Application of overexpression of long non-coding RNA CCTT in artificial aneuploid construction

Abstract

This invention discloses the application of overexpression of the long non-coding RNA CCTT in the construction of artificial aneuploidy. This invention claims protection for the use of substances capable of promoting lncRNA CCTT expression in the following P1 and / or P2: P1, constructing artificial aneuploid cells, or preparing products for constructing artificial aneuploid cells; P2, promoting cell proliferation, or preparing products for promoting cell proliferation. Overexpression of lncRNA CCTT can significantly induce aneuploidy, and more importantly, it does not affect or promote cell proliferation. This invention, to some extent, explains the "aneuploidy paradox," thereby ensuring that aneuploidy can be amplified and stably cultured over a long period, which is of great significance in the construction of artificial aneuploidy.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of overexpression of long non-coding RNA (lncRNA) CCTT in the construction of artificial aneuploids. Background Technology

[0002] Aneuploidy refers to cells containing an abnormal number of chromosomes (increased or missing). These cells are widely present in tumor tissues and are thought to play a role in promoting tumor development. However, research has shown that the proliferation of aneuploid cells is inhibited, leading to the long-standing "aneuploidy paradox"—why tumor cells can tolerate large numbers of aneuploid cells and overcome their proliferative inhibitory effects, maintaining vigorous proliferative capacity.

[0003] Artificial aneuploidy is an excellent material for studying genome stability and its regulatory mechanisms. Previous studies have created engineered artificial aneuploidy by adding a chromosome to cells. For example, mouse embryonic fibroblasts with extra chromosomes 1, 13, 16, or 19 have been created through Robertsonian translocations. However, the biggest challenge in constructing artificial aneuploidy is that aneuploid cells are inhibited in proliferation due to various stressors such as genomic instability, oxidative stress, and DNA damage, which may lead to cell death, making it difficult to culture aneuploid cells stably for a long time. Solving the "aneuploidy paradox" and revealing new mechanisms by which aneuploidy overcomes the proliferation inhibition effect may provide new methods and strategies for establishing artificial aneuploidy, showing great promise for application. Summary of the Invention

[0004] The purpose of this invention is to provide an application of overexpression of lncRNA CCTT in the construction of artificial aneuploids.

[0005] In a first aspect, the present invention claims the use of a substance capable of promoting lncRNA CCTT expression in the following P1 and / or P2:

[0006] P1. Constructing artificial aneuploid cells, or preparing products for constructing artificial aneuploid cells;

[0007] P2, promoting cell proliferation and / or growth, or preparing products for promoting cell proliferation and / or growth.

[0008] Secondly, the present invention claims protection for a method for constructing artificial aneuploid cells.

[0009] The method for constructing artificial aneuploid cells claimed in this invention may include the following steps: introducing a substance that can promote the expression of lncRNA CCTT into recipient cells to obtain aneuploid cells with increased lncRNA CCTT expression (the proportion of aneuploid cells is increased compared with that before introduction).

[0010] In a specific embodiment of the present invention, after obtaining aneuploid cells with increased lncRNA CCTT expression, the method further includes treating the aneuploid cells with colchicine to synchronize their cell cycle. The concentration of colchicine is 0.02 μg / ml. The treatment time with colchicine is 18 h.

[0011] In both of the above aspects, the substance that can promote the expression of lncRNA CCTT can be a DNA molecule that can be transcribed into lncRNA CCTT or an expression vector containing the DNA molecule.

[0012] In both of the above aspects, the lncRNA CCTT is the RNA shown in SEQ ID No. 1.

[0013] Furthermore, the DNA molecule capable of being transcribed into the lncRNA CCTT is shown at positions 11-749 of SEQ ID No. 2.

[0014] In a specific embodiment of the present invention, the expression vector containing the DNA molecule is a recombinant plasmid obtained by cloning the DNA fragment shown at positions 11-749 of SEQ ID No. 2 into the multiple cloning site of the pcDNA3.1(+) vector.

[0015] In this invention, the product may specifically be a reagent kit.

[0016] In both of the above aspects, the cells may be tumor cells or non-tumor cells.

[0017] Furthermore, the tumor cells may be colon cancer cells or lung cancer cells, etc.

[0018] Furthermore, the non-tumor cells may be endothelial cells or epithelial cells, etc.

[0019] Furthermore, the colon cancer cells may be HT116 cells; the lung cancer cells may be A549 cells.

[0020] Furthermore, the endothelial cells may be umbilical vein endothelial cells; the epithelial cells may be bronchial epithelial cells. In a specific embodiment of the present invention, the umbilical vein endothelial cells are human umbilical vein endothelial cells (HUVEC); the epithelial cells are bronchial epithelial cells (BEAS-2B).

[0021] Thirdly, the present invention claims protection for artificial aneuploid cells constructed using the method described in the second aspect above.

[0022] Experiments have shown that overexpression of lncRNA CCTT can significantly induce aneuploidy, and more importantly, it does not affect or promote cell proliferation. This invention, to some extent, explains the "aneuploidy paradox," thus ensuring that aneuploids can be amplified and cultured stably for a long time, which is of great significance in the construction of artificial aneuploids. Attached Figure Description

[0023] Figure 1 To overexpress lncRNA CCTT, qRT-PCR was used to detect the expression level of lncRNA CCTT in HCT116 (A) or A549 (B) cells transfected with pCMV-CCTT for 48 h, or in A549 cells stably overexpressing lncRNA CCTT (C). Unpaired t-test was used, **p<0.01. In the figure, Ctrl represents control cells transfected with the empty vector; OE-CCTT represents cells transfected with the pCMV-CCTT vector / stable overexpressing lncRNA CCTT.

[0024] Figure 2 Aneuploidy was induced by overexpression of lncRNA CCTT. HCT116 cells were overexpressed with lncRNA CCTT, and the aneuploidy rate was statistically analyzed by karyotype analysis after 72 hours. Unpaired t-test was performed, **p<0.01. In the figure, Ctrl represents control cells transfected with the pCMV empty vector; OE-CCTT represents cells transfected with the pCMV-CCTT vector.

[0025] Figure 3 The expression of lncRNA CCTT promotes the proliferation of A549 cells. A shows the growth curve of A549 cells overexpressing lncRNA CCTT. B shows the colony formation of A549 cells overexpressing lncRNA CCTT. Unpaired t-test, *p<0.05, **p<0.01, ***p<0.001. C shows tumor formation of A549 cells overexpressing lncRNA CCTT in nude mice. Tumor volume (middle) and mass (right) were measured 22 days after cell inoculation, n=10. Paired t-test, **p<0.01.

[0026] Figure 4Overexpression of lncRNA CCTT resists various cell proliferation inhibitory effects. A shows the 72-hour cell count of human endothelial HUVEC cells overexpressing lncRNA CCTT; B shows the 72-hour cell count of human bronchial epithelial BEAS-2B cells overexpressing lncRNA CCTT; C shows the growth curve of human colon cancer HCT116 cells overexpressing lncRNA CCTT. Unpaired t-test, ns, p>0.05, no significant difference; *p<0.05. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] The nucleotide sequence of the lncRNA CCTT involved in the following examples is shown in SEQ ID No. 1.

[0030] Example 1: LncRNA CCTT overexpression induces aneuploidy

[0031] I. Construction of LncRNA CCTT overexpression vector

[0032] Using genomic DNA from the human lung cancer A549 cell line as a template, the full-length lncRNA CCTT fragment was amplified by PCR and sequenced to confirm the absence of mutations (SEQ ID No. 2, the first and last ten positions are the restriction enzyme sites and protective bases). The amplified fragment was cloned into the EcoRI and BamHI restriction sites of the pcDNA3.1(+) expression plasmid, and the resulting recombinant plasmid was named pCMV-CCTT after being verified by sequencing.

[0033] PCR reaction procedure:

[0034] Pre-denaturation at 94℃ for 5 minutes;

[0035] 94℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 1min, 30 cycles;

[0036] Extend at 72℃ for 10 minutes;

[0037] Store at 4°C.

[0038] PCR primer sequences:

[0039] F: 5'-CGGGATCCCGCTTTTTGAGGCAGCTACCAGAAG-3';

[0040] R: 5'-CGGAATTCCGCTCAGTTTCTTTTTTAAATTTT-3'.

[0041] Structural description of recombinant plasmid pCMV-CCTT: The recombinant plasmid was obtained by inserting the DNA fragment shown in positions 11-749 of SEQ ID No. 2 between the EcoRI and BamHI restriction sites of the pcDNA3.1(+) expression plasmid.

[0042] II. Preparation of HCT116 aneuploid cells overexpressing lncRNA CCTT

[0043] The pCMV-CCTT constructed in step one was transfected into human colon cancer cells HT116. The expression level of lncRNA CCTT was detected by qRT-PCR 48 h after transfection.

[0044] The primers used to detect lncRNA CCTT are as follows:

[0045] F: 5'-CTAAGGATTCGGTGTTGG-3';

[0046] R: 5'-CCTGCCAGGTTAGAGGG-3'.

[0047] Using GAPDH as an internal reference, the primers are as follows:

[0048] F: 5'-ACCCAGAAGACTGTGGATGG-3';

[0049] R: 5'-CAGTGAGCTTCCCGTTCAG-3'.

[0050] The results showed that HCT116 cells overexpressing lncRNA CCTT were successfully obtained. Figure 1 ).

[0051] III. Cell Karyotype Analysis

[0052] 1. Inoculate HCT116 cells overexpressing lncRNA CCTT obtained in step 2 and control cells transfected with empty vector into 6-well plates, culture them until the cell confluence reaches about 70%, add colchicine (0.02 μg / ml) and incubate for 18 h to synchronize the cell cycle, and perform karyotype analysis after 72 h.

[0053] 2. Digest the cells, centrifuge, add 0.4% KCl dropwise to resuspend the cells, and treat with hypotonicity at 37°C for 10 min.

[0054] 3. Add an equal volume of Carnoy's fixative (ethanol: glacial acetic acid = 3:1, volume ratio) and fix at room temperature for 10 min.

[0055] 4. Centrifuge, discard the supernatant, resuspend the cells in Carnoy's fixative, and fix at room temperature for 10 min.

[0056] 5. Repeat step 4 once.

[0057] 6. Take the cell suspension and smear it onto a pre-cooled glass slide, add 10% Giemsa stain, and stain at room temperature for 20-30 minutes.

[0058] 7. Wash away the excess dye with distilled water, observe and photograph at 100x oil immersion, and count the number of chromosomes in each cell.

[0059] The results are as follows Figure 2 As shown, overexpression of lncRNA CCTT leads to aneuploidy.

[0060] The results of this embodiment show that overexpression of lncRNA CCTT in human colon cancer cells HT116 ( Figure 1 ), and it was found that it induces aneuploid cells ( Figure 2 ).

[0061] Example 2: LncRNA CCTT overexpression resists multiple cell proliferation inhibitions

[0062] I. Preparation of LncRNA CCTT overexpressing cells

[0063] 1. Transient overexpression

[0064] Following steps one and two of Example 1, human lung cancer cells A549, human umbilical vein endothelial cells (HUVEC), and human bronchial epithelial cells (BEAS-2B) overexpressing lncRNA CCTT were constructed. Step two of the following assay was performed using each lncRNA CCTT-overexpressing cell and its corresponding control cells (cells transfected with the pCMV empty vector). The A549 cells overexpressing lncRNA CCTT were shown below. Figure 1 As shown.

[0065] 2. Stable overexpression (construction of stable cell lines)

[0066] (1) The retrovirus pQCXIP-CCTT overexpressing lncRNA CCTT was packaged using the retroviral expression vector pQCXIP (Biovector, catalog number: 3558629).

[0067] (2) Vaccination 2×10 5 A549 cells were transferred to 6-well plates and allowed to adhere to the walls until they reached 70% confluence.

[0068] (3) Cells were infected with pQCXIP-CCTT retrovirus, with empty virus as a control (1 ml virus solution + 1 ml DMEM medium was incubated for 12 h and then replaced with DMEM complete medium).

[0069] (4) After 48 hours, puromycin (3 μg / ml) was added for screening, and the surviving cells were amplified and established after 3 days.

[0070] (5) The overexpression efficiency of lncRNA CCTT was detected by cellular RNA qRT-PCR.

[0071] The results showed that lncRNA CCTT overexpression efficiency was high, and a stable overexpression A549 cell line was successfully constructed. Figure 1 ).

[0072] II. Cell growth / proliferation detection

[0073] 1. Cell counting

[0074] 72 hours after transfection, the pCMV-CCTT cells were counted, with cells transfected with the pCMV empty vector serving as a control.

[0075] 2. Cell growth curve

[0076] (1) Select appropriate density to seed cells in 24-well plates according to different cell types, culture at 37°C and 5% CO2 concentration, and record the initial cell number (0d).

[0077] (2) At appropriate time points (such as 3d, 5d, 7d, etc.), the cell counts of the control group (cells transfected with pCMV empty vector) and the experimental group (cells transfected with pCMV-CCTT) were analyzed using a cell counter.

[0078] (3) Based on the number of cells at each time point, the cell growth curve was plotted and statistical analysis was performed through three independent replicate experiments.

[0079] 3. Cell clone formation experiment

[0080] (1) Take cells from each group in the logarithmic growth phase, including A549 cells that are stably overexpressing lncRNA CCTT and control A549 cells infected with empty virus, digest them with trypsin and pipette them into single cells, centrifuge them, and resuspend them in serum-containing culture medium.

[0081] (2) Cell counting: 50, 100 and 200 cells per dish were seeded into 10cm culture dishes to ensure uniform cell distribution. The cells were then cultured at 37℃ and 5% CO2 for 2-3 weeks.

[0082] (3) Observe and change the medium regularly. When visible clones appear, stop the culture. Discard the supernatant and wash carefully twice with PBS. Fix the cells with 4% paraformaldehyde and incubate at room temperature for 15 min.

[0083] (4) Discard the fixative, add an appropriate amount of crystal violet staining solution, stain at room temperature for 20 minutes, then slowly wash away the staining solution with running water and air dry.

[0084] (5) Count the number of clones in each group and calculate the clone formation rate = (number of clones / number of inoculated cells) × 100%.

[0085] III. Detection of cell tumorigenicity (tumor formation experiment in nude mice)

[0086] (1) Prepare 6-8 week old male BALB / c nude mice (Vitamin B1L) and house them in an SPF-grade animal room.

[0087] (2) Culture each cell line, including A549 cells that are stably transfected with lncRNA CCTT and control A549 cells infected with empty virus, in 15cm culture dishes and grow to a suitable density.

[0088] (3) Digest the cells and resuspend them in 1×PBS to prepare 1×10⁻⁶ cells. 7 / ml density suspension.

[0089] (4) Use a pre-cooled pipette tip to draw 100 μl of cell suspension, and add 100 μl of Matrigel to dilute and mix well.

[0090] (5) Anesthetize the nude mice and place them upright on the operating table. Use a microsyringe to inject the cell suspensions of the control group and the experimental group into the subcutaneous tissue at the symmetrical positions of the left and right armpits on the back of the nude mice. When a bulge is seen, prevent the fluid from flowing out.

[0091] (6) Observe the condition of nude mice: within 1 week of inoculation, the bulge is absorbed, and tumors begin to form after 2-3 weeks. The diameter of the tumor is measured regularly.

[0092] (7) At an appropriate time (when the nude mouse is in poor condition or the tumor diameter reaches 1 cm), the nude mouse is euthanized, the tumor in situ is dissected and observed, and the size and weight of the tumor are measured.

[0093] The results showed that overexpression of lncRNA CCTT exerted a pro-cancer effect in various tumor cells, including promoting A549 cell proliferation, colony formation, and tumorigenesis in nude mice. Figure 3Meanwhile, overexpression of lncRNA CCTT had no significant inhibitory effect on the proliferation of human endothelial cells HUVEC and bronchial epithelial cells BEAS-2B. Figure 4 It has a promoting effect on the growth and proliferation of human colon cancer cells HCT116. Figure 4 This suggests that its function has a certain degree of cellular universality.

[0094] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. <110> Academy of Military Medicine, Academy of Military Sciences of the Chinese People's Liberation Army; Institute of Biophysics, Chinese Academy of Sciences <120> Application of overexpression of long noncoding RNA CCTT in the construction of artificial aneuploidy <130> GNCLN212032 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 730 <212> RNA <213> Artificial sequence <400> 1 cuuuuugagg cagcuaccag aagggagcac caagcagaug cagcuccccu ucccucccau 60 uccaccaucc acugucccca gcaagaaccu gcgggagggu ggcccaaugg ggagaaaacu 120 aaggauucgg uguugggacc acuccugccc ugaccugccc ugugacuccg ucauacucuc 180 caaaggccag acccuccuag accagcugga accaccauca agaugucccc agccauguca 240 gacucugggg ccccaggcgg agggcaacca gaugucuuca gcuccaaguc uggccucucc 300 uccagcaag cagcaacug cagagaccuu ggaaggauc aaccaauac auguccaou 360 uccugcccuc uaaccuggca ggggagcaag gcccagccaa ggaguacag aacugaggc 420 uuggccaggc gugguggcuc acaccugshaa ucucagcacu gggaggccaa gguggggcaga 480 ucgcuugagc ccaggauu gagaccagcc cgaggaacau ggcgaaaccc caucucuaca 540 aaaaaaacag aaauuagcca agugugggg cacgugucug uaguuucagc uacucaggag 600 gcuuaggugg gaggaucacc gcuuaggugg gaggauugcu ugshaugagc gaggauugcu 660 cuccagccag ggagacagag ugagacccug uccaaaaaaaaaaaaaaaaaaaaaaa 720 730 <210> 2 <211> 759 <212> DNA <213> Artificial sequence <400> 2 cgggatcccg cttttgagg cagctaccag aagggagcac caagcagatg cagctcccct 60 tccctcccat tccaccatcc actgcccca gcagaacct gcgggaggt ggcccaatgg 120 ggagaaaact areatttcgg tgttgggacc actcctgccc tgacctgccc tgtgactccg 180 tcatactctc caaaggccag accctcctag accagctgga accaccatca agatgtcccc 240 agccatgtca gactctgggg ccccaggcgg agggcaacca gatgtcttca gctccaagtc 300 tggcctctcc tcccagcaag cagccaactg cagagacctt ggaaaggatc aaccatatac 360 aatgtccatt tcctgccctc taacctggca ggggagcaag gcccagccaa ggagttacag 420 aaactgaggc ttggccaggc gtggtggctc acacctgcaa tctcagcact gggaggccaa 480 ggtgggcaga tcgcttgagc ccaggagttt gagaccagcc cgaggaacat ggcgaaaccc 540 catctctaca aaaaatacag aaattagcca agtgtggtgg cacgtgtctg tagtttcagc 600 tactcaggag gcttaggtgg gaggatcacc tgagcctggg aggtagaggt tgcaatgagc 660 agagattgct ctccagccag ggagacagag tgagaccctg tcccaaaata aataaataa 720 tagaaataa atttaaaaaa gaaactgagc ggaattccg 759

Claims

1. Application of substances that can promote lncRNA CCTT expression in the following P1 and / or P2: P1. Construct artificial aneuploid cells, or prepare reagents or kits for constructing artificial aneuploid cells; P2, promoting cell proliferation and / or growth, or preparing reagents or kits for promoting cell proliferation and / or growth; The lncRNA CCTT is the RNA shown in SEQ ID No. 1; The substance that can promote the expression of lncRNA CCTT is a DNA molecule that can be transcribed into lncRNA CCTT or an expression vector containing the DNA molecule. The DNA molecule capable of being transcribed into lncRNA CCTT is shown at positions 11-749 of SEQ ID No. 2; The cells in question are tumor cells; The tumor cells are colon cancer cells or lung cancer cells.

2. Use according to claim 1, characterized in that: The expression vector containing the DNA molecule is a recombinant plasmid obtained by cloning the DNA fragment shown in positions 11-749 of SEQ ID No. 2 into the multiple cloning site of the pcDNA3.1(+) vector.

3. Use according to claim 1 or 2, characterized in that: The colon cancer cells were HCT116 cells; the lung cancer cells were A549 cells.

4. A method for constructing artificial aneuploid cells, comprising the following steps: introducing a substance capable of promoting the expression of lncRNA CCTT into recipient cells to obtain aneuploid cells with increased lncRNA CCTT expression; wherein the lncRNA CCTT is the RNA shown in SEQ ID No. 1; The substance that can promote the expression of lncRNA CCTT is a DNA molecule that can be transcribed into lncRNA CCTT or an expression vector containing the DNA molecule. The DNA molecule capable of being transcribed into lncRNA CCTT is shown at positions 11-749 of SEQ ID No. 2; The cells in question are tumor cells; The tumor cells are colon cancer cells or lung cancer cells.

5. The method of claim 4, wherein: The expression vector containing the DNA molecule is a recombinant plasmid obtained by cloning the DNA fragment shown in positions 11-749 of SEQ ID No. 2 into the multiple cloning site of the pcDNA3.1(+) vector.

6. The method according to claim 4 or 5, characterized in that: The colon cancer cells were HCT116 cells; the lung cancer cells were A549 cells.

Images