CN111893121B

CN111893121B - Circular RNA and application thereof

Info

Publication number: CN111893121B
Application number: CN202010854978.XA
Authority: CN
Inventors: 曾勇; 袁克非; 刘海灵; 徐琳; 兰天; 李慧
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-08-24
Filing date: 2020-08-24
Publication date: 2021-12-03
Anticipated expiration: 2040-08-24
Also published as: CN111893121A

Abstract

The invention discloses a circular RNA and application thereof, wherein the cDNA sequence of the circular RNA is shown as SEQ ID NO: 1, the structure of the circular RNA is SEQ ID NO: 1 is spliced after being transcribed to form an end-to-end circular structure. The invention detects the expression difference condition of the circDLC1 in the liver cancer tissue and the tissue beside the cancer through QPCR, finds that the expression of the circDLC1 in the liver cancer tissue is obviously lower than that of the tissue beside the cancer, shows that the circDLC1 can be used for liver cancer diagnosis, can confirm through migration and invasion experiments that the migration and invasion capacity of the liver cancer cell is obviously inhibited after the circDLC1 is over-expressed, shows that the circDLC1 can be used as a treatment target spot for diagnosis and treatment of hepatocellular carcinoma; by constructing a nude mouse subcutaneous tumor model, a nude mouse liver cancer in-situ model and a nude mouse late lung metastasis model, the circDLC1 overexpression is proved to have an inhibiting effect on the proliferation, invasion and metastasis of liver cancer cells in a nude mouse body.

Description

Circular RNA and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a circular RNA, a kit containing the circular RNA, a pharmaceutical composition and application of the circular RNA as a treatment target in diagnosis and treatment of hepatocellular carcinoma.

Background

Liver cancer is one of the most common malignant tumors worldwide. Primary Hepatocellular carcinoma (HCC) is the major form of liver cancer, accounting for approximately 90% of primary liver cancer. Postoperative recurrence and metastasis of liver cancer are the major causes of poor prognosis, with a five-year survival rate of less than 20%.

Circular RNAs (circRNAs) are a class of noncoding RNAs that do not have a 5 'terminal cap and a 3' terminal poly (A) tail and are covalently linked to form a loop structure. Since the circular RNA has a closed circular structure, it is not easily digested by RNase R enzyme and is more stably present in an organism than linear RNA. An increasing number of studies have demonstrated that circular RNA is aberrantly expressed and plays an important role in many types of cancer. However, the role of circular RNA in liver cancer is poorly understood, and it is necessary to search for circular RNA of hepatocellular carcinoma and screen circular RNA that can be used as a target for therapy to diagnose and treat hepatocellular carcinoma.

Disclosure of Invention

The invention aims to provide a circular RNA, a kit and a pharmaceutical composition containing the circular RNA and application of the circular RNA as a therapeutic target in diagnosis and treatment of hepatocellular carcinoma based on the function and mechanism research of circDLC1 in growth and metastasis of liver cancer cells by the inventor.

The invention is realized by the following technical scheme:

a circular RNA, wherein the cDNA sequence of the circular RNA is shown as SEQ ID NO: 1, the structure of the circular RNA is SEQ ID NO: 1 is spliced after being transcribed to form an end-to-end circular structure.

The circDLC1(CircBase ID: hsa _ circ _0135718) contains the 14 th to 16 th exons of the DLC1 gene, and the cyclized nucleotide sequence thereof has 552 bases, and no report on the function of the circDLC1 exists at present. The cDNA sequence corresponding to circDLC1 is shown as SEQ ID NO: 1, the sequence of circDLC1 is shown in SEQ ID NO: 2, respectively. The structure of circDLC1 is SEQ ID NO: 2 is a circular structure formed by connecting the nucleotide sequences shown in the specification end to end.

The inventor finds that circDLC1 is a circular RNA regulated by KIAA1429 through experimental research. KIAA1429 is an important component of the N6-methyladenosine (m6A) methyltransferase complex, and has been shown to promote the development of hepatocellular carcinoma by enhancing the level of m6A methylation in liver cancer. The tumor tissue of a liver cancer patient in Waxi hospitals proves that the expression of circDLC1 in the cancer tissue is obviously lower than that of a tissue beside cancer, and the fact that the circDLC1 possibly plays an important role in the occurrence and development of liver cancer is suggested. In addition, in vitro experiments show that the over-expressed circDLC1 can inhibit proliferation, migration and invasion of liver cancer cells. In vivo experiments show that the over-expression of circDLC1 can inhibit tumor growth, intrahepatic metastasis and pulmonary metastasis. The inventor discovers through mechanism research that circDLC1 can be combined with RNA binding protein HuR, thereby reducing the interaction of HuR and MMP1 mRNAs and further inhibiting the expression of MMP 1. The experimental results and the mechanism research prove that the circDLC1 plays a vital role in the growth and the transfer of the liver cancer, and therefore, the circDLC1 can be used as a potential drug action target spot for treating the liver cancer.

Based on the research of the circDLC1, the invention provides a kit for diagnosing liver cancer, which comprises a reagent for quantitatively detecting circular RNA hsa _ circ _ 0135718.

Further, the reagent comprises a primer capable of amplifying the circular RNA.

As a preferred embodiment of the primers for amplifying circDLC1 in the present invention, the primers include the following primer pairs:

F：5'-TCGAGACTACGTTGTTTTAAGGGTA-3'(SEQ ID NO：3)

R：5'-AGGGGCTTCAGCTCTTGTTC-3'(SEQ ID NO：4)

further, the kit also comprises an RNA extraction reagent and a reverse transcription reaction system. The RNA extraction reagent is a Trizol reagent, and the reverse transcription reaction system comprises reverse transcriptase, buffer solution, RNase inhibitor, Oligo, dNTPs and the like.

The invention also provides a pharmaceutical composition, which comprises the circular RNA hsa _ circ _0135718 and/or an agent for increasing the expression amount of the circular RNA, and a pharmaceutically acceptable carrier.

The invention further provides application of any one of the pharmaceutical compositions in preparing a medicament for inhibiting proliferation and migration of liver cancer cells or treating liver cancer.

The invention also provides application of the circular RNA in preparing a liver cancer diagnosis kit.

The invention also provides application of the circular RNA in preparing or screening a medicament for treating liver cancer.

The invention also provides application of the reagent for increasing the expression quantity of the circular RNA in preparing a medicament for treating liver cancer.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the invention designs a specific primer capable of amplifying circDLC1, after the primer is used for amplifying to obtain circular RNA circDLC1, a generation sequencing proves the splicing site of the circDLC1, and an RNase R digestion experiment proves that the circDLC1 is a circular RNA molecule with a closed circular structure;

2. the invention detects the expression difference condition of circDLC1 in liver cancer tissues and paracancerous tissues through QPCR, finds that the expression of circDLC1 in the liver cancer tissues is obviously lower than that of the paracancerous tissues, and shows that the circDLC1 can be used for liver cancer diagnosis;

3. according to the invention, the circDLC1 is constructed on a special lentiviral vector for circular RNA to establish a stable cell strain for over-expressing the gene, and a CCK8 proliferation experiment shows that the proliferation capacity of the liver cancer cell is obviously inhibited after the circDLC1 is over-expressed, and a Transwell migration and invasion experiment can prove that the migration and invasion capacity of the liver cancer cell is obviously inhibited after the circDLC1 is over-expressed; therefore, the circDLC1 gene and the expression product thereof can be used as a therapeutic target point for the treatment of hepatocellular carcinoma;

4. according to the invention, the influence of the over-expression of the circDLC1 on the proliferation, invasion and metastasis of liver cancer cells in a nude mouse is examined by constructing a nude mouse subcutaneous tumor model, a nude mouse liver cancer in-situ model and a nude mouse late lung metastasis model, and the over-expression of the circDLC1 gene and an expression product thereof is proved to play a role in inhibiting the proliferation, invasion and metastasis of liver cancer cells in an animal body.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:

FIG. 1 is a diagram of the characteristic structure of circDLC1 in one embodiment of the present invention;

FIG. 2 is a sequence diagram of the splicing sites of the amplification product of circDLC1 according to one embodiment of the present invention;

FIG. 3 is a graph comparing the digestion of RNase R on circDLC1 and mDLC1 in one embodiment of the present invention;

FIG. 4 is a graph showing the comparison of the results of QPCR detection of circDLC1 expression in liver cancer tissue and paracancerous tissue in one embodiment of the present invention;

FIG. 5 is a schematic diagram showing the cell proliferation curves of a hepatoma cell line overexpressing circDLC1 and a control cell line in one embodiment of the present invention, wherein the abscissa is the number of days and the ordinate is the absorbance at 450nm detected by a microplate reader;

FIG. 6 is a graph showing the results of experiments on invasion and migration of cells according to an embodiment of the present invention;

FIG. 7 shows the effect of over-expression of circDLC1 on the proliferation of hepatoma tumors in nude mice according to an embodiment of the present invention, wherein (a) the subcutaneous tumorigenesis of nude mice 28 days after subcutaneous injection of hepatoma cells over-expressing circDLC 1; (b) the curve is the subcutaneous tumor volume growth curve of the nude mice;

FIG. 8 is a graph of the effect of over-expression of circDLC1 on intrahepatic tumor metastasis in nude mice in one example of the present invention, wherein (a) is Fluorescence field (Brightfield Fluorescence) showing intrahepatic tumor metastasis results of hepatocellular carcinoma cells over-expressing circDLC1 injected under the liver capsule of nude mice; (b) taking a photo of HE staining of a liver metastatic tumor of a nude mouse; (c) a quantitative analysis chart of an intrahepatic neoplasia experiment is shown, wherein the ordinate is the number of tumors under a fluorescence visual field;

FIG. 9 is a graph of the effect of over-expressing circDLC1 on lung metastasis of tumors in nude mice in an example of the present invention, wherein (a) is a fluorescence field showing lung metastasis results of nude mice injected caudal vein with hepatoma carcinoma cells over-expressing circDLC 1; (b) the photo is a lung metastasis tumor HE staining photo of a nude mouse; (c) the quantitative analysis chart is a lung metastasis experiment, wherein the ordinate is the tumor number under a fluorescence visual field.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

The technology related to the invention is a conventional technical means of molecular cloning, wherein related enzymes, primers, reagents and reaction conditions can be reasonably selected according to the experience of a person skilled in the art without description, related reagent consumables belong to common products sold in the market, and related detection means and instruments are well known and skilled in the art.

All statistical analyses of the invention were performed using GraphPad Prism 8 software. Student's t-test, Wilcoxon signed ranks test, was performed appropriately. A p-value less than 0.05 indicates statistical significance.

Example 1:

in this example, the inventors designed a specific primer pair capable of amplifying circDLC1(circBase ID: hsa _ circ _0135718), amplified a circular RNA of the circDLC1 gene using the primer pair, confirmed the splice site (back-splice site) of the circular RNA by a one-generation sequencing method, and then confirmed that circDLC1 is a circular RNA molecule expressed as a closed circular structure by RNase R digestion experiments.

The specific experimental scheme is as follows:

1. extraction and concentration determination of total RNA of cells

(1) When the cell content of the 6-well plate exceeds 90%, discarding the old culture solution, washing the plate once with PBS, adding 1mL Trizol into each well, and transferring the plate to a 1.5mL EP tube;

(2) the EP tube was left at room temperature for 10 minutes to sufficiently separate the nucleic acid protein complex;

(3) adding 0.2mL of chloroform into an EP tube, shaking for about 10s, and then standing for 5min at room temperature; chloroform is a nonpolar molecule and can effectively inhibit the activity of RNA enzyme, when cell solution added with Trizol is mixed with chloroform, water molecules of protein are removed by the chloroform, so that the protein loses water and is denatured, and the separation of an aqueous phase and an organic phase is accelerated;

(4)12000g, centrifuging for 15min at 4 ℃, wherein the solution of the EP tube is divided into 3 layers, the bottom layer is red organic matter, the upper layer is colorless water phase, and RNA exists in the water phase;

(5) transferring the supernatant (about 450mL) to a new EP tube, adding isopropanol with the same volume, and standing at room temperature for 10 min; isopropanol can absorb water around RNA, so that the RNA is precipitated;

(6)12000g, centrifuging for 10min at 4 ℃, observing white RNA precipitates at the bottom and the side of the tube after centrifugation, and discarding supernatant;

(7) washing the RNA precipitate with 75% DEPC-ethanol solution, 7500g, centrifuging at 4 deg.C for 5min, and removing supernatant;

(8) putting the RNA precipitate in a biological safety cabinet for 5min, airing, adding 20 microliters of DEPC (diethylpyrocarbonate) water to dissolve the RNA, and operating the step on ice;

(9) the purity and concentration of the total RNA of the cells are detected by a ScanDrop100 ultramicro nucleic acid analyzer.

Reverse transcription of cDNA

Preparing a reverse transcription reaction system on ice (taking a 20-mu-L system as an example), and adding prepared cell total RNA for reverse transcription after the preparation is completed, wherein the reverse transcription reaction system is as follows:

the reverse transcription reaction conditions were as follows:

the reaction was carried out at 37 ℃ for 15 minutes, and then at 85 ℃ for 5 seconds. After the temperature is reduced to 4 ℃, the cDNA obtained by the reaction is diluted by 5 times, and then the cDNA is stored in a refrigerator at the temperature of minus 20 ℃.

3. Primer design

Primers were designed using webpage Primer 3.0 and verified using NCBI Blast.

The design principle of the primers is as follows: (1) the GC content of the primer is 50-60%; (2) the length of the primer is 17-25 bp; (3) the Tm of the primer is 57-63 ℃; (4) the position of the primer avoids the tertiary structure of the target sequence; (5) avoid G or C base repetition; (6) avoiding the base at the tail end of the primer as A; (7) the primers and the products avoid forming secondary structures; (8) the length of the product is between 100 and 150 bp; (9) the product was avoided to have 4 single base repeats.

Through the above design principle, a primer 10 pair is designed, preferably, the following primer pairs are adopted in this embodiment, and the primer pairs are synthesized by kyoto medley company:

F：5'-TCGAGACTACGTTGTTTTAAGGGTA-3'(SEQ ID NO：3)

R：5'-AGGGGCTTCAGCTCTTGTTC-3'(SEQ ID NO：4)

in one or more embodiments, amplification can also be performed using primer pairs other than those described above.

4. First generation sequencing

After the cDNA obtained by reverse transcription is amplified by the primer pair, the amplified product is subjected to first-generation sequencing. As shown in fig. 1 and 2, the circDLC1 was derived from

exons

14, 15 and 16 of the DLC1 gene, and the results of the first generation sequencing demonstrated that there was a reverse splicing ligation of circDLC1 and accurately displayed the splice site of the amplified product, indicating that the formation of circDLC1 was not due to a recombination mismatch in the genome.

RNase R digestion test

The RNase R enzyme is an RNase that can digest linear RNA but has little effect on circular RNA.

Mu.g of cellular total RNA was incubated with 10U of RNase R enzyme in a volume of 10. mu.L at 37 ℃ for 30 minutes, followed by warming to 75 ℃ and holding for 10 minutes to inactivate the RNase R enzyme, and finally the effect of RNase R addition on circDLC1 and mDCL1 was analyzed by RT-qPCR. The results of the experiment are shown in FIG. 3, wherein mock is a control group not treated with RNase R enzyme. As can be seen from FIG. 3, the expression of linear mDCL1 was significantly reduced after digestion with RNase R enzyme, whereas circDLC1 was resistant to digestion with RNase R enzyme, confirming that it has a cyclic structure.

Example 2:

in the embodiment, the expression difference of the circDLC1 in the liver cancer tissue and the corresponding para-cancer tissue is detected by fluorescent quantitative pcr (qpcr), and the expression of the circDLC1 in the liver cancer tissue is found to be obviously lower than that in the para-cancer tissue, which indicates that the circDLC1 can be used for liver cancer diagnosis.

The specific experimental scheme is as follows:

1. tissue total RNA extraction and concentration determination

Approved by the ethical review committee of university of Sichuan, written informed consent was provided to each patient according to the policy of the committee, and after the patients were completely informed and signed, 40 cases of liver cancer tissues and paired normal cancer-adjacent tissues after radical surgical resection in Waxi hospital were collected. After clinical collection, samples were stored in liquid nitrogen tanks by pathology laboratory staff and then transferred to a liver surgery experiment at-80 ℃ in a refrigerator.

The total RNA extraction and concentration determination experiment of the tissue comprises the following steps:

(1) adding 1mL Trizol into 1.5mL EP tube, pre-cooling with liquid nitrogen in a mortar, taking out soybean tissue from a refrigerator at-80 deg.C, grinding into powder, and transferring the tissue powder into EP tube;

(2) the EP tube was left at room temperature for about 10 minutes so that the nucleic acid protein complex could be sufficiently separated;

(3) adding 0.2mL of chloroform into an EP tube, shaking for about 10s, and then standing for 5min at room temperature;

(5) transferring the supernatant (about 450mL) to a new EP tube, adding isopropanol with the same volume, and standing at room temperature for 10 min;

(8) putting the RNA precipitate in a biological safety cabinet for 5min, airing, adding 20 mu L of DEPC water to dissolve the RNA, and operating the step on ice;

(9) the RNA purity and concentration were determined using a ScanDrop100 ultramicro nucleic acid analyzer.

QPCR amplification experiment

Tissue RNA reverse transcription was performed using the cDNA reverse transcription reaction in example 1.

The PCR reaction system was prepared on ice (20. mu.L for example), and the cDNA template obtained by reverse transcription was added after the preparation. Wherein, the PCR reaction system is as follows:

the primer pair used for amplifying the circular RNAcircDLC1 was the primer pair preferred in example 1:

F：5'-TCGAGACTACGTTGTTTTAAGGGTA-3'(SEQ ID NO：3)

R：5'-AGGGGCTTCAGCTCTTGTTC-3'(SEQ ID NO：4)

the reaction conditions include:

the first step is as follows: pre-denaturation

5 minutes at 95 ℃;

the second step is that: PCR reaction (40 cycles)

At 95 ℃ for 20 seconds; 60 ℃, 20 seconds; 72 ℃ for 20 seconds.

The third step: analysis of melting curves

5 seconds at 65 ℃; 95 ℃ for 5 seconds.

Quantitative analysis is carried out after PCR amplification. The calculation formula of the relative expression quantity of the target gene is as follows: 2- Δ Ct ═ 2- [ Δ Ct (Δ Ct) Test- (Δct) Control. Wherein, the delta Ct is Ct target-Ct housekeeping, the Ct target is a target gene Ct value, the Ct housekeeping is housekeeping gene Ct value, the delta Ct represents the phase Ct value of each sample target gene relative to housekeeping gene, the delta Ct is (delta Ct) Test- (. DELTA Ct) Control, which represents that the treatment group is normalized relative to the Control group, and 2-delta Ct represents the relative expression quantity of the treatment group relative to the Control group and represents the relative expression multiple of the target gene.

The results of the QPCR test are shown in FIG. 4. As can be seen from FIG. 4, the expression result of circDCL1 in the liver cancer clinical tissue sample 40 shows that the expression of circDC1 in the liver cancer tissue (Tumor tissue) is significantly lower than that of the tissue (Normal tissue) beside the cancer, which indicates that circDC1 has important significance in the occurrence and development of liver cancer, can be used as an ideal prognostic marker for hepatocellular carcinoma patients, and can play a positive role in the diagnosis of hepatocellular carcinoma.

Example 3:

in the embodiment, the circDLC1 is constructed on a special lentiviral vector for circular RNA to establish a stable cell strain for over-expressing the gene, and a CCK8 proliferation experiment shows that the proliferation capacity of the liver cancer cell is obviously inhibited after the circDLC1 is over-expressed, and a Transwell migration and invasion experiment can prove that the migration and invasion capacity of the liver cancer cell is obviously inhibited after the circDLC1 is over-expressed; the experiments show that the circDLC1 gene and the expression product thereof can be used as a treatment target point for treating hepatocellular carcinoma.

The specific experimental scheme is as follows:

1. lentivirus infection

(1) Taking out the lentivirus frozen at-80 ℃, and placing on ice to be melted;

(2) taking out a 24-pore plate paved with cells to be infected one day in advance, and discarding the old culture solution;

(3) adding 0.5mL of PBS into each hole for washing, discarding the PBS after full infiltration, and repeating the operation for 1 time;

(4) add 0.5mL of fresh DMEM medium to each well;

(5) adding appropriate amount of virus solution, mixing gently, and standing at 37 deg.C and 5% CO₂Culturing in a constant-temperature incubator;

(6) 12-16 hours after infection, remove virus-containing medium, add 0.5mL PBS for washing, discard PBS after sufficient infiltration, repeat the procedure 1 time, add 0.5mL fresh DMEM medium to each well, place at 37 deg.C with 5% CO₂Culturing in a constant-temperature incubator;

(7) the infection efficiency was judged by observing the green fluorescence under an inverted fluorescence microscope 48 hours after infection.

Screening of stably infected cell lines with puromycin:

(1) discarding the old culture medium in the 24-hole plate after 24 hours, adding 0.5mL of PBS into each hole for washing, discarding the PBS after full infiltration, and repeating the operation for 1 time;

(2) adding 0.5mL of 0.25% pancreatin solution into each hole, slightly shaking, placing in an incubator at 37 ℃, removing pancreatin when observing cell shrinkage and rounding under a microscope, adding 1mL of fresh DMEM culture solution to repeatedly wash the cells on the bottle wall to completely drop, and uniformly mixing cell suspension;

(3) transferring 250 mu L of cell suspension to a new 24-hole plate, and sequentially adding 4 holes;

(4) each well was supplemented with 500. mu.L of fresh DMEM medium and placed at 37 ℃ with 5% CO₂Culturing in a constant-temperature incubator;

(5) discarding the old culture medium in the 24-hole plate again after 24 hours, adding 0.5mL of PBS into each hole for washing, discarding the PBS after full infiltration, repeating the operation for 1 time, and adding 0.5mL of fresh DMEM culture solution;

(6) sequentially adding puromycin stock solution into 4 wells at concentrations of 0.5. mu.g/mL, 1.0. mu.g/mL, 2.0. mu.g/mL, and 5.0. mu.g/mL, mixing, standing at 37 deg.C and 5% CO₂Culturing in a constant-temperature incubator;

(7) observing the growth and death of cells in each well, selecting the wells with fewer dead cells for amplification culture, and simultaneously confirming that the infection efficiency is 100% under an inverted fluorescence microscope;

(8) and (4) carrying out passage and seed preservation on the stably transformed cell strain after the expanded culture.

CCK-8 cell proliferation assay

Dehydrogenases in mitochondria of living cells can interact with WST-8 compounds, and finally reduce the compounds to hydrophilic formazan dyes, which are yellow after dissolution, and the amount of yellow formazan generated is positively correlated with the number of living cells, i.e., the more the number of living cells, the higher the yellow degree of the solution. This feature is therefore exploited for cell proliferation assays.

(1) 3000 cell lines overexpressing circDLC1 and empty vector cell lines were seeded per well in 96-well plates;

(2) adding 10 mu L of CCK-8 reagent into the corresponding area hole at 1, 2, 3 and 4 days respectively;

(3) standing at 37 deg.C with 5% CO₂The constant temperature incubator is used for culturing for about 2 hours;

(4) finally, the absorbance value is measured at 450nm by using a microplate reader.

The results are shown in FIG. 5, in which LV-Vector is the control cell line infected with the empty Vector, and LV-circDLC1 is the cell line over-expressing circDLC 1. As can be seen from fig. 5, after circDLC1 is over-expressed, the proliferation ability of the liver cancer cells is significantly inhibited, and the proliferation rate of the liver cancer cells is significantly reduced.

3. Cell invasion assay

(1) Selecting a small chamber with the aperture of 8 mu m, washing the small chamber soaked in 75% ethanol in a biological safety cabinet by PBS, irradiating for 2h by ultraviolet, and placing all gun tips in a refrigerator for precooling;

(2) diluting the matrigel according to the dilution of the matrigel, namely DMEM serum-free medium which is 1:5, wherein the step is carried out on ice;

(3) uniformly spreading matrigel on a membrane of the cell, and marking the matrigel for an invasion experiment;

(4) placing the small chamber in a 24-pore plate, and placing the small chamber in an incubator at 37 ℃ to solidify matrigel;

(5) preparing cell suspension by adopting a cell passage method, re-suspending cells by using a culture medium without serum, counting by using a cell counting plate, and calculating the cell concentration;

(6) inoculation of 5X 10 in Chamber containing matrigel⁴Individual cell (migration experiment inoculation 3X 10)⁴Cells), 500 μ L of serum-free DMEM high-glucose medium was added to the chamber;

(7) add 600. mu.L of serum-containing medium to 24-well plates, transfer the chamber to 24-well plates, and place at 37 ℃ in 5% CO₂Culturing in an incubator for 24h, and detecting;

(8) taking out the 24-hole plate, sucking away liquid in the small chamber, and wiping off cells and matrix glue in the small chamber by using a cotton swab;

(9) add 500. mu.L PBS in the chamber and 24-well plate, wash the chamber 2 times;

(10) adding 500 μ L of 4% paraformaldehyde into 24-well plate, placing the chamber into the well containing paraformaldehyde, and keeping the room temperature constant for 20 min;

(11) after fixation, 500 μ L PBS was added to a clean 24-well plate and washed for 2 times, and staining was performed after drying the cells;

(12) adding 500 μ L crystal violet into 24-well plate for dyeing for 10 min;

(13) after dyeing, the chamber was lifted by tweezers and gently rinsed in PBS, and the image was taken after drying.

The experimental results are shown in fig. 6, which proves that the migration and invasion abilities of the liver cancer cells of the cell strain over-expressing circDLC1 are obviously inhibited compared with the cell strain of the control group with empty vector.

Example 4:

in this example, the influence of circDLC1 overexpression on proliferation, invasion and metastasis of liver cancer cells in nude mice was examined by constructing a nude mouse subcutaneous tumor model, a nude mouse liver cancer in-situ model and a nude mouse late stage lung metastasis model.

This example was conducted using 4 to 6 weeks of male nude mice purchased from Beijing Huafukang laboratory animals, Inc.

1. Over-expression of circDLC1 for inhibiting proliferation of liver cancer cells in nude mice

The liver cancer cell line stably overexpressing circDLC1 in example 3 and a control cell line were inoculated to the mid-posterior axillary region of a blood supply-rich region of nude mice. Tumor volume was measured once a week after inoculation (V ═ 1/2 × a × b)²A is a long axis, b is a short axis), the longest and shortest parts of the tumor are measured by a vernier caliper, and a tumor volume growth curve is drawn, so that as shown in figure 7(b), the tumor volume growth rate of the LV-circDLC1 group which over-expresses circDLC1 is obviously slower than that of the LV-Vector of the control group. Nude mice were sacrificed 28 days later and tumors were removed and photographed. As shown in FIG. 7(a), the tumor volume of LV-circDLC1 group overexpressing circDLC1 was significantly smaller than that of control LV-Vector. The experiments show that after the circDLC1 is over-expressed, the tumorigenicity capacity of the liver cancer cells under the skin of a nude mouse is obviously reduced.

2. Over-expression of circDLC1 for inhibiting liver cancer cell transfer in nude mouse liver

The anesthetized nude mice were mounted on a hot plate in a supine position. The skin of the nude mouse was cut transversely approximately 1cm under the xiphoid process of the nude mouse and the muscle layer was exposed, and after finding the leucorrhea line on the muscle layer, the muscle layer was cut transversely and an opening to the abdominal cavity was opened. After finding the liver midlobes of the nude mice, the hepatoma cell line stably overexpressing circDLC1 in example 3 and the control cell line were injected under the liver capsule membrane of the nude mice using an insulin injection needle, and the muscle layer and the skin layer of the nude mice were gradually sutured, and the mice were waited for revival. In the following 4 to 8 weeks, the development of tumor in the nude mice was closely observed using an ultrasonic device, and it was determined when the nude mice were sacrificed to take out the liver according to the healthy state of the nude mice and the tumor formation. From FIG. 8(a) it can be seen that the tumor profile of the liver of the LV-circDLC group overexpressing circDLC1 is significantly more localized and the fluorescence intensity is significantly weaker than that of the LV-Vector control group, the HE staining of FIG. 8(b) also shows that the number of intrahepatic metastases of the LV-circDLC1 group overexpressing circDLC1 is significantly less than that of the LV-Vector control group, and FIG. 8(c) is a statistical plot of intrahepatic metastases, counting the number of tumors per field of HE sections for the overexpressed circDLC1 and the control group. The above experiments show that the transfer capacity of the liver cancer cells in the liver is obviously inhibited after the circDLC1 is over-expressed.

3. Over-expression of circDLC1 for inhibiting lung metastasis of hepatoma carcinoma cells in nude mice

The liver cancer cell line stably overexpressing circDLC1 in example 3 and the control cell line were injected into the nude rat tail vein. In the later 6-8 weeks, the ultrasonic equipment is used for closely observing the occurrence and development of tumors in the nude mice, and determining when to sacrifice the nude mice and take out the lungs according to the health state of the nude mice and the tumor formation condition. From FIG. 9(a), it can be seen that the fluorescence intensity of lung of LV-circDLC1 group overexpressing circDLC1 is significantly weaker than that of LV-Vector of control group, and from the HE stained photograph of FIG. 9(b), it can also be seen that the number of lung metastases of LV-cirDLC1 group overexpressing circDLC1 is significantly smaller than that of LV-Vector of control group, and FIG. 9(c) is a statistical graph of lung metastasis tumors, counting the number of tumors per field per HE section of both the circDLC1 and control groups. The above experiments show that the lung transfer capacity of the liver cancer cells in vivo is obviously inhibited after the circDLC1 is over-expressed.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> Sichuan university Hospital in western China

<120> circular RNA and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 552

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 1

ggtaatgcaa agaaaacaaa gtttgggcaa accagatcag aaagatttga atgaaaacct 60

agctgccact caagggctgg cccatatgat cgccgagtgc aagaagcttt tccaggttcc 120

cgaggaaatg agccgatgtc gtaattccta taccgaacaa gagctgaagc ccctcactct 180

ggaagcactc gggcacctgg gtaatgatga ctcagctgac taccaacact tcctccagga 240

ctgtgtggat ggcctgttta aagaagtcaa agagaagttt aaaggctggg tcagctactc 300

cacttcggag caggctgagc tgtcctataa gaaggtgagc gaaggacccc ctctgaggct 360

ttggaggtca gtcattgaag tccctgctgt gccagaggaa atcttaaagc gcctacttaa 420

agaacagcac ctctgggatg tagacctgtt ggattcaaaa gtgatcgaaa ttctggacag 480

ccaaactgaa atttaccagt atgtccaaaa cagtatggca cctcatcctg ctcgagacta 540

cgttgtttta ag 552

<210> 2

<211> 552

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 2

gguaaugcaa agaaaacaaa guuugggcaa accagaucag aaagauuuga augaaaaccu 60

agcugccacu caagggcugg cccauaugau cgccgagugc aagaagcuuu uccagguucc 120

cgaggaaaug agccgauguc guaauuccua uaccgaacaa gagcugaagc cccucacucu 180

ggaagcacuc gggcaccugg guaaugauga cucagcugac uaccaacacu uccuccagga 240

cuguguggau ggccuguuua aagaagucaa agagaaguuu aaaggcuggg ucagcuacuc 300

cacuucggag caggcugagc uguccuauaa gaaggugagc gaaggacccc cucugaggcu 360

uuggagguca gucauugaag ucccugcugu gccagaggaa aucuuaaagc gccuacuuaa 420

agaacagcac cucugggaug uagaccuguu ggauucaaaa gugaucgaaa uucuggacag 480

ccaaacugaa auuuaccagu auguccaaaa caguauggca ccucauccug cucgagacua 540

cguuguuuua ag 552

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tcgagactac gttgttttaa gggta 25

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aggggcttca gctcttgttc 20

Claims

1. The application of the pharmaceutical composition in preparing the medicines for inhibiting the proliferation and migration of liver cancer cells or treating liver cancer is characterized in that the pharmaceutical composition is a circular RNA overexpression vector, and the cDNA sequence of the circular RNA is shown as SEQ ID NO: 1, the structure of the circular RNA is SEQ ID NO: 1 is spliced after being transcribed to form an end-to-end circular structure.

2. The application of the reagent for quantitatively detecting the circular RNA in preparing the liver cancer diagnostic kit is characterized in that the cDNA sequence of the RNA is shown as SEQ ID NO: 1, the structure of the circular RNA is SEQ ID NO: 1 is spliced after being transcribed to form an end-to-end circular structure.

3. The use according to claim 2, wherein the kit comprises reagents for the quantitative detection of the circular RNA.

4. Use according to claim 3, wherein said reagents comprise primers capable of amplifying said circular RNA.

5. The use according to claim 4, wherein the primers comprise:

F：5'-TCGAGACTACGTTGTTTTAAGGGTA-3' (SEQ ID NO：3)

R：5'- AGGGGCTTCAGCTCTTGTTC-3' (SEQ ID NO：4)。

6. the use according to any one of claims 2 to 5, wherein the kit further comprises an RNA extraction reagent and a reverse transcription reaction system.

7. The application of the circular RNA overexpression vector in the preparation of the medicine for treating liver cancer is characterized in that the cDNA sequence of the circular RNA is shown as SEQ ID NO: 1, the structure of the circular RNA is SEQ ID NO: 1 is spliced after being transcribed to form an end-to-end circular structure.