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 of circular RNA hsa_circ_0001741, recombinant engineering bacterium and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an expression vector of circular RNA hsa_circ_0001741, recombinant engineering bacteria and application thereof.

Background

Renal cell carcinoma (Renal cell carcinoma, RCC) is one of the common malignant tumors, and about 35 ten thousand people are diagnosed each year worldwide as WHO classifies renal cell carcinoma into clear cell carcinoma, papillary cell carcinoma, chromophobe cell carcinoma, collecting duct carcinoma, unclassified renal cell carcinoma, and the like, according to the tumor cell origin and the characteristics of its genetic change. Renal clear cell carcinoma (clear cell Renal Cell Carcinoma, ccRCC) is the most common clinical renal malignancy, with about 70% of renal cell carcinomas being renal clear cell carcinoma (ccRCC). Surgical excision is currently an effective method for treating kidney cancer, but approximately 1/3 of patients with kidney cancer have undergone distant metastasis at the time of diagnosis, and 20% -40% of patients undergo recurrence after surgery and develop metastatic kidney cancer. Metastatic renal cancer is insensitive to traditional radiotherapy, chemotherapy and endocrine treatment, and patient prognosis is poor.

Loop RNA (Circular RNA) is a type of non-coding RNA and is a class of loop structures that are covalently joined end-to-end, without either a 5 'end cap structure or a 3' end polyadenylation tail. With the increasing application of high-throughput sequencing technology in life science and medical research, more and more circular RNAs are discovered, which play an important role in the pathogenesis of diseases, particularly malignant tumors, and are potential new therapeutic targets and diagnostic markers. At present, partial researchers focus on the abnormal expression of circular RNAs in ccRCC and the relation between the circular RNAs and the development and metastasis of the ccRCC, and suggest that the circular RNAs have wide application prospects in the diagnosis, prognosis and treatment of the ccRCC.

Disclosure of Invention

A circular RNA hsa_circ_0001741 is located on chromosome 7 (chr 7): 128655032-128658211, the parent gene of which is the linear TNPO3 gene, and hsa_circ_0001741 has been reported to be associated with ovarian cancer resistance. The invention discovers that hsa_circ_0001741 reduces the expression of ccRCC in research, verifies the effect of hsa_circ_0001741 in the occurrence and development of ccRCC, and further provides a concept for preparing novel ccRCC therapeutic drugs.

The present invention first provides an expression vector for circular RNA hsa_circ_0001741 comprising a circular RNA expression sequence linked 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.

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

GTTCATGCATGGGAGATCTCAGACCAGTTGTTACAGATCCGGCAGGATGTGGAGTCATGCTATTTTGCTGCACAGACCATGAAAATGAAGATTCAGACCTCATTTTATGAGCTCCCCACAGACTCTCATGCCTCTTTACGGGACTCATTGCTAACCCATATCCAGAACTTGAAAGACTTGTCACCTGTTATTGTAACGCAGCTGGCTTTAGCAATAGCAGATCTTGCCCTACAGATGCCTTCCTGGAAGGGATGTGTGCAAACACTGGTGGAAAAATACAGCAATGATGTGACTTCTTTGCCTTTTTTGCTGGAGATCCTTACAGTGTTACCTGAAGAAGTACATAGTCGTTCCTTACGAATTGGAGCTAATCGGCGCACAGAAATTATAGAAGATTTGGCCTTCTACTCTAGTACAGTAGTATCTCTATTG。

In one embodiment according to the 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 and the like.

In another aspect, the present invention provides a method for preparing an expression vector for expressing the above circular RNA hsa_circ_0001741, comprising:

1) Designing a primer pair by taking human genome cDNA as a substrate and the paired sequences of exons 2 and 4 of a gene TNPO3 as templates, and obtaining a circular RNA expression sequence through PCR amplification;

2) And connecting the annular RNA expression sequence to an annular RNA expression plasmid to obtain an expression vector of hsa_circ_ 0001741.

In one embodiment according to the invention, specific restriction sites are added to each of the two primers of the primer pair, which specific restriction sites are matched to restriction sites in the circular RNA expression plasmid.

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

The invention further provides a recombinant engineering bacterium for amplifying the hsa_circ_0001741 expression vector, which comprises the expression vector; the recipient cell may be a prokaryotic recipient cell, preferably E.coli, or a eukaryotic recipient cell, preferably yeast; coli DH 5. Alpha. Competent cells are preferred in the present invention.

The invention also provides application of the annular RNAhsa_circ_0001741 or the expression vector in preparation of medicines for diagnosing, treating or prognosing renal cell carcinoma.

Furthermore, the invention also provides a diagnostic reagent for diagnosing renal cell carcinoma, which comprises a primer pair or a probe for detecting the hsa_circ_0001741 expression level in a sample; preferably, the primer pair is SEQ ID NO. 4 and SEQ ID NO. 5; more preferably, the expression level detection is performed by real-time fluorescent quantitative PCR.

Further, the invention also provides a medicament for prognosis or treatment of renal cell carcinoma, which comprises hsa_circ_0001741 or the expression vector and a targeting administration vector; preferably, the targeted drug delivery carrier is selected from the group consisting of liposomes, microspheres, microcapsules, nanoparticles, or nanocapsules.

The technical scheme of the invention has the following beneficial effects:

according to the invention, the hsa_circ_0001741 overexpression vector is constructed by using the pCD-ciR vector, after the hsa_circ_0001741 overexpression vector is transfected into ccRCC tumor cells, the metastasis of renal cancer cells can be obviously inhibited, secondly, siRNA of hsa_circ_0001741 is synthesized, and after the ccRCC tumor cells are transfected, the hsa_circ_0001741 knocked down is found to obviously increase the migration and proliferation capacity of the renal cancer cells, which indicates that hsa_circ_0001741 can effectively inhibit the migration and proliferation of the ccRCC cells, so that hsa_circ_0001741 has wide prospect in the aspect of treatment and prognosis of patients with renal cancer metastasis.

Drawings

FIG. 1 is a schematic diagram of the structure of hsa_circ_0001741 and a map of the fluorescence quantitative PCR detection hsa_circ_0001741, wherein A is a structure diagram of hsa_circ_0001741, and B is a map of the fluorescence quantitative PCR detection hsa_circ_ 0001741.

FIG. 2 is a detection profile identifying the circular RNA properties of hsa_circ_0001741; wherein A is a chart of results of a ribocleare R (Rnase R) digestion experiment; b is an Oligo dT primer reverse transcription experimental result graph; c is a genome amplification experimental result diagram; d is an actinomycin D experimental result diagram; e identifies hsa_circ_0001741 loop detection profile for Sanger sequencing.

FIG. 3 is a graph showing the detection of significant low expression of hsa_circ_0001741 in ccRCC tumor tissue; wherein A-B are: expression of hsa_circ_0001741 in ccRCC tissue.

FIG. 4 is a graph of the results of verifying the effect of hsa_circ_0001741 on ccRCC tumor cells; wherein, A-C are the influences on the migration capacity of the cells after the hsa_circ_0001741 is interfered or overexpressed in ccRCC tumor cells; d is the effect on the proliferation capacity of the cells after stable transformation in ccRCC cells interferes with hsa_circ_ 0001741.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.

Reagents useful in the present invention

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Example 1 fluorescent quantitative PCR detection of hsa_circ_0001741 expression

1. Designing hsa_circ_0001741 primer, wherein the primer sequence is as follows:

F：TAATCGGCGCACAGAAATTA(SEQ ID NO:4)

R：AATAGCATGACTCCACATCCTG(SEQ ID NO:5)

the specificity of the primer is verified by fluorescent quantitative PCR and agarose gel electrophoresis, and the specific steps are as follows:

1) Fluorescent quantitative PCR reaction system:

10 μL system, SYBR 5.0 μL, forward primer (5 μM) 0.5 μL, reverse primer (5 μM) 0.5 μ L, ddH2O 2.0 μ L, cDNAs 2.0.0 μL.

2) Reaction conditions:

95℃，30s；

95 ℃,10s (40 cycles);

58℃，10s；

fluorescence was collected at 72℃for 10s at 72℃in a cycle.

The dissolution profile was collected and increased from 60 ℃ to 95 ℃ by 0.5 ℃ for 71 cycles per cycle.

2. Agarose gel electrophoresis:

1) Preparing 2% agarose gel:

according to agarose: tee=1 g:50mL of the solution is prepared and mixed, heated in a microwave oven until the solution is clarified, nucleic acid dye (10000 x) is added, the concentration of the working solution of the nucleic acid dye is 1x, the mixed solution is poured into a mold after the mixed solution is uniformly mixed, and the mixed solution is taken out and placed into an electrophoresis apparatus after the liquid is cooled and solidified.

2) Electrophoresis:

taking the fluorescent quantitative PCR product, adding 1uL 10x Loading buffer into each hole, mixing uniformly, and then loading the sample, and carrying out electrophoresis for 120V for 30min. After the electrophoresis, the gel was taken out and developed.

3) Results:

as can be seen from FIG. 1, the agarose gel electrophoresis of the primers designed according to hsa_circ_0001741 structure showed that the product size was between 100bp and 150bp, consistent with the expected 128 bp.

Example 2 identification of circular RNA characteristics of hsa_circ_0001741

1. Ribonfclease R (RNase R) digestion identifies hsa_circ_0001741 as a loop:

after extracting Caki-1 and RCC-JF cell total RNA by Trizol method, the total RNA is digested according to the following system:

1) Digestive system:

mock group: 10 Xbuffer 1.5 mu L, RNA mu g, ddH ₂ O up to 15μL；

Rnase R group: 10 Xbuffer 1.5 mu L, RNA mu g, RNase R6U, ddH ₂ O up to15μL。

2) Reaction conditions:

37 ℃ for 10min;85 ℃,5s; preserving at 4 ℃.

After digestion, 5. Mu.L of the product was subjected to 1% agarose gel electrophoresis, and the effect of RNA digestion was examined. Another 5. Mu.L of digested product was subjected to reverse transcription according to the following system:

10. Mu.L System, 5X PrimeScript buffer, 2. Mu. L, random 6mers (100. Mu.M) 2.5. Mu.L

Enzyme 0.5μL、RNA 5μL。

Reaction conditions:

37℃，15min；

85℃，5s；

preserving at 4 ℃.

After dilution by adding 40. Mu.L of nuclease-free water to the reverse transcription product, the expression of hsa_circ_0001741 and GAPDH was quantitatively examined (dye method) in each of three wells as follows. 10 μL system, SYBR 5.0 μL, forward primer (5 μM) 0.5 μL, reverse primer (5 μM) 0.5 μ L, ddH2O 2.0 μ L, cDNAs 2.0.0 μL.

Reaction conditions:

95℃，30s；

95 ℃,10s (40 cycles);

58℃，10s；

fluorescence was collected at 72℃for 10s at 72℃in a cycle.

The dissolution profile was collected and increased from 60 ℃ to 95 ℃ by 0.5 ℃ for 71 cycles per cycle.

Synthesis of 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 identified hsa_circ_0001741 as a loop:

1) Reverse transcription (10 uL system) was performed with the same RNA samples in the following groupings:

group one: 5X PrimeScript buffer mu L, random mers (100. Mu.M) 2.5 mu L, enzyme 0.5 mu L, H O up to 10. Mu.L;

group II: 5X PrimeScript buffer. Mu.L, oligo dT Primer (50. Mu.M) 2.5. Mu. L, enzyme 0.5.5. Mu. L, H2O up to 10. Mu.L.

2) Reaction conditions:

37 ℃ for 15min;85 ℃,5s; preserving at 4 ℃.

To the reverse transcription product was added 40. Mu.L of nuclease-free water for dilution.

3. The expression of hsa_circ_0001741 and linear TNPO3 was quantitatively determined (dye method) according to the following system:

10 μL system, SYBR 5.0 μL, forward primer (5 μM) 0.5 μL, reverse primer (5 μM) 0.5 μ L, ddH2O 2.0 μ L, cDNAs 2.0.0 μL.

Reaction conditions:

95℃，30s；

95 ℃,10s (40 cycles);

58℃，10s；

fluorescence was collected at 72℃for 10s at 72℃in a cycle.

The dissolution profile was collected and increased from 60 ℃ to 95 ℃ by 0.5 ℃ for 71 cycles per cycle.

3) The final PCR product obtained after reverse transcription of the random primers was subjected to Sanger sequencing.

4. Genomic amplification experiments identified hsa_circ_0001741 as a loop:

DNA extraction kit (centrifugal column) (day root) Caki-1, RCC-JF cell DNA:

1) Pancreatin digests Caki-1, RCC-JF cells, and 500g was centrifuged for 5min after washing and the supernatant was discarded; 200. Mu.L of buffer GA was added thereto and mixed well.

2) Adding 20 mu L of proteinase K, and uniformly mixing; adding 200 mu L of buffer solution GB, mixing the materials evenly in a reverse way, incubating the materials at 70 ℃ for 10min, and then performing instantaneous centrifugation; adding 200 mu L absolute ethanol, fully oscillating and uniformly mixing for 15s, and performing instantaneous centrifugation.

3) Transferring the lysate to an adsorption column, centrifuging at 12000rpm for 30s at room temperature, and discarding the supernatant; to the adsorption column, 500. Mu.L of buffer GD was added, and the mixture was centrifuged at 12000rpm at room temperature for 30 seconds, and the supernatant was discarded.

4) Adding 600 μl of the rinse PW to the adsorption column CB3, centrifuging at 12000rpm at room temperature for 30s, discarding the supernatant, and repeating for one time; centrifuging at 12000rpm for 2min, discarding the waste liquid, and drying at room temperature for 5min.

5) Transferring the adsorption column into a new EP tube, adding 50 mu L of eluent, standing at room temperature for 5min, centrifuging at 12,000rpm for 2min, and collecting the solution into a centrifuge tube to obtain the total DNA of Caki-1 and RCC-JF cells.

6) The concentration and quality of DNA were determined by NanoDrop One.

5. Conventional PCR was performed according to the following reaction system to detect hsa_circ_0001741 and linear TNPO3 expression, respectively:

10. Mu.L of the system, rTaq 5.0. Mu.L, forward primer (5. Mu.M) 0.5. Mu.L, reverse primer (5. Mu.M) 0.5. Mu. L, ddH2O 2.0. Mu. L, cDNAs/gDNAs 2.0. Mu.L.

Reaction conditions:

95℃，3min；

95 ℃,30s (40 cycles);

58℃，30s；

72℃，1min；

72℃，5min；

4℃，∞。

the PCR products were subjected to 2% agarose gel electrophoresis.

6. Effect of actinomycin on stability of hsa_circ_ 0001741:

1) Caki-1, RCC-JF cells were plated in 12-well plates, 2.5X10 ⁵ And/or holes.

2) After overnight incubation, actinomycin (2. Mu.g/mL) was added and the control group was added with the same volume of DMSO and incubation continued.

3) After actinomycin is acted for 0h,3h,6h and 12h, total RNA of cells is extracted respectively, and expression conditions of hsa_circ_0001741 and linear TNPO3 are detected by qRT-PCR.

7. Results:

as shown in FIG. 2, hsa_circ_0001741 is more resistant to RNase digestion than the parent gene Linear-TNPO3, which proves that hsa_circ_0001741 is more stable; the Oligo dT primer reverse transcription experiment proves that Oligo dT can reverse transcribe Linear genes but can not reverse transcribe hsa_circ_0001741, and that compared with Linear-TNPO3, hsa_circ_0001741 lacks the poLy A tail, so that the Oligo dT reverse transcription product can not detect hsa_circ_0001741; the genome amplification experiments further illustrate the looping of hsa_circ_0001741, so that the use of primers for hsa_circ_0001741 in genomic DNA does not amplify the product; actinomycin D experiments demonstrated that hsa_circ_0001741 was more stable than Linear-TNPO3 and Sanger sequencing results demonstrated the looping of hsa_circ_ 0001741.

Example 3hsa_circ_0001741 was significantly underexpressed in ccRCC tumor tissue and was associated with tumor grading

1. Extracting total RNA of tissues or cells:

1) Kidney cancer and paracancerous tissue were carefully removed from liquid nitrogen, minced, and placed in a tissue homogenate tube containing 1mL TrizoL. Marking, and pre-cooling on ice.

2) The homogenization tube containing Trizol and tissue sample was placed symmetrically in the tissue homogenizer. The parameters were set to shake 5000rpm,15s. And after the primary action is finished, taking down the homogenizing pipe, placing the homogenizing pipe on ice for more than 2 minutes, and cooling the homogenizing pipe to the temperature.

3) Repeating step 2) at least 2 times until the tissue is substantially disrupted.

4) Lysates without tissue fragments were aspirated into new enzyme-free 1.5mL EP tubes. Cellular RNA extraction begins with this step and the subsequent steps are identical.

5) 200. Mu.L of chloroform was added to the Trizol-tissue/cell lysate, and the mixture was vortexed for 30 seconds and allowed to stand at room temperature for 10 minutes. Then centrifuged at 12000rpm and 4℃for 15min.

6) After centrifugation, the supernatant without the buffy coat was carefully aspirated into a new EP tube, an equal volume of isopropanol was added, immediately inverted and mixed, and allowed to act at room temperature for 10min. Then centrifuged at 12000rpm and 4℃for 10min.

7) After centrifugation, the bottom of the EP tube was observed for white precipitate, the supernatant was carefully discarded, and 1mL of 75% ethanol was added for washing. Then, the mixture was centrifuged at 7500rpm at 4℃for 5min.

8) After repeating 7) once, the supernatant was carefully discarded, and after ethanol was completely volatilized at room temperature, a volume of nuclease-free water was added. Packaging, and storing at-80deg.C.

2. Tissue or cellular RNA quality and concentration identification:

1) Starting a NanoDrop One instrument, and clicking RNA after the self-checking of the standby device is completed.

2) After blank detection with nuclease-free water, 1 μl of each sample was assayed. RNA concentration and A260/280, A260/230 ratio were observed and recorded.

3) After the measurement is finished, the instrument is cleaned by the non-ribozyme water, the measurement interface is exited, and the instrument is closed.

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

10. Mu.L system, 5X PrimeScript buffer. Mu.L, OLigo dT Primer (50. Mu.M) 0.5. Mu. L, random 6mers (100. Mu.M) 0.5. Mu. L, enzyme 0.5.5. Mu. L, RNA 1. Mu.g, ddH ₂ O up to 10. Mu.L. Reaction conditions: 37 ℃ for 15min;85 ℃,5s; preserving at 4 ℃.

To the reverse transcription product was added 40. Mu.L of nuclease-free water for dilution.

The expression of hsa_circ_0001741 was quantitatively detected (dye method) as follows:

10 μL system, SYBR 5.0 μL, forward primer (5 μM) 0.5 μL, reverse primer (5 μM) 0.5 μ L, ddH ₂ O 2.0μL、cDNAs 2.0μL。

Reaction conditions:

95℃，30s；

95 ℃,10s (40 cycles);

58℃，10s；

fluorescence was collected at 72℃for 10s at 72℃in a cycle.

The dissolution profile was collected and increased from 60 ℃ to 95 ℃ by 0.5 ℃ for 71 cycles per cycle.

3. Results:

as shown in fig. 3, hsa_circ_0001741 was significantly underexpressed in ccRCC kidney cancer tissue, and was found to be down-regulated by more than 2-fold after data analysis, and it was seen that hsa_circ_0001741 was correlated with tumor grade after arrangement according to clinical information of kidney cancer tissue, and that hsa_circ_0001741 expression was lower as tumor grade was higher.

EXAMPLE 4 construction of hsa_circ_0001741 overexpression vector and interference siRNA

1. hsa_circ_0001741 overexpression vector construction:

1) Primer design:

and finding out the gene sequence according to the gene information. According to the restriction enzyme sites of the vector multiple cloning sites, a proper restriction enzyme site is selected and added to the 5' end of the primer. Two enzymes KpnI and BamHI were selected for cleavage experiments based on the cleavage sites on the pCD-ciR vector.

The cloned primer sequence of hsa_circ_0001741 is as follows:

F(SEQ ID NO:2):

ggGGTACCTGAAATATGCTATCTTACAGGTTCATGCATGGGAGATCTCA

R(SEQ ID NO:3):

cgGGATCCTCAAGAAAAAATATATTCACCAATAGAGATACTACTGTAC

2) The target fragment was amplified according to the following reaction:

PrimeSTAR Max Premix (2X) 25 mu L, F-Primer (10. Mu.M) 2 mu. L, R-Primer (10. Mu.M) 2. Mu.L, template cDNA 2. Mu. L, ddH ₂ O add to 50μL。

Reaction conditions:

pre-denaturation at 98 ℃ for 2min, 32-34 amplification cycles (denaturation at 98 ℃ for 10s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 30-60 s (10 s/kb), extension at 72 ℃ for 10min, and observation of the result under ultraviolet light after 1% agarose gel electrophoresis (150V, 20-30 min) of the PCR product, and gel cutting and recovery of the target fragment.

3) And (3) glue recovery:

all target fragments and skeleton carriers in the test are recovered and purified according to the use instruction operation of the gel recovery kit (taking the qingke GE0101-200 gel recovery kit as an example):

cutting the target DNA fragment by a clean scalpel, and placing the target DNA fragment into a 1.5mL centrifuge tube (avoiding long-time ultraviolet irradiation); adding 500 μl Buffer GL, and water-bathing at 65deg.C for 5min, and gently turning the centrifuge tube up and down until the glue is completely melted; 250 mu L Buffer BL,12000g are added into an adsorption column EC, the solution is centrifuged for 1min, and waste liquid is discarded; transferring the solution in the centrifuge tube into an adsorption column, centrifuging for 1min at 12,000 g; removing waste liquid, adding 700 mu L Buffer W2, 12,000g, centrifuging for 1min; repeating the previous step, discarding the waste liquid, and carrying out air separation for 2min at a speed of 12,000 g; placing the adsorption column into a new clean 1.5mL centrifuge tube, and standing at room temperature for 2min; adding proper amount (35-50 mu L) of ddH preheated at 60-65 ℃ into the center of the membrane ₂ O, standing at room temperature for 3min, centrifuging for 2min at 12,000 g. The liquid in the centrifuge tube is the recovered DNA target fragment, and can be used immediately or stored at-20 ℃.

4) Preparation of expression vector skeleton and adhesive fragment:

the expression vector and the fragment recovered in 3) were subjected to the following systemDouble digestion was performed with 10 XK Buffer (2X) 5. Mu. L, bamH I2.5. Mu. L, kpn I2.5. Mu.L, vector (15-20. Mu.g)/fragment (total), ddH ₂ O up to 50. Mu.L. After 4 hours of water bath at 37 ℃, fragments are generally recovered by PCR cleaning, a result is observed under ultraviolet light after 1% agarose gel electrophoresis is carried out on a carrier skeleton, and target fragments are recovered by cutting gel. The glue recovery step is the same as before.

5) Ligation reaction:

the connection of the target fragment and the carrier skeleton is selected from a connection kit Solution I of TaKaRa company. The reaction system is as follows: sol μtion I5 μL, carrier skeleton 1 μL, gel recovery product 4 μL, 16 ℃ (PCR instrument/water bath), 2h.

6) Transformation of E.coli DH 5. Alpha. Competent cells with plasmid or ligation product:

taking out competent cells from a refrigerator at-80 ℃ and placing the competent cells on ice for melting; adding the connection product (5-15 mu L) to be converted into competent cells, gently mixing, and carrying out ice bath for 15-30 min; heat shock at 42 ℃ for 80-90 s; immediately ice for 2min; adding 400-800 mu L of empty LB culture medium into each tube in an ultra-clean workbench, placing the tubes in a shaking table at 37 ℃ and at 200r/min, and incubating for 30-45 min;12,000g, centrifuging for 1min; discarding the supernatant, and re-suspending the thalli by 50-100 mu L of liquid, transferring the thalli to LB solid culture medium containing corresponding antibiotics, and uniformly coating by using a sterilization coating rod; after inversion culture at 37 ℃ for 12-16 hours, single colony can appear.

7) Small-scale extraction of E.coli plasmid (kang is century endotoxin removal small-scale kit):

picking a single colony, inoculating the single colony into a 15mL centrifuge tube containing 5mL of liquid LB culture medium, and carrying out shaking culture at 37 ℃ and 200r/min for overnight (16-18 h); centrifuging the bacteria at 12,000g for 1min, and discarding the supernatant; the bacterial pellet was resuspended in 250. Mu.L Buffer P1; adding 250 mu L Buffer P2, gently inverting the centrifuge tube for several times, and standing at room temperature for 3-5 min; adding 250 mu L Buffer E3, gently inverting the centrifuge tube for several times, and standing at room temperature for 5min; centrifuging at 12,000g for 5min, transferring supernatant to Endo-reverse FM adsorption column, centrifuging at 12,000g for 1min; 200 mu LPS is added into a Spin CoLumns DM adsorption column, and the mixture is centrifuged for 1min at 12,000 g; adding 225 mu L of isopropanol into the solution from which endotoxin has been removed, uniformly mixing, adding into a Spin CoLumns DM adsorption column, and centrifuging at 12,000g for 1min; 700 μLBuufer PW was added and centrifuged at 12,000g for 1min; discarding the waste liquid, and carrying out air separation for 2min at 12,000 g; placing the adsorption column into a clean 1.5mL centrifuge tube, and standing for 5min at room temperature after opening the cover; adding 50-100 mu L of ddH2O preheated at 65 ℃ on the adsorption film, standing for 5min at room temperature, centrifuging for 2min at 12,000g, and preserving at-20 ℃ for later use.

8) And sequencing and detecting the extracted plasmid.

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

1) And (3) paving:

cells in good condition were packed in 4X 10 per well (6 well plate) ⁵ cells were plated (note: the number of plated wells was adjusted according to the cell size);

2) plasmid/siRNA transfection:

after 24h of adherent culture, the plasmid volume was calculated from the plasmid concentration (6 well plate should be plus plasmid mass 2. Mu.g). Two systems were divided according to transfection procedure A, B: a: 200. Mu.L opti-MEM+5. Mu.L Lipo2000; b:200 mu L opti-MEM+X mu L plasmid, and standing for 5min after the A system is uniformly mixed; adding the system B into the system A, uniformly mixing, and standing for 15min; siRNA transfection (siRNA was synthesized by Ji Ma): after 24h of adherent culture, the transfection reagents were mixed according to the following system: a: 200. Mu.L opti-MEM+5. Mu.LLipo 2000; b:200 mu L opti-MEM+5 mu LsiRNA, and standing for 5min after the A system is uniformly mixed; adding the system B into the system A, uniformly mixing, and standing for 15min;

3) The medium in the 6-well plate was discarded, and 1.6mL of opti-MEM medium was added to each well; 400 mu L of the above-mentioned mixed liquid was added to each well;

4) After the culture solution is evenly mixed, the 6-hole plate is placed at 37 ℃ and 5 percent CO ₂ Sterile culture was performed in a cell incubator. After 24h, the RNA from the cells was extracted by Trizol method, and reverse transcription and fluorescent quantitative PCR were performed on the RNA according to the method of example 3, to detect overexpression and knock-down hsa_circ_0001741 efficiency.

The sequence of siRNA of hsa_circ_0001741 is as follows:

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

the antisense strand 5'to 3' (SEQ ID NO: 11): CAUGCAUGAACCAAUAGAGTT.

3. Results:

as shown in A in FIG. 4, the efficiency of knocking down hsa_circ_0001741 can reach 90%, and the efficiency of over-expressing hsa_circ_0001741 can reach more than 3 times.

Example 5hsa_circ_0001741 inhibits migration and proliferation of ccRCC tumor cells

1. Establishment of sh-hsa_circ_0001741 stably transformed renal cancer cell line

1) At moi=1: 50 to Caki-1 cells, while PoLybrene (7. Mu.g/mL) was added. Fresh complete medium was changed 24h after infection.

The sh-hsa_circ_0001741 lentivirus used in the invention is synthesized by hantao, and the shRNA sequence is as follows:

sh-hsa_circ_0001741(SEQ ID NO:12)：

GTAGTATCTCTATTGGTTCATGCAT

sh-NC(SEQ ID NO:13)：

TTCTCCGAACGTGTCACGTAA

2) After 48h of infection, puromycin is added for screening, puromycin screening concentration: caki-1 is 1.5 mug/mL, one screening period is used until all wild type cells which are not infected by virus die, then the complete culture medium containing puromycin is replaced every 2-3 days, and after 3 continuous screening, stable transgenic strains are frozen.

3) And extracting total RNA of the stable transgenic cells and the control cells by using the TrioL method, and detecting the expression condition of hsa_circ_0001741 by using qRT-PCR according to the method in case 3.

2. Cell proliferation assay:

the proliferation experiment is carried out by using a CCK-8 kit, cells stably transformed hsa_circ_0001741 are taken for digestion, cell suspension is obtained, the cell quantity is set to 3000 cells/hole, the cells are respectively cultured for 24h,48h,72h,96h and 120h in a 37 ℃ incubator, and 3 repeats are carried out on the same sample. After 24h,48h,72h,96h and 120h of incubation, the original medium was discarded from each well, 100uL of CCK-8 mix (prepared according to medium: CCK-8 reagent=9:1) was added, and incubation was continued for 2 hours in a 37℃cell incubator. And finally, measuring the absorbance at 450nm on an enzyme-labeled instrument to obtain a 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, respectively, and the number of cells capable of penetrating the membrane after transfection was determined by cell migration analysis experiments. The specific operation steps are as follows:

1) Obtaining cells from a cell culture flask by a digestion method, and re-suspending the cells by using RPMI1640 culture medium without FBS, wherein the cell concentration is 5 multiplied by 105/mL;

2) 200 mu L of cell suspension is added into an upper chamber, 600 mu L of RPMI1640 cell culture medium containing 10% FBS is added into a lower chamber, and the culture is carried out for 20 to 24 hours at 37 ℃;

3) After the culture is finished, the non-invasive cells on the upper chamber are wiped off by a cotton swab, the transwells are removed, inverted and air-dried;

4) The chamber was placed in a 24-well plate with 500. Mu.L of crystal violet staining solution added thereto, and the membrane was immersed in the medium staining solution at 37℃for 10min;

5) The cells were removed, washed with PBS, photographed and counted.

4. Results:

as shown in fig. 4B-D, the cell migration ability after hsa_circ_0001741 after interference is significantly enhanced; in contrast, the ability of kidney cancer cells to migrate is significantly reduced after overexpression of hsa_circ_0001741; and the proliferation capacity of cells is obviously enhanced after stable transformation and interference of hsa_circ_0001741 in kidney cancer cells, and the results show that hsa_circ_0001741 can inhibit the proliferation and migration of ccRCC kidney cancer cells.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.

Sequence listing

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<141> 2021-08-25

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Claims (8)

1. Use of an expression vector of circular rnahsa_circ_0001741 or circular RNA hsa_circ_0001741, the circular RNA expression sequence of which is SEQ ID No. 1, for the preparation of a medicament for diagnosis, treatment or prognosis of renal cell carcinoma.

2. The use of claim 1, wherein the expression vector comprises a circular RNA expression sequence linked to a circular RNA expression plasmid.

3. The use according to claim 2, wherein the circular RNA expression plasmid is selected from any one of pCD-ciR, pCD2.1-ciR, pLC5-ciR, plcldh-ciR, pLO-ciR, pCE-RB-Mam or pcdna3.1 (+) CircRNA Mini Vector.

4. A medicament for prognosis or treatment of renal cell carcinoma, characterized by comprising an expression vector of hsa_circ_0001741 or a circular RNA hsa_circ_0001741, the circular RNA expression sequence being SEQ ID No. 1, and a targeted drug delivery vector.

5. The drug of claim 4, wherein the targeted drug delivery vehicle is selected from the group consisting of liposomes, microspheres, microcapsules, nanoparticles, and nanocapsules.

6. The medicament of claim 5, wherein the targeted drug delivery vehicle is liposomal lipo2000.

7. The drug of claim 4, wherein the expression vector comprises a circular RNA expression sequence linked to a circular RNA expression plasmid.

8. The drug of claim 7, wherein the circular RNA expression plasmid is selected from any one of pCD-ciR, pCD2.1-ciR, pLC5-ciR, pLCDH-ciR, pLO-ciR, pCE-RB-Mam or pcDNA3.1 (+) CircRNA Mini Vector.

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