Engineering HEK293T cell line by lentivirus to produce miR34a-loaded exosomes

RNA (ribonucleic acid) antisense is developing as a possible treatment option. As an RNA, miR-34a is involved in P53 function and cancer cell apoptosis. Although the therapeutic applications of miRNAs have several limitations, such as structural instability and susceptibility to nucleases. To resolve these issues, this study aims to apply exosomes as a delivery vehicle for miR-34a. This study aims to create a cell factory to generate miR34a-enriched exosomes. The produced nanoparticles act as a delivery system and improve the structural stability of miR34a. First exosome specific sequences were inserted into miR34a. The resulting miR34a oligonucleotide was transduced HEK293T cells genome with a lentiviral system. In the structure of miR34a oligonucleotide, six nucleotides were substituted to increase its packaging rate into exosomes. To maintain the secondary structure, stability, and expression of the miRNA gene, changes to the miR34a oligonucleotide were made using PCR (polymerase chain reaction) Extension. The forward-34a (5-TGGGGAGAGGCAGGACAGG-3) and Reverse-34a primers (5-TCCGAAGTCCTGGCGTCTCC-3) were used for amplification of the miR34a gene from DNA. The results confirmed that the changes in miR34a oligonucleotide do not affect its secondary structure. The energy level of the manipulated miR34a oligonucleotide was kept the same compared to the original one. Moreover, the loading of miR34a to the exosomes was increased. Our findings revealed that normal HEK293T did not express miR34a. However, lentiviral transduced miR34a oligonucleotide induced the loading of miR34a into the exosome. Moreover, replacing six nucleic acids in the 3’ end of miR34a increased the loading of miR34a to exosome.


Introduction
RNA (ribonucleic acid) antisense has attracted much attention as a new candidate for therapeutic drug development.These biomolecules occur naturally in all cells and regulate various genes through different signaling pathways [1].To this end, the microRNA (miRNA) family represses cancerrelated genes and has several members, including miR-34a.As a crucial mediator of p53 function, miR-34a targets cancer signaling pathways, such as down-regulation of cell proliferation, induction of apoptosis, cell cycle arrest, etc. miR-34a contributes to cell-cycle arrest by downregulating CDK4/6; Cyclin E2, MET, and Bcl-2 mRNAs to enrich seed-matching sequences in their 3'-UTRs directly [2].Moreover, it can also inhibit several pro-apoptotic proteins by translational repression of silent information regulator sirtuin 1 (SIRT1), a NAD-dependent deacetylase [3].To that end, restoration of miR-34a activity prevents chemotherapy resistance [4,5].
Biomolecules such as miR-34a suffer from structural instability and susceptibility to nucleases; hence, using different delivery systems could be helpful.Although several nanoparticle packaging systems were designed and applied for RNA interference (RNAi) based cancer therapy, the toxicity and immunogenicity of these approaches are still troublesome [6][7][8].Using nanocarriers such as exosomes with features similar to cells could resolve this problem.
Exosome is a naturally occurring nano-carrier for cell-tocell communication and exists in all biological systems.The exosome is a cup-like structure with a 30-100 nm diameter with bold features such as biocompatibility, no toxicity, and no immunogenicity; hence, the exosome is a proper candidate for RNAi delivery and targeted therapy [9,10].It is worth noting that the application of these nanoparticles also struggles with efficient cargo loading, isolation methods, purity, and microbial infection.Various studies tried to remove these obstacles [11].Exosomes, as a communication device of the cells, carry RNA, specifically miRNA.This sparks the idea of applying exosomes as a proper device for RNA delivery [12].The exosome's contents lack ribosomal RNA (rRNA) and have a high amount of small RNAs, messenger RNAs (mRNAs), and miRNAs, which are named exosomal shuttle RNAs (esRNAs) [13].Exosomes extracted from body fluids such as plasma, amniotic fluid, and saliva can also carry their specific esRNAs, which function in recipient cells [14].The cell transporting machinery distinguishes esRNAs from their cytosolic form due to the difference in their sequence with a not well-understood mechanism [15].Recognition of esRNA-specific sequences can be applied in creating a specific cell factory that produces targeted exosomes containing an identified exosomal miRNA.This study aims to design a particular miR34a expression system and create a stable cell line that releases exosomes with elevated miR34a cargo.

Software and primer designing
The sequence of the miR34a oligonucleotide was taken from the NCBI database (GenBank: EF609116.1)and was checked using CLC main version 5 software.RNA software also analyzed the secondary structures of the wild-type and mutant miR34a oligonucleotide.Gene Runner and OLIGO7 software were applied to design six primers to amplify the miR34a oligonucleotide, site-directed mutagenesis, and cloning.The primers have restriction site sequences compatible with the pCDH vector and spacer sequences.

miR34a oligonucleotide amplification and sitedirected mutation
Total Deoxyribonucleic acid (DNA) was extracted from whole blood cells by QIAamp DNA extraction kit (Qiagen, Germany).Two primer pairs were separately used to amplify the 250 bp DNA encoding miR34a by polymerase chain reaction (PCR).The forward-34a (5-TGGGGAGA-GGCAGGACAGG-3) and Reverse-34a primers (5-TCC-GAAGTCCTGGCGTCTCC-3) were used for amplification of the miR34a gene from DNA.PCR reactions were performed using Takara PFU polymerase kit for 30 cycles (reaction conditions were as follows: 30s at 95 ºC, 30s at 60 ºC, and 30s at 72 ºC).The first pair, 34a forward primer (5'-TGGGGAGAGGCAGGACAGG -3' and S2 (5' TTA CTA TTG CTC ACA A CA TCC TC C TAA GAC ACT GC 3′) were used for amplification of the first DNA fragment with the first set of six nucleotides deserted point mutation.
In comparison, the second pair S1 (5′CTCGAGTA-CAACTATGCGGCCGCAGCAGAATG-GGAGAT-GAATTTCA3′) and 34a reverse (5′ACGACGCGTCG TTAGTGTTACAGAGTCT-GATATCC3′) were used for amplification of the second fragment of DNA with a second set of six nucleated deserted point mutation encoding miR34a pre-miRNA.PCR reactions were performed using Takara puf kit for 30 cycles (reaction conditions were as follows: 30s at 95 ºC, 30s at 60 ºC, 30s at 72 ºC for the first reaction, and 30s at 95 ºC, 30s at 60 ºC, 30s at 72 ºC for the second reaction).Both S1 and S2 primers were designed to include an overlapping sequence.In the next step, these overlapping fragments were used to assemble two amplified sequences into full-length DNA through an overlap extension of PCR (30s at 95 ºC, 30s at 60 ºC, and 120s at 72 ºC).Finally, a primer pair composed of 34a forward and 34a reverse was used to amplify the assembled fragment following reaction conditions as the 30s at 95ºC, 30s at 60ºC, and 90s at 72º C).
Positive bacterial colonies were harvested and cultured overnight in an LB medium for plasmid extraction.DNA sequencing with universal primers was done to confirm the accuracy of the cloned product.

Lentiviral vector packaging, titration, and cell transduction
Lentiviral particles were produced in HEK293T cells as described by Kutner et al. with some modifications [18].Briefly, 24 h before transfection, 5 million HEK 293T cells were plated in a 10 cm dish in DMEM for confluency of 80-90% the next day.Cell culture medium was replaced with fresh complete DMEM two hours before transfection.Then, cells were transfected with Lenti ORF pcdh-miR34a (wild and mutant separately), pMD2G, and pSPAX by calcium phosphate method.The culture medium was replaced with fresh medium 14 h post-transfection.For virus isolation, the Culture medium was collected three times at 12 h intervals.
To increase virus particle concentration, cell debris was removed following centrifugation of culture medium at 300 g for 5 min, and the supernatant was subsequently filtrated through Millex-HV 0.45 μm PVDF filter (Millipore).Lentiviral particles were precipitated via ultracentrifugation at 45,000 g for 1.5 h, and the virus particles in the pellet were re-suspended in 1 mL of phosphate-buffered saline (PBS).Concentrated virus particles were used to transduce HEK 293T cells.
Three days post-transduction, flow cytometric analysis was performed for lentivirus titration [15].Briefly, serial dilution (0.5, 1, 5, 25, and 50) of concentrated virus particles was added to 100,000 HEK 293T cells and cultured in 500 μL of DMED medium.Three days later, cells were harvested and washed twice with PBS, and flow cytometry counted the GFP-positive cell population.
The concentrated virus was applied for viral transduction.Briefly, HEK293T cells (6.0 × 10 4 ) were cultured in 6 well plates.After 24 h, 10 μL of concentrated virus dissolved in 2 mL of FBS-free DMEM medium, and the final solution was added to each well.After 16 h, the medium was replaced with a fresh DMED complete medium.The GFP (Green fluorescent protein) fluorescent microscopy was applied for transduction rate detection after 72 h.

GFP fluorescent microscopy
Transfected and transduced cells were washed with warm PBS and 24 h after treating.The distribution of the fluorescence was analyzed on an Olympus fluorescence microscope with a 488 nm fluorescent filter.Wavelengths of 395 nm were used to excite GFP, respectively; emission spectra were collected with 540 nm bandpass filters.The fluorescence images of the cells transduced with pCDH lentiviral vector were recorded every 24 h.

Puromycin kill curve and stable cell line selection
First, HEK293T cells (6.0 × 10 4 , not infected with the virus) were cultured in 5 wells of a 24-well plate containing 1 mL of complete DMEM medium.On the next day, when the cells stick to the bottom of the plate, we replace the culture medium of the cells with 1 mL of the culture medium containing zero, 0.5, 1, 1.5, and 2 micrograms of antibiotic puromycin, respectively.On the first day after the treatment, the culture medium of the cells was replaced with the selected medium containing the antibiotic puromycin, and the number of living cells in each well was counted daily by vital staining.The best result is obtained 1 to 4 days after antibiotic treatment.The number of living cells for each concentration was shown as a curve during this period.The least amount of antibiotic that destroys the cells at this time was applied for the cell selection after transduction.

Isolation of exosomes
To produce miR34a enriched exosomes, HEK293T stable cells were cultured in DMEM supplied with 10% exosomefree FBS.The culture media were periodically collected at 48 h intervals, and then exosomes were isolated via sequential centrifugation and re-suspended in PBS.Aliquots of exosomes were stored at -80 ºC.To purify the crude exosome, 250 mL of cell culture media was centrifuged for 10 min at 300 g, 4 °C.The supernatant was collected and recentrifuged for 30 min at 10,000 g, 4 °C.Then the supernatant was transferred to a new ultracentrifuge bottle and spined for 70 min at 110,000 g, 4 °C.The pellet was resuspended in PBS and transferred the sample to a polyallomer conical tube and centrifuged for 2 h at 100,000 g, 4 °C.To wash the EV pellet the supernatant was discarded and the pellet was resuspend in 150 μL cold 1X PBS and store at − 80 °C [16].

Exosome characterization
Transmission electron microscopy (TEM), zeta sizer analysis, and western blot analysis were carried out to characterize the obtained exosome.For electron microscopy imaging, the ultra-concentrated exosomes were fixed with 4% paraformaldehyde in PBS.The selected samples were air-dried on Formvar-carbon coated TEM grids and stained sequence was manipulated for more expression, as illustrated in Fig. 1a.The bioinformatics analysis using RNA software indicated that the changes of miR34a did not change its secondary structure compared to wild-type miR34a (Fig. 1b).
The miR34a oligonucleotide amplification and sitedirected mutagenesis were performed by six primers (Fig. 2).At first, the 300-base pair (bp) of the human genome DNA fragment, including miR34a oligonucleotide, was amplified, sequenced fallow blast, and confirmed miR34a oligonucleotide.Two specific primers with restriction sites in the 5' end re-amplified this segment for gene cloning into pCDH lentiviral vector.After digestion of miR34a and pCDH, gene and vector ligated and pCDH-miR34aW was constructed and cloned into stbl4 (Fig. 2b).
We used the overlap extension PCR method to change six nucleotides in 3p and 5p coding sequences of miR34a.Therefore, two segments of miR34a were amplified and manipulated, then ligated by PCR, one PCR amplified half of the 5p segment of miR34a and stem-lope and changed the 5p miR34a by R soeing reverse primer.The second PCR was performed to amplify the 3p segment of miR34a and stem lope and introduce diversity in 3-p miR34a oligonucleotide (Fig. 2a); the stem lope has overlap function in the final PCR which soied to segment of mutated miR34a (Fig. 2b).This fragment digested and insert to pCDH and pCDH-miR34aM was constructed and cloned into stbl4 bacteria.
As the pCDH vector has a reporter gene to evaluate transfection efficiency, the GFP expression was used to transfect HEK293T cells.Up to 90% of the cells express the GFP reporter gene; HEK293T virus producer cells were also nucleated due to VSV-G protein expression during virus production (Fig. 2c).
The virus particles were concentrated by ultra-centrifugation, and the titer of lentiviral stock was determined by flow cytometry.Transduction of 8 × 10 4 HEK cells by 5 μL of the virus suspension induced GFP expression in the 8 × 10 3 cells [17].The lentiviral titer was estimated by flow cytometry as 1.6 × 10 9 TdU/mL.The electron microscopy result of the HEK293 T cells showed that 100% of the cells were transduced with pCDH-miR34a Vector (Fig. 2d).

HEK293T cells transduction and stable cell line production
The HEK293T cells were transduced with virus particles bearing miR34aW and miR34aM gene (MOI = 5).The puromycin kill curve for HEK293T (Fig. 3) showed that 1.5 μg/ ml of Puromycin is the minimum concentration of antibiotic that kills non-transduced HEK293T cells.To select a stable cell line containing miR34aM and miR34aW within its with 0.5% uranyl acetate in 30% ethanol for 10 min, followed by staining with lead citrate for 10 min.The sample was sequentially rinsed in 0.05 M NaOH and distilled water to remove lead precipitates on the grid sections.Then, the grids were dried at room temperature.The stained cells were scanned under the transmission electron microscope (Philips) operating at 150 kV.According to the manufacturer's instructions, the purified exosomes' dimensions were measured using a zeta sizer (Malvern Corp.).
The transduced cells and exosomes were lysed with RIPA buffer containing protease inhibitor cocktail (Roche, Germany), and the obtained total protein was subjected to Western Blot analysis.Briefly, the cells were washed twice with ice-cold PBS and centrifuged at 4000 rpm for 5 min.The cells (and exosomes) were treated with lysis buffer and incubated on ice for 20 min with shaking.Cell debris was precipitated by centrifugation at 13,000 rpm for 20 min, and the upper phase was collected as a source of the total protein.The number of total proteins was calculated using the Bradford assay.The extracted proteins (10 μg) were boiled for 5 min in 10X SDS buffer (1 μg) and resolved on 10% polyacrylamide gel for 2 h.Protein fragments were transferred to the nitrocellulose membrane under the constant current of 300 mA for 2 h, and the membrane was blocked with 5% nonfat dry skim milk in PBS for 2 h.The membrane was washed with PBS and treated with HRP-conjugated anti-His tag antibody for 16 h to detect LAMP-DARPin G3 chimeric protein.After washing with PBS three times, the related band was detected using an ECL solution.In addition, the expression of CD63 as an exosome marker was investigated by flow cytometry.

Reverse transcription and quantitative PCR
Total RNA was extracted using Qiagen RNeasy mini kit from purified exosomes three days post-transduction of HEK293T cells with miR-34a virus.Revert-Aid™ First Strand cDNA Synthesis Kit (Fermentas, Germany) was applied for cDNA synthesis according to the manufacturer's instructions.The relative quantitative PCR (qPCR) experiments were performed on the ABI thermal cycler in the total volume of 20 μL containing 10 μL of Qiagen qPCR Master Mix (Qiagen), 10 pmol of each primer, and 2 μL of each cDNA.16 s RNA was used as a reference gene.

miR34a oligonucleotide manipulation
The sequence of wild-type miR34a (miR34aW) was taken from the NCBI database (GenBank: EF609116.1),and its distribution obtained by DLS was in accordance with the images obtained by TEM (Fig. 4a).
Western blot analysis of the exosomes extracted from the medium of transduced cells indicated expression of CD63 but did not show calnexin expression (Fig. 5a and b).As illustrated in Fig. 5c immunophenotyping analysis, the exosomes were positive for CD81 (54.4 ± 0.5%) (Fig. 5c).

Expression of wild type and manipulated miR34a
According to the results of relative qPCR, miR34a expression extracted from non-transduced HEK293T, miR34aW lentiviral transduced HEK293T, and miR34aM lentiviral genome, Puromycin was added to kill non-transduced cells in the cell culture medium for two weeks.

Exosome characterization result
In the current study, we hypothesized that modified HEK293T cells with pCDH-miR34a vector secrete functional miR-34a into exosomes.We initially performed isolation and characterization of transduced HEK293T cell derives exosomes to confirm this hypothesis.According to the results, exosome fractions showed around 75 nm sizes in diameter (Fig. 4b).Exosomes demonstrated a spherical shape with a uniform particle size.The particle size Fig. 1 (a) The sequence of wild type and manipulated miR34a.The wild-type sequences were manipulated for more expression.(b) The bioinformatics analysis Infelunce of miR34a changes on its secondary structure compared to the wild-type miR34a Fig. 3 Puromycin kill curve for HEK293T.In the absent of puromycin the cells continue growing.The addition of puromycin halts the cells growth and the lowest concentration that kills the cells is 0.75 mg (p value < 0.01) Fig. 2 (a) overlap extension PCR including negative control (a1), 100 bp ladder (a2), amplification of 200 bp miR34a oligonucleotide which contains 5p segment, the 5' mutated fragment (was introduced in the S2 primer), overlapping fragment of miR34a were amplified by forward and S2 primer (a3), amplification of 200 bp miR34a oligonucleotide which contains 3p segment, the 3' mutated fragment (was introduced in the S1 primer), overlapping fragment of miR34a were amplified by reverse and S1 primer (a4).(b) miR34a oligonucleotide amplification: the miR34a mutant (soeing from two fragments of miR34a amplified from first PCR rection (a3 and a4 in the Fig. 2a)) (b1), the miR34a product (which is a wild type of miR34a amplified from human DNA) (b2), negative control (b3), and the ladder (b4) (c) florescent microscopy imaging of HEK293T transfected with pCDH miR34a-lentiviral vector for viral packaging.(d) florescent microscopy imaging of HEK293T transduced with miR34a-lentiviral vector 1 3 Fig. 5 (a) Western Blot The results showed the positive expression of exosomal markers and the negative expression of calnexin.Immunophenotyping results According to the effects of flow cytometry, the expression of exosomal marker CD63 was positive Fig. 4 TEM and DLS analysis of the produced exosome.(a) TEM of the produce exosome which shows spherical shape and diameter below 100 nm (b) DLS analyse of the produce exosome which shows the particles size distribution stability in body fluids, biocompatibility, safety, and nontoxicity [24].
This study aims to create a cell factory to generate miR34a-enriched exosomes.miR-34a is a member of the mir34 family involved in P53 regulation, cell signaling, and cell survival pathways.MiR34a stops the cell cycle at the G1 step and induces apoptosis or cell death.MiR34a is a tumor suppressor molecule, and the degradation of miR34a was observed in several types of cancers [25][26][27].
The expression of miR-34a is suppressed in malignancies and some molecular processes, such as cell differentiation and proliferation [27].miR34a affects osteosarcoma's metastasis, and decreases in its expression could lead to increased cell growth and metastasis [28].Applying mimetic miR34a as a candidate for cancer therapy has progressed to phase I of the clinical trial, and production and packaging of miR34a into liposome has been successfully done [29].
There are several methods for loading miRNAs into exosomes.The most straightforward procedure to generate miRNA-enriched EVs is to incubate them with miRNAsecreting cells.Using a concentration gradient for this method can be a natural process that does not create a risk factor for cells.However, this approach has the disadvantage that it cannot predict the concentration of loaded miRNAs in exosomes and has low efficiency due to the instability of miRNAs [30].Another method is electroporation.For the first time, L Alvarez-Erviti et al. [31] used exosomes to transport siRNA to the brain cells.They insert the desired siRNA into the exosomes using electroporation approaches.JH Wang et al. [32] used electroporation to load EVs with HChrR6 mRNA.But the results of this method seemed inadequate.Using electroporation to transfect nucleic acids inside exosomes is a simple, inefficient method.This method requires the purification and isolation transduced HEK293T cells.Then the total RNA transcribed into the complementary DNA (cDNA) sa the template for the quantitative PCR or real-time PCR reaction (qPCR).The amount of amplificated product was measured in each PCR cycle using fluorescence.The amplification curve was drawn by device, and each sample's Ct value was determined.Finally, the 2^-ΔΔCt method was applied to quantify the results.The results indicated that intact cells have no delectable miR34a expression while its expression on miR34aM transduced HEK293T cells was increased approximately 2.9-fold compared to miR34aW transduced HEK293T (p < 0.01) (Fig. 6).

Discussion
miRNAs, non-coding RNAs, affect various biological processes.They play a vital role in the growth and development of organisms [18].miRNAs have been introduced as novel drug candidates.Assessing their safety and effectiveness is the most crucial step in their entry into the clinical field [19].On the other hand, the low stability of miRNAs, difficult delivery to the target cell, rapid degradation in the blood by ribonucleases, clearance by the reticuloendothelial system, being trapped in the endosome and then degradation in the lysosome limit their clinical applications.Considering the development of nanoscience and the progress of nanoparticles in drug delivery, it can be said that the delivery of miRNAs by nanoparticles can be an effective method of transferring miRNAs [20][21][22].Due to nanoparticles, the entry of miRNA into the target cells is multiplied [23].
Conversely, unlike nanoparticles that are synthesized manually, exosomes as nanoparticles of endogenous origin have unique features such as better targeting capacity, other changes in other regions.For manipulating the 3' ends of mir5p, the manipulation of a complementary sequence at mir3p is also needed.The miR34a oligonucleotide-required changes were created using the overlap extension PCR method by four specific primers at two gene regions.One pair for insertion of GGAGAG sequence at 3' end of mir34-5p, and another pair for insertion of 6 nucleotides CTCTCC that is complementary to the first manipulated change to stabilize the secondary structure.The results confirmed the changes in the miR34a oligonucleotide, and bioinformatics analysis has also indicated that these changes do not affect the secondary structure of the molecule.The energy level of the manipulated molecule was not changed compared to the original one.
High expression of the desired RNA in exosome-producing cells via lentiviral vectors can lead to the enrichment of that RNA and its encoded protein in the produced exosomes [40].Lentiviral vectors can transduce both dividing and non-dividing cells, while retroviral vectors only transduce non-dividing cells [41].Therefore, in the present research, a lentiviral vector was chosen for the overexpression of miR34a and the creation of a stable cell line that produces miR34a-loaded exosomes and releases them to the medium.

Conclusion
The results indicate that normal HEK293T does not express miR34a in the exosomes.Transduction of HEK293T by lentiviral vector containing wild type miR34a leads to the overexpression of miR34a in HEK293T cell and induces the loading of miR34a into exosome.Replacing six nucleic acids in the 3' end of miR34a structure increases the exosome miR34a loading to 3-folds compared to the overexpressing wild type miR34a. of extracellular vesicles before and after transfection, and repeated purification can lead to the loss of exosomes [33].In another study, TN Lamichhane et al. [34] investigated the transport of siRNAs loaded by sonication in extracellular vesicles.Contrary to the high cellular uptake of extracellular vesicles (80%), the overall delivery of siRNA to cells was deficient (2.96%).Sonication also has limitations, such as changing the shape of the extracellular vesicle membrane, generating heat, destroying surface proteins, and not being suitable for delivering hydrophobic drugs [35].
Cell engineering is another approach to producing miRNA-enriched exosomes.Overexpression of the desired miRNA in exosome-producing cells by cellular machinery and mimicking the spontaneous miRNA loading and packaging system are two approaches used to generate miRNAloaded exosomes [36].In the present research, we used both systems.First, we inserted exosome-specific sequences into miR34a.Then we transduced the manipulated miR34a oligonucleotide via a lentiviral system into the HEK293T genome, which applied CMV promoter for high and permanent expression of miR34a oligonucleotide.
Various studies suggest a sorting sequence at the 3' end of miRNA sequences.It is predicted that the transfer of miRNA into the exosome is carried out by this sequence [37].This sequence can be used to transfer miRNA into the exosome.MF Bolukbasi et al. [38] investigated a 25-nt sequence containing "CTGCC" on a stem-loop structure and a miR-1289 binding site in the 3UTRs of many mRNAs enriched in MVs derived from GBM cells.Their findings indicate the critical role of the presence of the CTGCC sequence in the loop structure and the binding site of miR-1289 in the 3'UTR in increasing the integration of mRNAs in MVs.By RNA sequencing (RNA-seq) from a panel of human B cells and their secreted exosomes, D Koppers-Lalic et al. [39] demonstrated that miRNAs that are 3' adenylated were relatively enriched in cells, while 3' uridylated isoforms were overexpressed in exosomes.Therefore, the post-transcriptional modifications help to guide the sorting of ncRNA to EVs somewhat.Villarroya-Beltri et al. [15] described sequence motifs in miRNAs that control their localization into exosomes (EXOmiRNA).These specific sequences (GGAG) are present at the end of miRNA, leading the miRNA to the endosomes and finally to exosomes.Sumoylated hnRNPA2B1 binds to EXOmiRNA and controls its loading into exosomes.In this study, we substituted six nucleotides of miR34a oligonucleotide to increase its packaging rate into exosomes.Since replacing nucleotides at the 5' and 3' ends of the miR34a oligonucleotide could affect its expression, stability, and secondary structure, a 250 bp fragment capturing and containing miR34a sequences was selected for cloning in the present research.Moreover, any changes in one part of the gene may require manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
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Fig. 6
Fig. 6 Expression of miR34a 3-fold more loading of miR34a into the exosome than when miR34a was only overexpressed in the cell (p-value < 0.01)