Improved miR-OE skeleton as well as construction method and application thereof

Abstract

The invention relates to a framework based on a miR-30 framework, a construction method of the framework and application of the framework in RNAi (Ribonucleic Acid Interference). The skeleton construction method comprises the following steps: taking an existing shRNAmiR30 vector as a PCR (Polymerase Chain Reaction) template; the method comprises the following steps: designing two pairs of complementary overlapping primers of siRNA containing a target gene, namely primers F1 and R1 and primers F2 and R2, respectively performing amplification, and performing OE-PCR by taking the obtained P1 and P2 as templates and the primers F1 and R2 as upstream and downstream primers to obtain miR-OE. Furthermore, according to the construction method disclosed by the invention, reverse mutation of a stem-loop structure in a miR-OE skeleton and an EcoRI restriction enzyme cutting site is also carried out. The primers required to be synthesized in the miR-OE skeleton and miR-OE-EL construction method are short chains with the length of less than 60nt, the synthesis difficulty and cost are effectively reduced, and the primer design is more flexible; moreover, the interference efficiency of the miR-OE-EL skeleton on the target gene is obviously improved, and the miR-OE-EL skeleton can be well applied to preparation of shRNA drugs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an improved miR-OE skeleton and a construction method and application thereof. Background Art

[0002] RNA interference (RNAi) refers to the gene silencing phenomenon in eukaryotes induced by double-stranded RNA and targeted degradation of homologous complementary mRNA. At present, there are three commonly used gene knockdown tools: small interfering RNA (siRNA) chemically synthesized in vitro. siRNA is transiently expressed in cells and can only produce a short-term gene inhibition effect; short hairpin RNA (shRNA) stably expressed by plasmid vectors is processed by intracellular Dicer enzyme to generate siRNA. Although it can maintain long-term stable gene silencing, most shRNA vectors rely on RNA polymerase III promoter (Pol III, such as U6, H1, etc.) to drive. This type of promoter is a constitutive high-expression promoter that cannot regulate gene expression in specific tissues and its overexpression will non-specifically compete and inhibit the processing elements of endogenous microRNA (miRNA), leading to serious toxic side effects; the shRNA system (shRNAmiR) based on endogenous miRNA precursor (primary miRNA, pri-miRNA) uses pri-miRNA as a skeleton to replace the mature sequence of miRNA with the interference sequence of the target gene. It can be driven by various types of promoters and produce siRNA for gene silencing through the endogenous miRNA processing pathway.

[0003] Compared with the other two types of knockdown tools, the shRNAmiR knockdown system has many advantages; it can be transcribed through a variety of RNA polymerase II or III (Pol II, III) promoters, including constitutive promoters, tissue-specific promoters and inducible promoters; specific promoters such as muscle tissue-specific promoters MHCK7 and ACTA1, liver-specific promoters TBG and ALB, etc., can achieve target gene silencing in specific tissues or cells in vivo; and inducible promoters such as the tetracycline-induced TRE promoter can reversibly regulate gene silencing through the binding or debinding of small molecule drugs. In addition, the Pol II promoter can effectively transcribe and synthesize longer RNA, which can achieve polycistronic tandem expression of two or more shRNAmiRs and knock down multiple target genes at the same time; and the target gene can be co-expressed with polycistronic shRNAmiRs to achieve a single vector and multifunctional application. Based on the above-mentioned spatiotemporal specificity and fine regulation of polycistronic expression, shRNAmiR can effectively improve the efficiency of target gene silencing; in addition, compared with the Pol III promoter, the transcriptional activity of the Pol II promoter is lower, which can avoid the production of oversaturated shRNA and reduce cytotoxicity.

[0004] Although the shRNAmiR knockdown system is more flexible and efficient, its complex construction method seriously restricts its application in vivo and in vitro. The most commonly used shRNAmiR vector is constructed using the miR-30 backbone, and the miR-30 backbone is constructed by introducing XhoI and EcoRI restriction sites into the conservative sequences at the 5′ and 3′ ends of the human endogenous miR-30 (Endo-miR-30) backbone to facilitate cloning and adding targeting sequences, and in order to construct the overhanging structure of the two bases at the 3′ end, the two conservative base pairs before and after the loop structure (Loop) are changed from CU / GG to UA / UA ( Figure 1 A, B); Johanners Zuber's team further relocated the EcoRI restriction site of miR-30 to the non-conserved region at the 3′ distal end, restoring the conserved region at the 3′ proximal end to the natural sequence, which is the "miR-E" skeleton ( Figure 1C), its RNAi efficiency is significantly enhanced compared to miR-30, but it also makes the target sequence that needs to be connected longer. The shRNA sequences of miR-30 and miR-E backbones both contain flanking regions, specific target siRNA sequences and top loop sequences. The commonly used construction method is to chemically synthesize complementary long-chain oligonucleotide chains of more than 110nt, and then use oligonucleotide annealing to form small fragments, and then access the vector through XhoI and EcoRI restriction sites; some studies use PCR amplification method, which requires the synthesis of complementary long-chain oligonucleotide chains of about 100nt as PCR templates, and the synthesis of upstream and downstream primers containing corresponding restriction sites. After amplification, it is cloned through restriction digestion, purification, connection and other steps. The operation is more complicated; it is worth noting that the primer synthesis is greater than 60nt, the synthesis is more difficult, the error probability is higher, and the price is more expensive. Dapeng Wang and other researchers developed a modified miR-AB backbone based on miR-E ( Figure 1 B), it introduces BamHI and Apal restriction sites in the stem, so that the shRNA sequence only contains specific siRNA sequences and circular structures, and two complementary oligonucleotides of 75nt and 67nt are required to be synthesized for cloning, and its RNAi interference effect is not significantly different from that of the miR-E backbone. Although miR-AB shortens the length of the fragment accessed to the vector, the required synthesized oligonucleotide chain is still greater than 60nt; in addition, the restriction site modification in the conservative sequence will have a certain effect on the RNAi structure; and BamHI and Apal restriction sites are introduced on the basis of XhoI and EcoRI restriction sites, making the subsequent multiple cloning site selection for other genes in series less; and there are the same problems as miR-30 and miR-E, the restriction sites of their access fragments are fixed, so the shRNAmiR cloning vector cannot be selected at will, and the subsequent steps of replacing the vector are increased. Therefore, based on the miR-30 backbone, how to control the synthesized oligonucleotide chain required for cloning the shRNA target sequence to a short chain within 60nt, and to change the cloning site at will while retaining the shRNA conservative sequence to the maximum extent, so as to reduce the difficulty and cost of synthesis, make the cloning process more flexible and efficiently silence the target gene, is a key problem that the shRNAmiR system needs to solve. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide an improved miR-OE framework of shRNAmiR based on the miR-30 framework and a construction method thereof, as well as the application of the improved framework in RNAi.

[0006] The technical solutions for achieving the above-mentioned purpose include the following.

[0007] The first aspect of the present invention is to provide a method for constructing a miR-OE framework, comprising the following steps:

[0008] The shRNAmiR vector with miR-30 as the backbone was used as the PCR template;

[0009] Design two pairs of complementary overlapping primers containing siRNA of the target gene, namely primers F1 and R1, and primers F2 and R2, wherein F1 and R2 are respectively provided with restriction sites and can amplify the sequences of 5′UTR and 3′UTR of the miR-30 backbone in the template; the R1 is composed of 19 nucleotides of the fixed loop sequence of the miR-30 backbone, the siRNA of the target gene and the corresponding part of the 5′UTR sequence; the F2 is composed of 19 nucleotides of the fixed loop sequence of the miR-30 backbone, the siRNA of the target gene and the corresponding part of the 3′UTR sequence; the primers F1 and R1 can amplify the full length of the 5′UTR, and the primers F2 and R2 can amplify the full length of the 3′UTR;

[0010] Amplification is performed separately, the primers F1 and R1 are used to amplify to obtain amplification product P1, and the primers F2 and R2 are used to amplify to obtain amplification product P2.

[0011] OE-PCR was performed using amplified products P1 and P2 as templates and primers F1 and R2 as upstream and downstream primers to obtain the full-length fragment of pri-miRNA miR-OE.

[0012] In some of the embodiments, the overhang structures of the two bases on the left and right sides of the 19 nucleotides of the fixed loop sequence in R1 are ta and ta respectively, and the overhang structures of the two bases on the left and right sides of the 19 nucleotides of the fixed loop sequence in F2 are ta and ta respectively.

[0013] In some of the embodiments, the overhang structures of the two bases on the left and right sides of the 19 nucleotides of the fixed loop sequence in R1 are cc and ag, respectively, and the overhang structures of the two bases on the left and right sides of the 19 nucleotides of the fixed loop sequence in F2 are ct and gg, respectively.

[0014] In some embodiments, the 19 nucleotide sequence of the fixed loop sequence in R1 is such as SEQ ID NO: 39 and the 19 nucleotide sequence of the fixed loop sequence of the miR-30 backbone in F2 is such as SEQ ID NO: 40; or

[0015] The 19 nucleotide sequence of the fixed loop sequence in R1 is shown in SEQ ID NO: 37 and the 19 nucleotide sequence of the fixed loop sequence of the miR-30 backbone in F2 is shown in SEQ ID NO: 38.

[0016] In some embodiments, the partial 3′UTR sequence in F2 is shown as SEQ ID NO:41.

[0017] In some embodiments, the partial 3′UTR sequence in F2 is shown as SEQ ID NO:42.

[0018] In some embodiments, the restriction sites of F1 and R2 are XbaI and BamHI, respectively. In the miR-30 skeleton construction method of the present invention, other restriction sites can be replaced according to actual needs without affecting the successful construction of the miR-30 skeleton. For example, the restriction sites can be replaced with: BglII, BstBI, ClaI, EcoRI, EcoRV, HindIII, HpaI, KpnI, MluI, NdeI, NheI, NotI, PacI, PmeI, SalI, SmaI, SpeI, XhoI and the like.

[0019] In some embodiments, the sequence of F1 is shown as SEQ ID NO:1.

[0020] In some embodiments, the sequence of R2 is shown in SEQ ID NO:4.

[0021] The restriction enzyme cleavage sites in the sequences of F1 and R2 can be replaced with other corresponding restriction enzyme cleavage sites as needed.

[0022] The R1 is shown in SEQ ID NO: 25, and the F2 is shown in SEQ ID NO: 26; or

[0023] The R1 is shown in SEQ ID NO: 27, and the F2 is shown in SEQ ID NO: 28; or

[0024] The R1 is shown in SEQ ID NO:25, and the F2 is shown in SEQ ID NO:33; or

[0025] The R1 is shown in SEQ ID NO:27, and the F2 is shown in SEQ ID NO:33.

[0026] The second aspect of the present invention is to provide the miR-OE skeleton obtained according to any of the above-mentioned construction methods.

[0027] In some of the embodiments, the miR-OE backbone is the miR-OE-E backbone as shown in SEQ ID NO:34.

[0028] In some of the embodiments, the miR-OE backbone is the miR-OE-L backbone as shown in SEQ ID NO:35.

[0029] In some of the embodiments, the miR-OE backbone is the miR-OE-EL backbone as shown in SEQ ID NO:36.

[0030] The third aspect of the present invention is to provide the application of the miR-OE skeleton in siRNA.

[0031] The fourth aspect of the present invention is to provide the use of the miR-OE skeleton in the preparation of shRNA drugs.

[0032] The present invention provides a new method for constructing a skeleton based on a miR-30 skeleton. The method utilizes OE-PCR to design corresponding primers. The primers required for synthesis are all short chains with a length of less than 60 nt. The difficulty and cost of synthesis are effectively reduced, and the miR-OE skeleton can be obtained efficiently and at low cost. Moreover, through the construction method of the present invention, especially after the site of the reverse mutation, the interference efficiency of the obtained shRNA on the target gene is significantly improved, and the primer design is more flexible. The restriction sites in F1 and R2 can be replaced at will, making the cloning process more flexible and simple. Through the construction method of the present invention, a variety of miR-OE skeletons can be flexibly obtained, including miR-OE-E skeleton, miR-OE-L skeleton, and miR-OE-EL skeleton. The miR-OE skeleton obtained by the present invention effectively improves the interference efficiency of RNA on the target gene by reverse mutation of the sites of EcoRI and the ring structure in the existing miR-30 skeleton, and can be well used to prepare the corresponding shRNA drug, realize RNAi, and obtain the desired therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of skeleton structure; A: Endo-miR30 skeleton and miR-30 skeleton;

[0034] B: miR-E skeleton and miR-AB skeleton; C: miR-OE skeleton construction method and miR-OE skeleton.

[0035] Figure 2 The map and sequence position of pTRIPZ-RFP-shRNAmiR30_CD274; A is the map of pTRIPZ-RFP-shRNAmiR30_CD274; B is the design of two pairs of primers F1 / R1 and F2 / R2 and their positions in the plasmid sequence.

[0036] Figure 3 This is a schematic diagram of the electrophoresis results of miR-OE amplification results in Example 1.

[0037] Figure 4 This is the sequence alignment result of the interference vector in Example 2.

[0038] Figure 5 This is the sequencing peak diagram of the interference vector in Example 2.

[0039] Figure 6 Schematic diagram of the interference effect of shRNAmiR carried by the miR-OE skeleton on the target gene in Example 2.

[0040] Figure 7 Schematic diagram of the new skeleton structure, where A: new skeleton miR-OE-X; B: new skeleton miR-OE-L; C: new skeleton miR-OE-E.

[0041] Figure 8 This is the sequence alignment result of the interference vector in Example 3.

[0042] Fig. 9 This is the sequencing peak diagram of the interference vector shRNAmiR-SQSTM1-EL1 / EL2 / EL3 / EL4 in Example 3.

[0043] Fig.10 This is the sequencing peak diagram of the interference vector shRNAmiR-TAX1BP1-EL1 / EL2 / EL3 / EL4 in Example 3.

[0044] Fig.11 This is a schematic diagram of the interference effect of shRNAmiR on the target gene after the XhoI, EcoRI and loop structure sites are reversely mutated in Example 3, wherein A: SQSTM1, B: TAX1BP1.

[0045] Fig.12 This is a schematic diagram of the interference effect of shRNAmiR on the target gene after the EcoRI and loop structure sites are simultaneously reverse mutated in Example 4, wherein A: SQSTM1, B: TAX1BP1.

[0046] Fig.13 Schematic diagram of the structure of the new skeleton miR-OE-EL.

[0047] Fig.14 This is the structure of the miR-OE-E skeleton.

[0048] Fig.15 This is the structure of the miR-OE-L skeleton.

[0049] Fig.16 This is the structure of the miR-OE-EL skeleton. DETAILED DESCRIPTION

[0050] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0051] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0052] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0053] Definitions To facilitate understanding of this technology, certain terms and phrases are defined below.

[0054] Overlap extension polymerase chain reaction (overlap extention PCR, OE-PCR), hereinafter referred to as overlapping PCR.

[0055] RNAi: Introducing antisense RNA corresponding to mRNA into cells can cause specific degradation of mRNA, leading to silencing of its corresponding gene. This post-transcriptional gene silencing mechanism (PTGS) is called RNA interference. In RNAi, the antisense strand of siRNA is used to cut the target mRNA to achieve gene silencing. One siRNA strand that is completely complementary to the target RNA in the siRNA is the guide strand, and the other siRNA strand is the passenger strand. The siRNA on the RISC complex is unwound into a single strand, the passenger strand will be cleaved, and the guide strand will be retained.

[0056] The present invention adopts overlap extension polymerase chain reaction (OE-PCR) technology to develop a novel shRNAmiR construction method based on miR-30 skeleton.

[0057] The shRNAmiR construction method includes using an existing shRNAmiR vector containing a miR-30 backbone as a PCR template, using 19 nucleotides of a fixed loop sequence of the miR-30 backbone as an overlapping complementary sequence for OE-PCR, introducing a 21nt target sequence of a silencing gene in the middle of a primer, and using a fixed matching sequence of the miRNA backbone at the tail end of the primer. The miRNA fragment can be obtained by overlapping PCR with front and rear primers, and then can be cloned into any plasmid vector by enzyme cutting.

[0058] The present invention is further described in detail below with reference to specific embodiments.

[0059] Example 1: Construction method of miR-OE skeleton

[0060] Take the construction of shControl (derived from the TurboGFP target sequence of copepod green fluorescent protein variant) as an example, as shown in Table 1 and Figure 1 As shown, the 19nt loop sequence is the overlapping part (the shaded part in Table 1, Figure 1 The orange part in the middle) was extended to the two stem ends, and TurboGFP siRNA (boxed part) and the adjacent part of the miR-30 backbone UTR matching sequence were added to one forward and one antisense sequence in the two pairs of primers respectively, and two complementary overlapping primers were designed as R1 and F2; a complementary matching sequence was designed in the 5′UTR and extended outward to introduce the restriction enzyme site XbaI (wavy part) as the F1 forward primer, and a complementary matching sequence was designed at the end of the 3′UTR region and extended outward to introduce the restriction enzyme site BamHI (wavy part) as the R2 reverse primer; the primers required for synthesis above were all short-chain oligonucleotides (<60nt).

[0061] According to the reaction system and conditions in Table 2, pTRIPZ-RFP-shRNAmiR30_CD274 (plasmid map see Figure 2 A, the design of the two pairs of primers F1 / R1 and F2 / R2 and their positions in the plasmid sequence are shown in Figure 2 A and Figure 2 B) as template, primers F1 and R1 were used to amplify the full length of 5′UTR, siRNA and loop region to obtain P1 (180 bp), and primers F2 and R2 were used to amplify the full length of 3′UTR, siRNA and loop region to obtain P2 (196 bp); OE-PCR was performed using P1 and P2 as templates and outer primers F1 and R2 as upstream and downstream primers, and P1 and P2 were extended and spliced ​​by overlapping chains to form a full-length pri-miRNA fragment, called miR-OE (376 bp) ( Figure 1 C), the electrophoresis results of the amplification are as follows Figure 3As shown; after digestion with XbaI and BamHI, it was cloned into the pCDH-EF1α-MCS-IRES-Puro vector (purchased from SBI System Biosciences, product number CD532A-2) to obtain the shRNA miR negative control plasmid pCDH-EF1-mir30_shControl-IRES-Puro (hereinafter abbreviated as shCtrl).

[0062] Table 1. Primer sequences for synthesis of TurboGFP shRNA (shControl)

[0063]

[0064] Table 2. OE-PCR reaction system and conditions

[0065]

[0066]

[0067] Note: Prime MaxDNA Polymerase was purchased from Takara (Takara R045A).

[0068] In the process of constructing the miR-OE skeleton described in the present invention, only short-chain primers with a length of less than 60 nt need to be synthesized, and the difficulty and cost of synthesis are significantly reduced; and the two restriction enzyme cleavage sites are outside the full-length region of pri-miRNA, without changing its conservative sequence and natural structure, and the restriction enzyme cleavage sites can be changed at will according to the needs of the cloning vector, and the cloning operation is simpler, more convenient and flexible.

[0069] Example 2 Validation of miR-OE Skeleton

[0070] Further, in order to detect the RNAi effect mediated by the miR-OE backbone, the shRNA sequences of 5 target genes (CALCOCO2, NBR1, OPTN, SQSTM1, TAX1BP1) were designed by software, and 4 target sequences were selected respectively (Table 3). As shown in Table 4, the target sequences and their reverse complementary sequences in Table 3 were respectively filled into the overlapping primers R1 and F2 boxes of the corresponding control vectors (R1_P and F2_G were obtained, wherein the boxes were the sense and antisense sequences of siRNA, which could be sequences of 19-21 bases, i.e., NNNNNNNNNNNNNNNNNNNN represents a siRNA sequence of 19-21 bases in length, and the same applies to the following tables); using the OE-PCR method, the reaction system and conditions for amplification were the same as in Table 2: the first round of PCR used the pCDH-EF1-mir30_shContr described in Example 1 ol-IRES-Puro was used as a template (other existing shRNAmiR plasmids containing miR-30 backbones could also be used as templates), and primers F1 and R1_P, F2_G and R2 were used to amplify two fragments respectively; the second round of PCR used the two amplified fragments as templates, and F1 and R2 were used as upstream and downstream primers to amplify the corresponding target sequence of miR-OE; the interference vectors were obtained by restriction digestion and cloned into pCDH-EF1α-MCS-IRES-Puro, and named as shRNAmiR-CALCOCO2-1 / 2 / 3 / 4, shRNAmiR-NBR1-1 / 2 / 3 / 4, shRNAmiR-OPTN-1 / 2 / 3 / 4, shRNAmiR-SQSTM1-1 / 2 / 3 / 4 and shRNAmiR-TAX1BP1-1 / 2 / 3 / 4; and the accuracy of the sequences was then verified by sequencing (sequence alignment results are shown in Figure 4 , sequencing peak diagram see Figure 5 The above interference vector and its corresponding target gene expression plasmid (Table 5) were co-transfected into cells for interference effect detection. The specific steps are as follows (taking shRNAmiR-CALCOCO2-1 as an example): 1) HEK293T cells were counted and expressed at 5×10 5 12 cells / well were plated in a 12-well plate. After 12 h, shRNA miR-CALCOCO2-1 (2 μg) and overexpression plasmid pCMV-CALCOCO2 (human)-3×Flag-Neo (abbreviated as Flag-CALCOCO2) (0.5 μg) were added to 60 μL Opti-MEM (Gibco TM, Cat.No.31985070) was diluted, and then PEI25000 (Polysciences, Inc.23966) was immediately added at a ratio of 1:3 of plasmid (μg): transfection reagent (μL). After mixing, the mixture was allowed to stand at room temperature for 30 minutes and then added to HEK293T cells. After culturing at 37°C for 6 hours, the mixture was replaced with fresh complete medium. After culturing for 48 hours, the cells were lysed to extract total protein; 2) The knockdown level of CALCOCO2 was detected by Western Blot using a Flag tag antibody. Figure 6 As shown, the shRNAmiR carried by the miR-OE skeleton can exert a certain interference effect on its target gene. Since miR-OE is amplified based on the miR-30 skeleton, the right wing conservative region still retains the introduction of the EcoRI restriction site and the mutation of the conservative base pairs on both sides of the loop, and the XhoI restriction site introduced in the left wing region does not affect the conservative sequence but also mutates the skeleton sequence; the mutation of these three sites may affect the target gene interference effect of shRNAmiR; and the miR-OE skeleton construction method of the present invention is not based on the restriction connection of XhoI and EcoRI cloning sites, and the middle Loop region as a complementary pairing sequence of overlapping PCR can also be easily changed through primer design. We speculate that restoring these three mutations to the original miR-30 skeleton sequence may improve its gene silencing efficiency.

[0071] Table 3. shRNA target sequences of each target gene

[0072]

[0073] Table 4. Primer sequences of shRNAs for each target gene

[0074]

[0075]

[0076] Table 5. Overexpression plasmids corresponding to each interference vector in the co-transfection system

[0077]

[0078] Note: All overexpression plasmids in the table were purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0079] Example 3 Construction of miR-OE-X, miR-OE-L and miR-OE-E skeletons and verification of RNAi effects

[0080] In this example, the two target genes SQSTM1 and TAX1BP1 are used as examples to repair these three mutation sites back to the natural sequence and detect their effects on the RNAi effect. As shown in Table 6, the two mutated base pairs TA / TA in the loop structure of overlapping primers R1_P and F2_G are changed back to the natural structure CT / GG (marked in italics), and overlapping primers R1_L and F2_L are obtained; as shown in Table 7, the two bases CG at the XhoI mutation site are changed back to AA (marked in italics), and the 23 bases upstream and downstream of the site are used as complementary overlapping sequences (bold parts) to extend outward to obtain primers R1_X and F2_X; using the constructed target gene shRNAmiR30 vectors as templates, refer to the amplification reaction system and conditions in Table 2, respectively, combined with the outer primers OE-PCR was performed on F1 and R2, and the vectors with loop sequence mutation and XhoI site reversion mutation were obtained (i.e., shRNAmiR-SQSTM1-L1 / L2 / L3 / L4, shRNAmiR-TAX1BP1-L1 / L2 / L3 / L4 and shRNAmiR-SQSTM1-X1 / X2 / X3 / X4, shRNAmiR-TAX1BP1-X1 / X2 / X3 / X4), and the backbones were named miR-OE-L and miR-OE-X, respectively. Figure 7 A, B).

[0081] Since the EcoRI site is relatively close to the stem-loop structure, pCDH-EF1-mir30_shControl-IRES-Puro was used as a template. As shown in Table 8, the base A at the EcoRI mutation site was mutated back to C (in italics), and the 21 bases upstream and downstream of the site were used as complementary overlapping sequences (in bold). Primers R1_CE and F2_CE were extended outwards, and OE-PCR and cloning were performed in combination with the outer primers F1 and R2 to obtain pCDH-EF1-miR30_shControl_E-IRES-Puro; it was used as a control template, and the loop structure was obtained. The structure was an overlapping complementary sequence (shaded part), and the forward overlapping primer F2_E of each target gene was designed (Table 9, the bold font is the site that has been reversed in the control template), and the reverse overlapping primer was R1_P in Table 4. Then, OE-PCR was performed using the outer primers F1 and R2, and cloned into pCDH-EF1α-MCS-IRES-Puro to obtain the interference vectors of each target gene with EcoRI site mutation repaired (i.e., shRNAmiR-SQSTM1-E1 / E2 / E3 / E4 and shRNAmiR-TAX1BP1-E1 / E2 / E3 / E4), and the backbone was named miR-OE-E ( Figure 7 C).

[0082] The above reverse mutation interference vectors have been sequenced to confirm the accuracy of the sequence (sequence comparison results are shown in Figure 8 , sequencing peak diagram see Fig. 9 and Fig.10 ). Further Western Blot was used to detect the silencing level of the target gene, and it was found that the site reversion mutation of EcoRI and the loop structure significantly increased the silencing effect of each shRNA on SQSTM1 ( Fig.11 A) and TAX1BP1( Fig.11 B) interference efficiency; and since XhoI did not affect the conservative sequence, the reverse mutation of its site did not significantly affect the silencing effect of the target gene.

[0083] Table 6. Primer sequences for loop sequence reversion mutation

[0084]

[0085] Table 7. Primer sequences for XhoI site reverse mutation

[0086]

[0087] Table 8. Primer sequences for shControlEcoRI site reversion

[0088]

[0089] Table 9. Primer sequences for EcoRI site reverse mutation

[0090]

[0091] Among them, the 19 nucleotide sequences of the fixed loop sequence are as follows:

[0092] tacatctgtggcttcactaSEQ ID NO:37, tagtgaagccacagatgtaSEQ ID NO:38,

[0093] cccatctgtggcttcacagSEQ ID NO:39,ctgtgaagccacagatgggSEQ ID NO:40.

[0094] Example 4 Construction of miR-OE-EL skeleton and verification of RNAi effect

[0095] In order to further verify the importance of the high conservation of pri-miRNA sequences to gene expression regulation, the EcoRI and loop structure sites were simultaneously reversed. As shown in Table 10, the primer R1_L for loop sequence mutation repair was used to design the corresponding overlapping primer F2_EL for adding EcoRI site repair, and each shRNAmiR30 vector of the SQSTM1 and TAX1BP1 target genes was used as a template, combined with the primers F1 and R2 on both sides, and OE-PCR was performed according to the reaction system and conditions in Table 2, and cloned into pCDH-EF1α-MCS-IRES-Puro (i.e., shRNAmiR-SQSTM1-EL1 / EL2 / EL3 / EL4 and shRNAmiR-TAX1BP1-EL1 / EL2 / EL3 / EL4) and sequenced for verification (sequence alignment results are shown in Figure 8 , sequencing peak diagram see Fig. 9 and Fig.10 ). Western Blot results showed that the co-repair of EcoRI and loop structure sites in shRNA significantly improved the interference efficiency of the two target genes SQSTM1 and TAX1BP1 compared with the separate repair of the two sites, further illustrating that the conservative sequence of pri-miRNA plays an important role in the generation of miRNA ( Fig.12 ). The two pairs of primers in Table 10 were used for OE-PCR. The oligonucleotide chains required for synthesis were all short chains with a length of less than 60 nt, which effectively reduced the difficulty and cost of synthesis. The cloning sites in F1 and R2 were not in the conserved sequence and could be replaced at will, making the cloning process more flexible and simple. The EcoRI and loop structure sites in the existing miR-30 skeleton could be reverse mutated by R1_L and F2_EL, which effectively improved the interference efficiency of RNA on the target gene. The new skeleton obtained by OE-PCR was called miR-OE-EL ( Fig.13 ). Among them, the right segment sequence of F2_EL without reversion mutation is ttgcctactgcctcggaatSEQ ID NO:41, and the right segment sequence of F2_EL with reversion mutation is ttgcctactgcctcggactSEQ ID NO:42.

[0096] Table 10. Primer sequences for reverse mutation of EcoRI site and loop sequence site

[0097]

[0098] The resulting miR-OE-E backbone structure is shown in Fig.14 and SEQ ID NO:34, the miR-OE-L backbone structure is shown in Fig.15 and SEQ ID NO:35, the composition of miR-OE-EL skeleton is shown in Fig.15and SEQ ID NO:36. Figures 14 to 16 In the figure, blue is XbaI restriction site, rose red is XhoI restriction site, yellow is BamHI restriction site, bold is EcoRI site or its reverse mutation, and the wavy subscript is Figure 1 C and Figure 7 The contents with three ellipsis marks on the left and right sides of the three skeleton schematics are red words representing ring structures or their reverse mutations, and the boxes are the sense and antisense sequences of siRNA, which can be sequences of 19-21 bases, that is, NNNNNNNNNNNNNNNNNNNN represents a siRNA sequence with a length of 19-21 bases.

[0099] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for constructing a miR-OE skeleton, characterized in that: The construction method comprises the following steps: The shRNAmiR vector with miR-30 as the backbone was used as the PCR template; Design two pairs of complementary overlapping primers containing siRNA of the target gene, namely primers F1 and R1, and primers F2 and R2, wherein F1 and R2 are sequences with restriction sites, which can amplify the 5′UTR and 3′UTR of the miR-30 backbone in the template; the R1 is composed of 19 nucleotide sequences of the fixed loop sequence of the miR-30 backbone, the siRNA of the target gene and the corresponding part of the 5′UTR; F2 is composed of 19 nucleotide sequences of the fixed loop sequence of the miR-30 backbone, the siRNA of the target gene and the corresponding part of the 3′UTR; primers F1 and R1 can amplify the full length of the 5′UTR, and primers F2 and R2 can amplify the full length of the 3′UTR; Amplification is performed separately, the primers F1 and R1 are used to amplify to obtain amplification product P1, and the primers F2 and R2 are used to amplify to obtain amplification product P2. OE-PCR was performed using amplification products P1 and P2 as templates and primers F1 and R2 as upstream and downstream primers to obtain the PCR product, namely the miR-OE skeleton.

2. The construction method according to claim 1, characterized in that: The overhang structures of the two bases on the left and right sides of the 19 nucleotides of the fixed loop sequence in R1 are ta and ta respectively, and the overhang structures of the two bases on the left and right sides of the 19 nucleotides of the fixed loop sequence in F2 are ta and ta respectively; or The overhang structures of the two bases on the left and right sides of the 19 nucleotides of the fixed loop sequence in R1 are cc and ag, respectively, and the overhang structures of the two bases on the left and right sides of the 19 nucleotides of the fixed loop sequence in F2 are ct and gg, respectively.

3. The construction method according to claim 2, characterized in that: The 19 nucleotide sequence of the fixed loop sequence in R1 is such as SEQ ID NO: 39 and the 19 nucleotide sequence of the fixed loop sequence of the miR-30 backbone in F2 is such as SEQ ID NO: 40; or The 19 nucleotide sequence of the fixed loop sequence in R1 is shown in SEQ ID NO: 37 and the 19 nucleotide sequence of the fixed loop sequence of the miR-30 backbone in F2 is shown in SEQ ID NO:

38.

4. The construction method according to any one of claims 1 to 3, characterized in that: The corresponding partial 3′UTR sequence in F2 is shown in SEQ ID NO:42 or SEQ ID NO:

41.

5. The construction method according to any one of claims 1 to 3, characterized in that: The R1 is shown in SEQ ID NO: 25, and the F2 is shown in SEQ ID NO: 26; or The R1 is shown in SEQ ID NO: 27, and the F2 is shown in SEQ ID NO: 28; or The R1 is shown in SEQ ID NO:25, and the F2 is shown in SEQ ID NO:33; or The R1 is shown in SEQ ID NO:27, and the F2 is shown in SEQ ID NO:

33.

6. The construction method according to any one of claims 1 to 3, characterized in that: The sequence of F1 is as shown in SEQ ID NO: 1, or the sequence of F1 is as shown in SEQ ID NO: 1, but the restriction enzyme cleavage site is different; and / or The sequence of R2 is as shown in SEQ ID NO: 4, or the sequence of R2 is as shown in SEQ ID NO: 4, but the restriction enzyme cleavage site is different.

7. The miR-OE skeleton obtained according to any one of the construction methods of claims 1-6.

8. The miR-OE backbone according to claim 7, wherein The miR-OE backbone is the miR-OE-E backbone as shown in SEQ ID NO: 34; or The miR-OE backbone is the miR-OE-L backbone as shown in SEQ ID NO: 35; or The miR-OE skeleton is the miR-OE-EL skeleton shown in SEQ ID NO:

36.

9. Use of the miR-OE skeleton according to claim 7 or 8 in RNAi.

10. Use of the miR-OE skeleton according to claim 7 or 8 in the preparation of shRNA drugs.

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