A LncRNA for antagonizing replicative senescence of human mesenchymal stem cells and its applications

Abstract

This invention relates to the field of biomedical technology, specifically to a lncRNA used to antagonize the replicative senescence of human mesenchymal stem cells and its applications. The lncRNA is ENST00000585537.1, located on human chromosome Chr17q24. Compared with existing technologies, this lncRNA has the following advantages: The lncRNA described in this application has extremely high species conservation, being expressed only in certain human tissues and organs, with its expression level gradually increasing with aging. Knockdown of this lncRNA can significantly reduce chronic inflammatory secretion in senescent mesenchymal stem cells in vitro, improve the apoptotic state and differentiation capacity of senescent mesenchymal stem cells, suggesting that it may play an important role in delaying the senescence of human mesenchymal stem cells, possessing high application potential and providing high reference value for in vivo experiments on anti-aging.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a lncRNA used to antagonize the replicative senescence of human mesenchymal stem cells and its applications. Background Technology

[0002] Stem cells are undifferentiated cells with a high capacity for self-renewal and the ability to regenerate various tissues and organs, thus holding broad prospects for research and application in tissue engineering and regenerative medicine. In mammals, stem cells can be divided into embryonic stem cells and adult stem cells. Embryonic stem cells are derived from the inner cell mass of the blastocyst, while adult stem cells originate from various tissues. With advancements in science and technology, stem cell research has received increasing attention, and stem cells hold an extremely important position in the field of biology.

[0003] Mesenchymal stem cells (MSCs) are an important type of adult stem cell found in the stroma of various tissues and organs, particularly bone marrow and subcutaneous fat. They possess multi-lineage differentiation potential, capable of differentiating into osteoblasts, adipocytes, chondrocytes, and neurons. In tissue engineering and regenerative medicine research and applications, MSCs are considered important seed cells due to their wide availability, low immunogenicity, diverse differentiation potential, and lack of ethical controversy. In vitro, MSCs exhibit fibroblast-like adherent growth and express various cell surface markers. However, like other normal somatic cells, MSCs have a limited number of passages for in vitro expansion. After a certain number of mitotic divisions, they enter a replicative senescent state, characterized by increased size, decreased cell viability, reduced proliferation and differentiation capacity, and disordered immune regulation. This poses challenges to scientific research, tissue engineering, and regenerative medicine applications of MSCs. Therefore, exploring ways to improve the in vitro replicative senescence of mesenchymal stem cells is crucial.

[0004] Long non-coding RNAs (lncRNAs) are non-coding RNAs in the transcriptome that are longer than 200 nt and mostly possess secondary structures. In mammalian genomes, 4%–9% of sequences produce transcripts that are lncRNAs. These characteristics enable lncRNAs to target chromatin, mRNA, and proteins through various mechanisms to regulate intricate biological processes, such as cell lineage differentiation, immune regulation, and cancer development. Interestingly, most lncRNAs exhibit low species conservation, low expression abundance, and highly specific spatiotemporal expression patterns. This means that the discovery of lncRNAs that regulate aging and improve aging phenotypes, and the elucidation of their regulatory mechanisms, will not only have great potential as specific anti-aging targets for humans, but also hold promise for clinical translation into non-coding RNA therapies for aging-related treatments. However, due to the poor species conservation of lncRNAs, most of the lncRNAs discovered in recent years related to delaying aging and improving aging phenotypes are based on mouse or cell lines, significantly reducing the guiding value of these studies for human anti-aging research. Therefore, there is an urgent need to discover LncRNAs related to the anti-aging effects of human MSCs and to conduct in-depth research on their functions and mechanisms. Summary of the Invention

[0005] To address the aforementioned technical problems in the existing technology, this invention provides a lncRNA for antagonizing the replicative senescence of human mesenchymal stem cells and its application. Specifically, this is achieved through the following technical solution:

[0006] A lncRNA for antagonizing the replicative senescence of human mesenchymal stem cells, wherein the lncRNA, ENST00000585537.1, is highly conserved only in humans and located on human chromosome Chr17q24, as shown in the attached image. Figure 1 As shown.

[0007] The use of substances that knock down LncRNAs used to antagonize replicative senescence of human mesenchymal stem cells in the preparation of products for inhibiting apoptosis.

[0008] The use of the substance described above, which is used to antagonize the replicative senescence of human mesenchymal stem cells, in the preparation of products for inhibiting the cellular senescence secretory phenotype.

[0009] The use of substances that knock down LncRNAs used to antagonize the replicative senescence of human mesenchymal stem cells in the preparation of products for improving the osteogenic differentiation capacity of senescent mesenchymal stem cells.

[0010] The application of substances that overexpress the described LncRNA, which is used to antagonize the replicative senescence of human mesenchymal stem cells, in the preparation of products for promoting apoptosis.

[0011] Application of substances overexpressed to antagonize replicative senescence of human mesenchymal stem cells in the preparation of products for promoting cellular senescence secretory phenotypes.

[0012] The application of substances that overexpress the LncRNA described above, which is used to antagonize the replicative senescence of human mesenchymal stem cells, in the preparation of products for reducing the osteogenic differentiation capacity of senescent mesenchymal stem cells.

[0013] Furthermore, the inhibition of apoptosis refers to the inhibition of apoptosis in senescent human mesenchymal stem cells.

[0014] A drug that reduces chronic inflammatory secretion in senescent mesenchymal stem cells and improves the apoptotic state and differentiation capacity of senescent cells, characterized in that it contains a substance that knocks down the LncRNA of claim 1, which is used to antagonize the replicative senescence of human mesenchymal stem cells.

[0015] Furthermore, the inhibition of apoptosis refers to the inhibition of apoptosis in senescent human mesenchymal stem cells.

[0016] Compared with the prior art, the technical effects of this invention are reflected in:

[0017] This invention discloses a lncRNA for antagonizing replicative senescence of human mesenchymal stem cells. The lncRNA, ENST00000585537.1, is located on human chromosome Chr17q24. Compared with existing technologies, this lncRNA has the following advantages: The lncRNA described in this application has extremely high species conservation, being expressed only in certain human tissues and organs, with its expression level gradually increasing with aging. Knockdown of this lncRNA can significantly reduce chronic inflammatory secretion in senescent mesenchymal stem cells in vitro, improve the apoptotic state and differentiation capacity of senescent mesenchymal stem cells, suggesting that it may play an important role in delaying the senescence of human mesenchymal stem cells, possessing high application potential and providing high reference value for in vivo experiments on anti-aging.

[0018] This LncRNA plays an important role in improving the senescent secretory phenotype, differentiation, and apoptosis of human adipose-derived mesenchymal stem cells, and is expected to become an effective target for delaying and improving the quality of life of aging in clinical practice. It achieves clinical translation and application through non-coding RNA therapy, overcoming the shortcomings and defects mentioned in the background technology. Attached Figure Description

[0019] Figure 1This study analyzes the location and species conservation of LncRNA ENST00000585537.1 in the human genome.

[0020] Figure 2 This invention provides an aging model for the in vitro replicative aging of human adipose-derived mesenchymal stem cells in Example 1 of the present invention.

[0021] Figure 3 The expression trend of LncRNA ENST00000585537.1 in the in vitro replicative aging process of human adipose-derived mesenchymal stem cells is shown in Example 2 of this invention.

[0022] Figure 4 In Example 3 of this invention, the production of the secretory phenotype of replicative senescence in human adipose-derived mesenchymal stem cells was inhibited after siRNA knockdown of LncRNA ENST00000585537.1.

[0023] Figure 5 In Example 4 of this invention, the production of the senescence secretion phenotype of replicative senescence in human adipose-derived mesenchymal stem cells was promoted after overexpression of LncRNA ENST00000585537.1 by lentivirus.

[0024] Figure 6 In Example 5 of this invention, after knocking down LncRNA ENST00000585537.1 with shRNA, apoptosis in the replicative senescence of human adipose-derived mesenchymal stem cells was inhibited.

[0025] Figure 7 In Example 6 of this invention, the apoptosis of replicative senescence in human adipose-derived mesenchymal stem cells was promoted after overexpression of LncRNA ENST00000585537.1 by lentivirus.

[0026] Figure 8 In Example 7 of this invention, the osteogenic differentiation capacity of human adipose-derived mesenchymal stem cells was improved after knocking down LncRNA ENST00000585537.1 with shRNA. Detailed Implementation

[0027] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0028] Example 1

[0029] Culture and induction of human adipose-derived mesenchymal stem cells:

[0030] Primary human adipose-derived mesenchymal stem cells (hAMSCs) were obtained from adipose tissue extracted during liposuction. Liposuction fat is normally medical waste; these cells were obtained from the Department of Plastic Surgery, Chinese Academy of Medical Sciences, to ensure the extraction method did not cause additional harm to the patient and did not compromise patient privacy. Informed consent was obtained from the patient, and an application was submitted to and approved by the Ethics Review Committee of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The cells were cultured in complete culture medium (DMEM / F12 + 2% fetal bovine serum + penicillin 100 IU / mL + streptomycin 100 μg / mL) at 37°C in a 5% CO2 incubator. The culture medium was changed every two days, and the cells were passaged when the confluence reached 80%-90%. Passage 3 (P3) and 14 (P14) human adipose-derived mesenchymal stem cells were used for aging modeling validation and were subjected to viral knockdown or overexpression treatment for various experiments.

[0031] Experimental results are as follows Figure 2 As shown, Figure 2 A shows the SA-β-gal staining results of the 3rd and 14th generation hAMSCs. The positive rate of SA-β-gal staining in the P14 group was significantly higher than that in the P3 group. Figure 2 B shows the expression results of aging-related marker genes in passages 3 and 14 of hAMSCs. Western blot images show the protein expression of GAPDH (as an internal control), P16, P21, and Lamb1. The expression levels of P14 cells were significantly different from those of P3 cells in both mRNA and protein. The results indicate that P14 cells had a significantly higher proportion of SA-β-gal positive cells and significantly higher expression levels of p16, p21, and Lamb1 genes than P4 cells. Therefore, P14 cells exhibit a clear aging phenotype and can be used as an aging control group for subsequent experiments. (Scale bar: 100 μm.)

[0032] Example 2

[0033] Expression trend analysis of LncRNA ENST00000585537.1:

[0034] When the cell growth density reaches approximately 90%, the cells are passaged normally, and the third and fourteenth generation hAMSCs are harvested. RNA is extracted using standard procedures for qRT-PCR experiments.

[0035] Experimental results are as follows Figure 3 As shown, Figure 3The expression trend of lncRNA ENST00000585537.1 during the replicative senescence of hAMSCs. Figure 3 It can be seen that the expression level of LncRNA ENST00000585537.1 is significantly increased during the replicative senescence of hAMSCs.

[0036] Example 3

[0037] Knockdown of lncRNA ENST00000585537.1 suppressed the expression of the senescence secretory phenotype in replicative senescent hAMSCs.

[0038] Third-generation normally cultured MSCs were used for meaningless control sequence knockdown (Young MSCs, Y-siRNA-NC), and fourteenth-generation hAMSCs (Replicative senescence MSCs, RS-) were used to effectively knock down lncRNA ENST00000585537.1 using two pairs of small interfering RNAs (siRNAs). All siRNAs were purchased from Suzhou Jima Biotechnology Co., Ltd., and transfection was performed using the laboratory-standard liposome transfection method. hAMSCs were harvested 24 hours after transfection, and RNA was extracted according to standard procedures for qRT-PCR experiments to detect knockdown efficiency. When cell density reached approximately 90%, RNA was extracted and cell supernatant was collected for qRT-PCR and ELISA experiments to detect the expression of senescence-related secretory phenotypes IL-1β, IL-6, MCP1, and TNF-α. All experiments showed that knockdown of lncRNA ENST00000585537.1 inhibited the expression of the senescence secretory phenotype in replicative senescent hAMSCs.

[0039] The specific sequences of the two pairs of small interfering RNAs (siRNAs) are as follows:

[0040] si-MEK6-AS1-1

[0041] F:GCUGUGACCAAUGAACAAUTT

[0042] R:AUUGUUCAUUGGUCACAGCTT

[0043] si-MEK6-AS1-2

[0044] F:GUGGUUCCCAUCUCAAGAATT

[0045] R:UUCUUGAGAUGGGAACCACTT

[0046] Experimental results are as follows Figure 4 As shown. Figure 6 A represents the knockdown efficiency of siRNA against LncRNA ENST00000585537.1; Figure 4 B and C show the relative expression of aging-related secretory phenotypic markers in the aging control group, aging knockdown experimental group, and young control group, as detected by qRT-PCR and Western blotting. Figure 4 It was found that, compared with the aging control group, knocking down LncRNA resulted in a decrease in the mRNA and protein levels of cellular senescence-related secretory phenotypic markers. (Note: RS-siRNA-NC is a meaningless control sequence, while RS-siRNA-1 and RS-siRNA-2 are specific sequences designed for ENST00000585537.1.)

[0047] Example 4

[0048] Overexpression of lncRNA ENST00000585537.1 promoted the expression of the senescence secretory phenotype in replicative senescent hAMSCs:

[0049] Third-generation (Young MSCs, Y-Lenti-NC) cells cultured normally were used for overexpression of a meaningless control sequence. Fourteenth-generation hAMSCs (Replicative Senescence MSCs, RS-) were used for efficient overexpression of lncRNA ENST00000585537.1 using a lentiviral vector. Both the overexpression plasmid and lentiviral vector were purchased from Suzhou Jima Biotechnology Co., Ltd., and infection was performed using a commonly used laboratory lentiviral infection method. Forty-eight hours after infection, puromycin was used for selection. After selection, hAMSCs were harvested, and RNA was extracted for qRT-PCR experiments to detect overexpression efficiency. Experiments were conducted when cell density reached approximately 90%, and subsequent experimental treatments were completely consistent with Example 3. All experiments showed that overexpression of lncRNA ENST00000585537.1 promoted the expression of the senescent secretory phenotype in replicative senescent hAMSCs.

[0050] Experimental results are as follows Figure 5 As shown. Figure 5 A represents the overexpression efficiency detection of LncRNA ENST00000585537.1; Figure 5 B and C show the relative expression of aging-related secretory phenotypic markers in the aging control group, the aging overexpression experimental group, and the young control group, as detected by qRT-PCR and Western blotting. Figure 5It was found that, compared with the aging control group, overexpression of LncRNA led to an increase in the RNA and protein levels of cellular senescence secretory phenotypic markers. (Note: RS-Lenti-NC is a meaningless control sequence, and RS-Lenti-LncRNA is an overexpression-specific sequence designed for ENST00000585537.1.)

[0051] Example 5

[0052] Knockdown of LncRNA ENST00000585537.1 significantly inhibited apoptosis in replicative senescent hAMSCs.

[0053] Third-generation hAMSCs from normal culture were used for meaningless control sequence knockdown. Fourteenth-generation hAMSCs were then used to effectively knock down the lncRNA ENST00000585537.1 using a lentiviral vector. Both the knockdown plasmid and the lentiviral vector were purchased from Suzhou Jima Biotechnology Co., Ltd., and infection was performed using a commonly used laboratory lentiviral infection method, following the same procedure as in Example 3. When the cell growth density reached approximately 90%, experimental procedures were performed, including collecting cell proteins and conducting flow cytometry to detect apoptosis.

[0054] Cells were loaded at a rate of 1×10 5 Cells were seeded at a density of [number] cells / well in 6-well plates. After good cell adhesion, the old culture medium was discarded and replaced with fresh medium containing / without the drug. After 72 hours, the cells were detected according to the instructions of the Annexin V-FITC / PI apoptosis detection kit. The principle is as follows: In normal living cells, phosphatidylserine (PS) is located on the inner side of the cell membrane. However, in early apoptotic cells, PS flips from the inner side of the cell membrane to the surface and is bound by Annexin-V with high affinity. Propidium iodide (PI) is a nucleic acid dye that cannot penetrate the intact cell membrane of normal cells or early apoptotic cells, but can penetrate the cell membrane of late apoptotic and necrotic cells, staining the nucleus red. This is used to distinguish between surviving early apoptotic cells and necrotic or late apoptotic cells. Therefore, when Annexin V and PI are used in combination and detected by flow cytometry, the vast majority of late apoptotic cells exhibiting replicative senescence are located on the right side of the coordinate axis.

[0055] Experimental results are as follows Figure 6 As shown. Figure 6 A represents the results of flow cytometry analysis of apoptotic cells; Figure 6 B represents the detection of apoptosis-related markers in replicative senescent hAMSCs. Figure 6 It can be seen that, compared with the control group, knocking down LncRNA significantly inhibited apoptosis of replicative senescent hAMSCs.

[0056] Example 6

[0057] Overexpression of lncRNA ENST00000585537.1 significantly promoted apoptosis in replicative senescent hAMSCs: The lentiviral infection process for overexpressing lncRNA ENST00000585537.1 was identical to that in Example 4. Experiments were performed when cell density reached approximately 90%, including cell protein collection and flow cytometry analysis for apoptosis detection. Subsequent experimental treatments were identical to those in Example 5.

[0058] Experimental results are as follows Figure 7 As shown. Figure 7 A represents the results of flow cytometry analysis of apoptotic cells; Figure 7 B represents Western blot analysis of apoptosis-related markers in replicative senescent hAMSCs. Figure 7 It was found that, compared with the control group, overexpression of LncRNAENST00000585537.1 significantly promoted apoptosis of replicative senescent hAMSCs.

[0059] Example 7

[0060] LncRNA ENST00000585537.1 significantly improved the osteogenic differentiation ability of replicating senescent hAMSCs: Third-generation hAMSCs were used for a meaningless control sequence knockdown. Fourteenth-generation hAMSCs were then used to effectively knock down LncRNA ENST00000585537.1 using a lentiviral vector. Both the knockdown plasmid and the lentiviral vector were purchased from Suzhou Jima Biotechnology Co., Ltd., and infection was performed using a commonly used laboratory lentiviral infection method, identical to that in Example 4. When the cell density reached approximately 90%, experiments were conducted using osteogenic differentiation induction medium (DMEM + 10% fetal bovine serum + penicillin 100 IU / mL + streptomycin 100 μg / mL + dexamethasone 10⁻⁸ M + sodium β-glycerate 10⁻³ M + ascorbic acid 10⁻⁴ M) for induction and various experiments. The experimental results are as follows: Figure 8 As shown. Figure 8 A represents ALP staining of cells in each group on day 6. After knockdown, the number of ALP-positive cells in replicative senescent hAMSCs increased significantly. Figure 8 B represents the detection of mRNA expression levels of key osteogenic-related genes RUNX2, ALP, COL1A1, and OPN. (Scale bar: 100 μm) Figure 8 It can be seen that knockdown of LncRNAENST00000585537.1 can promote osteogenic differentiation of replicative senescent hAMSCs.

[0061] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. The application of a reagent that knocks down the expression level of LncRNA ENST00000585537.1, which antagonizes the replicative senescence of human mesenchymal stem cells, in inhibiting the senescence of human mesenchymal stem cells, characterized in that, The reagent specifically contains the following two pairs of small interfering RNAs: si-MEK6-AS1-1 F: GCUGUGACCAAUGAACAAUTT R: AUUGUUCAUUGGUCACAGCTT si-MEK6-AS1-2 F: GUGGUUCCCAUCUCAAGAATT R: UUCUUGAGAUGGGAACCACTT The LncRNA ENST00000585537.1 has a full-length nucleotide sequence of 584 nt, as follows: GTTTGACCACGCGTATCGATGTCGACTTGTAGATTTCTGCAGATCGCAACAAGCCTCGACTGCTTATTGGCACAGTCTGCAAAGGGCCTCCTCCGTTCCAAAGTTGCTCATCCTAAAACCTGAAAAAAAAAATGACTCAGAAGTGGTTCCCATCTCAAGAACTACCCACGAGTCTCAGTGGACACATGTGAGTATGAGATTCCCCGATGCAGCCTTCCAATCCAGCAGAGGTTCCAACCAACTTCCAAACAGGTGCAACTTGATGGCACCTCATTTTTGGCTGTACCAGATGCCTTCTATCTCTGGACTCCTGCCCTCAGACTTCTCCGACACCTGAAGAAAGCTTCTGGGACAGCTGACATGCACAACCTGGGAGCTAAAACATGTGGGACCAGCTGTGACCAATGAACAATGATGGACAGGAGACGATAACCCCATTAGTTCCTTACAGTGCTGAGATAAATTTCATGAGTCTCCTAGAAGATCTGATGGGATTGAACATCAGTTGTACCTAGCATGGCCGCTGTACTGGCTTTCCTTCCTTCCCTATTTCATCCCCCCTCGTCCCTCCTACTCCTTGGGATCACATTGACAAATAAGTCTCTTGCATGCAAAAAAAAAAAAAAAAAAAAA。

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