Cashmere goat lncRNA, interference vector and application thereof in regulation and control of hair follicle development of cashmere goat

Abstract

The invention belongs to the technical field of animal gene engineering, and particularly relates to a down producing goat lncRNA, an interference vector and application of the interference vector to regulation and control of down producing goat hair follicle development, the down producing goat lncRNA is lncRNA TCONS-00237214 and is used for regulating and controlling down producing goat hair follicle development; the nucleotide sequence of the lncRNA TCONS-00237214 is as shown in SEQ ID NO. 1 (sequence identifier number 1). According to the invention, the hair papilla cells are selected for subsequent experimental verification, and the cell cycle, cell proliferation and cell migration of the hair papilla cells are detected by constructing an interference lncRNA TCONS-00237214 stable transformation hair papilla cell line. Results of the invention show that melatonin can regulate and control expression of lncRNA TCONS-00237214, participate in cell cycle transformation, promote proliferation and migration of hair papilla cells and inhibit apoptosis of the hair papilla cells, thereby affecting generation and development of hair follicles.

Description

Technical Field

[0001] The invention belongs to the technical field of animal genetic engineering, and specifically relates to a cashmere goat lncRNA, an interference vector and an application thereof in regulating the development of cashmere goat hair follicles. Background Art

[0002] Long non-coding RNA, or lncRNA, is a type of non-coding RNA with a length of more than 200 nucleotides. Studies have suggested that lncRNA may be involved in regulating the growth and development of organisms, and plays a key role in the development and regeneration of the skin. It is also related to a series of physiological phenomena such as the development of the epidermis and hair follicles, pigment formation, and the periodic growth of hair follicles. As the "signaling center" of hair follicles and the "supply station" of nutrients, hair papilla cells are the targets of many hormones and small molecules. They can affect the periodic growth of hair follicles by regulating hormone-like small molecules, and have an important regulatory effect on the development and regeneration of hair follicles. Therefore, it is necessary to explore the molecular mechanism of lncRNA in regulating hair follicle development in hair papilla cells. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a cashmere goat lncRNA, an interference vector and applications thereof in regulating the development of cashmere goat hair follicles.

[0004] The present invention is achieved through the following technical solutions: A cashmere goat lncRNA, wherein the cashmere goat lncRNA is lncRNA TCONS-00237214; the nucleotide sequence of the lncRNA TCONS-00237214 is shown in SEQ ID NO.1, and is used for regulating the development of cashmere goat hair follicles.

[0005] An interference vector, wherein the interference vector is a shRNA interference vector of the lncRNA TCONS-00237214.

[0006] The interference vector is used in inhibiting the development of cashmere goat hair follicles. The interference vector is used to inhibit the expression of the cashmere goat lncRNA, thereby inhibiting the development of cashmere goat hair follicles.

[0007] Preferably, the inhibiting of cashmere goat hair follicle development includes any one of the following situations.

[0008] Inhibits the cell cycle of dermal papilla.

[0009] Inhibits the proliferation of dermal papilla cells.

[0010] Inhibits dermal papilla cell migration.

[0011] Promote apoptosis of dermal papilla cells.

[0012] Inhibit the expression of mRNA related to the proliferation or migration of hair papilla cells, the mRNA includes COL11A1, COL1A1 .

[0013] Preferably, the hair papilla cells are true fur papilla cells of cashmere goats.

[0014] Preferably, the interference vector is used to prepare a product for inhibiting the proliferation or migration of hair papilla cells.

[0015] Preferably, the interference vector is used to prepare a product that promotes apoptosis of hair papilla cells.

[0016] Application of the cashmere goat lncRNA or the interference vector in promoting the development of cashmere goat hair follicles.

[0017] Preferably, the cashmere goat hair follicle development is promoted by promoting the expression of the cashmere goat lncRNA or inhibiting the expression of the interference vector.

[0018] Preferably, melatonin is added to promote the expression of lncRNA in cashmere goats or inhibit the expression of the interference vector.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a cashmere goat lncRNA, an interference vector and an application thereof in regulating the development of cashmere goat hair follicles. The cashmere goat lncRNA provided by the present invention is lncRNA TCONS-00237214, whose nucleotide sequence is shown in SEQ ID NO.1, and is used to regulate the development of cashmere goat hair follicles. The research results of the present invention show that lncRNA TCONS-00237214 has an effect on the development of cashmere goat hair follicles. COL11A1, COL1A1 The lncRNA TCONS-00237214 can inhibit cell proliferation and migration by regulating the cell cycle process. The lncRNA TCONS-00237214 can promote cell apoptosis. The present invention selects the dermal papilla cells for subsequent experimental verification. Based on the lncRNA TCONS-00237214 constructed by the research institute COL11A1 / COL1A1 The present invention constructs a stable transfection dermal papilla cell line by interfering with lncRNA TCONS-00237214, and uses qRT-PCR to detect the expression of lncRNA TCONS-00237214 and COL11A1 / COL1A In terms of regulatory relationship at the cellular level, DNA staining, CCK8 method and scratch test were used to detect the cell cycle, cell proliferation and cell migration of dermal papilla cells.

[0020] 2. The results of the present invention found that melatonin can regulate the expression of lncRNA TCONS-00237214, participate in cell cycle transformation, promote the proliferation and migration of hair papilla cells, inhibit the apoptosis of hair papilla cells, and thus affect the occurrence and development of hair follicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 This is a diagram showing the morphological observation results of hair papilla cells of the present invention.

[0023] Figure 1 In the figure, A is the morphological observation picture of hair papilla cells at 10 days; B is the morphological observation picture of hair papilla cells at 15 days; C is the morphological observation picture of hair papilla cells after passage; D is the morphological observation picture of hair papilla cells after adding melatonin.

[0024] Figure 2 This is a diagram of the interference vector construction of the present invention.

[0025] Figure 2 In the figure, A is the construction diagram of the negative control vector; B is the construction diagram of the interference lncRNA TCONS-00237214 vector.

[0026] Figure 3 A stable dermal papilla cell line interfering with lncRNA TCONS-002372144 was constructed for the present invention.

[0027] Figure 3 In the figure, A is the bright field cell image of the NC group after transfection; B is the bright field detail image of the lncRNA TCONS-00237214 interference group after transfection; C is the bright field cell image of the MT+lncRNA TCONS-00237214 interference group after transfection; D is the fluorescence field cell image of the NC group after transfection; E is the fluorescence field cell image of the lncRNA TCONS-00237214 interference group after transfection; F is the fluorescence field detail image of the MT+lncRNA TCONS-00237214 interference group after transfection; G is the qRT-PCR relative expression result graph of the NC group, the lncRNA TCONS-00237214 interference group and the MT+lncRNA TCONS-00237214 interference group.

[0028] Figure 4 For the present invention COL11A1 / COL1A1Relative expression in dermal papilla cell lines.

[0029] Figure 4 In the figure, A is the target gene after interference with lncRNA TCONS-00237214 COL11A1 B is the expression level diagram of target gene after interference with lncRNA TCONS-00237214 COL1A1 Expression level diagram.

[0030] Figure 5 This is a diagram of cell proliferation detected by the CCK8 method of the present invention.

[0031] Figure 6 This is a diagram of cell migration detected by the cell scratch test of the present invention.

[0032] Figure 6 In the figure, A is the result of cell scratch experiment; B is the result of bright field cell migration rate of hair papilla cell line; C is the result of fluorescent cell migration rate of hair papilla cell line.

[0033] Figure 7 This is a diagram showing the results of detecting the cell cycle of hair papilla by flow cytometry in the present invention.

[0034] Figure 7 In the figure, A is the cycle analysis result of flow cytometry in the NC group; B is the cycle analysis result of flow cytometry in the MT+interference lncRNA TCONS-00237214 group; C is the cycle analysis result of flow cytometry in the interference lncRNA TCONS-00237214 group; D is the statistical analysis chart of the cell cycle of dermal papilla.

[0035] Figure 8 This is a graph showing the results of detecting apoptosis of dermal papilla cells by flow cytometry in the present invention.

[0036] Figure 8 In the figure, A is the four-quadrant diagram of cell apoptosis in the NC group; B is the four-quadrant diagram of cell apoptosis in the MT+interference lncRNA TCONS-00237214 group; C is the four-quadrant diagram of cell apoptosis in the interference lncRNA TCONS-00237214 group; D is the result of qRT-PCR detection of apoptosis rate in the NC, MT+interference lncRNA TCONS-00237214 and interference lncRNA TCONS-00237214 groups. DETAILED DESCRIPTION

[0037] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in the present invention. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0038] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0039] The beneficial effects of the present invention are described below by means of specific embodiments: The main instruments and equipment used in the present invention are as follows: The equipment includes a carbon dioxide constant temperature incubator, purchased from Eppendorf; an ultracentrifuge, purchased from Eppendorf; a fluorescence microscope, purchased from Nikon; an ELISA reader, purchased from Bio-Tek; a Nano Drop 2000 UV spectrophotometer, purchased from Thermo; an ultra-low temperature refrigerator, purchased from Thermo Fisher Scientific; an agarose gel electrophoresis tank; an inverted fluorescence microscope; a mini centrifuge; an automatic ultrapure water instrument; a pressure cooker and an autoclave; a -80°C ultra-low temperature refrigerator; and a flow cytometer, purchased from Beckman CytoFLEX.

[0040] The main reagents used in the present invention were purchased from Bio-Rad Biotech Sales Department, as shown below: DMEM culture medium, purchased from Tecono; fetal bovine serum, purchased from Tecono; triple antibiotic penicillin + streptomycin + amphotericin, purchased from gibco; 0.25% trypsin, containing EDTA; PBS buffer, purchased from gibco; 0.25% trypsin, without EDTA; DMSO; CCK8 kit; melatonin, M5250, purchased from Sigma; FITC Annexin VApoptosis, purchased from BD Pharmingen TM; PI staining solution, purchased from MCE.

[0041]

[0042] Example 1 1.1 Test methods 1.1.1 Culture of dermal papilla cells (1) After the collected tissue is trimmed in PBS buffer supplemented with the third antibody, it is cut into evenly sized tissue blocks of the size of a match head and placed in PBS buffer for later use.

[0043] (2) Digest the tissue blocks in 0.25% pancreatic enzyme for 45 min.

[0044] (3) After terminating the digestion with culture medium supplemented with 20% fetal bovine serum, the cells were spread on a culture dish and placed upside down in a carbon dioxide incubator for 6 hours.

[0045] (4) After the tissue adheres to the wall, slowly add culture medium and continue culturing.

[0046] (5) While waiting for the primary cells to crawl out, change the culture medium at irregular intervals to observe the cell growth.

[0047] (6) After the primary cells have grown all over the culture dish, aspirate the culture medium and wash twice with PBS buffer supplemented with the third antibody.

[0048] (7) Add 1 ml of 0.25% pancreatic enzyme and digest for 5 min in a 30°C CO2 incubator.

[0049] (8) Add serum-containing culture medium to terminate digestion. This step separates epithelial cells and a small amount of fibroblasts.

[0050] (9) Wash again twice with PBS buffer supplemented with the third antibody.

[0051] (10) Add 0.25% pancreatic enzyme and place in a 37°C CO2 incubator for 5 min. Then, add serum-containing culture medium to terminate digestion.

[0052] (11) Transfer the cell suspension in the culture medium into a 15 mL centrifuge tube and place it in a centrifuge at 1200 rpm for 5 min.

[0053] (12) The resulting precipitate is the dermal papilla cells. The supernatant is removed by aspiration, and a 10% volume fraction of fetal bovine serum culture medium is added and cultured in a 37°C carbon dioxide incubator.

[0054] 1.1.2 Construction of lentiviral interference vector The lentiviral vector was constructed by Shanghai GeneCare Gene Medical Technology Co., Ltd. First, the target gene was determined, and the siRNA target site was selected to construct the interference vector, which relies on RNA polymerase III promoters including U6 and Cbh, and manipulates a small hairpin RNA, the English name of which is short hairpinRNA, referred to as shRNA, to express it in the cell. Then a negative control was designed. The siRNA used as a negative control should have the same composition as the selected siRNA sequence. The usual practice is to disrupt the base sequence in the selected siRNA. Subsequently, the DNA single strand of the siRNA sequence was annealed and cloned into the downstream of the promoter of the corresponding vector. Starting from the transcription AUG start codon, the downstream AA sequence was searched, and the 19 nucleotides adjacent to the 3' end of each AA were recorded as candidate siRNA target sites. siRNA with a GC content of about 40% was more effective. Secondly, the potential sequence was compared with the corresponding genome database, human or mouse, rat, etc., to exclude those sequences that were homologous to other coding sequences / ESTs.

[0055] The sequence of the shRNA is shown in SEQ ID NO. 2, which is: ccggGCGCAATGAAGACAGGTTAGTctcgagACTAACCTGTCTTCATTGCGCtttttg.

[0056] 1.1.3 Lentiviral transfection of dermal papilla cells The fur papilla cells cultured using the 6-well plate method were cultured in a 37°C, 5% carbon dioxide incubator to achieve a fusion rate of 20%~30%; they were continued to be cultured using serum-free DMEM and HitransG-P virus enhancement solution was added; based on the preliminary experiment, the amount of the virus was calculated according to the MOI=10 method and cultured at 37°C, 5% carbon dioxide; after 12 hours, the serum-free DMEM was aspirated and replaced with complete culture medium containing serum and three antibodies, and then cultured for about 72 hours; the experimental group with better transfection effect was cultured for 24 hours at a final dosage of 5µg / mL under the action of puromycin.

[0057] Note: Lentiviral vectors were constructed and packaged by Shanghai GeneCare Gene Medical Technology Co., Ltd. Interference vectors of lncRNA TCONS-00237214 and shRNA negative controls were constructed.

[0058] 1.1.4 Total RNA extraction from dermal papilla cells Extract total RNA from the dermal papilla cell line when the cell confluence reaches 80%; discard the DMEM complete medium, add 1mL preheated PBS buffer to wash three times; add 1mL Trizol to lyse the cells; then add chloroform to mix, centrifuge and absorb the aqueous phase; add isopropanol equal to the volume of the aqueous phase to mix, centrifuge and discard the supernatant, add 75% ethanol to wash the precipitate; discard the supernatant after centrifugation, dry at room temperature, add RNase-free water to fully dissolve the RNA; the obtained RNA is stored at -70℃. Note: Extraction of total RNA dermal papilla cells were cultured in six-well plates.

[0059] 1.1.5 qRT-PCR detection Reverse transcription was performed using the Kangwei Reagent HiFiScript gDNA Removal RT MasterMix Kit, as shown in Table 1, to obtain a cDNA template; cDNA templates and primers were prepared according to Table 2, and GAPDH was used as an internal standard to quantify lncRNA and target genes, as shown in Table 3; 2 -△△CT The CT relative expression calculation formula was used to calculate the quantitative data of lncRNA and target genes. The relative expression of lncRNAs and target genes was calculated using the Pearson correlation analysis method, and visualized using Graphpad prisim9.0. The primers used were designed by Shanghai Bioengineering Co., Ltd., and the lncRNA and mRNA primer sequence information is shown in Table 4.

[0060] Table 1 Reverse transcription reaction system

[0061] Table 2 qRT-PCR reaction system

[0062] Table 3 qRT-PCR reaction procedure

[0063] Table 4 lncRNA and mRNA primer sequence information

[0064] 1.1.6 CCK8 assay for dermal papilla cell proliferation Three groups were set up, namely, negative control group NC, interference lncRNA TCONS-00237214 group and MT+interference lncRNA TCONS-00237214 group, and MT was melatonin. Cells were collected by enzyme digestion, 100 μL of cell suspension was inoculated into a 96-well plate, and the plate was placed in a constant temperature carbon dioxide incubator for 24 hours; at the same time every day, the 96-well culture plate of the corresponding time was taken out, 10 μL of CCK8 reagent was added to each well, and the absorbance at 450 nm was measured with an enzyme reader after 2 hours; the measured OD values ​​were sorted and calculated, and plotted using GraphPad 9.0.

[0065] 1.1.7 Scratch test to detect dermal papilla cell migration Before the scratch test, several marking lines with a spacing of 0.5 cm were drawn horizontally on the back of the six-well plate with a marker pen; the cells were inoculated in the six-well plate with the spacing lines and cultured at 37°C and 5% carbon dioxide until 100% fusion was achieved, and then the scratch test was performed, and the scratch was made perpendicular to the marking line with a 200μL gun tip; after the scratch was completed, it was washed three times with preheated PBS buffer; serum-free DMEM was cultured and placed in a 37°C, 5% carbon dioxide incubator. Photos were taken at the same position at 0 hours and 24 hours for observation, and the scratch area was counted using Imagej software. The calculation formula of mobility is: mobility = (0h blank area - 24h blank area) / 0h blank area.

[0066] 1.1.8 Flow cytometry detection of dermal papilla cell cycle Prepare a single cell suspension, centrifuge at 4°C and 1200rpm for 5 minutes; add pre-cooled 70% volume fraction ethanol to the cell pellet collected above, blow evenly, and keep at 4°C overnight; after removing the supernatant, resuspend it with 1mL of pre-cooled PBS buffer, centrifuge at 4°C and 1200rpm for 5 minutes, remove the supernatant and save it as a precipitate; add 1mL of pre-cooled 70% volume fraction ethanol to the cell pellet collected above, blow gently to make it even, and fix it at 4°C for more than 2h; centrifuge at 4°C and 1200rpm for 5 minutes, remove the supernatant, add 1mL of pre-cooled PBS buffer to resuspend it, centrifuge at 4°C and 1200rpm for 5 minutes, remove the supernatant and keep the cell pellet; prepare an appropriate PI staining working solution according to the amount of the test object, place it at 4°C, and it is effective within 24h; add 0.5mL PI dye solution was added to the suspended cells and cultured at 37°C in the dark for 40 minutes. Red fluorescence was detected at a laser wavelength of 488 nm using a flow cytometer, and light scattering was detected to analyze the DNA content and light scattering.

[0067] 1.1.9 Detection of apoptosis of dermal papilla cells by flow cytometry Prepare a single cell suspension, centrifuge at 4°C, 1200rpm for 5min; discard the supernatant, collect the cells, wash once with pre-cooled PBS buffer, gently resuspend the cells and count; take the resuspended cells, centrifuge at 4°C, 1200rpm for 5min, discard the supernatant; wash the cells once with PBS buffer, discard the supernatant after centrifugation, add 100μL diluted 1xAnnexin V Binding Buffer to resuspend the cells; add 5.0μL Annexin V-APC Reagent and 5.0μL PI Reagent to the cell suspension; vortex gently to mix, incubate at room temperature in the dark for 15min; add 400μL diluted 1xAnnexin V Binding Buffer to mix the sample; immediately test on the machine. If the test cannot be carried out in time, please keep it on ice in the dark and complete the test within 1 hour.

[0068] 1.2 Results and Analysis 1.2.1 Morphological observation of dermal papilla cells After 10 days, Figure 1 As shown in A, some cells can be seen growing from the edges of the tissue block. These cells are short spindle-shaped or irregular square-shaped and clustered together. Figure 1 As shown in B, these cells will begin to enter the logarithmic growth stage. During this process, a large amount of cytoplasm will appear. Soon, they will slowly lose their original shape. As shown in the figure, in the initial growth process, epidermal cells that are easier to grow will climb out. Most of them are irregular quadrilaterals and gather into clusters. It can be determined that they are relatively primitive hair papilla cells. The morphology of hair papilla cells after subculture is as follows Figure 1 As shown in C, the cell growth rate is accelerated after subculture. The morphology of hair papilla cells after adding melatonin is as follows Figure 1 As shown in D, after adding melatonin, the proliferation rate of hair papilla cells is faster and the cell morphology is more obvious, which can be used for subsequent experiments.

[0069] 1.2.2 Identification of lentiviral interference vectors Determine the siRNA sequences of the target gene and the negative control. The vector of the negative control NC is hU6-MCS-CMV-puro, and the vector of the interfering lncRNA TCONS-00237214 is hU6-MCS-CBh-gcGFP-IRES-puromycin. Figure 2The GC content of each of the two sequences is 47.62%, as shown in Table 5. After sequence alignment, it was found that there were only 16 consecutive bases of homology with two genes, and no other 16 consecutive bases of homology with any sequence in the genome. This target point construction was effective and had a good effect, indicating that the vector was successfully constructed and subsequent experiments could be carried out.

[0070] Table 5 Target sequences

[0071] 1.2.3 Construction of a stably transfected dermal papilla cell line The FITC protein fluorescence results of each test group showed that the cells in the test group all emitted green fluorescence and had normal morphology, indicating that the transfection effect was good. After the lncRNA TCONS-00237214 interference vector was successfully constructed, the third-generation hair papilla cells were transfected. The transfection efficiency was observed by inverted fluorescence microscopy 72 hours after transfection. The transfection efficiency was the highest and the cell morphology was the best, as shown in Figure 2. Figure 3 As shown in A to F in Figure 1, the transfection efficiency was as high as 80% and the cell death was low, which can be further studied. In addition, the qRT-PCR results showed that Figure 3 As shown in G, in the dermal papilla cells, the relative expression levels of the interference lncRNA TCONS-00237214 and melatonin MT+interference lncRNA TCONS-00237214 were significantly lower than those of the NC group. * indicates P<0.05 , indicating that the stable transfection of lncRNA TCONS-00237214 into dermal papilla cell line was successfully constructed.

[0072] 1.2.4 qRT-PCR detection COL11A1 / COL1A1 Expression at the dermal papilla cell level By using qRT-PCR COL11A1 / COL1A1 The results of expression in the stable dermal papilla lineage were as follows Figure 4 As shown, in the dermal papilla cell line with interference of lncRNA TCONS-00237214, COL11A1 / COL1A1 The relative expression of the NC group was significantly different from that of the corresponding group. ** indicates P<0.01 However, after adding melatonin, the expression of the target gene increased significantly. * indicates P<0.05 , and found that melatonin can increase the relative expression of target genes by regulating the expression of lncRNA, indicating that lncRNA TCONS-00237214 and COL11A1 / COL1A1 There is a positive correlation between melatonin and lncRNA, and melatonin promotes the expression of lncRNA and has a regulatory effect at the mRNA level.

[0073] 1.2.5 CCK8 assay for dermal papilla cell proliferation The results of the CCK8 assay showed that Figure 5 As shown in the figure, compared with the NC group, the cell proliferation level was significantly reduced after interfering with lncRNA TCONS-00237214 in the hair papilla cells. ** indicates P<0.01 ; After adding melatonin, the proliferation level of hair papilla cells that interfered with lncRNA TCONS-00237214 was also significantly reduced, ** indicates P<0.01 However, compared with the cell proliferation ability of lncRNA TCONS-00237214, the cell proliferation ability of lncRNA TCONS-00237214 was significantly increased after adding melatonin. * indicates P<0.05 , indicating that interference with lncRNA TCONS-00237214 can inhibit the proliferation of dermal papilla cells, but the addition of melatonin can promote the proliferation of dermal papilla cells.

[0074] 1.2.6 Scratch test to detect dermal papilla cell migration The results of cell scratch test showed that Figure 6 As shown, in the dermal papilla cells, the mobility of the NC group was greater than that of the lncRNA TCONS-00237214 interference group, and the difference between the two groups was extremely significant. P<0.01 ; After adding melatonin, the migration rate of dermal papilla cells interfering with lncRNA TCONS-00237214 was also significantly lower than that of the NC group. * indicates P<0.05 However, compared with the migration rate of dermal papilla cells interfered with lncRNA TCONS-00237214, the migration rate of dermal papilla cells interfered with lncRNA TCONS-00237214 after adding melatonin was significantly increased. * indicates P<0.05 , indicating that melatonin can promote the migration rate of hair papilla cells. The above studies show that interfering with lncRNA TCONS-00237214 inhibits the migration of hair papilla cells.

[0075] 1.2.7 Flow cytometry detection of dermal papilla cell cycle Flow cytometry analysis of the cell cycle of dermal papilla cells Figure 7 As shown in A to C in Figure 1, it was found that most cells interfered with lncRNA TCONS-00237214 stayed in the G0 / G1 phase, and the transition from the G0 / G1 phase to the S phase and G2 / M phase was inhibited, and the cell proliferation index was significantly reduced. Figure 7 As shown in D, after adding melatonin, the cycle ratio of the dermal papilla cells in the S phase and G2 phase of the lncRNA TCONS-00237214 interference group was also significantly lower than that of the NC group. ** indicates P<0.01However, compared with the hair papilla cell cycle interfered with lncRNA TCONS-00237214, the G0 / G1 phase ratio of hair papilla cells interfered with lncRNA TCONS-00237214 after adding melatonin was relatively reduced, and the degree of inhibition of the conversion of G0 / G1 phase to S phase and G2 / M phase was reduced, indicating that melatonin can accelerate the progression of hair papilla cell cycle. The results obtained by the above research are consistent with the experimental results of CCK8 detection of cell proliferation, indicating that interference with lncRNA TCONS-00237214 can inhibit the conversion of G0 / G1 phase to S phase and G2 / M phase, inhibit the cell cycle process, and thus inhibit cell proliferation.

[0076] 1.2.8 Detection of apoptosis of dermal papilla cells by flow cytometry After 72 hours of transfection, the dermal papilla cells were collected and the cell apoptosis was determined by flow cytometry using the Annexin V-APC / DAPI double staining method. Figure 8 As shown in the figure, compared with the NC group, interference with lncRNA TCONS-00237214 significantly promoted the apoptosis of hair papilla cells. P<0.01 ; After adding melatonin, interfering with lncRNA TCONS-00237214 also significantly inhibited the apoptosis of hair papilla cells. * indicates P<0.05 Compared with the lncRNA TCONS-00237214 interference group, the melatonin addition group reduced the proportion of early and late apoptosis and inhibited apoptosis, indicating that the addition of melatonin to hair papilla cells can inhibit apoptosis. The above studies show that interfering with lncRNA TCONS-00237214 has a role in promoting apoptosis in hair papilla cells.

[0077] In summary, the present invention established a stable dermal papilla cell line with interfering lncRNA TCONS-00237214. The qRT-PCR detection results showed that lncRNA TCONS-00237214 and COL11A1 / COL1A1 There is a positive correlation between melatonin and lncRNA, and melatonin promotes the expression of lncRNA and has a regulatory effect at the mRNA level. The results of CCK8 method, cell migration and flow cytometry experiments show that interference with lncRNA TCONS-00237214 can inhibit cell proliferation and cell migration and promote cell apoptosis by regulating the cell cycle process. Melatonin can regulate the expression of lncRNA TCONS-00237214, participate in cell cycle transformation, promote hair papilla cell proliferation and migration, inhibit hair papilla cell apoptosis, and thus affect the occurrence and development of hair follicles.

[0078] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0079] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A cashmere goat lncRNA, characterized in that: The cashmere goat lncRNA is lncRNA TCONS-00237214; the nucleotide sequence of the lncRNA TCONS-00237214 is shown in SEQ ID NO.1, and is used to regulate the development of cashmere goat hair follicles.

2. An interference vector, characterized in that: The interference vector is the shRNA interference vector of the lncRNA TCONS-00237214 according to claim 1, and the sequence of the shRNA is shown in SEQ ID NO.

2.

3. Use of the interference vector according to claim 2 in inhibiting the development of cashmere goat hair follicles, characterized in that: The interference vector is used to inhibit the expression of the cashmere goat lncRNA, thereby inhibiting the development of the cashmere goat hair follicles.

4. The use according to claim 3, characterized in that: The inhibition of cashmere goat hair follicle development includes any of the following situations: Inhibits the cell cycle of dermal papilla; Inhibit the proliferation of hair papilla cells; Inhibits dermal papilla cell migration; Promote apoptosis of dermal papilla cells; Inhibit the expression of mRNA related to the proliferation or migration of hair papilla cells, the mRNA includes COL11A1, COL1A1 .

5. The use according to claim 4, characterized in that: The hair papilla cells are real fur papilla cells of cashmere goats.

6. The use according to claim 5, characterized in that: The interference carrier is used for preparing a product for inhibiting proliferation or migration of hair papilla cells.

7. The use according to claim 5, characterized in that: The interference vector is used for preparing a product that promotes apoptosis of hair papilla cells.

8. Use of the cashmere goat lncRNA according to claim 1 or the interference vector according to claim 2 in promoting the development of cashmere goat hair follicles.

9. The use according to claim 8, characterized in that: The hair follicle development of cashmere goats is promoted by promoting the expression of the cashmere goat lncRNA or inhibiting the expression of the interference vector.

10. The use according to claim 9, characterized in that Melatonin is added to promote the expression of the cashmere goat lncRNA or inhibit the expression of the interference vector.

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