WO2019169710A1 - Tumor molecular marker circbirc6 and inhibitor and use thereof - Google Patents

Abstract

Provided are a tumor molecular marker circBIRC6 and an inhibitor and the use thereof. The expression level of the circBIRC6 gene in gastric cancer patients is significantly upregulated. By using the circBIRC6 inhibitor, the circBIRC6 in human gastric cancer cells is knocked down at the cellular level, such that the proliferation, invasion and migration abilities of gastric cancer cells are decreased, and the circBIRC6 inhibitor may also be suitable for a variety of cancers with abnormal expression of circBIRC6.

Description

Tumor molecular marker circBIRC6 and its inhibitor and use Technical field

The invention relates to the field of genetic engineering technology, in particular to a tumor molecular marker circBIRC6 and an inhibitor and use thereof.

Background technique

Gastric cancer (GC) is one of the most common malignancies worldwide. According to the latest World Health Organization cancer control project statistics, up to 7 million patients die of cancer every year in the world, including gastric cancer patients. 700,000. There are about 934,000 new cases of gastric cancer in the world each year, of which China's new cases account for about 40% of the global total (nearly 400,000), and its morbidity and mortality are about twice the world average. At present, the early diagnosis rate of gastric cancer is less than 10%. It is usually found to be advanced or metastasis. About 50%-70% of patients with advanced gastric cancer will have recurrence after operation. The five-year survival rate is less than 30. %. With the further elucidation of the pathogenesis of tumors and the development of molecular biology techniques at the cellular and molecular levels, tumor treatment has entered a new era of molecular targeted therapy. Compared with systemic radiotherapy, targeted therapy can effectively and selectively kill tumor cells, reduce damage to normal tissues, and have fewer adverse reactions. Therefore, exploring new therapeutic strategies and therapeutic targets has become one of the most concerned areas of gastric cancer research.

After the completion of the human genome sequencing program, scientists have found that protein-coding genes account for a much smaller proportion in the transcriptome than non-coding RNAs (ncRNAs). Sequencing results show that more than 80% of the transcripts are ncRNAs. . Most of the initial ncRNA was considered to be the "noise" of genomic transcription and did not attract the attention of researchers. However, with the development of sequencing technology and the deepening of research, more and more evidences show that ncRNA can not only act as a regulatory factor to control the expression of genes at multiple levels of cells, maintain the elongation of telomeres, but also guide the repair of molecules to some extent. It plays an important biological function in life activities and disease occurrence. The ncRNAs currently found to regulate gene expression in humans mainly include microRNAs (miRNAs), lncRNAs (long noncoding RNAs, lncRNAs) of >200 nt, and newly discovered circular RNAs (circRNAs). CircRNAs are a class of endogenous ncRNA molecules that are widely present in human cells that regulate gene expression by binding to miRNA or other molecules at the transcriptional and post-transcriptional levels and inhibiting their function. At present, it is considered that circRNA is produced by special variable shear, and its 3' and 5' ends are covalently linked to form a closed loop structure, so its structure is relatively stable and difficult to degrade, which makes it a class. The possibility of efficient ceRNA. Compared with other types of ceRNA, circRNA is not easily degraded by RNase and miRNA, and most circRNA expression levels are high. A single circRNA molecule may contain a large amount of MRE, which can bind or release a large number of miRNAs instantaneously. Therefore, circRNA can be very efficient and stable. Play its ceRNA function. Since circRNA regulates the proliferation and invasion of tumor cells, it plays an important role in the development of various tumors and can be used as a potential biomarker for the diagnosis and prediction of tumor growth and invasion. Recent studies have confirmed that hsa_circ_002059 expression is significantly down-regulated in gastric cancer tissues, and its expression level is not only related to gender and age of gastric cancer patients, but also related to TNM staging and distant metastasis. Therefore, circRNA may be a new target for precise diagnosis and treatment of gastric cancer.

Summary of the invention

OBJECT OF THE INVENTION: In view of the deficiencies in the prior art, it is an object of the present invention to provide a tumor molecular marker circBIRC6 that meets the needs of gastric cancer. Another object of the present invention is to provide a use of a circBIRC6 gene for the preparation of a reagent for the diagnosis of gastric cancer. Still another object of the present invention is to provide an inhibitor of the circBIRC6 gene and its pharmaceutical use.

Technical Solution: In order to achieve the above object, the technical solution adopted by the present invention is as follows:

A tumor molecular marker circBIRC6, the nucleotide sequence is shown in SEQ ID NO.

The tumor molecular marker circBIRC6, which is a circular RNA, hsa_circ_0053441, localizes the 32602655-3262020661 region of human chromosome 2, and is formed by transcription of the BIRC6 gene after transcription.

Specific reverse PCR primers for circBIRC6 detection are:

The upstream sequence is: 5'-ATCATCAGCCTGCCTCATCT-3';

The downstream sequence is: 5'-CTGGAGTTTGCAGAGCAGTG-3'.

The use of the circBIRC6 in the preparation of a tumor diagnostic reagent.

The tumor is a tumor that is abnormally expressed by circBIRC6.

The inhibitor of circBIRC6 is an siRNA molecule, and the sequence of the siRNA molecule is as follows (including overhangs):

F: 5'-GAACCUUGCUAAACCAGGU UU-3’;

R: 5'-ACCUGGUUUAGCAAGGUUC UU-3';

The use of the inhibitor to inhibit abnormal expression of circBIRC6 in tumors.

The use of the inhibitor in the preparation of an antitumor drug.

Advantageous Effects: Compared with the prior art, the present invention confirms the objective existence of a new circBIRC6 gene for the first time. By detecting the expression of the gene in gastric cancer patients, it is found that the expression level is significantly up-regulated, and the circBIRC6 can be used as a diagnostic marker for tumors. Things. Provide new ideas for accurate diagnosis and treatment of tumors. In addition, CCK8 method, Transwell chamber and other techniques were used to study the effects of down-regulation of circBIRC6 expression on the biological behavior of gastric cancer cells in vitro and in vitro. It was found that specific siRNA sequences can effectively inhibit the expression of circBIRC6 in human gastric cancer cells. circBIRC6 is a target for precise diagnosis and treatment of medical treatments and will be widely used in the preparation of diagnostic kits and therapeutic drugs for various tumors (such as gastric cancer, colon cancer, breast cancer, etc.).

DRAWINGS

Figure 1 is a cluster analysis diagram of the expression of circular RNA in gastric cancer tissues and paired benign margin tissues;

2 is a real-time quantitative PCR amplification curve of the circBIRC6 gene and the internal reference GAPDH gene;

Figure 3 is a real-time quantitative PCR dissolution profile of the circBIRC6 gene and the internal reference GAPDH gene;

Figure 4 is a graph showing the expression of circBIRC6 in gastric cancer and benign margin tissues by real-time fluorescent quantitative PCR;

Figure 5 is a graph showing the interference efficiency of siRNA on circBIRC6 in gastric cancer cells;

Figure 6 is a graph showing the growth curve of gastric cancer cells after siRNA interferes with circBIRC6;

Figure 7 is a graph showing the results of invasion and migration of gastric cancer cells after siRNA interferes with circBIRC6;

Detailed ways

The invention will be further described below in conjunction with specific embodiments.

Example 1

1. Sample collection and processing

Gastric cancer samples and corresponding benign margin tissues were obtained from patients with gastric cancer from Nantong University Hospital from 2015 to 2016. Surgical treatment, removal of diseased tissue. Cut a piece of soybean-like tissue at 5 cm away from the lesion of the gastric cancer and away from the lesion. Cut a small piece of tissue with a thickness of not more than 0.5 cm with a scalpel, soak it in 1 mL of RNA later preservation solution (Simofly), and place it in -80 °C ultra-low temperature freezer in the spare. Each sample was confirmed by histopathology.

2. Second generation sequencing

6 pairs of gastric cancer patients (according to Lauren classification: 3 cases of intestinal type gastric cancer, 3 cases of diffuse gastric cancer) fresh cancer tissue and corresponding benign mucosal epithelial tissue (using Agilent 2200 for quality control), through the second generation of the Hiseq platform Next generation sequencing (NGS) performed high-throughput analysis on the gene transcriptome of these 6 pairs of samples, and identified 4262 circRNAs. After counting by TMM, Median, DESeq and other algorithms, there were 125 fold changes>1.5. (The differential circRNA clustering analysis is shown in Figure 1). Among them, circBIRC6 is highly expressed in gastric cancer, and the cDNA sequence of the circBIRC6 gene is SEQ ID NO. 1, which is a circular RNA (hsa_circ_0053441), which locates human chromosome 2 32602655~ The 32620661 region is formed by reverse cleavage of the BIRC6 gene after transcription.

3. Tissue RNA extraction

(1) Take tissue specimens with a diameter of about 0.5 cm, add 1 mL of Trizol and grind quickly, and grind as much as possible. (1 mL of Trizol lysed cells were added to the flask or to the six-well plate and collected in the EP tube). (2) The centrifuged mixture is instantaneously centrifuged. (3) The liquid was mixed by pipetting, and 200 μL of chloroform (trichloromethane, Trizol: chloroform = 1:0.2) was added. (4) Close the lid, shake it vigorously (vortexing) for 15 s, and let it stand on ice for 2-3 min. (5) 12000 r/min, 15 min, centrifugation at 4 ° C, the sample was divided into three layers (lower layer: red organic phase protein; intermediate layer: DNA; upper layer: aqueous phase RNA). (Overall arrangement: 75% ethanol to be used later, 1mL 75% ethanol = 750μL absolute ethanol + 250μL DEPC water). (6) Carefully pipette the upper liquid phase (about 500 μL) and transfer to a 1.5 mL centrifuge tube. (7) 500 μL of isopropanol was added to precipitate RNA (1:1 with the aspirated upper liquid phase), mixed by inversion, and incubated on ice for 15 min. (8) 12000r/min, 10min, centrifugation at 4°C, it can be seen that there is a gelatinous precipitate at the bottom of the tube. (9) Discard the supernatant, leave a gelatinous precipitate, add 1 mL of 75% absolute ethanol in step 5, gently shake, and mix by inversion. (10) 7500r/min, 10min, 4°C centrifugation (coordinated arrangement: during the period, the oven is preheated and heated to 55 °C). (11) Discard the supernatant, use the blotting paper to remove the residual liquid as much as possible to retain the sediment, cover the lid and prepare to dry. (12) Open the lid of the centrifuge tube and place it in a preheated oven for 6-8 minutes until the pellet becomes transparent. (13) Cover the lid, take it out, add 30 μL of DEPC water in turn, mix thoroughly by pipetting, cover the lid and place it in an oven at 55 ° C for 6-8 min to obtain RNA stock solution. (14) The concentration and purity of the solution were measured by an ultraviolet spectrophotometer. The purity of the total RNA was considered to be reliable in the range of 1.8 to 2.0 for A260/A280. Store in a refrigerator at -80 °C for use after dispensing.

4. Total RNA reverse transcription

RNA samples with the required concentration and purity can be used for further testing. The reverse transcription reaction system was: 5×Reactive Buffer 4 μL, OligoDT primer 2 μL, dNTPs (10 mM) 2 μL, RNase inhibitor (20 μ/μL) 1 μL, Reverse Transcriptase (200 μ/μL) 1 μL, Total RNA according to the corresponding volume of RNA concentration. RNase-free H ₂ O was made up to 20 μL. The reverse transcription kit was purchased from Thermo Scientific, Inc., and was carried out according to the kit method.

5. Real-time fluorescent quantitative PCR detection

(1) Select GAPDH as an internal reference to control the difference between samples.

(2) The primer sequences are as follows:

circBIRC6 (divergent primer)

F: 5'-ATCATCAGCCTGCCTCATCT-3’

R: 5'-CTGGAGTTTGCAGAGCAGTG-3'

GAPDH

F: 5’-AACGGATTTGGTCGTATTGGG-3’

R: 5'-CCTGGAAGATGGTGATGGGAT-3'

The real-time fluorescent quantitative PCR reaction system was: SYBR GreenImix (Rox) 10 μL, cDNA 3 μL, Forward Primer 1 μL, Reverse Primer 1 μL, and RNase-free H ₂ O 5 μL. RNA Master SYBR Green I was purchased from Roche, Switzerland.

(3) After the system is configured, mix it and centrifuge it instantaneously (be careful to avoid light). The reaction conditions were as follows: 95 ° C for 10 min, 95 ° C for 15 s, 58 ° C for 30 s, and 72 ° C for 30 s.

(4) One 40 cycles, the specificity of the PCR reaction was evaluated by the dissolution curve.

(5) Finally, the amplification curve (Fig. 2) and the dissolution curve (Fig. 3) of the expression levels of circBIRC6 and GAPDH in the tissue samples were obtained. It can be understood from the dissolution curve that the curve is a narrow single peak without primer dimer. And the interference of the ribbon. The sample cDNA has a Ct value of ≤ 30, and the RNA (ie, cDNA) quality is acceptable.

6. Analysis of expression level of circBIRC6 in gastric cancer tissues

Using qRT-PCR technology to verify the expression of circBIRC6 in gastric cancer tissues and its matched benign margin tissues, it was found that circBIRC6 was highly expressed in gastric cancer tissues (Fig. 4).

Example 2

1. siRNA design

The siRNA sequence against human circBIRC6 and the negative control siRNA (negative control, NC) were designed and completed by Guangzhou Boxin Co., Ltd. The sequence is as follows:

siRNA sequence (including overhangs) circBIRC6

F: 5'-GAACCUUGCUAAACCAGGU UU-3’;

R: 5'-ACCUGGUUUAGCAAGGUUC UU-3';

Negative control siRNA sequence

F: 5'-UUCUCCGAACGUGUCACGU dTdT-3';

R: 5'-ACGUGACACGUUCGGAGAA dTdT-3'.

2. Cell resuscitation: (1) Remove the cryotubes of HS-578T, LCC, MDA-MB-453, MCF-7 and MCF-10A cells from a -80 °C ultra-low temperature freezer and place them in a 37 ° C water bath for rapid shaking. So that the cells are completely thawed within 1 min. (2) Take out the frozen tube, disinfect it with alcohol, and put it into the clean bench. (3) Pipette the cell suspension into a 15 mL centrifuge tube, add 10 mL of RPMI-1640 or DMEM medium, mix well, and centrifuge at 1000 rpm for 5 min. (4) Aspirate the supernatant, mix 2 mL of the culture medium containing 10% fetal bovine serum, add the cells, add to the cell culture flask, and add the appropriate amount of the culture solution in a 37 ° C, 5% CO ₂ cell culture incubator. The culture was changed once a day, and the culture was continued.

3. Cell subculture: (1) UV lamp disinfection clean bench for 30 minutes, the required liquid is taken out in advance, after the temperature rises to room temperature, the alcohol disinfection bottle mouth is placed in the ultra-clean table, and the alcohol lamp is turned on. (2) Discard the old culture solution in the cell-filled culture flask, rinse it once with PBS, add 1 mL of 0.02% EDTA + 0.25% trypsin digest, digest for 3 to 5 minutes, observe the cell state under the microscope, when the cells When the circle is rounded and the gap becomes large, the digestion is terminated by adding 5 mL of RPMI-1640 or DMEM containing 10% fetal bovine serum, and the bottom of the bottle is repeatedly blown with a straw. (3) The blown cell suspension was dispensed into a new culture flask, and an appropriate amount of the culture solution was added thereto, and the culture was continued at 37 ° C in a 5% CO ₂ cell incubator.

4. Cell counting method: (1) After digesting the cells, prepare a cell suspension, wipe the coverslip with an alcohol cotton ball, and place it on the cell counting plate. (2) Blow the cell suspension and mix it, and gently inject 10 μL into the junction of the coverslip and the counting plate. (3) Under the microscope, read the number of cells in the square of the four corners of the counting plate. When the cells are pressed to the line, only the left side and the upper side are counted, except for the right side and the lower side.

(4) Calculation: cell density (pieces/mL) = (sum of 4 large cells/4) × 10 ⁴ .

5. Cell cryopreservation: (1) Select the cells in the logarithmic growth phase and change the solution 24 hours before cryopreservation. (2) Conventional digestion of cells, preparation of cell suspension, centrifugation 1000r / min, 5min. (3) Aspirate the supernatant and add the frozen solution. The final density of the cells in the frozen solution is 5×10 ⁶ /mL, and mix gently by pipetting. (4) Dispense the cell suspension into a sterile cryotube, 1 mL per tube. (5) Seal the frozen tube and indicate the cell name, freezing time and other information. (6) Cryopreservation: placed at 4 ° C for 30 min, placed at -20 ° C for 30 min, placed in a -80 ° C refrigerator, can be stored for about 3 months, if long-term preservation, put into a liquid nitrogen tank.

6. Culture of gastric cancer cells: (1) Remove gastric cancer cells from the -80 °C refrigerator according to the above-mentioned resuscitation method, rapidly thaw at 37 ° C, aspirate the cell suspension, rinse with RPMI-1640 medium, centrifuge at low speed, add 10% fetal The cells were mixed with RPMI-1640 medium of bovine serum, transferred to a 25 cm ² flask, and cultured in a 37 ° C, 5% CO ₂ cell culture incubator. (2) The cell culture medium was replaced the next day, and the cells were further cultured until the cell growth reached 90% fusion, and the cells were subcultured. (3) After several passages, some cells can be frozen, and some cells continue to be cultured and ready for testing.

7. LipofectamineTM 3000 (purchased from Invitrogen, USA) transfected with siRNA (determination of transfection efficiency) (1) One day before transfection, gastric cancer cells in logarithmic growth phase were inoculated at a density of 4 to 5 × 10 ⁴ /well. On a six-well culture plate, 2 mL of RPMI-1640 medium containing 10% fetal bovine serum was added. (2) Cultured in a 37 ° C, 5% CO ₂ cell culture incubator, the gastric cancer cell fusion rate reached about 70% within 24 hours. (3) Dilute 5 μl of LipofectamineTM 3000 with 250 μl of RPMI-1640 and mix gently. (4) After incubation for 5 min at room temperature, the diluted siRNA and diluted LipofectamineTM 3000 were mixed, gently mixed and incubated for 20 min at room temperature to allow formation of the complex siRNA-LipofectamineTM 3000. (5) Discard the culture medium in 6-well plate, wash the cells twice with PBS, add the mixed complex to each well containing the cells, gently shake the plate and mix them thoroughly, and mix well. Add 2 mL of RPMI-1640 and place in the incubator. (6) After 6 hours, the solution was changed, and 2 mL of RPMI-1640 medium containing 10% fetal calf serum was added. (7) Incubate at 37 ° C, 5% CO _{2 for} 48 hours, and collect cells when the cells grow to about 80-90% of the plate area. The interference efficiency of siRNA sequence on the expression of circBIRC6 in gastric cancer cells was detected by qRT-PCR. The results showed that circBIRC6 Specific siRNA can effectively inhibit the expression of circBIRC6 (Fig. 5).

8. Cell proliferation experiment (CCK-8 experiment)

(1) Digestion Each group of cells 48 h after transfection was collected and centrifuged for use. (2) The cells were resuspended in 1640 complete medium, and the cell density was adjusted to 30,000 cells/mL. (3) Add 100 μL of cell suspension to each well, set 5 replicate wells in each group, and gently tap the 96-well plate to make the cells evenly distributed. (4) After the cells were attached (about 24h), add CCK-8 reagent (10μL per well) at 24, 48, 72, 96h, gently tap the 96-well plate, and put it into the incubator for 1h, then remove it. The absorbance at 450 nm was measured on the standard instrument, and the linear range of the microplate reader was noted. (5) Using Graphpad prism to statistically process the measured data and draw a line graph (Fig. 6). The results showed that the proliferation activity of gastric cancer cells decreased after siRNA interference with circBIRC6.

9. Cell migration and invasion experiments (transwell chamber method)

(1) Digestion Each group of cells 48 h after transfection was collected and centrifuged for use. (2) The cells were resuspended in 1640 base cultures, and the cell density was adjusted to 5 × 10 ⁴ /mL. (3) 600 μL of complete medium was added to a 24-well plate, placed in a chamber, and fully infiltrated, and 100 μL of the cell suspension was taken to the upper chamber of the Transwell chamber. (4) In the invasion experiment, 50 μL Matrigel (50 μL Matrigel Matrigel mixed in 300 μL 1640 PBS) was added to the upper chamber along the sidewall of the chamber one day in advance, and the 24-well plate was tapped to make it evenly distributed on the bottom surface of the chamber. The bubbles were then placed in the incubator the next day. (5) After routine culture for 48-72 hours, take out, wash twice with 1×PBS, fix 4% paraformaldehyde for 20 min, and wash twice with 1×PBS. (6) Add 500 μL of crystal violet dye solution to a 24-well plate, place it in a small chamber, take it out after 10 minutes, wash it twice in 1×PBS, invert the chamber, and gently wipe off the cells that have not passed through the chamber with a cotton swab. (7) Observed the results with an inverted microscope (take 5 fields, calculate the average), use the Graphpad prism to statistically process the measured data, and draw a line graph (Figure 7). The results show that the invasion and migration ability of gastric cancer cells after siRNA interference with circBIRC6 decline.

Claims (8)

1. A tumor molecular marker circBIRC6, the cNDA sequence of which is shown in SEQ ID NO.
2. The tumor molecular marker circBIRC6 according to claim 1, wherein the circBIRC6 is a circular RNA, hsa_circ_0053441, and a region of 32602655 to 32620661 of human chromosome 2 is located, which is formed by transcription of the BIRC6 gene after transcription.
3. The tumor molecular marker circBIRC6 according to claim 1, wherein the specific reverse PCR primer for detection of circBIRC6 is:

   The upstream sequence is: 5'-ATCATCAGCCTGCCTCATCT-3';

   The downstream sequence is: 5'-CTGGAGTTTGCAGAGCAGTG-3'.
4. Use of the circBIRC6 of claim 1 for the preparation of a tumor diagnostic reagent.
5. The use according to claim 4, wherein the tumor is a tumor abnormally expressed by circBIRC6.
6. An inhibitor of circBIRC6 according to any one of claims 1 to 3, wherein the inhibitor is an siRNA molecule, and the sequence of the siRNA molecule is as follows:

   F: 5'-GAACCUUGCUAAACCAGGU UU-3’;

   R: 5'-ACCUGGUUUAGCAAGGUUC UU-3'.
7. Use of the inhibitor of claim 6 for inhibiting abnormal expression of circBIRC6 in a tumor.
8. Use of the inhibitor of claim 6 for the preparation of an antitumor drug.

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