Ring-shaped RNA circ-NFATC3 and application thereof

[0030] Example 1: RT-PCR reaction to detect the expression of circ-NFATC3 gene in liver cancer tissue.

[0031] The specific experimental plan is as follows:

[0032] 1. RNA extraction

[0033] 1) Tissue treatment: Take about 10mg of tissue and add 1ml Trizol, homogenize with a homogenizer; centrifuge for 15 minutes, 12000g, and take the supernatant.

[0034] 2) Add 200ul of chloroform to the supernatant, mix up and down vigorously for half a minute, and let it stand for 3 minutes.

[0035] 3) Centrifuge at 12000g for 15 minutes at 4°C. At this time, it can be seen that the lysate is divided into three layers: the upper layer is water-phase RNA; the middle layer is DNA, lipids, etc.; the lower layer is cell residues, proteins, polysaccharides, etc.

[0036] 4) Take the supernatant into a new EP tube; add an equal volume of isopropanol, mix well, let stand for 10 minutes, and centrifuge at 12000g for 10 minutes at 4°C.

[0037] 5) Carefully remove the supernatant, taking care not to lose the RNA pellet, add 1ml of 75% ethanol and turn upside down to resuspend the pellet.

[0038] 6) Centrifuge at 12000g for 10 minutes at 4°C, carefully remove the supernatant, and try to suck up the liquid on the tube wall. Be careful not to lose the RNA precipitate. If the precipitate becomes loose, centrifuge again. Let dry for about 15 minutes until there is no liquid on the tube wall.

[0039] 7) Add an appropriate volume (20-30ul) of DEPC water to dissolve the RNA, and bath at 58°C for 10 minutes.

[0040] 8) Take out 2ul for quantification, measure buffer: 10mM TrisCl (pH 7.8), and perform reverse transcription based on the quantification result. (1A260=40μg/ml, A260/A280=1.8~2.1).

[0041] 2. cDNA reverse transcription

[0042] 1) Experimental system

[0043] M-MLV Reverse Transcriptase:

[0044]

[0045] 3. Primers: The circular RNA primers are reverse primers, and a pair of forward primers are designed as controls. A set of gDNA template controls are set up during RT-PCR to confirm that circRNA comes from post-transcriptional shearing, not mutations such as gene fusion. At the same time, the linear gene GAPDH was detected as a negative control. The primers used are listed below:

[0046] SEQ ID NO: 2

[0047] 4. PCR: GAPDH is used as an internal control. PCR reaction system: add 2μl 10×Buffer to each reaction tube, respectively add 2μl dNTP, 1μl forward primer, 1μl reverse primer, 1μl cDNA template, 0.2μl Taq enzyme, add water to 20μl. The PCR reaction conditions are as follows: 94°C, 5 minutes pre-denaturation; 94°C, 30 seconds denaturation; 55°C, 30 seconds annealing; 72°C, 30 seconds extension; 35 cycles, agarose gel electrophoresis to detect PCR amplification products, the results see figure 1 , figure 1 The middle circular RNA primer is a reverse primer (black triangle symbol), and a pair of forward primers are designed as a control (white triangle symbol). A set of gDNA template controls are set up during RT-PCR to confirm that circRNA comes from post-transcriptional shearing. It is not mutations such as gene fusion, and the linear gene GAPDH is also detected as a negative control.

[0048] Example 2: QPCR detection of circ-NFATC3 expression in liver cancer

[0049] 1. RNA extraction: same as Example 1;

[0050] 2. cDNA reverse transcription: the same as in Example 1;

[0051] 3. QPCR amplification experiment

[0052] 1) Experimental system:

[0053]

[0054] The circ-NFATC3divergent primer of Example 1 was used to amplify the circular RNA circ-NFATC3, and the hsaGAPDH convergent primer of Example 1 was used to amplify the internal reference gene.

[0055] 2) Reaction conditions:

[0056] The first step: 95℃2min

[0057] The second step (40 cycles): 95°C for 3 seconds, 60°C for 30 seconds

[0058] The third step 60-95 ℃ dissolution curve

[0059] 3) On the computer for target gene amplification

[0060] 4) qPCR relative quantitative results

[0061] The calculation formula for the relative expression of the target gene is: 2-△△Ct=2-[(△Ct)Test-(△Ct)Control]. The Ct target is the Ct value of the target gene, and the Ct housekeeper is the Ct value of the housekeeping gene. △Ct=Ct Purpose-Ct Steward, which means the relative Ct value of the target gene of each sample to the housekeeping gene, △△Ct=(△Ct)Test-(△Ct)Control, which means the treatment group is normalized to the control group, 2 -△△Ct represents the relative expression level of the treatment group relative to the control group, and represents the relative expression multiple of the target gene.

[0062] Total RNA was digested by adding RNaseR at a ratio of 3U/ug, and QPCR was used to detect the effect of RNaseR on circ-NFATC3 and GAPDH. figure 2 , RNaseR digestion confirmed that circ-NFATC3 is not sensitive to RNase. RNaseR is an RNase that can digest linear RNA, but has no effect on circular RNA. After digesting total RNA with RNaseR, QPCR detection is performed. The results show that adding or not adding RNaseR has no significant effect on the expression of circ-NFATC3, but linear genes The expression of GAPDH was significantly reduced after RNaseR digestion.

[0063] QPCR detects the expression levels of circ-NFATC3 and GAPDH in liver cancer and corresponding adjacent tissues. The results are shown in image 3 , The expression of circ-NFATC3 was detected in 24 pairs of liver cancer clinical tissue samples, and the results showed that circ-NFATC3 was significantly down-regulated in cancer tissues. Control: adjacent tissues, Tumor: liver cancer tissues.

[0064] Example 3: Overexpression of circ-NFATC3 lentivirus and its stable cell line construction

[0065] 1. Overexpression circ-NFATC3 lentiviral vector construction: Synthesize the circ-NFATC3 linear full sequence in Shanghai Jierui Company, the sequence is annealed into a double-stranded DNA fragment, inserted into the LVminiCirc vector through multiple cloning sites, and the recombinant plasmid is identified by sequencing , Control negative control is an empty LVminiCirc vector with no sequence inserted.

[0066] 2. Lentivirus packaging

[0067] (1) 24h before transfection, digest 293T cells in logarithmic growth phase with trypsin and pass them to a 10cm cell culture dish at 37℃, 5% CO 2 Cultivate in an incubator. It can be used for transfection when the cell density reaches 70% to 80% in 24 hours. Cell state is very important for virus packaging, so it is necessary to ensure good cell state and fewer passages.

[0068] (2) Change the cell culture medium to serum-free medium before transfection.

[0069] (3) Add each prepared DNA solution (LVminiCirc-Circ-NFATC3/LVminiCirc vector 10μg, pGag/Pol vector 5μg, pRev vector 5μg, pVSV-G vector 5μg) into a sterile centrifuge tube, and add the corresponding volume of Opti -MEM is mixed evenly and the total volume is adjusted to 1.5ml.

[0070] (4) Shake Lipofectamine 2000 reagent gently, take 60 μl Lipofectamine 2000 reagent and mix with 1.5 ml Opti-MEM in another tube, and incubate at room temperature for 5 minutes.

[0071] (5) Mix the diluted DNA with the diluted Lipofectamine 2000, gently invert and mix without shaking.

[0072] (6) After mixing, incubate at room temperature for 20 minutes to form a transfection complex of DNA and Lipofectamine 2000 dilution.

[0073] (7) Transfer the mixture of DNA and Lipofectamine 2000 to the culture medium of 293T cells, mix well, and incubate at 37°C, 5% CO 2 Culture in a cell incubator.

[0074] (8) After 6 hours of incubation, aspirate the medium containing the transfection mixture, add 10ml of cell culture medium containing 10% serum to each bottle of cells, and hold at 37°C, 5% CO 2 Continue to incubate for 48 hours in the incubator.

[0075] 3. Harvesting and concentration of viruses

[0076] (1) Collect the 293T cell supernatant at 48 hours and 72 hours after transfection (starting from 0 hours after transfection).

[0077] (2) Centrifuge at 4000g for 10 min at 4°C to remove cell debris.

[0078] (3) Filter the supernatant with a 0.45μm filter in a 50ml centrifuge tube.

[0079] (4) Add the crude virus extract sample to the filter cup (maximum 19ml), and close the lid. Insert the filter cup into the filtrate collection tube.

[0080] (5) After the combination is completed, balance it and place it on the rotor.

[0081] (6) Centrifuge at 5000×g to the required virus concentration volume. It usually takes 10-15 minutes.

[0082] (7) After centrifugation, the filter cup is the virus concentrate.

[0083] (8) Remove the virus concentrate and store it in virus tubes after aliquots. It can be stored at 4°C for a week or -80°C for long-term storage. Take one of them for virus biological titer determination.

[0084] 4. Cells infected by lentivirus:

[0085] (1) Collect the cells by centrifugation in a 1.5ml tube according to the amount of cells, and then dilute the cell pellet with 100-200ul of serum-free culture medium, subject to the cells being completely immersed in the culture medium.

[0086] (2) Aspirate the over-expressed circ-NFATC3 virus solution and add it to the cells, and place the 1.5ml tube in a 37°C incubator for 30 minutes. Another LVminiCirc empty vector control virus infection was used as a control cell line.

[0087] (3) Aspirate the mixed solution from the tube and add it to the petri dish or hole.

[0088] (4) Add a sufficient amount of fresh culture medium.

[0089] (5) Change the liquid after 12 hours.

[0090] (6) After 48 hours, 2ug/ml puromycin was added to screen for stable cell lines.

[0091] 5. Identification of stable cell lines: The constructed stable cell lines are photographed and observed under a fluorescence microscope, the positive rate of GFP> 95%, and some cells were collected at the same time for QPCR testing, confirming the overexpression efficiency of circ-NFATC3> 2 times. See the result Figure 4 , The circ-NFATC3 gene-specific sequence was constructed on the LVminiCirc lentiviral vector, and the Huh7-circ-NFATC3 stable cell line overexpressing circ-NFATC3 was constructed by lentivirus. The result showed that the GFP positive rate was> 95%, QPCR test confirmed that circ-NFATC3 was successfully overexpressed, and the overexpression effect reached 2 times.