CN113789340B - Expression vector of circular RNA hsa_circ_0001741, recombinant engineering bacterium and application thereof - Google Patents

Abstract

The invention provides an expression vector of circular RNA hsa_circ_0001741, which comprises a circular RNA expression sequence connected to a circular RNA expression plasmid; the circular RNA expression sequence is a deoxynucleotide sequence with the nucleotide sequence similarity of more than 90% which is sequentially connected with the 2, 3 and 4 exons of the gene TNPO 3. hsa_circ_0001741 can effectively inhibit migration and proliferation of ccRCC cells, so hsa_circ_0001741 has broad prospects in terms of treatment and prognosis as a patient with renal cancer metastasis.

Description

Expression vector, recombinant engineering bacteria and their applications of circular RNA hsa_circ_0001741

Technical Field

The present invention relates to the field of biomedicine technology, and in particular to an expression vector, a recombinant engineering bacterium and applications thereof of circular RNA hsa_circ_0001741.

Background Art

Renal cell carcinoma (RCC) is one of the common malignant tumors. About 350,000 people are diagnosed with renal cell carcinoma every year worldwide. WHO divides renal cell carcinoma into clear cell carcinoma, papillary cell carcinoma, chromophobe cell carcinoma, collecting duct carcinoma and unclassified renal cell carcinoma based on the origin of tumor cells and their genetic changes. Clear cell renal cell carcinoma (ccRCC) is the most common renal malignancy in clinical practice, of which about 70% of renal cell carcinomas are clear cell renal cell carcinoma (ccRCC). Surgical resection is currently an effective method for treating renal cancer, but nearly 1/3 of renal cancer patients still have distant metastasis at the time of diagnosis, and 20% to 40% will relapse after surgery and develop into metastatic renal cancer. Metastatic renal cancer is insensitive to traditional radiotherapy, chemotherapy and endocrine therapy, and patients have a poor prognosis.

Circular RNA is a type of non-coding RNA. It is a type of circular structure that is covalently bound from head to tail and has neither a 5' cap structure nor a 3' polyadenine tail. As high-throughput sequencing technology is gradually applied to life science and medical research, more and more circular RNAs have been discovered. They play an important role in the onset of diseases, especially malignant tumors, and are potential new therapeutic targets and diagnostic markers. At present, some researchers have focused on the abnormal expression of circular RNA in ccRCC and its relationship with the occurrence, development and metastasis of ccRCC, suggesting that they have broad application prospects in the diagnosis, prognosis and treatment of ccRCC.

Summary of the invention

A circular RNA hsa_circ_0001741 is located on chromosome 7 (chr7): 128655032–128658211, and its parent gene is the linear TNPO3 gene. It has been reported that hsa_circ_0001741 is associated with ovarian cancer drug resistance. The present invention found that the expression of hsa_circ_0001741 decreased in ccRCC, and verified the role of hsa_circ_0001741 in the occurrence and development of ccRCC, and further provided a concept for preparing a new ccRCC therapeutic drug.

The present invention first provides an expression vector of circular RNA hsa_circ_0001741, which comprises a circular RNA expression sequence connected to a circular RNA expression plasmid; the circular RNA expression sequence is a deoxynucleotide sequence with a nucleotide sequence similarity of more than 90% that is sequentially connected to exons 2, 3 and 4 of gene TNPO3.

In one embodiment according to the present invention, the circular RNA expression sequence is SEQ ID NO: 1

GTTCATGCATGGGAGATCTCAGACCAGTTGTTACAGATCCGGCAGGATGTGGAGTCATGCTATTTTGCTGCACAGACATGAAAATGAAGATTCAGACCTCATTTTATGAGCTCCCCACAGACTCTCATGCCTCTTTACGGGACTCATTGCTAACCCATATCCAGAACTTGAAAGACTTGTCACCTGTTATTGTAACGCAGCTGGCTTTAGCAATAGCAGATCTTGCCCTACAGATGCCTTCCTGGAAGGGATGTGTGC AAACACTGGTGGAAAAATACAGCAATGATGTGACTTCTTTGCCTTTTTTGCTGGAGATCCTTACAGTGTTACCTGAAGAAGTACATAGTCGTCCTTACGAATTGGAGCTAATCGGCGCACAGAAATTATAGAAGATTTGGCCTTCTACTCTAGTACAGTAGTATCTCTATTG.

In one embodiment of the present invention, the circular RNA expression plasmid is selected from any one of circular RNA overexpression vectors such as pCD-ciR, pCD2.1-ciR, pLC5-ciR, pLCDH-ciR, pLO-ciR, pCE-RB-Mam, pcDNA3.1(+)CircRNA Mini Vector, etc.

Another aspect of the present invention provides a method for preparing an expression vector expressing the circular RNA hsa_circ_0001741, comprising:

1) Using human genomic cDNA as a substrate and the paired sequences of exons 2 and 4 of the gene TNPO3 as a template, a primer pair was designed, and the circular RNA expression sequence was obtained by PCR amplification;

2) Connecting the circular RNA expression sequence to the circular RNA expression plasmid to obtain the expression vector of hsa_circ_0001741.

In one embodiment of the present invention, specific restriction enzyme cleavage sites are added to the two primers of the primer pair respectively, and the specific restriction enzyme cleavage sites match the restriction enzyme cleavage sites in the circular RNA expression plasmid.

In one embodiment of the present invention, the primer pair is SEQ ID NO: 2 and SEQ ID NO: 3; the restriction enzyme cutting sites are KpnⅠ and BamHI; and the circular RNA expression plasmid is preferably a pCD-ciR vector.

The present invention further provides a recombinant engineered bacterium for amplifying an expression vector of hsa_circ_0001741, comprising the above-mentioned expression vector; the receptor cell can be a prokaryotic receptor cell or a eukaryotic receptor cell, the prokaryotic receptor cell is preferably Escherichia coli, and the eukaryotic receptor cell is preferably yeast; in the present invention, Escherichia coli DH5α competent cells are preferred.

The present invention also provides the use of circular RNAhsa_circ_0001741 or the above-mentioned expression vector in the preparation of a drug for the diagnosis, treatment or prognosis of renal cell carcinoma.

Furthermore, the present invention also provides a diagnostic reagent for diagnosing renal cell carcinoma, comprising a primer pair or a probe for detecting the expression level of hsa_circ_0001741 in a sample; preferably, the primer pair is SEQ ID NO:4 and SEQ ID NO:5; more preferably, the expression level is detected by real-time fluorescence quantitative PCR.

Furthermore, the present invention also provides a drug for the prognosis or treatment of renal cell carcinoma, which comprises hsa_circ_0001741 or the above-mentioned expression vector, and a targeted drug delivery vector; preferably, the targeted drug delivery vector is selected from liposomes, microspheres, microcapsules, nanoparticles or nanocapsules.

The beneficial effects of the above technical solution of the present invention are as follows:

The present invention constructs an hsa_circ_0001741 overexpression vector by using the pCD-ciR vector, and after transfection into ccRCC tumor cells, the metastasis of renal cancer cells can be significantly inhibited. Secondly, by synthesizing siRNA of hsa_circ_0001741 and transfecting it into ccRCC tumor cells, it is found that knocking down hsa_circ_0001741 can significantly increase the migration and proliferation ability of renal cancer cells, which indicates that hsa_circ_0001741 can effectively inhibit the migration and proliferation of ccRCC cells. Therefore, hsa_circ_0001741 has broad prospects in the treatment and prognosis of patients with renal cancer metastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the structure of hsa_circ_0001741 and a map of hsa_circ_0001741 detected by fluorescence quantitative PCR, wherein A is a structural diagram of hsa_circ_0001741, and B is a map of hsa_circ_0001741 detected by fluorescence quantitative PCR.

Figure 2 is a detection map for identifying the circular RNA characteristics of hsa_circ_0001741; wherein A is a RibonucleaseR (Rnase R) digestion experiment result diagram; B is an Oligo dT primer reverse transcription experiment result diagram; C is a genome amplification experiment result diagram; D is an actinomycin D experiment result diagram; E is a Sanger sequencing identification hsa_circ_0001741 circularization detection map.

Figure 3 is a detection map of significant low expression of hsa_circ_0001741 in ccRCC tumor tissues; wherein, A to B are: the expression of hsa_circ_0001741 in ccRCC tissues.

Figure 4 is a graph showing the results of validating the effect of hsa_circ_0001741 on ccRCC tumor cells; A to C show the effects of interfering with or overexpressing hsa_circ_0001741 in ccRCC tumor cells on cell migration ability; and D shows the effect of stable transfection of hsa_circ_0001741 in ccRCC cells on cell proliferation ability.

DETAILED DESCRIPTION

In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

Reagents used in the present invention

Example 1 Detection of hsa_circ_0001741 expression by fluorescent quantitative PCR

1. Design the primers for hsa_circ_0001741. The primer sequences are:

F：TAATCGGCGCACAGAAATTA(SEQ ID NO:4)

R：AATAGCATGACTCCACATCCTG(SEQ ID NO:5)

Fluorescence quantitative PCR and agarose gel electrophoresis were used to verify the primer specificity. The specific steps are as follows:

1) Fluorescence quantitative PCR reaction system:

10μL system, SYBR 5.0μL, Forward primer (5μM) 0.5μL, Reverse primer (5μM) 0.5μL, ddH2O 2.0μL, cDNAs 2.0μL.

2) Reaction conditions:

95℃, 30s;

95°C, 10 s (40 cycles);

58°C, 10s;

72°C, 10 s, fluorescence was collected at 72°C in a cycle.

Melting curves were collected, and the temperature increased from 60°C to 95°C with an increase of 0.5°C in each cycle, for a total of 71 cycles.

2. Agarose gel electrophoresis:

1) Prepare 2% agarose gel:

Prepare and mix according to the ratio of agarose:TAE=1g:50mL, heat in a microwave oven until the solution is clear, add nucleic acid dye (10000x), the concentration of nucleic acid dye working solution is 1x, mix well and pour the mixture into a mold, take it out and place it in an electrophoresis instrument after the liquid cools and solidifies.

2) Electrophoresis:

Take the above fluorescent quantitative PCR product, add 1uL 10x Loading buffer to each well, mix well, load the sample, and perform electrophoresis at 120V for 30min. After the electrophoresis, take out the gel for development.

3) Results:

As shown in Figure 1, the primers designed according to the hsa_circ_0001741 structure, agarose gel electrophoresis showed that the product size was between 100bp-150bp, consistent with the expected 128bp.

Example 2 Identification of circular RNA characteristics of hsa_circ_0001741

1. Ribonuclease R (RNase R) digestion to identify the circularization of hsa_circ_0001741:

After extracting total RNA from Caki-1 and RCC-JF cells using the TrizoL method, digest the cells according to the following system:

1) Digestive system:

Mock group: 10×buffer 1.5μL, RNA 5μg, ddH ₂ O up to 15μL;

Rnase R group: 10×buffer 1.5μL, RNA 5μg, Rnase R 6U, ddH ₂ O up to 15μL.

2) Reaction conditions:

37℃, 10min; 85℃, 5s; store at 4℃.

After digestion, take 5 μL of the product for 1% agarose gel electrophoresis to detect the RNA digestion effect. Take another 5 μL of the digested product and perform reverse transcription according to the following system:

10μL system, 5×PrimeScript buffer 2μL, Random 6mers (100μM) 2.5μL

Enzyme 0.5μL, RNA 5μL.

Reaction conditions:

37°C, 15 min;

85℃, 5s;

Store at 4°C.

After adding 40 μL of nuclease-free water to the reverse transcription product for dilution, the expression of hsa_circ_0001741 and GAPDH was quantitatively detected (dye method) according to the following system, with three replicate wells in each group. 10 μL system, SYBR 5.0 μL, Forward primer (5 μM) 0.5 μL, Reverse primer (5 μM) 0.5 μL, ddH2O 2.0 μL, cDNAs 2.0 μL.

Reaction conditions:

95℃, 30s;

95°C, 10 s (40 cycles);

58°C, 10s;

72°C, 10 s, fluorescence was collected at 72°C in a cycle.

Melting curves were collected, and the temperature increased from 60°C to 95°C with an increase of 0.5°C in each cycle, for a total of 71 cycles.

Synthetic primer sequences

Primer name SEQ ID NO. Sequence 5'-3' Linear-TNPO3-F 6 AGATCTTGCCCTACAGATGCCTT Linear-TNPO3-R 7 AATTTCTGTGCGCCGATTAGCTC GAPDH-F 8 CCACTCCTCCACCTTTGAC GAPDH-R 9 ACCCTGTTGCTGTAGCCA

2. Oligo dT primer reverse transcription and Sanger sequencing to identify the hsa_circ_0001741 ring:

1) Perform reverse transcription with the same RNA sample in the following groups (10uL system):

Group 1: 5×PrimeScript buffer 2μL, Random 6mers (100μM) 2.5μL, Enzyme 0.5μL, H2O up to 10μL;

Group 2: 5×PrimeScript buffer 2μL, Oligo dT Primer (50μM) 2.5μL, Enzyme 0.5μL, H2O up to 10μL.

2) Reaction conditions:

37℃, 15min; 85℃, 5s; store at 4℃.

Add 40 μL of nuclease-free water to the reverse transcription product for dilution.

3. Quantitative detection of hsa_circ_0001741 and linear TNPO3 expression by the following system (dye method):

10 μL system, SYBR 5.0 μL, Forward primer (5 μM) 0.5 μL, Reverse primer (5 μM) 0.5 μL, ddH2O 2.0 μL, cDNAs 2.0 μL.

Reaction conditions:

95℃, 30s;

95°C, 10 s (40 cycles);

58°C, 10s;

72°C, 10 s, fluorescence was collected at 72°C in a cycle.

Melting curves were collected, and the temperature increased from 60°C to 95°C with an increase of 0.5°C in each cycle, for a total of 71 cycles.

3) The final PCR product obtained after reverse transcription with random primers is subjected to Sanger sequencing.

4. Genome amplification experiment to identify hsa_circ_0001741 ring:

DNA extraction kit (spin column type) (Tian Gen) was used to extract DNA from Caki-1 and RCC-JF cells:

1) Digest Caki-1 and RCC-JF cells with trypsin, wash, centrifuge at 500 g for 5 min, discard the supernatant, add 200 μL buffer GA, and mix thoroughly.

2) Add 20 μL of proteinase K and mix; add 200 μL of buffer GB, mix by inversion, incubate at 70°C for 10 min, and centrifuge immediately; add 200 μL of anhydrous ethanol, mix thoroughly by oscillation for 15 s, and centrifuge immediately.

3) The lysate was transferred to an adsorption column, centrifuged at 12000 rpm for 30 s at room temperature, and the supernatant was discarded; 500 μL of buffer GD was added to the adsorption column, centrifuged at 12000 rpm for 30 s at room temperature, and the supernatant was discarded.

4) Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 s at room temperature, discard the supernatant, and repeat once; centrifuge the empty column at 12000 rpm for 2 min, discard the waste liquid, and dry at room temperature for 5 min.

5) Transfer the adsorption column to a new EP tube, add 50 μL of elution solution, place at room temperature for 5 minutes, and then centrifuge at 12,000 rpm for 2 minutes. Collect the solution in a centrifuge tube, which is the total DNA of Caki-1 and RCC-JF cells.

6) Determine the concentration and quality of DNA using NanoDrop One.

5. Perform conventional PCR according to the following reaction system to detect the expression of hsa_circ_0001741 and linear TNPO3 respectively:

10μL system, rTaq 5.0μL, Forward primer (5μM) 0.5μL, Reverse primer (5μM) 0.5μL, ddH2O 2.0μL, cDNAs/gDNAs 2.0μL.

Reaction conditions:

95℃, 3min;

95°C, 30 s (40 cycles);

58℃, 30s;

72℃, 1min;

72℃, 5min;

4℃,∞.

The above PCR products were subjected to 2% agarose gel electrophoresis.

6. Effect of actinomycin on the stability of hsa_circ_0001741:

1) Caki-1 and RCC-JF cells were plated in 12-well plates at 2.5×10 ⁵ cells/well.

2) After overnight culture, actinomycin (2 μg/mL) was added to the cells, and the same volume of DMSO was added to the control group and culture was continued.

3) After 0h, 3h, 6h, and 12h of actinomycin treatment, total cell RNA was extracted and the expression of hsa_circ_0001741 and linear TNPO3 was detected by qRT-PCR.

7. Results:

As shown in Figure 2, after RNase digestion, hsa_circ_0001741 was more resistant to RNase digestion than its parent gene Linear-TNPO3, proving that hsa_circ_0001741 was more stable; the reverse transcription experiment with Oligo dT primers proved that Oligo dT could reverse transcribe the linear gene but could not reverse hsa_circ_0001741, proving that compared with Linear-TNPO3, hsa_circ_0001741 lacked the poLy A tail, so Oligo hsa_circ_0001741 could not be detected in the dT reverse transcription product; the genome amplification experiment further demonstrated the circularization of hsa_circ_0001741, so the product could not be amplified using the primers of hsa_circ_0001741 in genomic DNA; the actinomycin D experiment confirmed that hsa_circ_0001741 was more stable than Linear-TNPO3, and the Sanger sequencing results confirmed the circularization of hsa_circ_0001741.

Example 3 hsa_circ_0001741 is significantly underexpressed in ccRCC tumor tissues and is associated with tumor grade

1. Extract total RNA from tissues or cells:

1) Carefully remove the renal cancer and adjacent tissues from liquid nitrogen, chop them into small pieces, and place them into a tissue homogenate tube containing 1 mL of TrizoL. Label the tube and pre-cool it on ice.

2) Place the homogenization tubes containing TrizoL and tissue samples symmetrically in a tissue homogenizer. Set the parameters to oscillate at 5000 rpm for 15 seconds. After one action is completed, remove the homogenization tube and place it on ice for more than 2 minutes to cool down.

3) Repeat step 2) at least 2 times until the tissue is basically broken.

4) Pipette the lysate without tissue fragments into a new 1.5 mL EP tube without enzyme. Cell RNA extraction starts from this step, and the subsequent steps are the same.

5) Add 200 μL of chloroform to the TrizoL-tissue/cell lysate, vortex for 30 seconds, and place at room temperature for 10 minutes. Then centrifuge at 12000 rpm, 4°C for 15 minutes.

6) After centrifugation, carefully pipette the supernatant without the buffy coat into a new EP tube, add an equal volume of isopropanol, immediately invert and mix, and allow to stand at room temperature for 10 minutes. Then centrifuge at 12,000 rpm, 4°C for 10 minutes.

7) After centrifugation, observe whether there is white precipitation at the bottom of the EP tube, carefully discard the supernatant, add 1 mL of 75% ethanol for washing, and then centrifuge at 7500 rpm and 4°C for 5 minutes.

8) Repeat step 7) once, discard the supernatant carefully, wait for the ethanol to evaporate completely at room temperature, then add a certain volume of nuclease-free water. Store at -80℃ after aliquoting.

2. Identification of tissue or cell RNA quality and concentration:

1) Turn on the NanoDrop One instrument, wait for the machine to complete the self-check, and click RNA.

2) After blank detection with nuclease-free water, take 1 μL of each sample for measurement. Observe and record RNA concentration and A260/280, A260/230 ratios.

3) After the measurement, clean the instrument with nuclease-free water, exit the measurement interface, and turn off the instrument.

Tissue RNA was reverse transcribed into mRNA according to the following system:

10μL system, 5× PrimeScript buffer 2μL, OLigo dT Primer (50μM) 0.5μL, Random6mers (100μM) 0.5μL, Enzyme 0.5μL, RNA 1μg, ddH ₂ O up to 10μL. Reaction conditions: 37℃, 15min; 85℃, 5s; 4℃ storage.

Add 40 μL of nuclease-free water to the reverse transcription product for dilution.

The expression of hsa_circ_0001741 was quantitatively detected by the following system (dye method):

10 μL system, SYBR 5.0 μL, Forward primer (5 μM) 0.5 μL, Reverse primer (5 μM) 0.5 μL, ddH ₂ O 2.0 μL, cDNAs 2.0 μL.

Reaction conditions:

95℃, 30s;

95°C, 10 s (40 cycles);

58°C, 10s;

72°C, 10 s, fluorescence was collected at 72°C in a cycle.

Melting curves were collected, and the temperature increased from 60°C to 95°C with an increase of 0.5°C in each cycle, for a total of 71 cycles.

3. Results:

As shown in Figure 3, hsa_circ_0001741 was significantly underexpressed in ccRCC renal cancer tissues. After data analysis, it was found that the expression was downregulated by more than 2 times. According to the clinical information of renal cancer tissues, hsa_circ_0001741 was related to the grade of the tumor. The higher the grade of the tumor, the lower the expression of hsa_circ_0001741.

Example 4 Construction of overexpression vector and interfering siRNA of hsa_circ_0001741

1. Construction of hsa_circ_0001741 overexpression vector:

1) Primer design:

Find the gene sequence based on the gene information. According to the restriction site of the vector multiple cloning site, select the appropriate restriction site to add to the 5' end of the primer. According to the restriction site on the pCD-ciR vector, select KpnⅠ and BamHI enzymes for restriction digestion experiments.

The cloning primer sequences of hsa_circ_0001741 are as follows:

F (SEQ ID NO:2):

ggGGTACCTGAAATATGCTATCTTACAGGTTCATGCATGGGAGATCTCA

R (SEQ ID NO:3):

cgGGATCCTCAAGAAAAAATATATTCACCAATAGAGATACTACTGTAC

2) Amplify the target fragment according to the following reaction:

PrimeSTAR Max Premix (2×) 25 μL, F-Primer (10 μM) 2 μL, R-Primer (10 μM) 2 μL, template cDNA 2 μL, ddH ₂ O add to 50 μL.

Reaction conditions:

Pre-denaturation at 98°C for 2 min → 32-34 amplification cycles (denaturation at 98°C for 10 s → annealing at 56°C for 15 s → extension at 72°C for 30-60 s (10 s/kb) → post-extension at 72°C for 10 min). The PCR product was subjected to 1% agarose gel electrophoresis (150 V, 20-30 min), and the results were observed under ultraviolet light. The gel was cut to recover the target fragment.

3) Rubber recycling:

The recovery and purification of all target fragments and backbone vectors in the experiment were performed according to the instructions of the gel recovery kit (taking the Qingke GE0101-200 gel recovery kit as an example):

Cut the target DNA fragment with a clean scalpel and place it in a 1.5mL centrifuge tube (avoid prolonged UV exposure); add 500μL Buffer GL and place in a 65℃ water bath for 5min, gently turning the centrifuge tube upside down until the gel is completely melted; add 250μL Buffer BL to the adsorption column EC, centrifuge at 12000g for 1min, and discard the waste liquid; transfer the solution in the centrifuge tube to the adsorption column, centrifuge at 12,000g for 1min; discard the waste liquid, add 700μL Buffer W2, centrifuge at 12,000g for 1min; repeat the previous step, discard the waste liquid, centrifuge at 12,000g for 2min; place the adsorption column in a new clean 1.5mL centrifuge tube and let it stand at room temperature for 2min; add an appropriate amount (35-50μL) of 60-65℃ preheated ddH ₂ O to the center of the membrane, let it stand at room temperature for 3min, and centrifuge at 12,000g for 2min. The liquid in the centrifuge tube is the recovered DNA target fragment, which can be used immediately or stored at -20℃.

4) Preparation of expression vector backbone and sticky fragments:

The expression vector and the fragments recovered in 3) were double-digested according to the following system: 10×K Buffer (2×) 5μL, BamHI 2.5μL, KpnⅠ 2.5μL, vector (15-20μg)/fragment (total), ddH ₂ O up to 50μL. After 4 hours in a 37℃ water bath, the fragments were usually recovered by PCR cleaning, the vector backbone was subjected to 1% agarose gel electrophoresis and the results were observed under ultraviolet light, and the gel was cut to recover the target fragment. The gel recovery steps were the same as before.

5) Ligation reaction:

The target fragment and the vector backbone were connected using TaKaRa's ligation kit Solution I. The reaction system was as follows: Solution I 5 μL, vector backbone 1 μL, gel recovery product 4 μL, 16°C (PCR instrument/water bath), connection for 2 hours.

6) Transformation of plasmid or ligation product into E. coli DH5α competent cells:

Take out the competent cells from the -80℃ refrigerator and place them on ice to thaw; add the ligation product to be transformed (5-15μL) to the competent cells, mix gently, and place on ice for 15-30min; heat shock at 42℃ for 80-90s; immediately place on ice for 2min; in the clean bench, add 400-800μL of empty LB culture medium to each tube, place at 37℃, 200r/min shaker, incubate for 30-45min; centrifuge at 12,000g for 1min; discard the supernatant, resuspend the bacteria in the remaining 50-100μL liquid, and transfer to the LB solid culture medium containing the corresponding antibiotics, and spread evenly with a sterilized coating rod; culture upside down at 37℃, and single colonies may appear after 12-16h.

7) Small-scale extraction of E. coli plasmid (Kangwei Century Endotoxin Removal Small-scale Extraction Kit):

Pick a single colony and inoculate it into a 15mL centrifuge tube containing 5mL liquid LB medium, and culture it at 37℃, 200r/min shaking overnight (16-18h); centrifuge the bacteria at 12,000g for 1min, and discard the supernatant; resuspend the bacterial pellet in 250μL of Buffer P1; add 250μL Buffer P2, gently invert the centrifuge tube several times, and let it stand at room temperature for 3-5min; add 250μL Buffer E3, gently invert the centrifuge tube several times, and let it stand at room temperature for 5min; centrifuge at 12,000g for 5min, transfer the supernatant to the Endo-Remover FM adsorption column, and centrifuge at 12,000g for 1min; add 200μLPS to the Spin CoLumns DM adsorption column, and centrifuge at 12,000g for 1min; add 225μL isopropanol to the solution from which endotoxin has been removed, mix well, and add Spin CoLumns Place the DM adsorption column in a centrifuge at 12,000 g for 1 min; add 700 μL Buufer PW and centrifuge at 12,000 g for 1 min; discard the waste liquid and centrifuge at 12,000 g for 2 min; place the adsorption column in a clean 1.5 mL centrifuge tube, open the lid and let it stand at room temperature for 5 min; add 50-100 μL 65°C preheated ddH2O on the adsorption membrane, let it stand at room temperature for 5 min, centrifuge at 12,000 g for 2 min, and store at -20°C for later use.

8) The extracted plasmid is sequenced.

2. Overexpression vector and siRNA transfection cell experiments of hsa_circ_0001741:

1) Laying planks:

The cells in good condition were plated at 4×10 ⁵ cells per well (6-well plate) (Note: the number of wells to be plated should be adjusted according to the cell size);

2) Plasmid/siRNA transfection:

After 24 hours of adherent culture, calculate the plasmid volume according to the plasmid concentration (the mass of plasmid added to the 6-well plate should be 2μg). According to the transfection steps, it is divided into two systems, A and B: A: 200μL opti-MEM + 5μL Lipo2000; B: 200μL opti-MEM + XμL plasmid, the A system is mixed and allowed to stand for 5 minutes; the B system is added to the A system, mixed and allowed to stand for 15 minutes; siRNA transfection (siRNA is synthesized by Jima): After 24 hours of adherent culture, mix the transfection reagents according to the following system: A: 200μL opti-MEM + 5μL Lipo2000; B: 200μL opti-MEM + 5μL siRNA, the A system is mixed and allowed to stand for 5 minutes; the B system is added to the A system, mixed and allowed to stand for 15 minutes;

3) Discard the culture medium in the 6-well plate and add 1.6 mL of Opti-MEM culture medium to each well; add 400 μL of the above mixed liquid to each well;

4) After mixing the culture medium, the 6-well plate was placed in a 37°C, 5% CO ₂ cell culture incubator for sterile culture. After 24 hours, the RNA of the cells was extracted by the TrizoL method, and the RNA was reverse transcribed and subjected to quantitative fluorescence PCR according to the method of Example 3 to detect the efficiency of overexpression and knockdown of hsa_circ_0001741.

The sequence of the hsa_circ_0001741 siRNA is:

Sense strand 5' to 3' (SEQ ID NO: 10): CUCUAUUGGUUCAUGCAUGTT;

Antisense strand 5’ to 3’ (SEQ ID NO: 11): CAUGCAUGAACCAAUAGAGTT.

3. Results:

As shown in Figure 4A, the efficiency of knocking down hsa_circ_0001741 can reach 90%, and the efficiency of overexpressing hsa_circ_0001741 can reach more than 3 times, with both efficiencies being acceptable.

Example 5 hsa_circ_0001741 inhibits the migration and proliferation of ccRCC tumor cells

1. Establishment of sh-hsa_circ_0001741 stable renal cancer cell line

1) Infect Caki-1 cells at a ratio of MOI = 1:50, and add PoLybrene (7 μg/mL) at the same time. Replace with fresh complete medium 24 hours after infection.

The sh-hsa_circ_0001741 lentivirus used in the present invention was synthesized by HanBio, and its shRNA sequence is as follows:

sh-hsa_circ_0001741(SEQ ID NO:12)：

GTAGTATCTCTATTGGTTCATGCAT

sh-NC (SEQ ID NO: 13):

TTCTCCGAACGTGTCACGTAA

2) 48 hours after infection, puromycin was added for screening. The puromycin screening concentration was 1.5 μg/mL for Caki-1. One screening cycle was completed until all wild-type cells that were not infected with the virus died. Then, the complete medium containing puromycin was replaced every 2-3 days. After three consecutive generations of screening, the stable strains were frozen.

3) The total RNA of the stable transfected cells and the control group cells was extracted by TrioL method, and the expression of hsa_circ_0001741 was detected by qRT-PCR according to the method in Case 3.

2. Cell proliferation assay:

Proliferation experiments were performed using the CCK-8 kit. Stably transfected hsa_circ_0001741 cells were digested to obtain cell suspensions. The cell volume was set to 3000 cells/well and cultured in a 37°C incubator for 24h, 48h, 72h, 96h and 120h, respectively. The same sample was repeated 3 times. After 24h, 48h, 72h, 96h and 120h of culture, the original culture medium was discarded from each well, and 100uCCK-8 mixed solution (prepared according to the culture medium: CCK-8 reagent = 9:1) was added, and then continued to be cultured in a 37°C cell culture incubator for 2 hours. Finally, the absorbance at 450nm was measured on an ELISA instrument to obtain the corresponding OD value.

3. Cell migration assay:

RCC-JF and Caki-1 cells were transfected with si-hsa_circ_0001741/si-NC and overexpressed hsa_circ_0001741/empty vector, respectively, and the number of cells that could penetrate the membrane after transfection was determined by cell migration assay. The specific steps are as follows:

1) Obtain cells from the cell culture flask by digestion method, and resuspend the cells in RPMI1640 medium without FBS to a cell concentration of 5×105 cells/mL;

2) Add 200 μL of cell suspension to the upper chamber and 600 μL of RPMI1640 cell culture medium containing 10% FBS to the lower chamber, and culture at 37°C for 20-24 h;

3) After the culture is completed, wipe off the non-invasive cells on the upper chamber with a cotton swab, remove the transwells, invert, and air dry;

4) Place the chamber in a 24-well plate with 500 μL of crystal violet staining solution and immerse the membrane in the culture medium staining solution at 37°C for 10 min;

5) Take out the chamber, wash with PBS, take pictures and count.

4. Results:

As shown in Figure 4B to D, the cell migration ability was significantly enhanced after interference with hsa_circ_0001741; on the contrary, the migration ability of renal cancer cells was significantly weakened after overexpression of hsa_circ_0001741; and the proliferation ability of cells was significantly enhanced after stable transfection and interference with hsa_circ_0001741 in renal cancer cells. The above results indicate that hsa_circ_0001741 can inhibit the proliferation and migration of ccRCC renal cancer cells.

The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Sequence Listing

<110> Chongqing Medical University

<120> Expression vector, recombinant engineering bacteria and application of circular RNA hsa_circ_0001741

<141> 2021-08-25

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 432

<212> DNA

<213> Human chromosome 7 (human chr 7)

<400> 1

gttcatgcat gggagatctc agaccagttg ttacagatcc ggcaggatgt ggagtcatgc 60

tattttgctg cacagaccat gaaaatgaag attcagacct cattttatga gctccccaca 120

gactctcatg cctctttacg ggactcattg ctaacccata tccagaactt gaaagacttg 180

tcacctgtta ttgtaacgca gctggcttta gcaatagcag atcttgccct acagatgcct 240

tcctggaagg gatgtgtgca aacactggtg gaaaaataca gcaatgatgt gacttctttg 300

ccttttttgc tggagatcct tacagtgtta cctgaagaag tacatagtcg ttccttacga 360

attggagcta atcggcgcac agaaattata gaagatttgg ccttctactc tagtacagta 420

gtatctctat tg 432

<210> 2

<211> 49

<212> DNA

<213> Artificial Sequence

<400> 2

ggggtacctg aaatatgcta tcttacaggt tcatgcatgg gagatctca 49

<210> 3

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 3

cgggatcctc aagaaaaaat atattcacca atagagatac tactgtac 48

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 4

taatcggcgc acagaaatta 20

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 5

aatagcatga ctccacatcc tg 22

<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 6

agatcttgcc ctacagatgc ctt 23

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 7

aatttctgtg cgccgattag ctc 23

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 8

ccactcctcc acctttgac 19

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 9

accctgttgc tgtagcca 18

<210> 10

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 10

cucuauuggu ucaugcaugt t 21

<210> 11

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 11

caugcaugaa ccaauagagt t 21

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 12

gtagtatctctattggttca tgcat 25

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 13

ttctccgaac gtgtcacgta a 21

Claims (8)

1. The application of circular RNA hsa_circ_0001741 or the expression vector of circular RNA hsa_circ_0001741 in the preparation of medicines for the diagnosis, treatment or prognosis of renal cell carcinoma, the circular RNA expression sequence is SEQ ID NO:1. 2. The application according to claim 1, wherein the expression vector comprises a circular RNA expression sequence connected to a circular RNA expression plasmid. 3. application as claimed in claim 2, is characterized in that, described circular RNA expression plasmid is selected from pCD-ciR, pCD2.1-ciR, pLC5-ciR, pLCDH-ciR, pLO-ciR, pCE-RB- Any of Mam or pcDNA3.1(+) CircRNA MiniVector. 4. A medicine for the prognosis or treatment of renal cell carcinoma, characterized in that it comprises an expression vector of hsa_circ_0001741 or circular RNA hsa_circ_0001741, and a targeted drug delivery vector, and the circular RNA expression sequence is SEQ ID NO:1. 5. The medicine according to claim 4, wherein the targeted drug delivery carrier is selected from liposomes, microspheres, microcapsules, nanoparticles or nanocapsules. 6. The medicine according to claim 5, wherein the targeted drug delivery carrier is liposome lipo2000. 7. The medicine according to claim 4, wherein the expression vector comprises a circular RNA expression sequence connected to a circular RNA expression plasmid. 8. The medicine according to claim 7, wherein the circular RNA expression plasmid is selected from pCD-ciR, pCD2.1-ciR, pLC5-ciR, pLCDH-ciR, pLO-ciR, pCE-RB- Any of Mam or pcDNA3.1(+) CircRNA MiniVector.

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