer cell model constructed by the construction method according to claim 6 for the preparation of a medicament for the treatment or co-treatment of colorectal cancer;
optionally, the ACVR1C inhibitor comprises: an agent that inhibits ACVR1C protein activity and/or expression level;
optionally, the ACVR1C inhibitor comprises: at least one of an ACVR1C antibody, a gene editing agent that inhibits ACVR1C protein activity and/or expression level, and a gene interfering agent that inhibits ACVR1C protein activity and/or expression level;
optionally, the gene interference agent comprises: any one or more of siRNA, shRNA, lentiviral vector and recombinant lentivirus;
optionally, the shRNA is the shRNA molecule of claim 1;
optionally, the lentiviral vector is the lentiviral vector of claim 2;
optionally, the recombinant lentivirus is the recombinant lentivirus of claim 3.
8. Use of an ACVR1C inhibitor as set forth in claim 7 or a colorectal cancer cell model as set forth in claim 5 or a colorectal cancer cell model constructed by the construction method as set forth in claim 6 for the preparation of an agent that inhibits at least one of proliferation, migration and invasion of colorectal cancer cells.
9. The use of claim 8, wherein the colorectal cancer cells comprise: human colorectal cancer cell HCT116.
10. Use of the ACVR1C inhibitor of claim 7 or the colorectal cancer cell model of claim 5 or the colorectal cancer cell model constructed by the construction method of claim 6 for inhibiting at least one of proliferation, migration and invasion of colorectal cancer cells, said use not being for direct purposes of diagnosis or treatment of a disease;
optionally, the colorectal cancer cell comprises: human colorectal cancer cell HCT116.
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