AU2019433418B2

AU2019433418B2 - Long-chain non-coding RNA and use thereof

Info

Publication number: AU2019433418B2
Application number: AU2019433418A
Authority: AU
Inventors: Xiaosong Gu; Yan Liu; Jiancheng WU; Yumin Yang; Chun YAO; Bin Yu
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2019-03-13
Filing date: 2019-05-24
Publication date: 2021-08-19
Anticipated expiration: 2039-05-24
Also published as: AU2019433418A1; CN109897853B; CN109897853A; WO2020181663A1

Abstract

Provided is an LncRNA LOC680254 related to cell proliferation and cell cycle control. The LncRNA LOC680254 is obtained by analyzing different LncRNAs in different time point samples after sciatic nerve injury of SD rats by use of RNA-seq and may be used as miRNA molecular sponge to specifically bind miR-30d-3p, miR-671, miR-3594-3p or miR-3473 so as to resist miRNA functions and promote expression of Psrc1 and Ska1, thereby promoting cell proliferation.

Description

LONG-CHAIN NON-CODING RNA AND USE THEREOF TECHNICAL FIELD

The present invention belongs to the professional field of biomedicine, and relates to a long-chain non-coding RNA and use thereof.

BACKGROUND

A long-chain non-coding RNA (LncRNA) is a type of RNA having a length greater than 200 nt and having no coding function. Expression of the LncRNA has space-time speciality, and the LncRNA can adjust expression of genes at various levels such as chromatin remodeling, transcriptional control and post-transcriptional processing. The LncRNA is closely related to a variety of diseases, at present, most of researches on the LncRNA mainly focus on the relationship with cancer and its role as a tumor specific marker, while the researches on the LncRNA in other physiological activities or diseases are very limited. In multicellular organisms, there are very strict control systems for various cell cycles, which enables various cells to proliferate or remain in a static state according to the needs of the organisms. The cell cycle is not only affected by synthesis, degradation or activation of some substances in the cells, but also controlled by the type, intensity and duration of various signals inside and outside the cells. Cell cycle control is completed based on cascade translation in the cells after integrating different signals in the cells, any abnormal link may lead to excessive or defective cell proliferation, and often accompanied by abnormal cell differentiation. Excessive or defective cell proliferation and excessive or insufficient cell apoptosis are associated with a variety of diseases, such as neurodegenerative diseases, cardiovascular diseases, immune diseases and so on. Therefore, the study found that the LncRNA related to cell cycle control has great clinical value. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

SUMMARY

In one aspect, the present disclosure provides use of a long-chain non-coding RNA, miR d-3p, miR-671, miR-3594-3p and miR-3473 as molecular intervention targets in controlling cell proliferation, apoptosis and a cell cycle, wherein a cDNA sequence of the long-chain non coding RNA is as shown in SEQ ID NO: 1. In another aspect, the present disclosure provides use of a long-chain non-coding RNA, miR-30d-3p, miR-671, miR-3594-3p and miR-3473 in the manufacture of a medicament for treating a disease related to cell proliferation or apoptosis, wherein a cDNA sequence of the long-chain non-coding RNA is as shown in SEQ ID NO: 1. In another aspect, the present disclosure provides use of a long-chain non-coding RNA, miR-30d-3p, miR-671, miR-3594-3p and miR-3473 in the manufacture of a medicament for controlling proliferation of neurogliocytes and repairing peripheral nerve injury, wherein a cDNA sequence of the long-chain non-coding RNA is as shown in SEQ ID NO: 1. In the present invention, an LncRNA LOC680254 related to Schwann cell proliferation and cell cycle control is obtained by analyzing different LncRNAs in different time point samples after sciatic nerve injury of SD rats by use of RNA-seq, and the LncRNA LOC680254 has the potential to become a new target for repairing of peripheral nerve injury. The specific technical solutions of the present invention are as follows: A long-chain non-coding RNA (LncRNA) LOC680254 is provided, wherein a cDNA sequence thereof is as shown in SEQ ID NO: 1. In some examples, the present invention provides use of the above long-chain non-coding RNA, miR-30d-3p (SEQ ID NO: 2), miR-671 (SEQ ID NO: 3), miR-3594-3p (SEQ ID NO: 4) and miR-3473 (SEQ ID NO: 5) as molecular intervention targets in controlling cell proliferation, apoptosis and a cell cycle. The long-chain non-coding RNA of the present invention can be used as miRNA molecular sponge to specifically bind the miR-30d-3p, the miR-671, the miR-3594-3p or the miR-3473 so as to resist miRNA functions; the miR-30d-3p and the miR-671 can promote expression of Psrc1 after functions thereof are inhibited; and the miRNA-3594-3p and the miRNA-3473 can promote expression of Skal after functions thereof are inhibited, so that cell proliferation, apoptosis and the cell cycle are adjusted.

In some examples, the present invention provides use of the above long-chain non-coding RNA, the miR-30d-3p, the miR-671, the miR-3594-3p and the miR-3473 as the molecular intervention targets in preparing medicines for treating diseases related to cell proliferation or apoptosis. The above diseases include: diseases related to excessive cell proliferation, including tumor, liver fibrosis, pulmonary fibrosis, renal fibrosis, prostatic hypertrophy, essential thrombocytosis, familial polycythemia, rheumatoid arthritis, psoriasis, interstitial glomerulopathy, atherosclerosis and so on. Diseases related to defective cell proliferation include nerve cell regeneration, diabetic nephropathy, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis), aplastic anemia and so on. Diseases related to insufficient cell apoptosis include tumor, and autoimmune diseases caused by failure of effective clearing of T lymphocytes for autoantigens, and so on. Diseases related to excessive cell apoptosis include cardiovascular diseases such as myocardial ischemia-reperfusion injury, heart failure, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis), and virus infection (e.g., AIDS). Specific research use of the present invention is use of the long-chain non-coding RNA as the molecular intervention target in preparing repairing medicines for controlling proliferation of neurogliocytes (Schwann cells) and then repairing peripheral nerve injury. In the present invention, 30 differently-expressed LncRNAs are obtained by analyzing different LncRNAs in different time point samples after sciatic nerve injury of SD rats by use

2a of RNA-seq, wherein the LncRNA LOC680254 of the present invention is continuously up-regulated in expression after nerve injury. It is approved by qRT-PCR that expression change of the LncRNA LOC680254 is consistent with RNA-seq results. The complete nucleotide sequences of the LncRNA LOC680254 are obtained by RACE reaction, and are located at 13q22. Subcellular localization analyzes that the LncRNA LOC680254 is mainly expressed in cytoplasm of Schwann cells. It is shown by research of the present invention that by disturbing expression of the LncRNA LOC680254 in vitro, proliferation of the Schwann cells is remarkably inhibited, apoptosis of the Schwann cells is promoted, and development of cycles of the Schwann cells is hindered. Gene chip analysis and ceRNA prediction are combined with Luciferase detection to prompt that the LncRNA LOC680254 is combined with 4 miRNAs to adjust expression of target genes Psrcl and Skal related to cell proliferation, apoptosis and the cell cycle, wherein the miR-30d-3p and the miR-671 target the Psrcl, and the miRNA-3594-3p and the miRNA-3473 target the Skal. It is prompted by research of the present invention that the LncRNA LOC680254 has the possibility to affect proliferation and the cell cycle of the Schwann cells by adjusting gene expression related to the cell cycle, and has the potential to become the new target for repairing of peripheral nerve injury.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is expression tendency and distribution of an LncRNA LOC680254 of the present invention. (FIG. la: Expression change (GAPDH for internal reference) of LOC680254 in sciatic nerve tissue after sciatic nerve injury, detected by qRT-PCR. Compared with Od, expression of the LncRNA LOC680254 in an injured sciatic nerve is continuously up-regulated (**p<0.01). FIG. 1b: Relative expression levels of the LncRNA LOC680254 in cytoplasm and nucleus of Schwann cells, detected by qRT-PCR. U6 is used as internal reference in the nucleus, and p-actin is used as internal reference of the cytoplasm. Distribution of the LncRNA LOC680254 in the cytoplasm or the nucleus is represented by percentages thereof in a total RNA). FIG. 2 is influence of disturbing an LncRNA LOC680254 on proliferation, apoptosis and a cell cycle of Schwann cells in the present invention. (FIG. 2a: Influence of disturbing the LncRNA LOC680254 on proliferation of the Schwann cells, detected by an Edu cell proliferation experiment. A histogram on the lower right side is a proliferation rate of the Schwann cells after the LncRNA LOC680254 is disturbed by an siRNA, and proliferation of the Schwann cells is remarkably inhibited after LOC680254 is knocked out. FIG. 2b: Influence of disturbing the LncRNA LOC680254 on apoptosis of the Schwann cells, detected by Annexin V-TITC/PI staining and flow cytometry technology. A histogram on the lower right side is a statistical graph of apoptosis cell percentages of the Schwann cells of different experiment groups through flow cytometry detection. FIG. 2c: Apoptosis situations of the Schwann cells detected by Tunel after siRNA transfection and contrast for 48 h. A histogram on the lower right side is a statistical graph of apoptosis cell percentages of the Schwann cells of the different experiment groups through Tunel detection. FIG. 2d: Influence of disturbing the LncRNA LOC680254 on the cycle of the Schwann cells, detected by flow cytometry detection. The figure shows average percentages of the cells in each period of the cell cycle (*p<0.05, **p<0.01, ***p<0.001)). FIG. 3 is influence of the LncRNA LOC680254 of the present invention on expression of genes Psrcl and Skal by adjusting miR-30d-3p, miR-671, miRNA-3594-3p and miRNA-3473. (FIG. 3a: Expression of the genes Psrcl and Skal in Schwann cells after the LncRNA LOC680254 is disturbed, detected by qRT-PCR. FIG. 3b: Combination situations of the LncRNA LOC680254 with the miR-30d-3p, the miR-671, the miRNA-3594-3p and the miRNA-3473, detected by Luciferase reporter assays. FIG. 3c: Combination situations of the miR-30d-3p, the miR-671, the miRNA-3594-3p and the miRNA-3473 with 3'UTR of the Psrcl and Skal respectively, detected by Luciferase reporter assays. FIG. 3d: Expression situations of the Psrcl after transfection of the miR-30d-3p and the miR-671 in the Schwann cells, detected by qRT-PCR. FIG. 3e: Expression situations of the Skal after transfection of the miRNA-3594-3p and the miRNA-3473 in the Schwann cells, detected by qRT-PCR (**p<0.01, ***p<0.001)).

FIG. 4 is adjustment of proliferation of Schwann cells by the LncRNA LOC680254 of the present invention through Psrc1 and Skal. (FIG. 4a: Influence of over-expressing the LncRNA LOC680254 in the Schwann cells on proliferation of the Schwann cells, detected by an Edu cell proliferation experiment. FIG. 4b: Influence of disturbing the Psrcl and the Skal in the Schwann cells on over-expressing the LncRNA LOC680254 to adjust proliferation of the Schwann cells, detected by the Edu cell proliferation experiment (*p<0.05, **p<0.01)).

DETAILED DESCRIPTION

The specific steps of the present invention are described by the following embodiments, but are not limited to the embodiments.

The terms used in the present invention, unless otherwise stated, generally have the meaning commonly understood by a person of ordinary skill in the art. The present invention is further described below in detail with reference to specific embodiments and data. It should be understood that the embodiments are only for describing the present invention by using examples, but do not limit the scope of the present invention in any manner. In the following embodiments, various processes and methods that are not described in detail are common conventional methods in the art. The present invention is further described below with reference to the specific embodiments. Embodiment 1 Studying expression and distribution characteristics of LncRNA LOC680254 1. Sciatic nerve tissue RNA extraction and qRT-PCR Tissue with pinched proximal ends of sciatic nerves of a rat at 0 d, 1d, 4 d, 7 d and 14 d was taken, tissue RNAs were extracted according to the specification of TRIZOL© Reagent (Invitrogen), after reverse transcription, qRT-PCR was performed by using SYBR© PrimeScript RT-PCR Kit (Takara), and operations were performed according to the specification of the kit (GAPDH as the internal reference), wherein a reaction program of a PCR instrument was as follows: Stage 1: 95°C 2 min; Stage 2 (Cycle: 40): 95°C 15s, 60°C 30s; Stage 3: 95°C 15s, 60°C 1 min, and 95°C 15s. A primer sequence of the LncRNA LOC680254 is as shown in SEQ ID NOs: 6-7. Results of qRT-PCR are as shown in FIG. la, and the results show that compared with 0 d, expression of the LncRNA LOC680254 in the injured sciatic nerve tissue is continuously up-regulated. 2. Rapid amplification of cDNA end (RACE) As required by the specification of SMARTerM RACE (Clotech), Tm>70°C, the base number is about 28 nt, and 5'-GSP primers are designed for the LOC680254. Sciatic nerve tissue of SD rats was taken, high-quality RNAs were extracted by using RNeasy Mini Kit (Qiagen), and in strict accordance with a specification system of a SMARTerM RACE (Clotech) kit, First-strand cDNA was synthesized by using 5'-CDS primers respectively to obtain corresponding 5'-cDNA. As required by the specification, a sequence of the 5'-GSP primers LOC680254-5-GSP designed for the LncRNA LOC680254 is as shown in SEQ ID NO: 8. Then 5'-RACE was performed by using touchdown PCR to obtain the 5' end of the LOC680254. After sequencing was finished, results were spliced with original known sequences to obtain a cDNA complete nucleotide sequence of the LOC680254, the cDNA complete nucleotide sequence was compared with rat sequences in the UCSC (http://genome.ucsc.edu/) database for analysis, and it is shown that the LncRNA LOC680254 is located at 13q22. 3. Detection of subcellular localization of LncRNA by qRT-PCR According to PARIS T M Kit (Ambion company), equal RNAs in cytoplasm and nuclei were extracted from Schwann cells respectively. For 100 1 RNA solutions obtained above, the RNAs were purified according to an RNA cleanup (Qiagen) operation in RNeasy Mini Kit, the two parts of the RNAs were eluted with RNase-free water (30 l) of the same volume and were respectively named as a cytoplasm-RNA and a nucleus-RNA, and concentrations were measured. According to a reverse transcription kit (Takara RR047A), a cytoplasm-RNA solution and a nucleus-RNA solution of the same volume were reversely-transcribed into cDNA, and qRT-PCR was performed by adopting SYBR© PrimeScript RT-PCR Kit (Takara). The reaction program of a PCR instrument was as follows: Stage 1: 95°C 2 min; Stage 2 (Cycle: 40): 95°C 15s, 60°C 30s; Stage 3: 95°C 15s, 60°C 1 min, and 95°C 15s. Expression of the LncRNA LOC680254, U6 and p-actin was detected by qRT-PCR, compared with the U6 mainly expressed in nuclei, and the p-actin mainly expressed in the cytoplasm, results are as shown in FIG. 1b, and the results show that the LncRNA LOC680254 is mainly expressed in cytoplasm. Embodiment 2 Studying influence of disturbing expression of LncRNA LOC680254 on proliferation, apoptosis and cell cycle of Schwann cells 1. Culture of primary Schwann cells A plurality of sciatic nerves of 1 d-old SD rats were taken, 1 ml of 3 mg/ml collagenase was added, and tissue was cut into pieces and digested at 37°C for 30 min. Centrifuging was performed at 1200 rpm at room temperature for 5 min, the collagenase was discarded, 1 ml of pancreatin was added, and digestion was performed at 37°C for 10 min. 3 ml of a complete medium was added to terminate digestion, a screen was used for filtering, a supernatant was removed by centrifugation (1200 rpm, 5 min), and then 3 ml of the complete medium was added to wash twice. The cells were planted in a culture dish wrapped with PLL so as to be cultured (5% C02, 37C). In the second day, a complete medium containing Arac (10 M) was used instead to inhibit rapid proliferation of fibroblast. In the fourth day, a complete medium containing HRG (50 ng/ml) and Forskolin (2 M) was used instead to stimulate rapid growth of the Schwann cells. After the cells were fused, the cells were purified. The cells were digested by pancreatin first and transferred into a 5 ml centrifuge tube to be centrifuged (1200 rpm, 5 min) to remove a supernatant. A complete medium containing anti-thy1.1 (1:1000) was used to suspend the cells so as to perform incubation on ice for 2 h. Centrifuging was performed at 1200 rpm for 5 min to remove a supernatant, a complement (250 1 of a Rabbit complement + 750 1 of a DMEM) was added to perform incubation at 37°C for 0.5 h. 3 ml of a DMEM was used for washing three times. The cells were planted in a culture dish wrapped with PLL. Solution-replacing culture (containing HRG and Forskolin) was performed for 8-12 h, after the cells were fused, the purity was 95% or above, and the cells were used for a subsequent experiment. 2. Schwann cell transfection Small molecule RNA transfection: In the experiment, an siRNA (final concentration: 100 nM) and an miRNA minic (purchased from Guangzhou RiboBio Co., Ltd., final concentration: 20 nM) were transferred into the Schwann cells by using LipofectamineTM RNAiMAX. A complete medium was used instead in the second day, and a subsequent experiment was performed as required. Sequences siRNA 254-si-i and 254-si-2 for the LncRNA LOC680254 are as shown in SEQ ID NO: 9, namely 5' CCAGCUGUCCAAGAUCAGA dTdT 3' and SEQ ID NO: 10, namely 5' UGUGGUUCCUUCAUGACAA dTdT 3'. Plasmid DNA transfection: Cells were inoculated as required first. When the density of the Schwann cells reached 85% or above, LipofectamineTM 3000 was evenly dropwise added into a cell culture solution according to a concentration ratio of lip 3000:plasmid = 2 l:1 g and slightly mixed evenly. A new complete medium was used instead after 4-6 h. A new complete medium was used instead again in the second day, and a subsequent experiment was performed as required. 3. Edu cell proliferation experiment EdU labeling: An EdU solution (reagent A) was diluted by a cell culture medium according to a ratio of 1000:1, and an appropriate amount of 50 M EdU culture medium was prepared. 100 1 of the 50 M EdU culture medium was added into each well of a 96-well plate, the plate was incubated for 24 h in a 37°C incubator, and the culture medium was discarded. The cells were cleaned by a PBS 1-2 times, 5 min each time. Cell immobilization: 100 L of a cell immobilization solution (a PBS containing 4% paraformaldehyde) was added into each well for room-temperature incubation for 30 min, and the cell immobilization solution was discarded. 50 L of 2 mg/mL glycine was added into each well, and after incubation in an orbital shaker for 5 min, a glycine solution was discarded. 100 1 of a PBS was added into each well, cleaning in the orbital shaker was performed for 5 min, and the PBS was discarded. (enhancement) 100 1 of a penetrant (0.5% TritonX-100 PBS) was added into each well, and incubation was performed in the orbital shaker for 10 min. Cleaning by a PBS was performed once for 5 min. Apollo staining: 100 1 of a IX Apollo@ staining reaction solution was added into each well, and after incubation in the orbital shaker at room temperature in a light shading manner for 30 min, the staining reaction solution was discarded. 100 1 of a penetrant (0.5% TritonX-100 PBS) was added, cleaning in the orbital shaker was performed 2-3 times, 10 min each time, and the penetrant was discarded. (enhancement) 100 1 of methanol was added into each well each time for cleaning 1-2 times, 5 min each time. A PBS was used for cleaning once, 5 min each time. DNA staining: A reagent F was diluted by ddH20 according to a ratio of 100:1, and an appropriate amount of a IX Hoechst 33342 reaction solution was prepared and stored in a light shading manner. 100 1 of the IX Hoechst 33342 reaction solution was added into each well, and after incubation in the orbital shaker at room temperature in a light shading manner for 30 min, the staining reaction solution was discarded. 100 1 of a PBS was added into each well each time for cleaning 1-3 times. 100 1 of the PBS was added into each well, and a fluorescence microscope is adopted for photographing. The primarily-cultured Schwann cells were transfected by using the 2 specific siRNAs (254-si-i and 254-si-2) of the LncRNA LOC680254 and siRNA Negative Control (NC), EdU labeling was performed according to the above method 48 h after siRNA transfection, and Edu experiment results are as shown in FIG. 2a. The results show: compared with a control group, knockout of the LncRNA LOC680254 remarkably inhibits proliferation of the Schwann cells. 4. Annexin V-FITC cell apoptosis detection The Schwann cells were digested off with the pancreatin and transferred into a 5 ml centrifuge tube. 1000 g centrifuging was performed for 5 min, a supernatant was discarded, cells were collected, and the cells were re-suspended by an appropriate amount of PBS and counted. 50-100 thousand of the re-suspended cells were taken and subjected to 1000 g centrifuging for 5 min, a supernatant was discarded, and 195 1 of an Annexin V-FITC binding buffer was added to slightly re-suspend the cells. 5 1 of Annexin V-FITC was added and slightly mixed evenly. 10 1 of a PI (propidium iodide) staining solution was added and slightly mixed evenly. Light-shaded incubation was performed at room temperature (20-25°C) for 10-20 min, and then the cells were placed in an ice bath. Aluminum foil may be used for light shading. The cells may be re-suspended 2-3 times in the incubation process to improve the staining effect. Immediately, detection was performed with a flow cytometer, where Annexin V-FITC had green fluorescence and the propidium iodide (PI) had red fluorescence. Expression of the LncRNA LOC680254 in the Schwann cells was disturbed by the siRNAs

(254-si-i and 254-si-2) specific to the LncRNA LOC680254. In 48 h after siRNA transfection, the apoptosis situation of the Schwann cells was detected through the flow cytometry according to the above method. Flow cytometry results are as shown in FIG. 2b. The results show that compared with a negative control group siRNA Negative Control (NC), disturbing the LncRNA LOC680254 remarkably promotes apoptosis of the Schwann cells. 5. TUNEL staining (TMR Red) Air-dried Schwann cells were immobilized by a fresh immobilizing solution under 15-25°C for 1 h; a PBS was used for cleaning for 30 min. Ice-bathing was performed with a penetrant (2-8°C) for 2 min. Preparation of a Tunel reaction mixture: two tubes (tube 1: an enzyme concentrated solution, and tube 2: label solution) were taken for staining of 10 samples and 2 negative control groups, 50 1 of a Tunel mixed solution was used for each sample, and 50 1 of the label solution was used for each control group. 100 1 of the label solution (tube 2) was taken for the control groups. The total amount (50 l) of the enzyme concentrated solution (tube 1) was added into the 450 1 of the label solution remaining in tube 2 to prepare 500 1 of the Tunel mixed solution, and even mixing was performed to fully mix all compositions. Labeling: A PBS was used to clean a slide 3 times, 3 min each time, and liquid around the samples was sucked carefully. 50 1 of the Tunel mixed solution was added into the samples. Light-shaded incubation was performed at 37°C in a wet box for 60 min. The PBS was used for cleaning 3 times. Hoechst was used for nucleus staining for 30 min. The slide was sealed with an anti-fluorescence quenching slide sealing solution, and an inverted fluorescence microscope or a laser scanning confocal microscope was used for detection. Expression of the LncRNA LOC680254 in the Schwann cells was disturbed by the siRNAs (254-si-i and 254-si-2) specific to the LncRNA LOC680254. In 48 h after siRNA transfection, the apoptosis situation of the Schwann cells was detected through TUNEL staining according to the above method, as shown in FIG. 2c. The results show that compared with a negative control group siRNA Negative Control (NC), disturbing the LncRNA LOC680254 remarkably promotes apoptosis of the Schwann cells. 6. Flow cytometry detection of cell cycle Preparation of a cell sample: The Schwann cells were digested with pancreatin and transferred into a 5 ml centrifuge tube. Centrifuging was performed at about 1000 g for 5 min to settle the cells. A supernatant was discarded, 1 ml of a pre-cooled PBS was added, and the cells were re-suspended and transferred into a 1.5 ml centrifuge tube. 1000 g centrifuging was performed for 5 min to settle the cells, and a supernatant was carefully discarded. A small amount of the PBS may be left to avoid sucking away the cells. The bottom of the centrifuge tube was gently flicked to disperse the cells so as to avoid agglomeration of the cells. Cell immobilization: 1 ml of pre-cooled 70% ethanol was added into the centrifuge tube, a gun head was used for slight blow-beating for even mixing, and immobilization was performed at 4°C for 24 h. 1000 g centrifuging was performed for 5 min to settle the cells. A supernatant was discarded, 1 ml of a pre-cooled PBS was added, and the cells were re-suspended. 1000 g centrifuging was performed for 5 min to settle the cells, a supernatant was discarded, a small amount of the PBS may be left, and the bottom of the tube was gently flicked to disperse the cells so as to avoid agglomeration of the cells. Preparation of a PI staining solution: An appropriate amount of the PI staining solution was prepared according to the number of samples to be detected. Staining: 0.5 ml of the PI staining solution was added into each tube of cell samples, the gun head was used for slight blow-beating to fully re-suspend the cells, and light-shaded warm bathing was performed at 37°C for 30 min. The cells were stored at 4°C or in an ice-bathing light shading manner. Flow cytometry detection was completed in 24 h. Flow cytometry detection and analysis: Red fluorescence was detected by a flow cytometer at an excitation wavelength of 488 nm, and a light scattering condition was detected at the same time. Cell DNA content analysis and light scattering analysis were performed by adopting proper analysis software. The LncRNA LOC680254-specific siRNAs (254-si-i and 254-si-2) and the negative control siRNA Negative Control (NC) were transfected in the Schwann cells, and the cell cycle was subjected to flow cytometry detection after 48 h according to the above method. Results are as shown in FIG. 2d, and the results show that compared with the control group, by disturbing the LncRNA LOC680254, the number of the cells at the period S is remarkably reduced. Embodiment 3 Studying influence of LncRNA LOC680254 on expression of genes Psrc1 and Skal by adjusting miR-30d-3p, miR-671, miRNA-3594-3p and miRNA-3473 1. Target gene screening LncRNA LOC680254-specific siRNAs and Negative Control (NC) were transfected to primarily-cultured Schwann cells, a total RNA was extracted, expression profile analysis was performed by using an Agilent expression profile chip, and 548 genes down-regulated 2 times or above are screened. According to the complete nucleotide sequence of the LncRNA LOC680254, 191 target genes that could be adjusted by the LncRNA LOC680254 in a ceRNA manner and miRNAs were selected by using a method of predicting ceRNAs through bioinformatics, established by Tay Y et al. (Tay Y, Kats L, Salmena L, et al. Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs [J]. Cell, 2011, 147(2): 344-357.). There were 54 genes that were down-regulated 3 times or above in the 191 target genes, and there were 82 genes whose number of miRNA binding sites were not less than 2. 20 genes were obtained by an intersection of the two, where 12 genes were related to cell proliferation and apoptosis. The two target genes, namely the Psrcl and the Skal, were confirmed further through qRT-PCR and dual-luciferase reporter detection. 2. Detecting expression of genes Psrcl and Skal in Schwann cells by qRT-PCR after disturbing LncRNA LOC680254 The primarily-cultured Schwann cells were transfected by the specific siRNA 254-si-2 of the LncRNA LOC680254. Cells were collected 48 h after siRNA transfection. RNAs were extracted. Reverse transcription was performed. Expression of the genes Psrcl and Skal was detected by qRT-PCR. A primer sequence of the gene Psrc1 is as shown in SEQ ID NO: 11 and 12, and a primer sequence of the gene Skal is as shown in SEQ ID NOs: 13 and 14. Results are as shown in FIG. 3a, and the results show that expression of the Psrcl and the Skal is also lowered when the LncRNA LOC680254 in the Schwann cells is disturbed. It is indicated that the LncRNA LOC680254 is able to control expression of the Psrcl and the Skal. 3. Dual-luciferase reporter detection Detection was performed by using a Dual-Luciferase*Reporter Assay System (Promega) kit. 293FT cells continued to be cultured for 24 h after transfection, the cell state was observed, and a pre-cooled PBS was used for washing once. 100 1 of 1xPLB was added, and a culture plate shaker was violently shaken at room temperature for 15 min. Each well was subjected to blow-beating 5 times, and a lysis buffer was transferred into an EP tube. Centrifuging was performed at 4°C and 13000 rpm for 5 min. 20 1 of LABII was added into a 96-well plate, then 20 1 of a cell lysis buffer was added, each well was subjected to blow-beating 5 times with uniform operations, the plate was placed on a microplate reader, and the fluorescence intensity of Firefly luciferase (Firefly) was detected by software Gene 5. Then 20 1 of Stop&Glo was added, and the fluorescence intensity of Renilla luciferase (Renilla) was detected by the microplate reader. Two parts of fluorescence were compared (relative luciferase). 3 repeated holes were made each time in the experiment, 3 or more times of experiment repetitions were performed, and analysis results were counted. The complete nucleotide sequence of the LncRNA LOC680254 was constructed to a pmirGLO carrier. PmirGLO-LOC680254-full, 4 miRNAs (miR-30d-3p, miR-671, miRNA-3594-3p and miRNA-3473) and an miRNA mimic (MC) were totally transfected in the 293FT cells. After 24 h's cell culture, fluorescence values of Firefly and Renilla were detected by using a dual-luciferase reporter gene system according to the above method, relative luciferase (Firefly/Renilla) was calculated, and results are as shown in FIG. 3b. The results show: compared with the MC, in the experiment groups with miRNAs transfected, standardized luciferase activity is remarkably lowered, indicating that the LncRNA LOC680254 may be bound with the 4 miRNAs to a certain extent. Then through a luciferase experiment, binding conditions of the above 4 miRNAs with 3'-UTR of the corresponding target genes Psrc1 and Skal were detected, and results are as shown in FIG. 3c. The results show that compared with the MC, after miRNA transfection, the luciferase activity is remarkably down-regulated, the miR-30d-3p and the miR-671 may be bound with Psrcl, and the miR-3594-5p and the miR-3473 may be bound with Skal. 4. Detecting influence of miRNAs on target gene expression by qRT-PCR In the primarily-cultured Schwann cells, the miRNAs (miR-30d-3p, miR-671, miRNA-3594-3p and miRNA-3473) and the miRNA mimic (MC) were transfected respectively. The cells were collected 48 h after miRNA transfection, the RNAs were extracted, reverse transcription was performed, and expression of the genes Psrcl and Skal were detected by qRT-PCR according to the above method. Results of qRT-PCR detection are as shown in FIG. 3d and FIG. 3e, and the results show that after transfection of the miR-30d-3p and the miR-671, the Psrcl is remarkably down-regulated at the mRNA level, and after transfection of the miR-3473 and the miR-3594-3p, the Skal is also remarkably down-regulated at the mRNA level. This indicates that the miR-30d-3p, the miR-671, the miRNA-3594-3p and the miRNA-3473 may be bound with the 3'-UTR of the Psrcl and Skal to inhibit expression of the Psrcl and the Skal, and the LncRNA LOC680254 may be competitively bound with the miR-30d-3p, the miR-671, the miRNA-3594-3p and the miRNA-3473 to resist inhibition of the miR-30d-3p, the miR-671, the miRNA-3594-3p and the miRNA-3473 on the Psrc1 and the Skal. Embodiment 4 Adjusting Schwann cell proliferation by LncRNA LOC680254 through Psrc1 and Skal Primarily-cultured Schwann cells were infected with a lentivirus (LV-254, control virus: LV-con) packaged with the complete nucleotide sequence of the LncRNA LOC680254 (the virus was diluted with an enhanced infection solution (Eni.S), and infection was performed according to MOI = 20), the LncRNA LOC680254 was over-expressed in the Schwann cells, and a conventional medium was used after 12 h to continue culture. Psrcl- and Skal-specific siRNA sequences (SEQ ID NO: 15 5'ggaggagauc cuugaugaa dTdT 3' and SEQ ID NO: 16 5' gcaucuauga gcucuguga dTdT 3') and negative control (NC) were transfected in the Schwann cells with the LncRNA LOC680254 over-expressed, and an Edu cell proliferation experiment was performed according to the above method after 48 h. Results are as shown in FIG. 4a and FIG. 4b. The results show that after the LncRNA LOC680254 is over-expressed in the Schwann cells, proliferation of the Schwann cells is obviously increased (FIG. 4a), while disturbing expression of the Psrcl and the Skal can resist the influence of the LncRNA LOC680254 on proliferation of the Schwann cells, indicating that the LncRNA LOC680254 adjusts Schwann cell proliferation through the Psrc1 and the Skal (FIG. 4b).

Claims

CLAIMS What is claimed is:

1. Use of a long-chain non-coding RNA, miR-30d-3p, miR-671, miR-3594-3p and miR 3473 as molecular intervention targets in controlling cell proliferation, apoptosis and a cell cycle, wherein a cDNA sequence of the long-chain non-coding RNA is as shown in SEQ ID NO: 1.

2. The use according to claim 1, wherein the long-chain non-coding RNA is used as miRNAmolecular sponge to specifically bind the miR-30d-3p, the miR-671, the miR-3594-3p or the miR-3473 so as to resist miRNA functions.

3. The use according to claim 2, wherein the miR-30d-3p and the miR-671 are able to promote expression of Psrc1 after functions thereof are inhibited; and the miRNA-3594-3p and the miRNA-3473 are able to promote expression of Skal after functions thereof are inhibited, so that cell proliferation, apoptosis and the cell cycle are adjusted.

4. The use according to claim 1, wherein use of the long-chain non-coding RNA, the miR d-3p, the miR-671, the miR-3594-3p and the miR-3473 as the molecular intervention targets in preparing medicines for treating diseases related to cell proliferation or apoptosis is provided.

5. The use according to claim 4, wherein the diseases related to cell proliferation or apoptosis comprise nerve injury, nerve cell regeneration, tumor, liver fibrosis, pulmonary fibrosis, renal fibrosis, prostatic hypertrophy, essential thrombocytosis, familial polycythemia, rheumatoid arthritis, psoriasis, interstitial glomerulopathy, atherosclerosis, diabetic nephropathy, neurodegenerative diseases, aplastic anemia, autoimmune diseases caused by failure of effective clearing of T lymphocytes for autoantigens, myocardial ischemia reperfusion injury and heart failure.

6. The use according to claim 5, wherein use of the long-chain non-coding RNA as the molecular intervention target in preparing repairing medicines for controlling proliferation of neurogliocytes and then repairing peripheral nerve injury is provided.

7. Use of a long-chain non-coding RNA, miR-30d-3p, miR-671, miR-3594-3p and miR 3473 in the manufacture of a medicament for treating a disease related to cell proliferation or apoptosis, wherein a cDNA sequence of the long-chain non-coding RNA is as shown in SEQ ID NO: 1.

8. The use of claim 7 wherein the disease related to cell proliferation or apoptosis is selected from the group consisting ofnerve injury, nerve cell regeneration, tumor, liver fibrosis, pulmonary fibrosis, renal fibrosis, prostatic hypertrophy, essential thrombocytosis, familial polycythemia, rheumatoid arthritis, psoriasis, interstitial glomerulopathy, atherosclerosis, diabetic nephropathy, neurodegenerative diseases, aplastic anemia, autoimmune diseases caused by failure of effective clearing of T lymphocytes for autoantigens, myocardial ischemia reperfusion injury and heart failure.

9. Use of a long-chain non-coding RNA, miR-30d-3p, miR-671, miR-3594-3p and miR 3473 in the manufacture of a medicament for controlling proliferation of neurogliocytes and repairing peripheral nerve injury, wherein a cDNA sequence of the long-chain non-coding RNA is as shown in SEQ ID NO: 1.

FIG. 1

FIG. 2

FIG. 3

FIG. 4

Sequence Table

<110> Nantong University

<120> Long-chain non-coding RNA and use thereof

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1 <211> 785 <212> DNA <213> Rat

<400> 1 cttcgagctg ctggtggaag ctgagacggt gtggggaggc ggagagaaaa gggggtgcgg 60 gaaatttaaa aagatgggaa aggcgcttcc ctttcttctt agccccttcc ctgttgattt 120 ctggaccaat gcttctttct tgaacgaaga gcatgaagac gctgctttat ctaacagtca 180 ccttggagga aacgccatcg ttcttccaga agggagatct ggcccccggt cttggattct 240 gagatcagtg cgagatcccg gctctgcgcc ctgtccgccc tggaactgga tttatcatca 300 gtttagaata gcatggctac gatgtgtggt gtggatggat ggacgagccc cgcaagtgca 360 aacaggacag tccaggttac ctgacaaagc agccaccggt gcaccagctg tccaagatca 420 gacctaggtc aatcactgac atggctaaaa gcaccaagaa ggtctggatc attgtgctgc 480 ctcactccaa aaaatggtga caaaaattaa aatcaaccag cacgccagga tcctctgtgg 540 caagaccaag ataatgtggg ccactgtcat ctggcactgt ggttccttca tgacaacaat 600 ggctggtggg tcctgggttt actacaccac ttctgctgtc agtcaaactg gccatcagga 660 gactgaagga aatgaaagac catagaagtg ctactgtctg agcagccctg gcctccagta 720 aatgagtcat tcctatgaca aaattataat aaaaagtgtt ggttgagtaa aaaaaaaaaa 780 aaaaa 785

<210> 2 <211> 22 <212> RNA <213> Artificial Sequence

<400> 2 cuuucaguca gauguuugcu gc 22

<210> 3 <211> 81 <212> RNA <213> Artificial Sequence

<400> 3 caggaagagg aggaagcccu ggaggggcug gaggugaugg auguuuuccu ccgguucuca 60 gggcuccacc ucuuucgagc c 81

<210> 4 <211> 22 <212> RNA <213> Artificial Sequence

<400> 4 cacaccgccu cugcccgcua gu 22

<210> 5 <211> 67 <212> RNA <213> Artificial Sequence

<400> 5 aucacagucc uauuucugcc cucaaagaca aaaaugaugu cuagggcugg agagauggcu 60 aaguggg 67

<210> 6 <211> 20 <212> DNA <213> Artificial Sequence

<400> 6 ggtgtggatg gatggacgag 20

<210> 7 <211> 20 <212> DNA <213> Artificial Sequence

<400> 7 tttggagtga ggcagcacaa 20

<210> 8 <211> 27 <212> DNA <213> Artificial Sequence

<400> 8 ggagtgaggc agcacaatga tccagac 27

<210> 9 <211> 19 <212> DNA <213> Artificial Sequence

<400> 9 ccagctgtcc aagatcagatt 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence

<400> 10 tgtggttcct tcatgacaatt 19

<210> 11 <211> 20 <212> DNA <213> Artificial Sequence

<400> 11 gacagtagca agcccaccaa 20

<210> 12 <211> 20 <212> DNA <213> Artificial Sequence

<400> 12 ggatggctga gggaaatggt 20

<210> 13 <211> 21 <212> DNA <213> Artificial Sequence

<400> 13 tgacccagag ctcgactcat a 21

<210> 14 <211> 20 <212> DNA <213> Artificial Sequence

<400> 14 cgggatttca tgtacgcagg 20

<210> 15 <211> 19 <212> DNA <213> Artificial Sequence

<400> 15 ggaggagauc cuugaugaatt 19 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence

<400> 16 gcaucuauga gcucugugatt 19