CN114085815A - Engineered exosome carrying nucleic acid type iron death inducer, and construction method and application thereof - Google Patents

Engineered exosome carrying nucleic acid type iron death inducer, and construction method and application thereof Download PDF

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CN114085815A
CN114085815A CN202111205223.8A CN202111205223A CN114085815A CN 114085815 A CN114085815 A CN 114085815A CN 202111205223 A CN202111205223 A CN 202111205223A CN 114085815 A CN114085815 A CN 114085815A
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exosome
lamp2b
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高源�
李晓菊
张存
张英起
李萌
郭欣燕
于金正
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Air Force Medical University of PLA
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Abstract

The invention relates to an engineered exosome carrying a nucleic acid type iron death inducer, and a construction method and application thereof. The existing iron death inducer has no targeting property and poor biocompatibility, so that the in vivo application of the iron death inducer is greatly limited. The invention constructs an SP94-lamp2b-RRM fusion protein expression vector, then constructs an exosome with SP94-lamp2b-RRM fusion protein, and finally constructs an engineered exosome loaded with multi-siRNA, namely the engineered exosome loaded with the nucleic acid type iron death inducer. The engineered exosome contains RNA binding protein, and can wrap a large amount of RNA containing RNA binding protein binding site sequences into the exosome in the biosynthesis process so as to achieve the effect of drug loading; the multi-siRNA which simultaneously targets GPX4 and DHODH is loaded, so that the expression of GPX4 and DHODH in cells can be effectively reduced, and the death of the cells is induced; the composition can be used together with sorafenib to further induce the death of hepatoma cell iron and overcome the drug resistance of sorafenib.

Description

Engineered exosome carrying nucleic acid type iron death inducer, and construction method and application thereof
Technical Field
The invention relates to the field of biomedicine, in particular to an engineered exosome carrying a nucleic acid type iron death inducer and a construction method and application thereof.
Background
Hepatocellular carcinoma (HCC) is one of the most common malignancies, severely threatening the health and life of the patient. Early liver cancer can be effectively treated by tumor ablation, surgical resection and other methods. However, most liver cancer patients are diagnosed at the middle and late stage and miss the optimal treatment period, and no better treatment method exists at present. Sorafenib (sorafenib) is approved by FDA to be used as a first-line therapeutic drug for late-stage liver cancer and unresectable liver cancer, can inhibit the activity of multiple kinases, further inhibit the proliferation of tumor cells, promote the apoptosis of the tumor cells and achieve the effect of inhibiting the malignant progression of tumors. Although sorafenib showed good therapeutic effect in the early stage of liver cancer treatment, sorafenib-treated tumors rarely regress completely, and most patients develop recurrent drug resistance. Therefore, combination therapy may be a better treatment option in order to improve survival time and quality of life for patients with liver cancer.
Iron death (ferroptosis) is a newly described form of programmed cell death characterized by iron-dependent accumulation of lipid peroxides to lethal levels. There is increasing evidence that iron death can be induced by inhibition of cystine/glutamate transporter (system xc-) activity, down-regulation of GPX4 or DHODH, and accumulation of Reactive Oxygen Species (ROS). Recent reports indicate that sorafenib can induce iron death by inhibiting system xc-. Furthermore, numerous studies have shown that the anticancer activity of sorafenib is mainly dependent on the induction of iron death by inhibiting the activity of system xc-. However, tumor cells can escape the iron death process by means of high expression of iron death suppressing genes (such as GPX4, DHODH) or low expression of iron death inducing genes (such as ACSL4, TFRC) and the like, and the survival and growth of the tumor cells are improved. We found, by public database analysis and in vitro cytology experiments, that two key genes that inhibit iron death, GPX4 and DHODH, were enriched in sorafenib-resistant patients and cells and correlated with poor prognosis in HCC patients receiving sorafenib treatment. Therefore, further enhancing the antitumor effect of sorafenib from the perspective of iron death may be an effective strategy to improve the therapeutic effect of sorafenib and overcome sorafenib resistance.
Iron death is one of the important ways for sorafenib to play an anti-tumor role, so that the combined use of sorafenib and an iron death inducer is an effective way for overcoming the drug resistance of sorafenib in liver cancer and improving the life cycle and the life quality of patients. Based on the important role of iron death in tumor occurrence and development and treatment drug resistance, the safe and effective iron death inducer has wide application prospect in tumor treatment. However, most of the existing iron death inhibitors have poor solubility and strong systemic toxicity, and the main reasons for the defects are that the existing iron death inducers do not have targeting property and have poor biocompatibility, so that the application of the iron death inducers in vivo is limited.
Disclosure of Invention
The invention aims to provide an engineered exosome carrying a nucleic acid type iron death inducer, and a construction method and application thereof, wherein the exosome can effectively load multi-siRNA simultaneously targeting GPX4 and DHODH, and carry the multi-siRNA into liver cancer cells to induce the iron death process of the liver cancer cells, enhance the treatment effect of sorafenib, overcome the limitation of the in-vivo application of the existing iron death inducer (poor biocompatibility and targeting property and large toxic and side effects) and enhance the sensitivity of liver cancer to sorafenib treatment.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the construction method of the engineered exosome carrying the nucleic acid type iron death inducer is characterized in that:
the method comprises the following steps:
step 1: construction of SP94-lamp2b-RRM fusion protein expression vector:
inserting CDS region sequences of SP94, lamp2b and RRM into pcDNA3.1 eukaryotic expression vector by molecular cloning method to obtain SP94-lamp2b-RRM fusion protein expression vector, and transfecting the fusion protein expression vector into 293T cell;
step 2: constructing an exosome carrying the SP94-lamp2b-RRM fusion protein:
culturing the cells transfected in the step 1, and collecting supernatant to obtain exosomes containing SP94-lamp2b-RRM fusion protein;
and step 3: construction of engineered exosomes loaded with multi-siRNA:
SP94-lamp2b-RRM fusion protein expression vector and multi-RNA containing GPX4 and DHODH siRNA and RNA binding protein binding sequence were co-transfected into 293T cells, after which culture was performed and the supernatant was collected to obtain multi-siRNA-loaded engineered exosomes.
The CDS region sequence of SP94 is:
TCTTTCTCTATTATTCATACTCCTATTCTTCCTCTT。
the CDS region sequence of the lamp2b is as follows:
ATGGTGTGCTTCCGCCTCTTCCCGGTTCCGGGCTCAGGGCTCGTTCTGGTCTGCCTAGTCCTGGGAGCTG
TGCGGTCTTATGCATTGGAACTTAATTTGACAGATTCAGAAAATGCCACTTGCCTTTATGCAAAATGGCA
GATGAATTTCACAGTACGCTATGAAACTACAAATAAAACTTATAAAACTGTAACCATTTCAGACCATGGC
ACTGTGACATATAATGGAAGCATTTGTGGGGATGATCAGAATGGTCCCAAAATAGCAGTGCAGTTCGGAC
CTGGCTTTTCCTGGATTGCGAATTTTACCAAGGCAGCATCTACTTATTCAATTGACAGCGTCTCATTTTC
CTACAACACTGGTGATAACACAACATTTCCTGATGCTGAAGATAAAGGAATTCTTACTGTTGATGAACTT
TTGGCCATCAGAATTCCATTGAATGACCTTTTTAGATGCAATAGTTTATCAACTTTGGAAAAGAATGATG
TTGTCCAACACTACTGGGATGTTCTTGTACAAGCTTTTGTCCAAAATGGCACAGTGAGCACAAATGAGTT
CCTGTGTGATAAAGACAAAACTTCAACAGTGGCACCCACCATACACACCACTGTGCCATCTCCTACTACA
ACACCTACTCCAAAGGAAAAACCAGAAGCTGGAACCTATTCAGTTAATAATGGCAATGATACTTGTCTGC
TGGCTACCATGGGGCTGCAGCTGAACATCACTCAGGATAAGGTTGCTTCAGTTATTAACATCAACCCCAA
TACAACTCACTCCACAGGCAGCTGCCGTTCTCACACTGCTCTACTTAGACTCAATAGCAGCACCATTAAG
TATCTAGACTTTGTCTTTGCTGTGAAAAATGAAAACCGATTTTATCTGAAGGAAGTGAACATCAGCATGT
ATTTGGTTAATGGCTCCGTTTTCAGCATTGCAAATAACAATCTCAGCTACTGGGATGCCCCCCTGGGAAG
TTCTTATATGTGCAACAAAGAGCAGACTGTTTCAGTGTCTGGAGCATTTCAGATAAATACCTTTGATCTA
AGGGTTCAGCCTTTCAATGTGACACAAGGAAAGTATTCTACAGCCCAAGAGTGTTCGCTGGATGATGACA
CCATTCTAATCCCAATTATAGTTGGTGCTGGTCTTTCAGGCTTGATTATCGTTATAGTGATTGCTTACGT
AATTGGCAGAAGAAAAAGTTATGCTGGATATCAGACTCTG。
the CDS region sequence of RRM is as follows:
ATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGG
ATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACG
GAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCTGCGC
TCCATGCAGGGTTTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCCAAGACCGACTCAGATATCA
TTGCCAAGATGAAAGGCACCTTCGTGGAGCGGGAC。
the sequence of the multi-RNA is:
sense(5'-3')：GGAGUAACGAAGAGAUCAACAAUGGGCCAUAAAUUCCGAAAU；
antisense(5'-3')：AUUUCGGAAUUUAUGGCCCAUUGCACUUGAUCUCUUCGUUACUCC。
the engineered exosome carrying the nucleic acid type iron death inducer obtained by the construction method.
The application of the engineered exosome in preparing anti-liver cancer drugs.
The engineered exosome can deliver multi-RNA to liver cancer cells to induce iron death of the liver cancer cells.
The application of the engineered exosome combined with sorafenib in preparing anti-liver cancer drugs.
The engineered exosome can induce the death of the iron of the hepatoma cells by cooperating with sorafenib.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a novel siRNA drug loading mode, namely an engineered exosome: the engineering exosome membrane contains RNA binding protein, and can wrap a large amount of RNA containing RNA binding protein binding site sequences into exosomes in the biosynthesis process so as to achieve the effect of drug loading.
2. The invention effectively loads multi-siRNA targeting GPX4 and DHODH simultaneously: can effectively reduce the expression of GPX4 and DHODH in cells, thereby inducing cell iron death.
3. The invention relates to a multi-siRNA containing an RNA binding protein binding sequence: the multi-siRNA can be loaded in large quantities into the engineered exosomes described above and then delivered to the target cell to function.
4. The engineered exosome carries targeting peptide SP94 targeting hepatoma carcinoma cells, and can be specifically absorbed by the hepatoma carcinoma cells without influencing the functions of other organs.
5. The engineered exosome can be used together with sorafenib to further induce iron death of hepatoma cells, so that drug resistance of sorafenib is overcome.
6. The engineered exosome also has good biocompatibility, does not show obvious toxic or side effect in a mouse body, and has good safety and wide application prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings of the embodiments can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of the structure of an engineered exosome.
FIG. 2 is a schematic of engineered exosomes delivering loaded multi-siRNA to hepatoma cells.
FIG. 3 is the results of western blot of engineered exosomes loaded with multi-siRNA inducing reduced expression of GPX4 and DHODH in hepatoma cells.
FIG. 4 is the CCK8 results for engineered exosomes loaded with multi-siRNA to increase sensitivity of hepatoma cells to sorafenib.
FIG. 5 is a schematic diagram of engineered exosomes enriched at mouse liver cancer sites.
FIG. 6 is a schematic diagram of an engineered exosome loaded multi-siRNA in combination with sorafenib to inhibit in situ liver cancer growth in mice.
Fig. 7 is a graph of the statistical results of fig. 6.
Fig. 8 is a graphical representation of the survival results of tumor-bearing mice after multi-siRNA loaded engineered exosomes in combination with sorafenib treatment.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The invention constructs an engineered exosome capable of targeting a hepatoma cell, namely an engineered exosome carrying a nucleic acid type iron death inducer, which can effectively load multi-siRNA simultaneously targeting high-expression iron death inhibition genes GPX4 and DHODH, and carry the multi-siRNA into the hepatoma cell to induce the iron death process of the hepatoma cell.
The method for constructing the engineered exosome specifically comprises the following steps:
step 1: construction of SP94-lamp2b-RRM fusion protein expression vector:
the CDS region sequences of SP94, lamp2b and RRM are inserted into pcDNA3.1 eukaryotic expression vector by molecular cloning method to obtain SP94-lamp2b-RRM fusion protein expression vector, and the fusion protein expression vector is transfected into 293T cell.
The CDS region sequence of SP94 is SEQ ID NO: 1:
TCTTTCTCTATTATTCATACTCCTATTCTTCCTCTT。
the CDS region sequence of the lamp2b is SEQ ID NO: 2:
ATGGTGTGCTTCCGCCTCTTCCCGGTTCCGGGCTCAGGGCTCGTTCTGGTCTGCCTAGTCCTGGGAGCTG
TGCGGTCTTATGCATTGGAACTTAATTTGACAGATTCAGAAAATGCCACTTGCCTTTATGCAAAATGGCA
GATGAATTTCACAGTACGCTATGAAACTACAAATAAAACTTATAAAACTGTAACCATTTCAGACCATGGC
ACTGTGACATATAATGGAAGCATTTGTGGGGATGATCAGAATGGTCCCAAAATAGCAGTGCAGTTCGGAC
CTGGCTTTTCCTGGATTGCGAATTTTACCAAGGCAGCATCTACTTATTCAATTGACAGCGTCTCATTTTC
CTACAACACTGGTGATAACACAACATTTCCTGATGCTGAAGATAAAGGAATTCTTACTGTTGATGAACTT
TTGGCCATCAGAATTCCATTGAATGACCTTTTTAGATGCAATAGTTTATCAACTTTGGAAAAGAATGATG
TTGTCCAACACTACTGGGATGTTCTTGTACAAGCTTTTGTCCAAAATGGCACAGTGAGCACAAATGAGTT
CCTGTGTGATAAAGACAAAACTTCAACAGTGGCACCCACCATACACACCACTGTGCCATCTCCTACTACA
ACACCTACTCCAAAGGAAAAACCAGAAGCTGGAACCTATTCAGTTAATAATGGCAATGATACTTGTCTGC
TGGCTACCATGGGGCTGCAGCTGAACATCACTCAGGATAAGGTTGCTTCAGTTATTAACATCAACCCCAA
TACAACTCACTCCACAGGCAGCTGCCGTTCTCACACTGCTCTACTTAGACTCAATAGCAGCACCATTAAG
TATCTAGACTTTGTCTTTGCTGTGAAAAATGAAAACCGATTTTATCTGAAGGAAGTGAACATCAGCATGT
ATTTGGTTAATGGCTCCGTTTTCAGCATTGCAAATAACAATCTCAGCTACTGGGATGCCCCCCTGGGAAG
TTCTTATATGTGCAACAAAGAGCAGACTGTTTCAGTGTCTGGAGCATTTCAGATAAATACCTTTGATCTA
AGGGTTCAGCCTTTCAATGTGACACAAGGAAAGTATTCTACAGCCCAAGAGTGTTCGCTGGATGATGACA
CCATTCTAATCCCAATTATAGTTGGTGCTGGTCTTTCAGGCTTGATTATCGTTATAGTGATTGCTTACGT
AATTGGCAGAAGAAAAAGTTATGCTGGATATCAGACTCTG。
the CDS region sequence of the RRM (the domain responsible for RNA binding in the RNA binding protein SNRPA) is SEQ ID NO: 3:
ATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGG
ATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACG
GAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCTGCGC
TCCATGCAGGGTTTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCCAAGACCGACTCAGATATCA
TTGCCAAGATGAAAGGCACCTTCGTGGAGCGGGAC。
step 2: constructing an exosome carrying the SP94-lamp2b-RRM fusion protein:
culturing the cells transfected in the step 1 by using a culture medium without exosome serum, and collecting supernatant to obtain exosomes with SP94-lamp2b-RRM fusion protein;
and step 3: construction of engineered exosomes loaded with multi-siRNA:
SP94-lamp2b-RRM fusion protein expression vector and multi-RNA containing GPX4 and DHODH siRNA and RNA binding protein binding sequence were co-transfected into cells, and then cultured using a medium without exosome serum, and the supernatant was collected to obtain engineered exosomes loaded with multi-siRNA.
The sequence of the multi-RNA is:
sense(5'-3')：GGAGUAACGAAGAGAUCAACAAUGGGCCAUAAAUUCCGAAAU(SEQ ID NO：4)；
antisense(5'-3')：AUUUCGGAAUUUAUGGCCCAUUGCACUUGAUCUCUUCGUUACUCC(SEQ ID NO：5)。
in the above steps, first, the fusion protein expression vector is transfected into cells, and it is confirmed that corresponding exosomes containing the fusion protein can be obtained (step 2); the fusion protein expression vector and the multi-siRNA are then co-transfected into the cell (step 3), the purpose of which is to load the multi-siRNA into the exosomes.
The engineered exosome carrying the nucleic acid type iron death inducer is obtained by the construction method, the nucleic acid type iron death inducer specifically refers to multi-siRNA capable of simultaneously targeting GPX4 and DHODH, both GPX4 and DHODH are key molecules for inhibiting iron death in tumor cells, and reduction of expression of the two molecules can effectively induce cell iron death. The engineered exosome carries targeting peptide SP94 targeting a liver cancer cell, is loaded with GPX4 and DHODH siRNA, contains RNA binding protein, can be used for treating liver cancer, can deliver multi-RNA to the liver cancer cell, effectively reduces the expression of GPX4 and DHODH in the cell, and induces the death of the liver cancer cell iron; the engineered exosome can be used together with sorafenib, can cooperate with sorafenib to induce iron death of liver cancer cells, can further enhance the treatment effect of sorafenib, and overcomes drug resistance of the liver cancer cells to sorafenib.
Example 1: co-culturing the engineered exosome and the liver cancer cell:
as shown in fig. 2, after co-culturing the obtained engineered exosomes with hepatoma cells, the engineered exosomes were found to be able to deliver the loaded multi-siRNA to hepatoma cells using the confocal experiment.
Example 2: synergistic combination of engineered exosomes with sorafenib:
the engineered exosome and sorafenib-resistant HepG2 cells are co-cultured, real time PCR, western blot, clone formation experiment, CCK8 experiment and the like are used for detecting the expression condition and cell activity of GPX4 and DHODH in the cells, and the results are shown in fig. 3 and fig. 4, the expression level of GPX4 and DHODH in the cells treated by the engineered exosome carrying multi-siRNA is obviously reduced, and the sensitivity of the cells to sorafenib is enhanced.
Example 3: enrichment of engineered exosomes in model mouse liver cancer sites:
the engineered exosomes are dyed by using DIR dye, then the engineered exosomes are injected into a liver cancer model mouse through caudal veins according to the amount of 100 mug (protein level) of each mouse, the distribution condition of the exosomes in the mouse is detected by using a small animal living body imaging technology, and as can be seen from figure 5, the engineered exosomes with the targeting polypeptide sequence can be enriched in the liver cancer part of the mouse.
Example 4: the engineered exosomes and sorafenib were combined to act on model mice:
the sorafenib and the engineered exosomes are used for carrying out combined treatment on the sorafenib-resistant in-situ liver cancer mouse, the growth condition of tumors in the mouse is detected by a small animal living body imaging technology, and the result shows that the sorafenib cannot effectively inhibit the tumor development of the model mouse, but the tumor of the mouse in the sorafenib and engineered exosomes combined group is remarkably reduced, and the life cycle is remarkably prolonged, as shown in fig. 6, 7 and 8.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
SEQUENCE LISTING
<110> China people liberation military and military medical university
<120> engineered exosome carrying nucleic acid type iron death inducer, and construction method and application thereof
<130> 20211015
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 36
<212> DNA
<213> CDS
<400> 1
tctttctcta ttattcatac tcctattctt cctctt 36
<210> 2
<211> 1230
<212> DNA
<213> CDS
<400> 2
atggtgtgct tccgcctctt cccggttccg ggctcagggc tcgttctggt ctgcctagtc 60
ctgggagctg tgcggtctta tgcattggaa cttaatttga cagattcaga aaatgccact 120
tgcctttatg caaaatggca gatgaatttc acagtacgct atgaaactac aaataaaact 180
tataaaactg taaccatttc agaccatggc actgtgacat ataatggaag catttgtggg 240
gatgatcaga atggtcccaa aatagcagtg cagttcggac ctggcttttc ctggattgcg 300
aattttacca aggcagcatc tacttattca attgacagcg tctcattttc ctacaacact 360
ggtgataaca caacatttcc tgatgctgaa gataaaggaa ttcttactgt tgatgaactt 420
ttggccatca gaattccatt gaatgacctt tttagatgca atagtttatc aactttggaa 480
aagaatgatg ttgtccaaca ctactgggat gttcttgtac aagcttttgt ccaaaatggc 540
acagtgagca caaatgagtt cctgtgtgat aaagacaaaa cttcaacagt ggcacccacc 600
atacacacca ctgtgccatc tcctactaca acacctactc caaaggaaaa accagaagct 660
ggaacctatt cagttaataa tggcaatgat acttgtctgc tggctaccat ggggctgcag 720
ctgaacatca ctcaggataa ggttgcttca gttattaaca tcaaccccaa tacaactcac 780
tccacaggca gctgccgttc tcacactgct ctacttagac tcaatagcag caccattaag 840
tatctagact ttgtctttgc tgtgaaaaat gaaaaccgat tttatctgaa ggaagtgaac 900
atcagcatgt atttggttaa tggctccgtt ttcagcattg caaataacaa tctcagctac 960
tgggatgccc ccctgggaag ttcttatatg tgcaacaaag agcagactgt ttcagtgtct 1020
ggagcatttc agataaatac ctttgatcta agggttcagc ctttcaatgt gacacaagga 1080
aagtattcta cagcccaaga gtgttcgctg gatgatgaca ccattctaat cccaattata 1140
gttggtgctg gtctttcagg cttgattatc gttatagtga ttgcttacgt aattggcaga 1200
agaaaaagtt atgctggata tcagactctg 1230
<210> 3
<211> 315
<212> DNA
<213> CDS
<400> 3
atggcagttc ccgagacccg ccctaaccac actatttata tcaacaacct caatgagaag 60
atcaagaagg atgagctaaa aaagtccctg tacgccatct tctcccagtt tggccagatc 120
ctggatatcc tggtatcacg gagcctgaag atgaggggcc aggcctttgt catcttcaag 180
gaggtcagca gcgccaccaa cgccctgcgc tccatgcagg gtttcccttt ctatgacaaa 240
cctatgcgta tccagtatgc caagaccgac tcagatatca ttgccaagat gaaaggcacc 300
ttcgtggagc gggac 315
<210> 4
<211> 42
<212> RNA
<213> Artificial Synthesis
<400> 4
ggaguaacga agagaucaac aaugggccau aaauuccgaa au 42
<210> 5
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<212> RNA
<213> Artificial Synthesis
<400> 5
auuucggaau uuauggccca uugcacuuga ucucuucguu acucc 45

Claims (10)

1. The construction method of the engineered exosome carrying the nucleic acid type iron death inducer is characterized in that:
the method comprises the following steps:
step 1: construction of SP94-lamp2b-RRM fusion protein expression vector:
inserting CDS region sequences of SP94, lamp2b and RRM into pcDNA3.1 eukaryotic expression vector by molecular cloning method to obtain SP94-lamp2b-RRM fusion protein expression vector, and transfecting the fusion protein expression vector into 293T cell;
step 2: constructing an exosome carrying the SP94-lamp2b-RRM fusion protein:
culturing the cells transfected in the step 1, and collecting supernatant to obtain exosomes containing SP94-lamp2b-RRM fusion protein;
and step 3: construction of engineered exosomes loaded with multi-siRNA:
SP94-lamp2b-RRM fusion protein expression vector and multi-RNA containing GPX4 and DHODH siRNA and RNA binding protein binding sequence were co-transfected into 293T cells, after which culture was performed and the supernatant was collected to obtain multi-siRNA-loaded engineered exosomes.
2. The construction method according to claim 1, characterized in that:
the CDS region sequence of SP94 is:
TCTTTCTCTATTATTCATACTCCTATTCTTCCTCTT。
3. the construction method according to claim 2, wherein:
the CDS region sequence of the lamp2b is as follows:
ATGGTGTGCTTCCGCCTCTTCCCGGTTCCGGGCTCAGGGCTCGTTCTGGTCTGCCTAGTCCTGGGAGCTG
TGCGGTCTTATGCATTGGAACTTAATTTGACAGATTCAGAAAATGCCACTTGCCTTTATGCAAAATGGCA
GATGAATTTCACAGTACGCTATGAAACTACAAATAAAACTTATAAAACTGTAACCATTTCAGACCATGGC
ACTGTGACATATAATGGAAGCATTTGTGGGGATGATCAGAATGGTCCCAAAATAGCAGTGCAGTTCGGAC
CTGGCTTTTCCTGGATTGCGAATTTTACCAAGGCAGCATCTACTTATTCAATTGACAGCGTCTCATTTTC
CTACAACACTGGTGATAACACAACATTTCCTGATGCTGAAGATAAAGGAATTCTTACTGTTGATGAACTT
TTGGCCATCAGAATTCCATTGAATGACCTTTTTAGATGCAATAGTTTATCAACTTTGGAAAAGAATGATG
TTGTCCAACACTACTGGGATGTTCTTGTACAAGCTTTTGTCCAAAATGGCACAGTGAGCACAAATGAGTT
CCTGTGTGATAAAGACAAAACTTCAACAGTGGCACCCACCATACACACCACTGTGCCATCTCCTACTACA
ACACCTACTCCAAAGGAAAAACCAGAAGCTGGAACCTATTCAGTTAATAATGGCAATGATACTTGTCTGC
TGGCTACCATGGGGCTGCAGCTGAACATCACTCAGGATAAGGTTGCTTCAGTTATTAACATCAACCCCAA
TACAACTCACTCCACAGGCAGCTGCCGTTCTCACACTGCTCTACTTAGACTCAATAGCAGCACCATTAAG
TATCTAGACTTTGTCTTTGCTGTGAAAAATGAAAACCGATTTTATCTGAAGGAAGTGAACATCAGCATGT
ATTTGGTTAATGGCTCCGTTTTCAGCATTGCAAATAACAATCTCAGCTACTGGGATGCCCCCCTGGGAAG
TTCTTATATGTGCAACAAAGAGCAGACTGTTTCAGTGTCTGGAGCATTTCAGATAAATACCTTTGATCTA
AGGGTTCAGCCTTTCAATGTGACACAAGGAAAGTATTCTACAGCCCAAGAGTGTTCGCTGGATGATGACA
CCATTCTAATCCCAATTATAGTTGGTGCTGGTCTTTCAGGCTTGATTATCGTTATAGTGATTGCTTACGT
AATTGGCAGAAGAAAAAGTTATGCTGGATATCAGACTCTG。
4. the construction method according to claim 3, wherein:
the CDS region sequence of RRM is as follows:
ATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGG
ATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACG
GAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCTGCGC
TCCATGCAGGGTTTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCCAAGACCGACTCAGATATCA
TTGCCAAGATGAAAGGCACCTTCGTGGAGCGGGAC。
5. the construction method according to claim 4, wherein:
the sequence of the multi-RNA is:
sense(5'-3')：GGAGUAACGAAGAGAUCAACAAUGGGCCAUAAAUUCCGAAAU；
antisense(5'-3')：AUUUCGGAAUUUAUGGCCCAUUGCACUUGAUCUCUUCGUUACUCC。
6. the engineered exosome carrying the nucleic acid-type iron death inducer obtained by the construction method according to claim 5.
7. The use of the engineered exosome of claim 6 in the preparation of an anti-liver cancer drug.
8. Use according to claim 7, characterized in that:
the engineered exosome can deliver multi-RNA to liver cancer cells to induce iron death of the liver cancer cells.
9. The use of the engineered exosome of claim 6 in combination with sorafenib in the preparation of an anti-liver cancer medicament.
10. Use according to claim 9, characterized in that:
the engineered exosome can induce the death of the iron of the hepatoma cells by cooperating with sorafenib.
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US20190263824A1 (en) * 2016-11-14 2019-08-29 Virginia Commonwealth University Inhibitors of cancer invasion, attachment, and/or metastasis
CN107890567A (en) * 2017-10-20 2018-04-10 南方医科大学南方医院 CDO1 is being prepared and the application in the dead related curing gastric cancer medicine of iron
CN111826443A (en) * 2020-07-03 2020-10-27 清华大学深圳国际研究生院 Application of serum exosome micro RNAs and liver cancer detection kit
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