Method for chimeric expression of recombinant small RNA in escherichia coli by using active tRNA and application

Abstract

The invention discloses a method for chimeric expression of recombinant small RNA (Ribonucleic Acid) in escherichia coli by utilizing active tRNA (Ribonucleic Acid) and application, and the method is characterized in that target small RNA is expressed in escherichia coli by taking an active tRNA chimeric functional miRNA precursor as a bracket. In addition, the invention also discloses application of the recombinant small RNA produced by the method in preparation of a diagnostic reagent, a scientific research reagent, a prodrug, a drug, a bulk drug or a pharmaceutical composition. According to the invention, the human tRNA is used as the stent, so that the toxicity and immunogenicity are possibly lower; in addition, the active tRNA is used as a bracket, so that the effect of targeted treatment of diseases can be achieved, and a synergistic interaction effect can be achieved with carried small RNA. The cell toxicity is reduced, the expression quantity is improved, and a tRNA scaffold with activity is screened, so that the tRNA scaffold and chimeric small RNA synergistically exert a treatment effect.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and medical technology, specifically relating to a method and application of chimeric expression of recombinant small RNA in Escherichia coli using active tRNA. Background Technology

[0002] In recent years, RNA therapy has become a novel treatment approach attracting attention from scientific research and industry due to its advantages such as simple design, short development cycle, and abundant candidate targets. A growing body of research has found that tRNAs, in addition to participating in protein synthesis, are also widely involved in the development and progression of various diseases. The cleavage of mature or precursor tRNAs can produce small RNAs derived from tRNAs (tsRNAs), and tsRNAs have been detected as being abnormally expressed in various diseases, suggesting the potential role of tRNAs and tsRNAs in disease treatment.

[0003] Previous studies have shown that tRNA can be successfully used as a scaffold to produce small ncRNAs in Escherichia coli (Luc Ponchon, et al., Recombinant RNA technology: the tRNA scaffold[J]. Nature Methods, 2007, 4(7): 571-576; Luc Ponchon, et al., Ageneric protocol for the expression and purification of recombinant RNA in Escherichia coli using atRNA scaffold[J]. Nature Protocols, 2007, 4(6): 947-959). Ponchon et al.'s method involves directly chimerating small ncRNA precursors with methionyl tRNA and human lysine tRNA for expression. After the recombinant RNA is cleaved by RNase H, the mature small ncRNA fragment is purified by ion exchange chromatography. However, this biological expression method has been shown to have limited applicability, and many small RNAs cannot be expressed or are expressed at very low levels under this strategy. Qian et al. from Northwestern Polytechnical University have provided a method for producing recombinant small RNAs by expressing short RNA fragments using human serine tRNA chimeric precursor microRNA (CN110964726B). Qian et al.'s method is more universal than that of Luc Ponchon et al. Human serine tRNA is a human-derived tRNA, making it a promising scaffold for the production of recombinant small RNAs. Since human cells naturally contain serine tRNA but lack bacterial methionyl tRNA, this invention reduces toxicity and immunogenicity. Several small RNAs that cannot be expressed by directly chimeric precursor microRNAs can be expressed using Qian et al.'s method. In the above methods, tRNA as a scaffold enhances the expression level and stability of recombinant small RNAs but does not possess biological activity. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method and application for chimeric expression of recombinant small RNA in Escherichia coli by utilizing active tRNA, which addresses the shortcomings of the prior art.

[0005] This invention utilizes human active tRNA chimeric hsa-miR-34a precursor. The development of recombinant small RNAs produced using human tRNA as a scaffold shows greater promise because human cells naturally contain this tRNA; therefore, using human tRNA as a scaffold may result in lower toxicity and immunogenicity. Furthermore, using active tRNA as a scaffold can achieve targeted disease treatment and synergistically enhance the therapeutic effect with the carried small RNA. This invention not only reduces cytotoxicity and increases expression levels but also obtains an active tRNA scaffold that synergistically exerts therapeutic effects with the chimeric small RNA.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for expressing recombinant small RNA in Escherichia coli using active tRNA, the method comprising: expressing target small RNA in Escherichia coli using active tRNA-chimeric functional miRNA precursor as a scaffold.

[0007] In the method described in this invention, the small RNA is an RNA with a length of less than 200 nt.

[0008] In the method of the present invention, the tRNA sequence is a sequence with a similarity of more than 90% to the human glutamate tRNA sequence, and the human glutamate tRNA sequence is SEQ ID NO.2.

[0009] In the method of the present invention, the sequence of the miRNA precursor is a sequence with a similarity of more than 90% to the hsa-miR-34a precursor sequence, and the hsa-miR-34a precursor sequence is SEQ ID NO.3.

[0010] In the method of the present invention, the miRNA precursor is embedded in the anticodon loop of the tRNA, and the embedded sequence is a sequence with a similarity of more than 90% to the sequence of SEQ ID NO.1.

[0011] Specifically, the method described in this invention includes the following steps:

[0012] Step 1: Using the restriction enzyme site at the anticodon loop of the tRNA in the pBSMrnaSeph plasmid, the expression sequence of the hsa-miR-34a precursor is inserted into the pBSMrnaSeph plasmid to construct the expression vector;

[0013] Step 2: Replace the mature sequence portion of miR-34a in the constructed expression vector with the target small RNA sequence to construct a chimeric target sequence expression vector; transform the chimeric target sequence expression vector into competent E. coli.

[0014] Step 3: After E. coli culture and amplification, total RNA was extracted from the bacteria using the saturated phenol method, and the target small RNA was isolated and purified by FPLC.

[0015] In the method described in this invention, the small RNA is shRNA, miRNA, siRNA, or an RNA aptamer.

[0016] In the method of the present invention, the Escherichia coli in step two is either strain HST08 or strain DH5α.

[0017] In the method described in this invention, in step three, Escherichia coli is cultured in TB medium until the OD reaches 1.1-1.7. The ratio of the volume of saturated phenol used to extract total RNA from the bacteria to the mass of the bacterial sludge is (2-6):1, where the volume unit is mL and the mass unit is g.

[0018] In addition, the present invention provides a method for constructing a tRNA scaffold using a human glutamate tRNA chimeric hsa-miR-34a precursor, and for preparing recombinant small RNA using the scaffold.

[0019] The present invention further provides the use of recombinant small RNA produced by the method of the present invention in the preparation of diagnostic reagents, research reagents, prodrugs, drugs, active pharmaceutical ingredients or drug compositions.

[0020] The following is a further explanation of the nouns and terms used in this invention:

[0021] Active tRNA: In addition to performing transport functions, tRNA also has other functional activities. The active tRNA mentioned in this invention refers to tRNA that can serve as a source of short non-coding RNA, i.e., small RNA derived from tRNA, and then tRNA exerts its regulatory function by being degraded into small RNA.

[0022] Chimeric expression: A method in which a miRNA sequence is inserted into a certain region of a tRNA and expressed together.

[0023] Small RNA: A class of non-coding RNA molecules with a length of about 20-30 nucleotides.

[0024] Human glutamate tRNA: Derived from human tRNA responsible for transporting glutamate.

[0025] miRNA precursor sequence: miRNA sequence containing hairpin structure.

[0026] hsa-miR-34a precursor sequence: the sequence corresponding to the hsa-miR-34a hairpin structure.

[0027] tRNA anticodon loop: A loop structure, i.e., an anticodon loop, is usually formed at the anticodon position in the tRNA cloverleaf structure.

[0028] pBSMrnaSeph plasmid: The name of a plasmid vector.

[0029] The mature sequence of miR-34a: the sequence obtained by precise cleavage of the hsa-miR-34a precursor sequence by an endonuclease.

[0030] Competent Escherichia coli: A specific form of Escherichia coli that readily absorbs exogenous DNA attached to the surface of E. coli cells when treated with CaCl2 or electroporation.

[0031] The Escherichia coli strain HST08 is a strain that lacks mrr, hsdRMS, mcrBC, and mcrA (these genes are essential for E. coli to excise exogenous methylated DNA).

[0032] The *E. coli* strain is DH5α: DH5α is a mutagenic strain with the following basic characteristics: Rˉ, Mˉ, and AMPˉ. Mˉ indicates that the DH5α strain lacks DNA modification, meaning its own DNA cannot be modified. Rˉ indicates that it lacks an "immune" mechanism and will not cleave introduced foreign DNA. AMPˉ indicates its sensitivity to ampicillin.

[0033] The raw materials and reagents are all available on the market or their sequences can be directly found in NCBI.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] 1. A tRNA scaffold for expressing recombinant small RNA was constructed using human glutamate tRNA chimeric with hsa-miR-34a precursor. Human glutamate tRNA is a human-derived tRNA, and using it as a scaffold for the production of recombinant small RNA has better development prospects because human cells themselves contain glutamate tRNA. Therefore, using human glutamate tRNA as a scaffold may result in lower toxicity and immunogenicity, thereby reducing cytotoxicity and increasing expression levels. Furthermore, human glutamate tRNA has therapeutic activity for breast cancer. Therefore, this invention enables both the recombinant tRNA portion and the small RNA portion it carries to exert biological activity, resulting in a dual therapeutic effect.

[0036] 2. This invention designs and prepares recombinant small RNAs using bioengineering technology, which has advantages such as simple equipment, rapid production, high yield, low cost, and good functionality.

[0037] 3. This invention also optimizes the culture conditions of Escherichia coli by replacing LB or 2×YT with TB medium, which prolongs the time for Escherichia coli to enter the growth and death phase and increases the total amount of bacteria obtained per unit volume. In addition, the optimal bacterial concentration for collecting Escherichia coli after culture is determined to be OD = 1.1 to 1.7. Under these density and culture conditions, the most total RNA can be obtained, which increases the yield of recombinant small RNA.

[0038] 4. This invention also optimizes the volume of water-saturated phenol added during bacterial total RNA extraction, and finds the minimum volume of water-saturated phenol required to obtain the best RNA extraction efficiency, thereby optimizing the cost-benefit ratio.

[0039] 5. Testing using various techniques has shown that the method of this invention can construct active recombinant small RNAs, and these recombinant small RNAs possess superior biological functions. The recombinant small RNAs produced meet the needs of scientific research and drug development.

[0040] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0041] Figure 1 Example 2 of this invention utilizes denaturing polyacrylamide gel electrophoresis to detect the expression of recombinant small RNAs in Escherichia coli.

[0042] Figure 2 This is Embodiment 4 of the present invention utilizing Bio-Rad NGC. TM The recombinant small RNA was purified using a chromatography system, and the purity of the collected fractions was identified by denaturing polyacrylamide gel electrophoresis.

[0043] Figure 3 This is the recombinant has-tRNA of Example 5 of the present invention. Glu Inhibitory effect of miR-34a on MDA-MB-231 cells.

[0044] Figure 4 In Example 6 of this invention, the intracellular levels of the corresponding tRNAs were detected using qPCR after different recombinant miR-34a were transfected into MDA-MB-231 cells.

[0045] Figure 5 Example 6 of this invention uses qPCR technology to detect the processing of recombinant miR-34a into mature form in MDA-MB-231 cells (values ​​are expressed as "mean ± standard deviation").

[0046] Figure 6 Example 7 of this invention utilizes CCK8 to detect hsa-tRNA. Glu IC of -miR-34a 50 value.

[0047] Figure 7 Example 8 of this invention utilizes the Chou-Talalay method to analyze has-tRNA. Glu Figure showing the synergistic effect of the stent and miR-34a.

[0048] Figure 8 Example 9 of this invention uses a cell colony formation experiment to determine...

[0049] hsa-tRNA Glu Figure 1 shows the effect of miR-34a on the proliferation capacity of MDA-MB-231 single cells.

[0050] Figure 9 Example 10 of this invention uses immunofluorescence technology to detect hsa-tRNA. Glu Figure showing the effect of miR-34a on cell apoptosis.

[0051] Figure 10 Example 11 of this invention detects hsa-tRNA through a scratch healing assay. Glu Figure showing the effect of miR-34a on cell migration ability.

[0052] Figure 11 Example 12 of this invention utilizes the Transwell invasion assay to detect hsa-tRNA. Glu Figure showing the effect of miR-34a on cell invasion ability.

[0053] Figure 12 Example 13 of this invention utilizes small animal in vivo imaging to detect hsa-tRNA at the animal level. Glu The therapeutic effect of miR-34a on a mouse model of breast cancer. Detailed Implementation

[0054] The following specific embodiments illustrate the implementation of the present invention. Unless otherwise stated, the experimental methods disclosed in the present invention all adopt conventional techniques in this technical field.

[0055] The design expression of recombinant miR-34a will be used as an example for illustration.

[0056] Example 1: A literature search was conducted in GtRNAdb (http: / / gtrnadb.ucsc.edu / ) for human tRNAs with anti-breast cancer activity. Glu Sequence, chimeric miR-34a precursor, and design of recombinant has-tRNA Glu Expression plasmid for -miR-34a (E-miR-34a).

[0057] The designed human active tRNA was predicted using the CentroidFold (http: / / www.ncrna.org / centroidfold) online prediction tool. GluThe secondary structure of recombinant miR-34a expressed by scaffold chimerism was predicted. By adjusting the position of the scaffold chimerism with miR-34a and adjusting individual nucleotides, the sequence with the optimal secondary structure was selected to construct the recombinant expression plasmid.

[0058] Example 2: Recombinant expression of hsa-miR-34a using an active tRNA scaffold

[0059] (1) Extract a small amount of recombinant E-miR-34a expression plasmid

[0060] 100 ng of recombinant E-miR-34a expression plasmid was transformed into HST08 or DH5α competent bacteria, and then cultured overnight at 37°C with shaking at 200 rpm in 5 mL LB medium. The bacterial culture was centrifuged at 10000 g for 2 min, and the precipitate was collected. The precipitate was resuspended in 180 μL of 10 mM magnesium acetate-Tris HCl solution, followed by 200 μL of saturated phenol, and the mixture was shaken at room temperature for 20–60 min. After centrifugation at 10000 g for 10 min, the aqueous phase was collected, and 0.1 times the volume of the aqueous phase of 5 M NaCl was added to precipitate macromolecular impurities. The supernatant was then added to 2 volumes of anhydrous ethanol, centrifuged at 10000 g for 10 min, and the supernatant was discarded. Residual ethanol was absorbed with absorbent paper, and after the RNA dried, DEPC water was added to dissolve the RNA. The concentration was determined, and the RNA was stored at -80°C.

[0061] (2) Identification by denaturing polyacrylamide gel electrophoresis

[0062] Mix the RNA sample with RNA loading buffer and add it to the wells of the denaturing gel. Electrophoresis at 80–100V for 50–80 min, then place the gel in a solution containing 0.5 μg / mL ethidium bromide and gently shake for 10–30 min. Observe the gel under a gel imaging system, take pictures and save them.

[0063] Figure 1 This invention utilizes denaturing polyacrylamide gel electrophoresis to detect the expression of recombinant E-miR-34a in *E. coli*. The synthesized recombinant plasmid was transformed into *E. coli* HST08 competent cells, and the expression level was identified by urea-denaturing polyacrylamide gel electrophoresis, with the expression level determined using bacterial methionine tRNA (btRNA). Met ) scaffold expressing recombinant miR-34a (btRNA) Met -miR-34a (i.e., M-miR-34a) was used as a positive control. For example... Figure 1As shown, if *E. coli* transformed with the recombinant miR-34a expression plasmid shows an additional band between 150-300 nt, it indicates successful expression of the recombinant small RNA; the thickness of the band reflects the expression level. The results showed that the E-miR-34a recombinant plasmid exhibited expression bands in both *E. coli* HST08 and DH5α, indicating that the recombinant E-miR-34a expression plasmid can efficiently express recombinant small RNA in *E. coli*. The effective sequence in the recombinant E-miR-34a plasmid is SEQ ID NO.1.

[0064] Example 3: Optimization of Escherichia coli culture conditions and total RNA extraction method

[0065] (1) TB medium was used instead of LB medium in “Example 2 (1)”, and the bacteria were cultured until the bacterial density reached OD of 1.1 or higher, and then collected by centrifugation. By comparison, the amount of bacteria harvested by this culture scheme was more than twice that of the LB medium culture method.

[0066] (2) The amount of water-saturated phenol used in “Example 2(1), extraction of total RNA from E. coli by lysis of E. coli with water-saturated phenol” was optimized. A more standardized lysis buffer ratio was used for E. coli cell lysis to find the minimum volume of water-saturated phenol required for sufficient bacterial lysis and to optimize the cost-effectiveness ratio. By comparison, when the ratio of saturated phenol to bacterial sludge is 2-6 (v / w) (e.g., 2 mL of water-saturated phenol is added to 1 g of collected bacterial sludge), the total RNA extraction rate can be increased from 3.5% to >7%.

[0067] Example 4: FPLC purification of recombinant E-miR-34a

[0068] (1) Using Bio-Rad NGC TM Chromatography System, using ion exchange column (ENrich) TM Q10×100 Column) Purification of recombinant E-miR-34a.

[0069] Mobile phase A: 10 mM NaH2PO4 solution, pH 7.0.

[0070] Mobile phase B: 10 mM NaH2PO4 solution, 1 M NaCl solution, pH 7.0.

[0071] The flow rate was 2.0 mL / min. The column was alternately washed with DEPC water, mobile phase A, and mobile phase B for approximately 1 h each time. Each wash consisted of 5 column volumes.

[0072] Total RNA was separated using the following procedures: 0–8.9 min (0% B), 8.9–13.7 min (55% B), 13.7–53.7 min (55–75% B), 73.7–83.7 min (100% B), and 83.7–93.7 min (0% B). RNA was detected at 260 nm absorbance, and the peak corresponding to recombinant RNA was collected. Purity was determined by denaturing polyacrylamide gel electrophoresis.

[0073] (2) RNA sample processing methods

[0074] The total RNA extraction procedure is the same as above. The extracted total RNA was centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was filtered through a 0.45 μm microporous membrane, and 5–10 mg was injected each time.

[0075] (3) FPLC component collection and concentration desalting

[0076] The purity of the collected fractions was determined by denaturing polyacrylamide gel electrophoresis. RNA was precipitated from the mixed fractions with two volumes of anhydrous ethanol and incubated at -80°C for approximately 1 hour. RNA was collected by centrifugation at 10000g at 4°C for 10 minutes. The RNA precipitate was dissolved in DEPC water and centrifuged at 7500g for 10 minutes at 4°C using Ultra-2 mL Centrifugal Filters. The filtrate was discarded. This step was repeated until all solutions were centrifuged. The filters were then inverted, centrifuged at 2000g for 2 minutes, and the resulting solution was collected. The concentration was determined, and the solution was stored at -80°C.

[0077] Figure 2 This invention utilizes Bio-Rad NGC TM The recombinant miR-34a was purified using a chromatography system, and the purity of the collected fractions was identified by denaturing polyacrylamide gel electrophoresis. Figure 2 Chromatography A shows the location of the target peak and the collected components. The results indicate that recombinant E-miR-34a was well separated from other endogenous RNAs in E. coli. Further urea-denaturing polyacrylamide gel electrophoresis identification results showed ( Figure 2 B) Compared with the unpurified total RNA, the purified recombinant E-miR-34a showed a single band without any extraneous bands or tailing, proving that the recombinant E-miR-34a had good purity. That is, pure recombinant E-miR-34a can be obtained after FPLC purification.

[0078] Example 5: Inhibitory effect of recombinant has-Glu-miR-34a on MDA-MB-231 cells

[0079] The effects of recombinant E-miR-34a on the proliferation and viability of MDA-MB-231 breast cancer cells were detected using the CCK8 cell proliferation and toxicity assay kit. The specific experimental procedures are as follows:

[0080] (1) Seed cells into 96-well plates, with 6 replicate wells per group. After the cells adhered, RNA transfection was performed according to the experimental groups.

[0081] (2) After transfection for 24h, 48h, 72h and 96h, 10% CCK8 detection solution was added, and the absorbance value of each well at 450nm was detected by microplate reader for data analysis.

[0082] like Figure 3 As shown, recombinant E-miR-34a significantly inhibited cell activity at 72h and 96h time points.

[0083] btRNA was constructed using a validated bacterial methionine tRNA that can be used for efficient small RNA recombination expression. Met- miR-34a and human serine tRNA to construct hsa-tRNA Ser -miR-34a, using recombinant miR-34a carried by these two "inert" tRNA scaffolds (since no literature reports significant anti-breast cancer activity of these two tRNAs, they are relatively inactive tRNAs in the breast cancer disease of this invention, and are referred to as inert in this article) as a control; to investigate whether the tRNA scaffold has a synergistic therapeutic effect with miRNA, a control was constructed using the tRNA scaffold. (From btRNA) Met hsa-tRNA Ser and hsa-tRNA Glu Scaffold-constructed control recombinant RNA (NC) Met NC Ser NC Glu Therefore, the subsequent experimental groups were set as follows: Blank, Mock, NC Met btRNA Met -miR-34a (M-miR-34a), NC Ser hsa-tRNA Ser -miR-34a (S-miR-34a), NC Glu and hsa-tRNA Glu -miR-34a(E-miR-34a).

[0084] Example 6: Intracellular processing and maturation of recombinant miR-34a

[0085] (1) Transfection of MDA-MB-231 cells

[0086] Cells were seeded at 10,000 cells / well in 24-well plates. Following the Engreen (Entranster) method... TM Cell transfection was performed according to the instructions for the R4000 transfection reagent. The transfection concentration of recombinant miR-34a was 50 nM.

[0087] (2) RNA extraction (EZNA miRNA Kit, OMEGA miRNA extraction kit)

[0088] Follow the kit instructions. After determining the concentration of the obtained miRNA, store it at -80°C for later use.

[0089] (3) Detection of intracellular processing and maturation of recombinant miR-34a by quantitative real-time PCR (q-PCR).

[0090] The extracted RNA was reverse transcribed into cDNA using the TAKARA reverse transcription kit. The reverse transcription conditions were: 37℃ for 15 min; 80℃ for 15 s. Using cDNA from each group as templates and U6 as an internal control, the expression level of miR-34a in cells was detected by qPCR. The qPCR reaction conditions were: 95℃ for 30 s denaturation; 95℃ for 10 s; 60℃ for 30 s, for 44 cycles. The primer sequences used are shown in Table 2 (the sequences are listed in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16).

[0091] Table 2 Primer sequences used in recombinant miR-34a reverse transcription and Q-PCR detection.

[0092]

[0093] like Figure 4 As shown, compared to the Mock group, E-miR-34a and NC Glu hsa-tRNA Glu All scaffolds showed significant expression. The expression of the other two tRNA scaffolds also showed a consistent trend, demonstrating that the three recombinant small RNAs could be successfully transfected into cells.

[0094] Figure 5This invention uses qPCR technology to detect the processing and maturation of recombinant miR-34a in MDA-MB-231 cells (values are expressed as "mean ± standard deviation"). Compared with the Mock group, the levels of mature miR-34a in all three groups of recombinant miR-34a were significantly increased; compared with the NC Glu group, the level of mature miR-34a expressed in cells after transfection with E-miR-34a was significantly increased, and it was significantly higher than the other two recombinant miR-34a (M-miR-34a and S-miR-34a). There was no significant difference between the three tRNA scaffold controls and the Mock group. Therefore, the results indicate that the recombinant miR-34a constructed using the novel humanized tRNA scaffold hsa-tRNA Glu carrying miR-34a can be successfully transfected into cells and is processed and matured in cells.

[0095] Example 7: Determination of the IC Glu value of hsa-tRNA 50 -miR-34a in inhibiting MDA-MB-231.

[0096] The cell proliferation toxicity detection kit CCK8 was used to detect the effect of recombinant miR-34a on the proliferation and viability of breast cancer cells MDA-MB-231. The specific experimental operations are as follows:

[0097] (1) Cells were seeded into 96-well plates at 3.5×10 3 cells per well, with 6 replicate wells in each group, and placed in a cell culture incubator for culture;

[0098] (2) After waiting for the cells to adhere, according to the experimental grouping: Blank, Mock, NC Glu , E-miR-34a, NC Ser , S-miR-34a, NC Met , M-miR-34a for RNA transfection;

[0099] (3) After 72 h, 10% CCK8 detection solution was added and cultured in a 37°C incubator for 2 h;

[0100] (4) The microplate was gently shaken, and the absorbance values of each well at 450 nm were detected by an ELISA reader for data analysis.

[0101] As Figure 6 and Table 3, the IC 50 value of the E-miR-34a group was 51.44 nM < the IC 50 value of S-miR-34a (56.39 nM) < the IC 50The value (60.39 nM) indicates that E-miR-34a exerts the strongest inhibitory effect on breast cancer cells MDA-MB-231; the IC50 of the three tRNA scaffold control groups was [missing value]. 50 The values ​​are all relatively large, and the results are sorted as follows: NC Met (111.9nM) <NC Ser (116nM) <NC Glu (118.9 nM), indicating that the tRNA scaffold's effect on cell proliferation is weaker than that of the corresponding recombinant miR-34a. The IC50 of each recombinant RNA pair... 50 The ratio is IC 50 Translation value (IC) 50 A shift greater than 1.5 is considered a significant IC (internal shift) indicator. 50 Translation, E-miR-34a and NC Glu The IC50 shift value is 2.31, S-miR-34a and NC Ser IC 50 The translation value is 2.06, M-miR-34a and NC Met IC 50 The shift value was 1.825, indicating that recombinant miR-34a produced a more significant inhibitory effect compared to its tRNA scaffold control. This means that miR-34a is bioactive when integrated into all three tRNA scaffolds, demonstrating the universality of the biosynthetic recombinant small RNA strategy. Furthermore, E-miR-34a and NC... Glu IC 50 The largest translation value indicates that miR-34a and hsa-tRNA are related. Glu The combination exhibits optimal activity and delivers better results.

[0102] Table 3. IC50 of recombinant miR-34a in inhibiting MDA-MB-231 cells 50 value

[0103]

[0104]

[0105] Example 8: has-tRNA in recombinant miR-34a Glu Synergistic effect of stent and miR-34a

[0106] Chou-Talalay is currently a widely accepted method for quantitative analysis of drug synergistic effects. The formula for the two-drug combination index is as follows. In the formula, D is the single dose and Dx is the combined dose.

[0107] Qualitatively and quantitatively describe the drug combination effect based on the dose-effect relationship data obtained by CCK8. Calculate the CI value according to the formula using CompuSyn software. The strength and nature of the interaction between drugs can be quantitatively judged by the CI value (CI > 1 indicates antagonistic effect, CI = 1 indicates additive effect, CI < 1 indicates synergistic effect). Meanwhile, CompuSyn software will draw the Fa-CI graph of the equivalent dose-effect ratio based on the obtained data, which can visually judge the change trend of the combined drug effect. Figure 7 The analysis results showed that the CI value of E-miR-34a < 1, proving that has-tRNA Glu scaffold and miR-34a have a strong synergistic effect.

[0108] Example 9: Effect of hsa-tRNA Glu -miR-34a on the proliferation ability of MDA-MB-231 cells

[0109] The colony formation assay is an experimental method that uses crystal violet staining solution to stain and count cell colonies, which can reflect the proliferation ability of cells. The specific operation steps are as follows:

[0110] (1) According to the experimental grouping: Blank, Mock, NC Met , M-miR-34a, NC Ser , S-miR-34a, NC Glu , G-miR-34a, chemically synthesized miR-34a precursor and mature form, perform RNA transfection. After 12 h of transfection, digest, pipette, and resuspend the cells. Seed 500 MDA-MB-231 cells per well into a 6-well plate, with 3 replicate wells for each group;

[0111] (2) Observe the cell status, change the medium every 3 days until cell colonies appear, which takes about 14 days;

[0112] (3) Fix the cells and then stain them with crystal violet solution;

[0113] (4) Scan and take pictures, and count the number of cell colonies.

[0114] Figure 8 The results showed that compared with the chemically synthesized miR-34a mature form, the cell colony formation of each group of biosynthetic recombinant miR-34a was significantly reduced, indicating that the bioactive recombinant miR-34a has stronger activity than the chemically synthesized miR-34a. The number of cell colonies was ranked as E-miR-34a < S-miR-34a < M-miR-34a, indicating that E-miR-34a inhibits cell proliferation the most. NC Met 、NC Ser and NCGlu It also has a certain ability to inhibit cell clone formation, among which NC Glu The effects were stronger. Meanwhile, the results showed that all three recombinant miR-34a variants were superior to their corresponding tRNA scaffold control groups, demonstrating that miR-34a, when chimeric with the tRNA scaffold, synergistically inhibits cell proliferation.

[0115] Example 10: hsa-tRNA Glu The effect of miR-34a on apoptosis in MDA-MB-231 cells

[0116] The best recognized biochemical marker for the early and late stages of apoptosis is the activation of caspases. Caspase 3, an evolutionarily conserved member of the caspase family, plays a well-defined role in apoptosis. The molecular mechanism of apoptosis is triggered by proteolytic enzymes belonging to the caspase 3 family. When inducible caspase cleaves and is activated into cleaved caspase-3, it enters the nucleus and cleaves chromosomal DNA into ladder-like fragments at the nucleosome level, thus initiating apoptosis. Therefore, detecting cleaved caspase-3 is crucial for detecting apoptosis. After RNA transfection of cells according to experimental groups, immunofluorescence staining was performed, and the expression of cleaved caspase-3 protein in each group was detected under a fluorescence microscope. Figure 9 ).

[0117] (1) Cell climbing: The cells were seeded into 24-well plates with 14 mm glass slides placed in advance. After the cells adhered and recovered their normal morphology, RNA transfection was performed according to the experimental groups. After culturing for 48 h, the original culture medium was discarded, and the cells were washed twice with preheated PBS and fixed with preheated 4% paraformaldehyde for 15 min.

[0118] (2) Wash with PBS 3 times, 3 min each time;

[0119] (3) Incubate with 0.5% Triton-X100 for 10 min;

[0120] (4) Clean with 0.1% Triton-X100 3 times, 5 minutes each time;

[0121] (5) Seal with sealing solution for 10 minutes;

[0122] (6) Following the antibody instructions, dilute the antibody with blocking buffer at the appropriate ratio, add 80 μL of antibody dilution buffer to each well, and incubate overnight at 4°C.

[0123] (7) Clean with 0.1% Triton-X100 three times, 5 minutes each time;

[0124] (8) Dilute the fluorescently labeled secondary antibody with blocking buffer at the appropriate ratio according to the instructions. Add 80 μL of antibody dilution buffer to each well and incubate in the dark for 2 hours.

[0125] (9) Clean with 0.1% Triton-X100 three times, 5 minutes each time;

[0126] (10) Dilute Hochest to 5 μg / mL with PBS, add 100 μL to each well, and stain at room temperature for 10 min;

[0127] (11) Clean with 0.1% Triton-X100 three times, 5 minutes each time;

[0128] (12) Remove the cell slide with the cells facing down and fix it onto a glass slide using a mounting medium;

[0129] (13) Take photos under a fluorescence microscope within two weeks to prevent fluorescence quenching.

[0130] Experimental results showed that both the biosynthesized recombinant miR-34a and the corresponding tRNA scaffold control exhibited varying degrees of fluorescence intensity, with the fluorescence intensity ranked from strongest to weakest as follows: E-miR-34a > NC. Glu >M-miR-34a>NC Met >S-miR-34a>NC Ser Among them, E-miR-34a and NC Glu The fluorescence intensity was strongest and mostly found within the nucleus, indicating that E-miR-34a and NC... Glu The recombinant miR-34a significantly upregulated caspase-3, effectively promoting apoptosis. Furthermore, the upregulation effect of recombinant miR-34a was superior to that of the tRNA scaffold control groups, validating the synergistic therapeutic effect of the tRNA scaffold and miR-34a. In contrast, no significant fluorescence signal was observed in the Blank, Mock, chemically synthesized miR-34a precursor, and chemically synthesized mature miR-34a groups, suggesting that the biosynthetic recombinant miR-34a has a superior ability to upregulate cleaved caspase-3 compared to the chemically synthesized miR-34a.

[0131] Example 11: hsa-tRNA Glu The effect of miR-34a on the migration of MDA-MB-231 cells

[0132] The scratch healing assay is a simple method for studying cell migration in vitro. It simulates the migration process of cells during the healing process in vivo. The basic steps are to create a "wound" in a cell monolayer, capture images periodically during cell migration, and compare the images to determine the cell migration rate.

[0133] (1) Use a marker pen to draw two straight horizontal lines on the bottom of the 24-hole plate to divide the holes into three equal parts for subsequent photo positioning.

[0134] (2) Seed about 70% of the cells into the well plates with the lines drawn, and then transfect them with RNA according to the groups.

[0135] (3) After the cell monolayer has covered the bottom of the well plate, use the pipette tip to draw a line vertically to the bottom of the plate. Use preheated PBS to wash away the detached cells, add serum-free culture medium, and place the plate in a cell culture incubator to continue culturing.

[0136] (4) After 0h and 24h, take pictures under an optical microscope and use ImageJ software to calculate the cell migration rate.

[0137] Experimental results show that ( Figure 10 Compared with the Blank group, the chemical synthesis of miR-34a precursor, chemical synthesis of mature miR-34a, and NC Met M-miR-34 did not significantly inhibit cell migration; compared with NC Met In comparison, the other two tRNA scaffold control groups (NC) Ser NC Glu It has a significant inhibitory effect on cell migration, among which NC Glu The best results suggest the presence of has-tRNA. Glu The scaffold exhibits a strong inhibitory effect on cell migration. For recombinant miR-34a, the order of migration inhibition ability is as follows: E-miR-34a > S-miR-34a > M-miR-34a, with E-miR-34a showing the best inhibitory effect. Furthermore, S-miR-34a has a stronger migration-inhibiting effect than NC. Ser E-miR-34a showed stronger migration inhibition than NC. Glu To reiterate, miR-34a can have a synergistic effect when it binds to an active tRNA scaffold.

[0138] Example 12: hsa-tRNA Glu Regulation of MDA-MB-231 cell invasion by miR-34a

[0139] The Transwell invasion assay is the most commonly used method for detecting the invasive ability of tumor cells. The principle is to separate the high-nutrient culture medium and the low-nutrient culture medium with a polycarbonate membrane. Cells are seeded into the low-nutrient culture medium, and the membrane pores are covered with Matrigel to mimic the extracellular matrix. Tumor cells must secrete hydrolytic enzymes and undergo amoeboid movements to pass through the Matrigel-lined membrane. The number of cells entering the lower chamber can reflect the invasive ability of tumor cells.

[0140] (1) Add the diluted Matrigel to the membrane on the upper layer of the transwell chamber and let it solidify in a 37°C incubator for 0.5-1h. Add 500μL of complete culture medium to the lower chamber.

[0141] (2) Digest the pre-transfected RNA cells, resuspend them in serum-free culture medium, add 100 μL of cell suspension to the upper chamber, making sure there are no air bubbles between the upper and lower chambers, and place them in a cell culture incubator to continue culturing.

[0142] (3) After culturing for 18 hours, remove the chamber, discard the supernatant, and gently wipe away the uninvaded cells inside the chamber with a cotton swab; wash the chamber with PBS and fix with 4% paraformaldehyde for 15 minutes.

[0143] (4) Discard the fixative, stain the cells in the lower layer of the chamber with 0.1% crystal violet staining solution for 10 min, wash with PBS 3 times, wait for the chamber to dry, and then take pictures under an optical microscope.

[0144] Experimental results show that ( Figure 11 Compared with the Mock group, the chemically synthesized miR-34a precursor, the chemically synthesized mature miR-34a, and NC Met Neither hsa-tRNA nor M-miR-34a showed significant inhibitory effects on cell invasion. Glu (NC Glu ) and hsa-tRNA Ser (NC Ser The scaffold significantly inhibited the invasion of MDA-MB-231 cells, and its corresponding recombinant miR-34a (S-miR-34a and E-miR-34a) exhibited even stronger inhibitory effects, validating the synergistic therapeutic effect of tRNA scaffold chimeric miR-34a. Furthermore, E-miR-34a and NC... Glu The inhibitory ability is the best, which also shows that the biosynthetic miR-34a is more effective than the chemically synthesized miR-34a.

[0145] Example 13: hsa-tRNA Glu Therapeutic effects of miR-34a on mouse orthotopic breast cancer model

[0146] To further investigate the therapeutic effect of E-miR-34a at the animal level, we constructed an orthotopic mouse model of breast cancer and administered the treatment.

[0147] 5×10 6MDA-MB-231 cells stably expressing GFP and luciferase were injected into the mammary pads (fourth pair of mammary glands) of 8-9 week old female BALB / cnu mice to establish an orthotopic animal model of breast cancer. Ten days after modeling, mice were randomly divided into 7 groups according to tumor size: saline group, NC group, and so on. Met Group, NC Ser Group, NC Glu Mice were divided into four groups: M-miR-34a, S-miR-34a, and E-miR-34a, with eight mice in each group. Each group received the corresponding drug treatment. The drugs were administered every three days for a total of six treatments, at a dosage of 1 mg / kg based on the mouse's body weight. Tumor volume was monitored during treatment, and the intensity of in situ tumor fluorescence signal was measured in vivo after treatment to evaluate the therapeutic effect of recombinant small RNA at the animal level. Experimental results showed ( Figure 12 A, Small animal live imaging Figure 12 B, Statistical analysis of bioluminescent signal intensity), compared with the Saline group, NC Met The bioluminescent signal intensity at the tumor site of the first group of mice showed no significant change, while the bioluminescent signal intensity at the tumor site of the other five groups of mice decreased significantly; compared with NC Met Compared to the control group, the other two tRNA scaffolds (NC) Ser NC Glu The bioluminescent signal intensity at the tumor site was significantly downregulated in the NC group of mice. Glu The treatment was most effective, suggesting that has-tRNA Glu The scaffold has the effect of inhibiting tumor growth; for recombinant miR-34a, the bioluminescent signal intensity at the mouse tumor site was ranked as E-miR-34a < S-miR-34a < M-miR-34a, with E-miR-34a showing the best therapeutic effect; at the same time, the bioluminescent signal intensity of the E-miR-34a group was lower than that of the NC group. Glu The treatment effect in the E-miR-34a group was stronger than that in the NC group. Glu This suggests that at the animal level, miR-34a also has a synergistic effect when it binds to the active tRNA scaffold.

[0148] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.

Claims

1. A method for chimeric expression of recombinant small RNA in *Escherichia coli* using active tRNA, characterized in that, include: Using active tRNA-chimeric functional miRNA precursors as a scaffold, target small RNAs were expressed in Escherichia coli.

2. The method for chimeric expression of recombinant small RNA in Escherichia coli using active tRNA according to claim 1, characterized in that, The small RNA is an RNA with a length of less than 200 nt.

3. The method for chimeric expression of recombinant small RNA in *E. coli* using active tRNA according to claim 1, characterized in that, The tRNA sequence is more than 90% similar to the human glutamate tRNA sequence, which is SEQ ID NO.

2.

4. The method for chimeric expression of recombinant small RNA in *E. coli* using active tRNA according to claim 3, characterized in that, The sequence of the miRNA precursor is a sequence with a similarity of more than 90% to the hsa-miR-34a precursor sequence, which is SEQ ID NO.

3.

5. The method for chimeric expression of recombinant small RNA in *E. coli* using active tRNA according to claim 4, characterized in that, The miRNA precursor is embedded in the anticodon loop of the tRNA, and the embedded sequence is a sequence with a similarity of more than 90% to the sequence of SEQ ID NO.

1.

6. The method for chimeric expression of recombinant small RNA in *E. coli* using active tRNA according to claim 5, characterized in that, Includes the following steps: Step 1: Using the restriction enzyme site at the anticodon loop of the tRNA in the pBSMrnaSeph plasmid, the expression sequence of the hsa-miR-34a precursor is inserted into the pBSMrnaSeph plasmid to construct the expression vector; Step 2: Replace the mature sequence portion of miR-34a in the constructed expression vector with the target small RNA sequence to construct a chimeric target sequence expression vector; transform the chimeric target sequence expression vector into competent E. coli. Step 3: After the E. coli culture and amplification, total RNA was extracted from the bacteria using the saturated phenol method, and the target small RNA was isolated and purified by FPLC.

7. A method for chimeric expression of recombinant small RNA in *E. coli* using active tRNA according to claim 1, 2, 3, 4, 5, or 6, characterized in that, The small RNA is shRNA, miRNA, siRNA, or RNA aptamer.

8. A method for chimeric expression of recombinant small RNA in *E. coli* using active tRNA according to claim 6, characterized in that, The Escherichia coli mentioned in step two is strain HST08 or DH5α.

9. A method for chimeric expression of recombinant small RNA in *E. coli* using active tRNA according to claim 6, characterized in that, In step three, E. coli was cultured in TB medium until the OD reached 1.1–1.

7. The ratio of the volume of saturated phenol used to extract total RNA from the bacteria to the mass of the bacterial sludge was (2–6):1, where the volume unit is mL and the mass unit is g.

10. The use of a recombinant small RNA produced by a method for chimeric expression of recombinant small RNA in Escherichia coli using active tRNA as described in claim 1, 2, 3, 4, 5 or 6 in the preparation of diagnostic reagents, research reagents, prodrugs, drugs, active pharmaceutical ingredients or pharmaceutical compositions.

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