CN116463340A - shRNA (short hairpin ribonucleic acid) of targeted annular RNA, lentivirus expression vector, construction method and application thereof - Google Patents
shRNA (short hairpin ribonucleic acid) of targeted annular RNA, lentivirus expression vector, construction method and application thereof Download PDFInfo
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- CN116463340A CN116463340A CN202211178593.1A CN202211178593A CN116463340A CN 116463340 A CN116463340 A CN 116463340A CN 202211178593 A CN202211178593 A CN 202211178593A CN 116463340 A CN116463340 A CN 116463340A
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- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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Abstract
The invention discloses shRNA for targeting circular RNA, which is characterized in that the circular RNA is circular RNA hsa_circ_0005571, and the shRNA comprises a sense strand, an antisense strand, a stem-loop structure and a termination signal. The invention also discloses a lentiviral expression vector of the shRNA and a construction method thereof. The shRNA of the targeting annular RNA hsa_circ_0005571 can be used for down regulating the expression of hsa_circ_0005571 by transfecting the shRNA, so that the activity of cancer cells is obviously inhibited, and a foundation is provided for clinical treatment and scientific research of the cervical cancer related circRNA.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to shRNA (short hairpin ribonucleic acid) targeting annular RNA, a lentivirus expression vector, a construction method and application thereof.
Background
Cervical cancer has become an important public health problem as one of the most common gynaecological malignancies. In recent years, along with the use of Human Papillomavirus (HPV) vaccines and the popularization of cervical cancer screening, the incidence of cervical cancer is obviously reduced in western developed countries, and the incidence of cervical cancer in China is in an increasing trend. According to the current latest incidence report of cervical cancer, the number of worldwide latest incidence cases is about 60 ten thousand per year, and the number of death cases is about 34 ten thousand per year. The number of new cases per year in China is approximately 11 ten thousand, the death number is approximately 5.9 ten thousand, the incidence rate of cervical cancer in China is approximately 1/5 of the global, and the cervical cancer is still a disease seriously endangering the health of women. Cervical treatment still presents a significant challenge while enhancing cervical cancer prevention. At present, surgery and chemoradiotherapy are main treatment means of cervical cancer, but the curative effect of most patients with advanced cervical cancer is not ideal, the prognosis of advanced recurrent cervical cancer is poor, and an effective treatment method is still lacking. Therefore, further research into the pathogenesis of cervical cancer is urgent, and exploration of new methods for treating cervical cancer is urgent.
Circular RNA (circularRNAs, circRNAs) is a single stranded closed circular RNA molecule formed by reverse splicing of a pre-mRNA. The research at present proves that various circRNAs are differentially expressed in cervical cancer cells, and widely participate in tumor cell growth, invasion, migration, metastasis and the like by competing for binding microRNAs (miRNAs) to exert sponge adsorption, interacting with RNA binding proteins, even translating functional peptides and the like. However, in general, research on the gircRNAs in cervical cancer is still in a starting stage, the detailed molecular action mechanism of the gircRNAs is not yet fully elucidated, and a large research space exists on the aspects of whether the gircRNAs can be used as a cervical cancer treatment target and the like.
The circular RNA hsa_circ_0005571, which is located on human chromosome 19 (18285849-18286507), originates from exons 1-3 of its linear parent gene IFI 30. There is currently little report on hsa_circ_0005571, and only one study suggests that hsa_circ_0005571 may be involved in regulating malignant biological processes such as proliferation, invasion, migration, and apoptosis of triple negative breast cancer cells (PMID: 35378000). However, there is no research report on the function of hsa_circ_0005571 in cervical cancer, and it is not known whether there is a correlation with the occurrence and development of cervical cancer.
Disclosure of Invention
Aiming at the problems, the invention aims to provide shRNA of targeted annular RNA hsa_circ_0005571 and application thereof, wherein the annular RNA is related to the proliferation capacity of cervical cancer cells, and shRNA designed for the shRNA can be applied to preparation of anti-cervical cancer drugs.
The aim and the technical problems of the invention are realized by adopting the following technical proposal.
One aspect of the present invention provides a shRNA targeting a circular RNA, the circular RNA being circular RNA hsa_circ_0005571, the shRNA comprising a sense strand, an antisense strand, a stem-loop structure, and a termination signal;
the sense strand has a base sequence shown in a sequence table SEQ.ID.No.2, and the antisense strand has a base sequence shown in a sequence table SEQ.ID.No.3;
the base sequence of the stem-loop structure is CTCGAG;
the base sequence of the termination signal is TTTTT or TTTTTTT.
In another aspect, the present invention also provides a lentiviral expression vector for shRNA targeting a circular RNA, wherein the vector is: pLKO.1-puro-hsa_circ_0005571-shRNA lentiviral vector.
In still another aspect, the present invention provides a method for constructing a lentiviral expression vector targeting shRNA of a circular RNA, the lentiviral expression vector being obtained by cloning the synthesized shRNA into a lentiviral expression vector plko.1-puro, the method comprising the steps of:
(1) Designing shRNA targets: the nucleotide sequence of the nucleotide sequence is shown as a sequence table SEQ.ID.No.1;
(2) DNA oligo sequence synthesis: designing an shRNA interference sequence according to the shRNA target, wherein the sense strand of the shRNA interference sequence has a base sequence shown in a sequence table SEQ.ID.No.2, and the antisense strand has a base sequence shown in a sequence table SEQ.ID.No.3;
(3) Annealing of DNA oligo sequence: uniformly mixing a sense strand and an antisense strand, adding an annealing buffer solution for annealing to form an shRNA template, and then diluting for later use;
(4) Linearizing a carrier: carrying out double enzyme digestion reaction on a lentiviral expression vector pLKO.1-puro at 37 ℃ for 3 hours, carrying out electrophoresis on a product, recovering linearization pLKO.1-puro, and diluting for later use;
(5) The target fragment is connected with a carrier: connecting the obtained linearization pLKO.1-puro with the obtained shRNA template at 16 ℃ overnight to obtain a connecting product;
(6) PCR identification and sequencing of transformed and positive clones: adding the connection product into competent cells of Escherichia coli, ice-bathing for 30min, and heat-shocking at 42 ℃ for 90s, and ice-bathing for 2min; adding an LB liquid culture medium without antibiotics for culture; uniformly smearing bacterial liquid on LB solid culture medium containing Amp, culturing overnight in an incubator at 37 ℃, selecting single colony the next day, carrying out bacterial liquid PCR identification, selecting positive clone for sequencing, and identifying the positive clone as the successfully constructed pLKO.1-puro-hsa_circ_0005571-shRNA lentiviral vector.
Further, the shRNA interfering sequence in step (2) further comprises a stem-loop structure with a CTCGAG as a base sequence, and a termination signal with TTTTT or TTTTTT as a base sequence.
Further, the concentration ratio of the sense strand to the antisense strand in step (3) is 1:1.
Further, the annealing procedure in step (3) is: 95 ℃ for 30s;72 ℃ for 2min;37 ℃ for 2min;25 ℃ for 2min.
Further, the enzyme used in the double cleavage reaction in the step (4) is AgeI/EcoRI, and the cleavage system is:
further, the connection system in step (5) is:
the invention also provides an application of shRNA of the targeted annular RNA in preparing anti-cervical cancer drugs.
The invention also provides an application of the shRNA lentiviral expression vector for targeting the annular RNA in preparing an anti-cervical cancer drug.
Compared with the prior art, the invention has obvious advantages and beneficial effects: the shRNA of the targeted annular RNA hsa_circ_0005571 disclosed by the invention can be used for down-regulating the expression of hsa_circ_0005571 by transfecting the shRNA, so that the activity of cancer cells is obviously inhibited, and a foundation is provided for clinical treatment and scientific research of the cervical cancer related circRNA.
The foregoing description is only an overview of the present invention, and is presented in terms of preferred embodiments of the present invention and detailed description below in conjunction with the accompanying drawings, in order to provide a more clear understanding of the technical means of the present invention.
Drawings
FIG. 1 shows the effect of lentiviral-mediated shRNA on hsa_circ_0005571 gene inhibition in cervical cancer cells;
FIG. 2 shows the effect of detecting the interference hsa_circ_0005571 on cervical cancer cell proliferation using CCK 8;
FIG. 3 is a graph showing the effect of the interference hsa_circ_0005571 on the tumor stem of cervical cancer cells.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description is presented by way of example only and is not intended to limit the invention.
Construction of shRNA lentiviral expression vector of example 1 hsa_circ_0005571
1. shRNA target design
hsa_circ_0005571 belongs to the exon circularization circRNA, and an interference target sequence is designed for the sequence before and after the cleavage site (backsplice junction). Through online design of websites: after https:// circinteractive. Nia. Nih. Gov/index. Html evaluation determination, the following sequences were selected as interference targets: GCCACTAACAGGCAATCTATA (SEQ ID NO: 1).
2. DNA oligo sequence Synthesis
The synthesized DNA oligo contains shRNA interference sequence, stem-loop structure and termination signal. shRNA interfering sequences comprise a sense strand and an antisense strand.
And designing a shRNA interference sequence according to the selected target sequence, and adding proper restriction enzyme cutting sites at two ends to complete the construction of the vector. In addition, TTTTT or TTTTTT transcription termination signals are added at the 3 '-end of the sense strand, and termination signal complementary sequences are added at the 5' -end of the sense strand; the stem-loop sequence (CTCGAG) is located in the center of the oligonucleotide linking the two oligonucleotides.
Meanwhile, a missense sequence was designed as a control group, and a single-stranded DNA oligo was synthesized by Souzhou Jin Weizhi Biotechnology Co.
The shRNA interference sequence is as follows:
hsa_circ_0005571-shRNA sense strand:
CCGGGCCACTAACAGGCAATCTATACTCGAGTATAGATTGCCTGTTAGTGGCTTTTTG(SEQ ID NO:2)；
hsa_circ_0005571-shRNA antisense strand:
AATTCAAAAAGCCACTAACAGGCAATCTATACTCGAGTATAGATTGCCTGTTAGTGGC(SEQ ID NO:3)；
the control missense sequences are as follows:
NC-shRNA sense strand (SEQ ID NO: 4):
CCGGGATGCTAGCTGCAACTGTCTACTCGAGTAGACAGTTGCAGCTAGCATCTTTTTG
NC-shRNA antisense strand (SEQ ID NO: 5):
AATTCAAAAAGATGCTAGCTGCAACTGTCTACTCGAGTAGACAGTTGCAGCTAGCATC
3. construction of shRNA lentiviral expression vector of hsa_circ_0005571
(1) Annealing of DNA oligo sequence
Mixing sense strand (100. Mu.M) and antisense strand (100. Mu.M) DNA oligo1:1 uniformly to make its final concentration be 50. Mu.M, adding annealing buffer (10 mM Tris, pH 7.5-8.0,50mM NaCl,1mM EDTA), annealing on PCR instrument (annealing procedure: 95 ℃,30s;72 ℃,2min;37 ℃,2min;25 ℃,2 min) to form DNA double strand, namely shRNA template. The annealed shRNA template was diluted with RNase-free water to a final concentration of 200nM for the next ligation reaction.
(2) Carrier linearization
The lentiviral expression vector pLKO.1-puro was subjected to double digestion with AgeI/EcoRI at 37℃for 3 hours, the vector was linearized, and the digested vector was subjected to 1% agarose gel electrophoresis, and the desired fragment was recovered and diluted to 50 ng/. Mu.L with RNase-free water. The cleavage system is shown in Table 1 below:
TABLE 1 enzyme digestion system
| Component (A) | Dosage of |
| Vector | 4μg |
| AgeI | 1μL |
| EcoRI | 1μL |
| Buffer(10×) | 5μL |
| ddH 2 O | Make up to 50 mu L |
(3) Ligation of the fragment of interest with the vector
The linearized plko.1-puro obtained in the above step was ligated to the obtained shRNA template, and ligation was performed overnight at 16 ℃ using T4 ligase, followed by transformation experiments. The connection system is shown in table 2 below:
table 2 connection system
(4) PCR identification and sequencing of transformed and positive clones
Adding 5 μl of the ligation product into 50 μl of E.coli competent cells, ice-bathing for 30min, heat-shocking at 42deg.C for 90s, and ice-bathing for 2min; adding 500 mu L of LB liquid medium without antibiotics, and shake culturing at 200rpm at 37 ℃ for 1h; the bacterial liquid is evenly smeared on LB solid medium containing Amp, and cultured overnight in a 37 ℃ incubator. The next day, single colonies are selected, bacterial liquid PCR identification is carried out, positive clones are selected for sequencing (Jin Weizhi Biotechnology Co., st.) and are subjected to sequencing comparison, and the identified positive clones are the successfully constructed pLKO.1-puro-hsa_circ_0005571-shRNA lentiviral vector.
(5) Lentivirus packaging and titer determination
HEK-293T cells (Wohpnuosai life technologies Co., ltd.) are transfected together with the extracted recombinant plasmid pLKO.1-puro-hsa_circ_0005571-shRNA, helper plasmid psPAX2 and PMD2.G (Biovector plasmid vector cell Gene preservation center), virus supernatants are collected at 48h and 72h respectively, cell morphology and green fluorescent protein GFP expression are observed, the extracted viruses are purified and concentrated, virus titer is measured by a gradient dilution method, and the prepared virus concentrate is packaged at-80 ℃ for preservation.
Acquisition of the Scramble virus: the pLKO.1-puro plasmid containing the missense sequence, the auxiliary plasmid psPAX2 and the PMD2.G are transfected into HEK-293T cells together, virus supernatants are collected respectively for 48 hours and 72 hours, the cell morphology and green fluorescent protein GFP expression are observed, the extracted viruses are purified and concentrated, the virus titer is measured by a gradient dilution method, and the prepared virus concentrate is split-packed at the temperature of minus 80 ℃ for preservation.
Example 2 hsa_circ_0005571-shRNA lentivirus infection of human cervical cancer cells and establishment of stable Transit
1. Taking Siha and Hela cells in logarithmic growth phase, and adjusting cell concentration to 2×10 5 Well seeded in 6-well plate;
2. the next day, NC-shRNA and hsa_circ_0005571-shRNA virus solution are respectively added into DEME high sugar culture medium (the components and the content are that L-glutamic acid is 4mM, naHCO) 3 3700mg/L, 4500mg/L of D-glucose, 1mM of sodium pyruvate, 15mg/L of phenol red indicator and pH of 7.2-7.4), 8 mug of transfection enhancing reagent polybrene is added into each hole, and the mixture is uniformly mixed by shaking;
3. the DEME high sugar culture medium containing the virus diluent (the components and the content are that L-glutamic acid is 4mM and NaHCO) is replaced after culturing for 12 hours 3 3700mg/L, 4500mg/L of D-glucose, 1mM of sodium pyruvate, 15mg/L of phenol red indicator and pH of 7.2-7.4), and the same method is repeated for 2 times; fluorescence and infection efficiency were observed under a microscope. If the fluorescence efficiency is more than 80%, the cell state is good, and the cell fusion degree is more than 80%, the infection is successful;
4. thereafter, DEME high sugar culture medium (component and content: L-glutamic acid 4mM, naHCO) containing puromycin (concentration 1. Mu.g/mL) was used 3 3700mg/L, 4500mg/L of D-glucose, 1mM of sodium pyruvate, 15mg/L of phenol red indicator, pH 7.2-7.4). The culture solution is replaced every 72 hours, and stable transfer cell lines can be obtained 3 times continuously.
EXAMPLE 3 hsaVerification of interference efficiency of circ 0005571-shRNA lentivirus
1. Experimental grouping: infecting an NC-shRNA group and infecting an hsa_circ_0005571-shRNA group;
2. collecting each group of cells, and extracting total RNA of the cells according to the instruction of a total RNA extraction kit (purchased from Shanghai Fei Biotechnology Co., ltd.);
3. cDNA reverse transcription was performed using the HiFiScript cDNA first Strand Synthesis kit (purchased from Jiangsu kang, century Biotech Co., ltd.) and experimental procedures were performed according to the product instructions. The obtained cDNA is stored in a refrigerator at the temperature of minus 20 ℃ for standby;
4. Real-Time PCR amplification detection, the procedure is: the mixture was pre-denatured at 95℃for 10min, then denatured at 95℃for 15s, annealed at 60℃for 30s, and extended at 72℃for 30s, and the total was subjected to 38 cycles. The ABI 7500 fluorescent quantitative PCR instrument selects a melting curve program, and continuously collects sample fluorescent signals during the ramp up process to obtain a melting curve. Real-Time PCR Using 2 -△△Ct The method is used for relative quantitative analysis. The primer sequences are shown in Table 3:
TABLE 3 specific amplification primer sequences and related information
FIG. 1 shows the effect of lentiviral-mediated shRNA on hsa_circ_0005571 gene inhibition in cervical cancer cells. As shown in FIG. 1, the hsa_circ_0005571-shRNA of the experimental group significantly reduced the expression level of hsa_circ_0005571 compared to the NC-shRNA group.
Example 4 hsa_circ_0005571-shRNA inhibits proliferation of cervical cancer cells
1. Experimental grouping: infecting an NC-shRNA group and infecting an hsa_circ_0005571-shRNA group;
2. taking the above groups of logarithmic phase cells, adjusting cell concentration to 3×10 3 Well inoculated in 96-well plates, 3 complex wells per group;
3. the effect of inhibiting hsa_circ_0005571 expression on cell proliferation was examined using CCK-8 experiments for 1-4 days in culture.
FIG. 2 shows the effect of the detection of the interference hsa_circ_0005571 on cervical cancer cell proliferation using CCK 8. The ability of the cervical cancer cell line to proliferate was inhibited by interfering with hsa_circ_0005571 expression as shown in FIG. 2.
Example 5 hsa_circ_0005571-shRNA inhibits tumor Stem of cervical cancer cells
1. Experimental grouping: infecting an NC-shRNA group and infecting an hsa_circ_0005571-shRNA group;
2. taking the above groups of logarithmic phase cells, adjusting cell concentration to 5×10 2 Inoculating the wells into 6-well plates, wherein each group comprises 3 compound wells;
3. the culture was continued for 10-14 days, and the effect of hsa_circ_0005571 on tumor dryness was examined using a plate cloning experiment.
FIG. 3 is a graph showing the effect of the interference hsa_circ_0005571 on the tumor stem of cervical cancer cells. The interference of hsa_circ_0005571 expression as shown in FIG. 3 inhibits the clonogenic capacity of cervical cancer cell lines, i.e. tumor stem.
In conclusion, the shRNA of the targeting annular RNA hsa_circ_0005571 can be used for down-regulating the expression of hsa_circ_0005571 by transfecting the shRNA, so that the activity of cancer cells is obviously inhibited, and a foundation is provided for clinical treatment and scientific research of the cervical cancer related circRNA. Therefore, the invention can provide the application of shRNA of the targeted annular RNA in preparing the anti-cervical cancer drugs, and the application of the slow virus expression vector of shRNA of the targeted annular RNA in preparing the anti-cervical cancer drugs.
While the invention has been described with respect to preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and that any such changes and modifications as described in the above embodiments are intended to be within the scope of the invention.
Claims (10)
1. A shRNA targeting a circular RNA, wherein the circular RNA is circular RNA hsa_circ_0005571, and the shRNA comprises a sense strand, an antisense strand, a stem-loop structure, and a termination signal;
the sense strand has a base sequence shown in a sequence table SEQ.ID.No.2, and the antisense strand has a base sequence shown in a sequence table SEQ.ID.No.3;
the base sequence of the stem-loop structure is CTCGAG;
the base sequence of the termination signal is TTTTT or TTTTTTT.
2. The lentiviral expression vector of shRNA targeting a circular RNA of claim 1, wherein the vector is: pLKO.1-puro-hsa_circ_0005571-shRNA lentiviral vector.
3. The method for constructing a lentiviral expression vector of shRNA targeting a circular RNA according to claim 2, wherein the lentiviral expression vector is obtained by cloning the synthesized shRNA into a lentiviral expression vector pLKO.1-puro, the method comprising the steps of:
(1) Designing shRNA targets: the nucleotide sequence of the nucleotide sequence is shown as a sequence table SEQ.ID.No.1;
(2) DNA oligo sequence synthesis: designing an shRNA interference sequence according to the shRNA target, wherein the sense strand of the shRNA interference sequence has a base sequence shown in a sequence table SEQ.ID.No.2, and the antisense strand has a base sequence shown in a sequence table SEQ.ID.No.3;
(3) Annealing of DNA oligo sequence: uniformly mixing a sense strand and an antisense strand, adding an annealing buffer solution for annealing to form an shRNA template, and then diluting for later use;
(4) Linearizing a carrier: carrying out double enzyme digestion reaction on a lentiviral expression vector pLKO.1-puro at 37 ℃ for 3 hours, carrying out electrophoresis on a product, recovering linearization pLKO.1-puro, and diluting for later use;
(5) The target fragment is connected with a carrier: connecting the obtained linearization pLKO.1-puro with the obtained shRNA template at 16 ℃ overnight to obtain a connecting product;
(6) PCR identification and sequencing of transformed and positive clones: adding the connection product into competent cells of Escherichia coli, ice-bathing for 30min, and heat-shocking at 42 ℃ for 90s, and ice-bathing for 2min; adding an LB liquid culture medium without antibiotics for culture; uniformly smearing bacterial liquid on LB solid culture medium containing Amp, culturing overnight in an incubator at 37 ℃, selecting single colony the next day, carrying out bacterial liquid PCR identification, selecting positive clone for sequencing, and identifying the positive clone as the successfully constructed pLKO.1-puro-hsa_circ_0005571-shRNA lentiviral vector.
4. The method of claim 3, wherein the shRNA interfering sequence in step (2) further comprises a stem-loop structure with CTCGAG as a base sequence and a termination signal with TTTTT or TTTTTT as a base sequence.
5. The method of constructing a lentiviral expression vector for targeting shRNA of circular RNA according to claim 3, wherein the concentration ratio of sense strand to antisense strand in step (3) is 1:1.
6. A method of constructing a lentiviral expression vector for targeting shRNA of circular RNA according to claim 3, wherein the annealing procedure in step (3) is: 95 ℃ for 30s;72 ℃ for 2min;37 ℃ for 2min;25 ℃ for 2min.
7. A method of constructing a lentiviral expression vector for targeting shRNA of circular RNA according to claim 3, wherein the enzyme used in the double cleavage reaction in step (4) is agoi/EcoRI, the cleavage system is:
ddH 2 o was made up to 50. Mu.L.
8. A method of constructing a lentiviral expression vector for targeting shRNA of circular RNA according to claim 3, wherein the ligation system in step (5) is:
ddH 2 o was made up to 15. Mu.L.
9. The use of shRNA targeting a circular RNA according to claim 1 in the preparation of an anti-cervical cancer medicament.
10. The use of a lentiviral expression vector of shRNA targeting a circular RNA according to claim 2 in the preparation of an anti-cervical cancer medicament.
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