CN112266932A - Construction of human RNF20 gene overexpression plasmid vector and cancer cell inhibition effect thereof - Google Patents
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Abstract
The invention relates to construction of a plasmid vector for over-expression of a human RNF20 gene and an effect of the plasmid vector in inhibiting cancer cells. The invention discloses the function of the human RNF20 gene in inhibiting colon cancer cells through in vitro cell experimental research of constructing an over-expression vector aiming at the human RNF20 gene. The human RNF20 gene overexpression vector constructed by the invention can efficiently infect target cells, further inhibit the proliferation and migration of colon cancer cells, promote the apoptosis of the colon cancer cells, and has important significance in the treatment of colon cancer and the research and development of related anti-cancer drugs.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a construction process of a human RNF20 gene overexpression plasmid vector and an effect of the vector in inhibiting cancer cells.
Background
In yeast and mammals, ubiquitin is a highly conserved biomacromolecule containing 76 amino acid polypeptide chains with a molecular weight of 8.5 kDa. Histone ubiquitination is an energy-driven enzyme-linked reaction process that requires the activation of three classes of enzymes in sequence: ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. It is now well studied that histone H2B is monoubiquitinated (H2Bub1), H2Bub1 is regulated by E3 ubiquitin ligase Bre1 in yeast, occurs on lysine 123 of histone H2B, and in mammals, occurs on lysine 120 of histone H2B, mainly under the control of heterodimer complex RNF20/RNF 40.
RNF20(RING finger protein 20) is a RING finger protein consisting of 976 amino acid residues, located at position 9q31.1 of human chromosome, belonging to the RING domain family of ubiquitin ligases, and plays an important role in both initiation and extension of transcription. RNF20 catalyzes the formation of H2bu 1, and then H2bu 1 binds to cryo-EM structures of COMPASS and Dot1, respectively, COMPASS and Dot1, as a DNA methyltransferase, catalyze the methylation of H3K4 and H3K79, respectively, thereby activating transcription. Furthermore, RNF20 interacts with subunit Cdc73 of the PAF1 complex, and then the PAF1 complex interacts with TF iis to relax RNA polymerase ii, thereby promoting transcriptional elongation.
Immunohistochemical analysis shows that a large number of primary tumors, namely H2Bub1, are down-regulated or deleted, the deletion of H2Bub1 accelerates the tumor progression, and RNF20 gene catalyzing the formation of H2Bub1 can selectively regulate gene expression and is known as a tumor suppressor. In view of the effect of the human RNF20 gene in tumors, a plasmid vector capable of highly expressing the human RNF20 gene is constructed, and the research on the action mechanism of the plasmid vector in the tumorigenesis and development is very important. At present, no report related to the construction of a human RNF20 gene overexpression vector exists, and the invention constructs a human RNF20 gene overexpression plasmid vector through a molecular correlation technology, and finds that the human RNF20 gene overexpression plasmid vector plays an important role in inhibiting the proliferation and migration of cancer cells and promoting the apoptosis of the cancer cells. The invention provides a new idea for preventing and treating cancers and a new scheme for screening and preparing anti-cancer drugs or preparations by applying related technologies of molecular biology.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a construction method of a human RNF20 gene overexpression vector and a research on the effect of the vector on inhibiting colon cancer cells. The plasmid vector is constructed by utilizing the existing double enzyme digestion technology and constructing the over-expression plasmid vector of the human RNF20 gene through gene modification, and in vitro cell experiments show that the human RNF20 gene plays an important role in inhibiting the proliferation and migration of cancer cells and promoting the apoptosis of the cancer cells.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a human RNF20 gene overexpression plasmid vector is constructed by the following steps:
(1) combining a pcMV-Tag2B plasmid and a human RNF20 fragment gene map, designing an upstream primer SEQ ID NO.1 and a downstream primer SEQ ID NO.2 of a human RNF20 gene, and amplifying the sequence by using cDNA of human mesenchymal stem cells HMSC as a template and utilizing a PCR technology to obtain the human RNF20 gene.
(2) The pcMV-Tag2B plasmid is cut by adopting a double enzyme digestion method to be linearized, and then the human RNF20 gene fragment and the linearized pcMV-Tag2B plasmid are spliced and recombined by using T4 ligase to construct a human RNF20 gene overexpression plasmid vector.
The constructed human RNF20 gene plasmid vector is characterized in that the high expression of the downstream target fragment human RNF20 gene is driven under the action of a CMV promoter and a CMV enhancer on a pcMV-Tag2B plasmid vector by using related molecular technology.
In order to further explore the role of the human RNF20 gene in the development of cancer, human colon cancer HCT116 and HT29 were selected as model cells.
The invention has the advantages and positive effects that:
1. the invention constructs a human RNF20 gene overexpression plasmid vector by using a double enzyme digestion method, can obviously improve the expression quantity of a tumor suppressor human RNF20 gene at the mRNA level and the protein level, can be efficiently introduced into colon cancer HCT116 and HT29 cells with the assistance of a transfection reagent TurboFect, specifically promotes the transcription and translation of the human RNF20 gene, increases the expression level of the human RNF20 gene, obviously inhibits the proliferation and migration capacity of the colon cancer cells, and promotes the apoptosis of the colon cancer cells.
2. The over-expression plasmid vector of the human RNF20 gene constructed by the invention plays an important role in inhibiting cancer cells, can be used as a potential target for cancer treatment, is used for researching related signal paths in the occurrence and development of cancer, provides a new idea for the prevention and treatment of cancer, and provides a new scheme for the screening and preparation of anti-cancer drugs or preparations.
Drawings
FIG. 1 is a DNA map of the pcMV-Tag2B plasmid;
FIG. 2 is a DNA map of human RNF20 gene;
FIG. 3 is an agarose gel electrophoresis of pcMV-Tag2B plasmid and successfully constructed over-expression plasmid of human RNF20 gene;
FIG. 4 shows that the expression level of RNF20 mRNA is significantly increased after the human RNF40 gene overexpression plasmid is transfected into human colon cancer HT29 cells for 24 h;
FIG. 5 shows that human RNF20 gene overexpression plasmid transfects human colon cancer HCT116 and HT29 cells for 24h, and then significantly inhibits the proliferation of the cells;
FIG. 6 shows that human RNF20 gene overexpression plasmid transfects human colon cancer HCT116 and HT29 cells for 24h, and then significantly inhibits migration of the cells;
FIG. 7 shows that human RNF20 gene overexpression plasmid transfects human colon cancer HCT116 and HT29 cells for 24h, and then remarkably promotes apoptosis of the cells.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments, which are provided for illustration only and are not intended to limit the scope of the present invention.
The experimental procedures used in the following examples are conventional, unless otherwise specified. The reagents and instruments used in the following examples are, unless otherwise specified, common reagents and instruments used in the laboratory and can be purchased from conventional sources.
The design idea of the invention is as follows: a human RNF20 gene overexpression plasmid vector is constructed by a double enzyme digestion method, is incubated with a transfection reagent TurboFect together, and is transfected into colon cancer cells by a transient transformation method, so that after the expression level of the human RNF20 gene is improved, the proliferation and migration of the colon cancer cells can be effectively inhibited, and the apoptosis of the colon cancer cells is promoted.
Example 1: preparation of plasmid vector for overexpression of human RNF20 Gene
1. Preparation of human RNF20 Gene
The coding region sequence of human RNF20 (NM-019592.7) gene is selected from Genbank of NCBI website, Primer5 software is used, according to pcMV-Tag2B plasmid vector map, in combination with the existing restriction endonuclease in the laboratory, two enzyme cutting sites of BamH I and HindIII are selected, and the upstream Primer and the downstream Primer of human RNF20 gene are designed. The cDNA of the human mesenchymal stem cell HMSC is used as a template, and the sequence is amplified by utilizing the PCR technology to obtain a target gene, namely the human RNF20 gene. Wherein, the sequence of the upstream Primer of the human RNF20 gene designed by the Primer5 software is SEQ ID NO.1, the sequence of the downstream Primer is SEQ ID NO.2, the reaction system for amplifying the sequences by using the PCR technology is shown in Table 1, and the reaction conditions for amplifying the sequences by using the PCR technology are shown in Table 2.
Table 1: PCR amplification sequence reaction system
Table 2: PCR amplification sequence reaction conditions
2. Preparation of recombinant plasmid
The plasmid pcMV-Tag2B was digested with restriction enzymes BamHI and Hind III at 37 ℃ for 2h, linearized, ligated with purified human RNF20 gene double-stranded DNA Oligo by T4 ligase overnight at 16 ℃, the ligation product was transformed into fresh E.coli DH 5. alpha. competent cells, spread on an LB plate containing kanamycin, and strains containing recombinant plasmids were selected.
Selecting single monoclonal antibody, placing into LB liquid culture medium containing kanamycin, placing into 37 deg.C shaking table to culture for 12-16h, collecting Escherichia coli thallus containing recombinant plasmid, and extracting plasmid. And (2) verifying by using a double enzyme digestion method, sending the verified positive recombinant plasmid to a company for sequencing, comparing a sequencing result with a sequence of the human RNF20 gene by using DNAMAN software, and obtaining a positive recombinant plasmid which is a human RNF20 gene over-expression plasmid vector which is named as pcMV-Tag2B-RNF20 and stored at-20 ℃, and placing the recombinant bacterium in 15-30% of glycerol and storing at-80 ℃.
Wherein, the reaction system of double enzyme digestion is shown in Table 3, the connection system of T4 ligase is shown in Table 4, pcMV-Tag2B is a publicly obtained plasmid, the DNA map is shown in figure 1, the RNF20 gene is a public gene, the DNA map is shown in figure 2, and the agarose gel electrophoresis images of pcMV-Tag2B and pcMV-Tag2B-RNF20 are shown in figure 3.
Table 3: double enzyme digestion reaction system
Table 4: t4 ligase ligation system
Example 2: high expression efficiency of human RNF20 gene after transfection of pcMV-Tag2B-RNF20 plasmid vector by real-time fluorescent quantitative RT-PCR detection
Human HT29 cells in logarithmic growth phase were digested with pancreatin and cell suspensions were prepared in D-MEM/F-12 medium containing 10% AusGeneX serum at 1.2X 10 per well6The individual cells were seeded in 6-well plates and then placed at 37 ℃ in 5% CO2Culturing in the incubator to make the cells adhere to the wall. To be tested for cellsTransfection can be initiated when the density grows to 70-80%, and the transfection mixture is prepared according to the transfection system, according to the plasmid concentration and instructions of the transfection reagent TurboFect. Among them, 6-well plate transfection systems are shown in Table 5.
Table 5: 6-well plate transfection system
The transfection mixture was incubated at 37 ℃ with 5% CO2The cells were incubated in the incubator of (1) for 20min, then transferred to HT29 cells, supplemented with a basal medium to 2mL per well, cultured for 6h, washed with D-MEM/F-12 medium containing 10% AusGeneX serum, and placed at 37 ℃ with 5% CO2The culture box is continuously cultured for 24 hours, and cells are collected. Total RNA was extracted according to instructions for Trizol reagent. According to the instruction of M-MLV reverse transcriptase, RNA is reversely transcribed into cDNA, the cDNA is reacted for 1h in a water bath at 37 ℃, and then the reverse transcriptase is inactivated in a water bath at 70 ℃ for 10 min. Among them, the reverse transcription system is shown in Table 6.
Table 6: reverse transcription system
An upstream RT primer with a sequence of SEQ ID NO.3 and a downstream RT primer with a sequence of SEQ ID NO.4 of the RNF20 gene are designed on line through a website, in addition, an upstream RT primer with a sequence of SEQ ID NO.5 and a downstream RT primer with a sequence of SEQ ID NO.6 of the GAPDH gene are designed, the GAPDH gene is used as an internal reference gene, and an RT-PCR instrument is used for real-time quantitative detection of the human RNF20 gene. The RT-PCR reaction system is shown in Table 7, and the RT-PCR instrument reaction conditions are shown in Table 8.
Table 7: RT-PCR reaction system
The RT-PCR instrument was programmed to: pre-denaturation at 95 deg.C for 2 min; then 40 cycles are carried out, wherein each cycle comprises denaturation at 95 ℃ for 10sec, annealing extension at 60 ℃ for 30 sec; after PCR is finished, denaturation is carried out for 1min at 95 ℃, and then cooling is carried out to 55 ℃ so as to ensure that the DNA double strands are fully combined; starting at 55 ℃ to 95 ℃, increasing by 0.3 ℃ in each step, and keeping for 1min while reading the absorbance. The delta. DELTA. Ct assay was used to calculate the mRNA level of the human RNF20 gene, and the results are shown in FIG. 4, which is a 3250-fold increase in the mRNA level of cells transfected with pcMV-Tag2B-RNF20 compared to cells transfected with pcMV-Tag2B plasmid.
Example 3: detection of proliferation Capacity of cancer cells transfected with pcMV-Tag2B-RNF20 plasmid
Human HCT116 and HT29 cells in logarithmic growth phase were digested with pancreatin, and cell suspensions were prepared in 4-5X 10 per well using D-MEM/F-12 medium containing 10% AusGeneX serum and DMEM high-sugar medium containing 10% AusGeneX serum, respectively4The individual cells were seeded in 96-well plates and then placed at 37 ℃ in 5% CO2Culturing in the incubator to make the cells adhere to the wall. Transfection can be initiated when the cell density grows to 70-80%, and the transfection mixture is prepared according to the transfection system, according to the plasmid concentration and instructions of the transfection reagent TurboFect. Among them, the 96-well plate transfection system is shown in Table 8.
Table 8: 96-well plate transfection system
The transfection mixture was incubated at 37 ℃ with 5% CO2After culturing for 6 hours, the cells were washed off with D-MEM/F-12 medium containing 10% AusGeneX serum and DMEM high-sugar medium containing 10% AusGeneX serum, and the cells were incubated at 37 ℃ and 5% CO for 20min2After further culturing for 24h, 10 mu of LMTT solution is added into each hole in a dark place, wrapped by tinfoil paper and placed at 37 ℃ with 5% CO2After further culturing for 4 hours, the culture medium was discarded, and 100. mu.L of dimethyl sulfoxide (DMSO) solution was added to each well in the dark. An enzyme-linked immunosorbent assay instrument is used,the absorbance was measured by setting the instrument parameters to 490nm absorbance and shaking for 10sec, and the data was processed and plotted, the results of which are shown in FIG. 5.
As shown in the results of FIG. 5, compared with the control cancer cell transfected with the pcMV-Tag2B plasmid, the HCT116 and HT29 colon cancer cells transfected with the pcMV-Tag2B-RNF20 plasmid have obviously inhibited the proliferation of the cancer cells after being cultured for 24h, which is far lower than the proliferation rate of the cancer cells in the control group, and the over-expression of the human RNF20 gene leads to the inhibition of the proliferation capacity of the cancer cells.
Example 4: testing the migration ability of cancer cells transfected with pcMV-Tag2B-RNF20 plasmid
Human HCT116 and HT29 cells in logarithmic growth phase were digested with pancreatin, and cell suspensions were prepared in 1.2X 10 per well using D-MEM/F-12 medium containing 10% AusGeneX serum and DMEM high-sugar medium containing 10% AusGeneX serum, respectively6The individual cells were seeded in 6-well plates and then placed at 37 ℃ in 5% CO2Culturing in the incubator to make the cells adhere to the wall. Transfection was initiated when the cell density reached 70-80%, and transfection mixtures were prepared according to the transfection system shown in Table 5, according to the plasmid concentration and the instructions of the transfection reagent TurboFect.
The transfection mixture was incubated at 37 ℃ with 5% CO2The cells were incubated in the incubator for 20min, transferred to HCT116 and HT29 cells, supplemented with D-MEM/F-12 basal medium and DMEM high-sugar basal medium to 2mL per well, cultured for 6h, scratched with a small gun head at each well of a 6-well plate, washed with D-MEM/F-12 medium containing 10% AusGeneX serum and DMEM medium containing 10% AusGeneX serum, the migration of the cells after 0h of transfection was photographed with a microscope, and the photographed cell positions were recorded, and then placed at 37 ℃, 5% CO, and the like2The cells were cultured in the incubator (2), and after 12, 24, and 48 hours of culture, the microscope was used to photograph the cells, and the migration of cancer cells was recorded, as shown in FIG. 6.
As shown in the results of FIG. 6, compared with the control cancer cell transfected with the pcMV-Tag2B plasmid, the HCT116 and HT29 colon cancer cells transfected with the pcMV-Tag2B-RNF20 plasmid have obviously inhibited the migration of the cancer cells after 12, 24 and 48 hours of culture, which is much lower than the migration speed of the control cancer cells, and the over-expression of the human RNF20 gene leads to the inhibition of the migration capability of the cancer cells.
Example 5: the effect of the pcMV-Tag2B-RNF20 plasmid on cancer cell apoptosis is detected
Human HCT116 and HT29 cells in logarithmic growth phase were digested with pancreatin, and cell suspensions were prepared in 1.2X 10 per well using D-MEM/F-12 medium containing 10% AusGeneX serum and DMEM high-sugar medium containing 10% AusGeneX serum, respectively6The individual cells were seeded in 6-well plates and then placed at 37 ℃ in 5% CO2Culturing in the incubator to make the cells adhere to the wall. Transfection was initiated when the cell density reached 70-80%, and transfection mixtures were prepared according to the transfection system shown in Table 5, according to the plasmid concentration and the instructions of the transfection reagent TurboFect.
The transfection mixture was incubated at 37 ℃ with 5% CO2The cultured cells were cultured for 20min, transferred into HCT116 and HT29 cells, supplemented with D-MEM/F-12 basal medium and DMEM high-sugar basal medium to 2mL per well, cultured for 6h, washed with D-MEM/F-12 medium containing 10% AusGeneX serum and DMEM high-sugar medium containing 10% AusGeneX serum, and then placed at 37 ℃ and 5% CO2After further culturing for 24h, the cells were collected and washed with PBS, then Annexin V-FITC and PI were added, incubated at room temperature in the dark and then detected using a flow cytometer, the results of which are shown in FIG. 7.
As shown in the results of FIG. 7, HCT116 and HT29 colon cancer cells transfected with the pcMV-Tag2B-RNF20 plasmid showed an increase in cancer cell apoptosis after 24h culture, compared to control cancer cells transfected with the pcMV-Tag2B plasmid, indicating that the overexpression of the human RNF20 gene promotes cancer cell apoptosis.
A sequence table:
SEQ ID NO.1：5’-CGCGGATCCATGTCAGGAATTGGAAATAAAAGAGCAGC-3’
SEQ ID NO.2：5’-CCCAAGCTTTCAACCAATGTAGATGCGATGAAAATC-3’
SEQ ID NO.3：5’-CAGTCTGTATGGCGGCACAA-3’
SEQ ID NO.4：5’-TCCGCAATTCCACCTTCAGC-3’
SEQ ID NO.5：5’-CGAGATCCCTCCAAAATCAA-3’
SEQ ID NO.6：5’-TTCACACCCATGACGAACAT-3’
although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Sequence listing
<110> Tianjin science and technology university
<120> construction of a plasmid vector for overexpression of human RNF20 gene and its effect of inhibiting cancer cells
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 38
<212> DNA
<213> nucleotide sequence encoding human RNF20 Gene (Unknown)
<400> 1
cgcggatcca tgtcaggaat tggaaataaa agagcagc 38
<210> 2
<211> 36
<212> DNA
<213> nucleotide sequence encoding the empty vector pcMV-Tag2B (Unknown)
<400> 2
cccaagcttt caaccaatgt agatgcgatg aaaatc 36
<210> 3
<211> 20
<212> DNA
<213> upstream RT primer of RNF20 Gene (Unknown)
<400> 3
cagtctgtat ggcggcacaa 20
<210> 4
<211> 20
<212> DNA
<213> downstream RT primer of RNF20 Gene (Unknown)
<400> 4
tccgcaattc caccttcagc 20
<210> 5
<211> 20
<212> DNA
<213> upstream RT primer of GAPDH Gene (Unknown)
<400> 5
cgagatccct ccaaaatcaa 20
<210> 6
<211> 20
<212> DNA
<213> downstream RT primer of GAPDH Gene (Unknown)
<400> 6
ttcacaccca tgacgaacat 20
Claims (5)
1. The constructed over-expression plasmid vector of the human RNF20 gene plays a role in inhibiting colon cancer cells.
2. A vector for increasing the expression of human RNF20 gene in colon cancer cells, the vector comprising:
the nucleotide sequence of the coding human RNF20 gene is SEQ ID NO. 1;
the nucleotide sequence of the coding blank vector pcMV-Tag2B is SEQ ID NO. 2.
3. The oligonucleotide of claim 2 encoding the human RNF20 gene of claim 1, wherein:
the nucleotide encoding the human RNF20 gene comprises a sense strand and an antisense strand, which are complementary to each other and, after transfection into cells, are integrated into the genome of the cells by DNA replication.
The blank vector pcMV-Tag2B comprises a CMV promoter and a CMV enhancer and can drive the expression of a downstream RNF20 gene.
4. The blank vector pcMV-Tag2B according to claim 2, wherein the blank vector comprises a NeoR/KanR resistance that can be screened.
5. A human RNF20 gene overexpression vector, which is transferred into a target cell under the assistance of a transfection reagent Thermo TurboFect according to the requirements of claims 2 to 4.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102906249A (en) * | 2010-05-25 | 2013-01-30 | 独立行政法人国立癌症研究中心 | Induced malignant stem cells or pre-induction cancer stem cells capable of self-replication outside of an organism, production method for same, and practical application for same |
| WO2018204764A1 (en) * | 2017-05-05 | 2018-11-08 | Camp4 Therapeutics Corporation | Identification and targeted modulation of gene signaling networks |
-
2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102906249A (en) * | 2010-05-25 | 2013-01-30 | 独立行政法人国立癌症研究中心 | Induced malignant stem cells or pre-induction cancer stem cells capable of self-replication outside of an organism, production method for same, and practical application for same |
| WO2018204764A1 (en) * | 2017-05-05 | 2018-11-08 | Camp4 Therapeutics Corporation | Identification and targeted modulation of gene signaling networks |
Non-Patent Citations (3)
| Title |
|---|
| NM_019592.7: "Homo sapiens ring finger protein 20 (RNF20), mRNA", 《GENBANK》 * |
| OHAD TARCIC: "RNF20 Links Histone H2B Ubiquitylation with Inflammation and Inflammation-Associated Cancer", 《CELL REP》 * |
| 崔雅妮: "慢病毒介导的低表达RNF20肝癌稳转细胞系的构建及其生物学功能分析", 《中国细胞生物学学报》 * |
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