CN115029789A - Construction of shRNA (short hairpin ribonucleic acid) library of DC-SIGN (dendritic cell-derived signal) and application of shRNA library in primary liver cancer - Google Patents

Construction of shRNA (short hairpin ribonucleic acid) library of DC-SIGN (dendritic cell-derived signal) and application of shRNA library in primary liver cancer Download PDF

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CN115029789A
CN115029789A CN202210609884.5A CN202210609884A CN115029789A CN 115029789 A CN115029789 A CN 115029789A CN 202210609884 A CN202210609884 A CN 202210609884A CN 115029789 A CN115029789 A CN 115029789A
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胡宝英
韩潇
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Nantong University
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Abstract

The invention discloses a construction method of a shRNA library of DC-SIGN and application thereof in primary liver cancer, wherein the construction method comprises the following steps: designing and synthesizing an oligonucleotide sequence of shRNA of DC-SIGN, and obtaining double-stranded oligonucleotide through high-temperature denaturation and slow cooling annealing; then connecting the double-stranded oligonucleotide to pSilencer 3.0-H1 subjected to restriction enzyme digestion linearization treatment, mixing, and forming a recombinant plasmid under the action of T4DNA ligase; and mixing the obtained recombinant plasmids according to equal proportion to form a DC-SIGN mixed shRNA library. The shRNA library can effectively silence DC-SIGN expression in macrophages, can be used as a technical means for targeting liver cancer immune tolerance to promote tumor immune activation, and has very wide application and development prospects for clinical treatment of liver cancer.

Description

Construction of shRNA library of DC-SIGN and application of shRNA library in primary liver cancer
Technical Field
The invention relates to construction of a shRNA library of DC-SIGN and application of the shRNA library in primary liver cancer, belonging to the technical field of biological engineering.
Background
Primary liver cancer (liver cancer for short) is one of the high-incidence malignant tumors in China, the incidence rate is the fifth place among all tumors in China, and the mortality rate is the second place. At present, the clinical treatment means of liver cancer is limited, and mainly comprises traditional surgical treatment, radiotherapy and chemotherapy. Although some new tumor treatment schemes such as targeted therapy and immunotherapy have been developed in recent years to achieve better therapeutic effects in melanoma and the like, it is still difficult for liver cancer patients to benefit from the above treatments. The mechanism of development of liver cancer is complex, and immune abnormalities play a crucial role in liver cancer development. Tumor immune tolerance is a precursor condition for inducing liver cancer, immune cells in the tumor can create a microenvironment for maintaining immune suppression, tumor-related macrophage cells and the like can express and secrete cytokines such as PD-L1, TGF-beta, IL-10 and the like, and the function inactivation and apoptosis of killer T cells are induced, so that the killing of an immune system on the liver cancer cells is inhibited. The immune tolerance mechanism of liver cancer is very complex, and the development of immunotherapy targets of liver cancer is limited.
Macrophages are a very critical class of immunoregulatory cells in liver cancer development. The macrophage is a population with proinflammatory and immune killing and can also be differentiated into a population with immunologic tolerance promotion, and the proportion of the two different macrophage populations plays a significant role in the liver cancer immune microenvironment. We have recently found through research that DC-SIGN/CD209 is a specific macrophage marker in liver cancer, and DC-SIGN is only expressed in immune-tolerogenic macrophages. The macrophage with positive DC-SIGN can secrete a large amount of immune tolerance cell factors such as PD-L1, TGF-beta, IL-10 and the like, so that the inactivation of killer T cells and tumor immune tolerance are caused, and the occurrence of liver cancer is promoted.
Disclosure of Invention
In order to solve the technical problems, the invention provides the following technical scheme:
a method for constructing shRNA library of DC-SIGN comprises the following steps:
designing and synthesizing an oligonucleotide sequence of shRNA of DC-SIGN, and obtaining double-stranded oligonucleotide through high-temperature denaturation and slow cooling annealing; then the double-stranded oligonucleotide is connected to pSilencer 3.0-H1 which is subjected to restriction enzyme digestion linearization treatment, and is mixed to form a recombinant plasmid under the action of T4DNA ligase; and mixing the obtained recombinant plasmids according to equal proportion to form a DC-SIGN mixed shRNA library, introducing the library into macrophages by using liposomes, silencing the expression of DC-SIGN in macrophage cells and converting the immunotolerance-promoting activity of the DC-SIGN.
Further, the oligonucleotide sequence of the shRNA comprises a plurality of shRNA targeting sequences:
shRNA-1:5’-CAGAACCTGACCCAGCTTAAA-3’；
shRNA-2:5’-TGGGTGAGCTCTCAGAGAAAT-3’；
shRNA-3:5’-GATGGGACTTTCAGATCTAAA-3’；
shRNA-4:5’-GAGGAAGACTGCGCGGAATTT-3’。
further, the restriction enzymes include BamHI and HindIII.
Further, the double-stranded oligonucleotide obtained by the high-temperature denaturation and slow cooling annealing specifically comprises: the oligonucleotide sequence was heated to 98 ℃ and cooled to 25 ℃ at a rate of 0.5 ℃/min.
The expression method of the shRNA library of the DC-SIGN comprises the following steps:
1) marking the liver cancer single cell suspension by using DC-SIGN and CD68 antibodies marked by fluorescent groups, and further separating the DC-SIGN by using a flow cytometer + CD68 + Macrophages are cultured in DMEM/F12 conditioned medium;
2) transfecting successfully cloned recombinant plasmid to separated and cultured DC-SIGN in liver cancer tissue by using liposome transfection reagent + CD68 + In macrophages, 36 hours after transfection, cell samples were collected and analyzed by flow cytometry for DC-SIGN expression on the surface of macrophage cells.
Further, the specific preparation method of the successfully cloned recombinant plasmid comprises the following steps:
a plurality of independent shRNA sequences interfering the expression of the DC-SIGN gene are cloned into a pSilencer 3.0-H1 eukaryotic expression vector, and PCR and DNA sequencing analysis prove that the construction of the recombinant clone is successful.
Further, the step 2) specifically comprises the following steps:
21) mu.g of plasmid was dissolved in 100. mu.l of opti-mem medium and 12. mu.l of lipofectin and 100. mu.l of opti-mem medium were added to another EP tube and incubated for 5min at room temperature;
22) mixing the plasmid and liposome solution, and incubating at room temperature for 15 min;
23) addition of DC-SIGN + CD68 + Macrophage, and centrifugation at 2000rpm for 60 min;
24) the transfected DC-SIGN + CD68 + The macrophages were re-incubated in the incubator.
The shRNA library of DC-SIGN is applied to the preparation of the medicine for treating the primary liver cancer.
Advantageous effects
In addition, the invention provides a method for constructing and expressing shRNA of DC-SIGN, which comprises the steps of synthesizing an oligonucleotide sequence of the shRNA, performing high-temperature denaturation and slow cooling annealing to obtain double-stranded oligonucleotide, then connecting the double-stranded oligonucleotide to pSilencer 3.0-H1 mixed after restriction enzyme digestion linearization treatment by BamHI and HindIII, forming recombinant plasmids under the action of T4DNA ligase, mixing the obtained recombinant plasmids according to equal proportion to form a DC-SIGN mixed shRNA library, introducing the DC-SIGN mixed shRNA library into macrophages by utilizing liposome, silencing the expression of DC-SIGN in the macrophages and converting the tolerizing activity of the DC-SIGN.
The invention utilizes a biological engineering technology to clone a plurality of independent shRNA sequences interfering DC-SIGN gene expression into a pSilencer 3.0-H1 eukaryotic expression vector. PCR and DNA sequencing analysis prove that the recombinant clone is successfully constructed, the eukaryotic expression vector is transfected into tumor-related macrophages, the silencing effect of the shRNA library on DC-SIGN expression in the macrophages is determined by flow cytometry, and the secretion of various immune tolerance cytokines in the macrophages can be inhibited. The research proves that the DC-SIGN interference shRNA library has the function of specifically inhibiting DC-SIGN positive macrophage (DC-SIGN) in the aspect of cell biology + CD68 + Macrophages) are capable of tolerating cytokine secretion.
In the invention, a shRNA mediated gene silencing technology is utilized to construct a plurality of shRNAs specifically targeting DC-SIGN and the shRNAs are mixed to form a shRNA library, and the shRNA library can overcome the problems of incomplete interference, off-target effect and the like of a single shRNA gene and play a stable and long-acting DC-SIGN interference capability. We find that the shRNA library can effectively silence the DC-SIGN expression in macrophages, can be used as a technical means for targeting liver cancer immune tolerance to promote the immune activation of tumors, and has very wide application and development prospects for clinical treatment of liver cancer.
Drawings
FIG. 1 shows DC-SIGN in hepatocarcinoma + CD68 + Macrophages secrete multiple immune-tolerant cytokines;
FIG. 2 shows that the shRNA library of DC-SIGN can inhibit the expression of DC-SIGN in macrophages;
FIG. 3 is a graph of the shRNA library of DC-SIGN inhibiting the secretion of immune-tolerant cytokines in macrophages;
FIG. 4 shows that library transduction of shRNA by DC-SIGN promotes the activation of killer T cells;
FIG. 5 shows that BDFACSAria flow cytometry detects the change of the apoptosis level of the liver cells.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
The invention adopts the following technical scheme for solving the technical problems:
FIG. 1 isolation and phenotypic analysis of DC-SIGN-positive macrophages in liver cancer tissue
Collecting fresh liver cancer tissue 100mg, washing with normal saline to remove blood, and cutting into 1-2mm under aseptic condition 3 The excised tissue RPMI-1640 medium was incubated at 37 ℃ for 2 hours with 1mg/mL of collagenase, 0.01% of hyaluronidase and 0.002% of DNase I, and the digested cell suspension was filtered through a 70 μm cell filter and resuspended in BD staining bufferIn the solution, the cells were incubated with fluorescent-labeled antibodies against DC-SIGN, CD68, PD-L1, TGF-. beta.and IL-10 for 30min, and then analyzed with a BD flow Analyzer for DC-SIGN-and DC-SIGN + CD68 + The expression of each immune tolerance cell factor in macrophage shows that DC-SIGN + CD68 + There was significant high expression of PD-L1, TGF-. beta.and IL-10 by macrophages (FIG. 1).
FIGS. 2-4
First, constructing shRNA library expression vector of DC-SIGN
And designing and synthesizing shRNA target positive strand and reverse strand oligonucleotide sequences of the DC-SIGN. After mixing the plus and minus strand oligonucleotides at a concentration of 5. mu.M, the mixed sample was heated to 98 ℃ and cooled to 25 ℃ at a rate of 0.5 ℃/min. Then mixed with pSilencer 3.0-H1 vector after being treated by BamHI and HindIII restriction enzyme digestion linearization, in a molar ratio of 3:1, added with 1 XT 4DNA ligase buffer and 1 mu l T4DNA ligase, incubated overnight at 16 ℃, transformed into DH5 alpha competent cells, plated into Amp resistant LB culture dish, and incubated overnight at 37 ℃ in an incubator. After the colonies are formed, selecting bacteria, identifying positive clones by using PCR, and verifying the positive clones by using sanger sequencing. And uniformly mixing the positive vectors of the four different target spots in a ratio of 1:1:1:1 to prepare a shRNA library of the DC-SIGN.
The second step is that: shRNA library vector transfection and expression detection of DC-SIGN
Marking the liver cancer single cell suspension by using DC-SIGN and CD68 antibodies marked by fluorescent groups, and further sorting out the DC-SIGN by using a flow cytometer + CD68 + Macrophages were cultured in DMEM/F12 conditioned media. Transfecting the successfully cloned recombinant plasmid into macrophages separated and cultured in liver cancer tissues by using a liposome transfection reagent:
1. mu.g of plasmid was dissolved in 100. mu.l of opti-mem medium and 12. mu.l of lipofectin and 100. mu.l of opti-mem medium were added to another EP tube and incubated for 5min at room temperature;
2. mixing the plasmid and the liposome solution, and incubating at room temperature for 15 min;
3. addition of DC-SIGN + CD68 + Macrophage, and centrifuging at 2000rpm for 60 min;
4. the transfected DC-SIGN + CD68 + The macrophages are incubated in the incubator again;
36 hours after transfection, cell samples were collected and analyzed by flow cytometry for DC-SIGN expression on the macrophage surface, indicating that pools of shRNA transduced with DC-SIGN significantly inhibited macrophage-indicative DC-SIGN expression (FIG. 2).
The third step: shRNA library of DC-SIGN for inhibiting immune tolerance cytokine expression in macrophage
Transfection of shRNA library of DC-SIGN into isolated DC-SIGN by Liposome transfection into hepatoma cells + CD68 + In macrophages, after 36h of transfection, cell samples were collected and resuspended in BD staining buffer, and 1:1000 of anti-PD-L1, TGF-beta and IL-10 antibody labeled by a fluorescent group was added for incubation for 30min, and the expression change of the immune tolerance cytokine after the shRNA library transfection without or with DC-SIGN was detected by using a BDFACSAria flow cytometer, and the result shows that the shRNA library transfected with DC-SIGN can inhibit the expression of the immune tolerance cytokine in macrophages (FIG. 3).
The fourth step: shRNA library of DC-SIGN for promoting activation of killer T cells in liver cancer tissues
Example 1: shRNA library of DC-SIGN for promoting increase of expression of killer T cell effector molecules
DC-SIGN transfected with shRNA library without or with DC-SIGN was prepared according to the same method as described above + CD68 + After macrophages, the macrophages are mixed with the liver cancer tissue single cell suspension and continuously cultured for 24h, then the single cell suspension is collected and resuspended in BD staining buffer, CD8, TCR and T cell effector molecule (GZMB, IFN gamma and PRF1) antibodies marked by 1:1000 fluorescent group are incubated for 2h at room temperature, then the cells are collected and used for detecting the expression change of effector molecules in CD 8T cells by using a BDFACSAria flow cytometer, and the result shows that the shRNA library transfected with DC-SIGN can inhibit the expression of various immune effector molecules in CD 8T cells (FIG. 4).
Example 2: shRNA library of DC-SIGN for promoting lethal T cell mediated liver cancer cell apoptosis
DC-SIGN transfected with shRNA library without or with DC-SIGN was prepared according to the same method as described above + CD68 + After macrophages are treated, the macrophages and the liver cancer tissue single cell suspension are mixed and continuously cultured for 24h, then the single cell suspension is collected and resuspended in BD staining buffer, the Annexin V/7-AAD apoptosis kit is used for incubating the cell suspension, then the collected cells are used for detecting the apoptosis level change of liver cells by a BDFACSAria flow cytometer, and the result shows that the shRNA library of the transfected DC-SIGN can promote the killing of immune cells on liver cancer cells (figure 5).
The invention researches and invents a key cell of tumor immunity, namely DC-SIGN, caused by shRNA intervention based on the characteristic that a tumor immune microenvironment can protect liver cancer cells from immune attack + CD68 + Macrophage to activate the tumor killing activity in liver cancer and promote the immune elimination of liver cancer cell. Research on cell and molecular level shows that the DC-SIGN shRNA library is introduced to obviously interfere DC-SIGN expression and prevent the secretion of immune tolerance factors in macrophages, so that DC-SIGN is relieved + CD68 + The inhibition activity of macrophages on CD 8T cells causes CD 8T cell mediated liver cancer cell killing, and the liver cancer treatment effect is achieved. The invention provides a pre-clinical specific target shRNA library for immunotherapy of liver cancer, and has important application prospects for clinical therapy of liver cancer and development of targeted drugs.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Sequence listing
<110> university of southeast Tong
Construction of shRNA library of <120> DC-SIGN and application of shRNA library in primary liver cancer
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cagaacctga cccagcttaa a 21
<210> 2
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgggtgagct ctcagagaaa t 21
<210> 3
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gatgggactt tcagatctaa a 21
<210> 4
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gaggaagact gcgcggaatt t 21

Claims (8)

1. A method for constructing shRNA library of DC-SIGN is characterized by comprising the following steps:
designing and synthesizing an oligonucleotide sequence of shRNA of DC-SIGN, and obtaining double-stranded oligonucleotide through high-temperature denaturation and slow cooling annealing;
then connecting the double-stranded oligonucleotide to pSilencer 3.0-H1 subjected to restriction enzyme digestion linearization treatment, mixing, and forming a recombinant plasmid under the action of T4DNA ligase; and mixing the obtained recombinant plasmids according to equal proportion to form a DC-SIGN mixed shRNA library.
2. The method for constructing a shRNA library of DC-SIGN as claimed in claim 1, wherein the oligonucleotide sequence of the shRNA comprises a plurality of shRNA targeting sequences:
shRNA-1:5’-CAGAACCTGACCCAGCTTAAA-3’；
shRNA-2:5’-TGGGTGAGCTCTCAGAGAAAT-3’；
shRNA-3:5’-GATGGGACTTTCAGATCTAAA-3’；
shRNA-4:5’-GAGGAAGACTGCGCGGAATTT-3’。
3. a method of constructing a shRNA library of DC-SIGN as claimed in claim 1, wherein the restriction enzymes comprise BamHI and HindIII.
4. The method for constructing the shRNA library of DC-SIGN according to claim 1, wherein the obtaining of the double-stranded oligonucleotide by the high-temperature denaturation and the slow cooling annealing specifically comprises: the oligonucleotide sequence was heated to 98 ℃ and cooled to 25 ℃ at a rate of 0.5 ℃/min.
5. The method for expressing the shRNA library of DC-SIGN according to claim 1, comprising the steps of:
1) marking the liver cancer single cell suspension by using DC-SIGN and CD68 antibodies marked by fluorescent groups, and further separating the DC-SIGN by using a flow cytometer + CD68 + Macrophages were cultured in DMEM/F12 conditioned medium;
2) transfecting successfully cloned recombinant plasmid to separated and cultured DC-SIGN in liver cancer tissue by using liposome transfection reagent + CD68 + In macrophages, 36 hours after transfection, cell samples were collected and analyzed for DC-SIGN expression on the macrophage surface using flow cytometry.
6. The method for expressing the shRNA library of DC-SIGN according to claim 5, wherein the specific preparation method of the successfully cloned recombinant plasmid comprises the following steps:
a plurality of independent shRNA sequences interfering the expression of the DC-SIGN gene are cloned into a pSilencer 3.0-H1 eukaryotic expression vector, and PCR and DNA sequencing analysis prove that the construction of the recombinant clone is successful.
7. The method for expressing the shRNA library of DC-SIGN according to claim 5, wherein the step 2) specifically comprises the following steps:
21) mu.g of plasmid was dissolved in 100. mu.l of opti-mem medium and 12. mu.l of lipofectin and 100. mu.l of opti-mem medium were added to another EP tube and incubated for 5min at room temperature;
22) mixing the plasmid and liposome solution, and incubating at room temperature for 15 min;
23) addition of DC-SIGN + CD68 + Macrophage, and centrifuging at 2000rpm for 60 min;
24) the transfected DC-SIGN + CD68 + The macrophages were re-incubated in the incubator.
8. The use of the shRNA library of DC-SIGN of claim 1 in the preparation of a medicament for treating primary liver cancer.
CN202210609884.5A 2022-05-31 2022-05-31 Construction of shRNA (short hairpin ribonucleic acid) library of DC-SIGN (dendritic cell-derived signal) and application of shRNA library in primary liver cancer Pending CN115029789A (en)

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