A kind of Inner Mongolia cashmere goat lncRNA, interference vector and its application in regulating the hair follicle development of Inner Mongolia cashmere goats

Abstract

The present invention belongs to the technical field of animal genetic engineering, and specifically relates to a cashmere goat lncRNA, an interfering vector and their application in regulating the hair follicle development of cashmere goats. The cashmere goat lncRNA is lncRNA TCONS-00237214, which is used to regulate the hair follicle development of cashmere goats; the nucleotide sequence of the lncRNA TCONS-00237214 is as shown in SEQ ID NO.1. The present invention selects dermal papilla cells for subsequent experimental verification. By constructing a stable transfected dermal papilla cell line interfering with lncRNA TCONS-00237214, the cell cycle, cell proliferation and cell migration of dermal papilla cells are detected. The results of the present invention show that melatonin can regulate the expression of lncRNA TCONS-00237214, participate in cell cycle transformation, promote the proliferation and migration of dermal papilla cells, and inhibit the apoptosis of dermal papilla cells, thereby affecting the occurrence and development of hair follicles.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal genetic engineering, and particularly relates to a cashmere goat lncRNA, an interfering vector and their application in regulating the hair follicle development of cashmere goats. Background Art

[0002] Long non-coding RNA, namely lncRNA, is a class of non-coding RNA with a length exceeding 200 nucleotides. Some studies suggest that lncRNA may be involved in regulating the growth and development of organisms, plays a key role in the development and regeneration of the skin, and is related to a series of physiological phenomena such as the development of the epidermis, hair follicles, pigment formation and the periodic growth of hair follicles. As the "signal center" of hair follicles and the "supply station" of nutrients, dermal papilla cells are the targets of many hormones and small molecules, and can affect the periodic growth of hair follicles by regulating hormonal small molecules, and have an important regulatory role in the development and regeneration of hair follicles. Therefore, it is necessary to explore the molecular mechanism of lncRNA in regulating hair follicle development in dermal papilla cells. Summary of the Invention

[0003] To solve the above problems, the present invention provides a cashmere goat lncRNA, an interfering vector and their application in regulating the hair follicle development of cashmere goats.

[0004] The present invention is achieved by the following technical solutions:

[0005] 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 it is used for regulating the hair follicle development of cashmere goats.

[0006] An interfering vector, wherein the interfering vector is a shRNA interfering vector of the lncRNA TCONS-00237214.

[0007] The application of the interfering vector in inhibiting the hair follicle development of cashmere goats, wherein the expression of the cashmere goat lncRNA is inhibited by the interfering vector, thereby inhibiting the hair follicle development of cashmere goats.

[0008] Preferably, the inhibition of the hair follicle development of cashmere goats includes any of the following situations.

[0009] Inhibiting the dermal papilla cell cycle.

[0010] Inhibiting the proliferation of dermal papilla cells.

[0011] Inhibiting the migration of dermal papilla cells.

[0012] Promoting the apoptosis of dermal papilla cells.

[0013] Inhibit the expression of related mRNAs that inhibit the proliferation or migration of dermal papilla cells, and the mRNAs include COL11A1, COL1A1 .

[0014] Preferably, the dermal papilla cells are true dermal papilla cells of cashmere goats.

[0015] Preferably, the interfering vector is used to prepare a product for inhibiting the proliferation or migration of dermal papilla cells.

[0016] Preferably, the interfering vector is used to prepare a product for promoting the apoptosis of dermal papilla cells.

[0017] Application of the cashmere goat lncRNA or the interfering vector in promoting the development of cashmere goat hair follicles.

[0018] Preferably, promote the development of cashmere goat hair follicles by promoting the expression of the cashmere goat lncRNA or inhibiting the expression of the interfering vector.

[0019] Preferably, promote the expression of the cashmere goat lncRNA or inhibit the expression of the interfering vector by adding melatonin.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a cashmere goat lncRNA, an interfering vector and their application in regulating the development of cashmere goat hair follicles. The cashmere goat lncRNA provided by the present invention is lncRNA TCONS-00237214, and its nucleotide sequence is shown in SEQ ID NO.1, which is used to regulate the development of cashmere goat hair follicles. The research results of the present invention show that: lncRNA TCONS-00237214 has a significant positive regulatory effect on COL11A1, COL1A1 All have significant positive regulatory effects in dermal papilla cells. Interfering with lncRNA TCONS-00237214 can inhibit cell proliferation and migration by regulating the cell cycle process. Interfering with lncRNA TCONS-00237214 promotes cell apoptosis. The present invention selects dermal papilla cells for subsequent experimental verification. Based on the co-expression regulatory network constructed by the research, the present invention constructs a stable transfected dermal papilla cell line interfering with lncRNA TCONS-00237214, and uses qRT PCR to detect the regulatory relationship between lncRNA TCONS-00237214 and COL11A1 / COL1A1 At the cellular level, DNA staining, CCK8 method and scratch test are used to detect the cell cycle, cell proliferation and cell migration of dermal papilla cells. COL11A1 / COL1A

[0022] 2. The results of the present invention show that melatonin can regulate the expression of lncRNA TCONS-00237214, participate in cell cycle transformation, promote the proliferation and migration of dermal papilla cells, inhibit the apoptosis of dermal papilla cells, and thus affect the hair follicle development. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 It is a morphological observation result diagram of the dermal papilla cells of the present invention.

[0025] Figure 1 In it, A is the morphological observation diagram of dermal papilla cells at 10 days; B is the morphological observation diagram of dermal papilla cells at 15 days; C is the morphological observation diagram of dermal papilla cells after passage; D is the morphological observation diagram of dermal papilla cells after adding melatonin.

[0026] Figure 2 It is a construction diagram of the interference vector of the present invention.

[0027] Figure 2 In it, A is the construction diagram of the negative control vector; B is the construction diagram of the vector interfering with lncRNA TCONS-00237214.

[0028] Figure 3 It is the stable transfected dermal papilla cell line constructed by interfering with lncRNA TCONS-00237214 of the present invention.

[0029] Figure 3 In it, A is the bright field cell diagram of the NC group after transfection; B is the bright field cell diagram of the group interfering with lncRNA TCONS-00237214 after transfection; C is the bright field cell diagram of the MT + interfering with lncRNA TCONS-00237214 group after transfection; D is the fluorescence field cell diagram of the NC group after transfection; E is the fluorescence field cell diagram of the group interfering with lncRNA TCONS-00237214 after transfection; F is the fluorescence field cell diagram of the MT + interfering with lncRNA TCONS-00237214 group after transfection; G is the qRT-PCR relative expression quantity result diagram of the NC group, the group interfering with lncRNA TCONS-00237214 and the MT + interfering with lncRNA TCONS-00237214 group.

[0030] Figure 4 For the present invention COL11A1 / COL1A1Relative expression levels in dermal papilla cell lines.

[0031] Figure 4 In [description of the figure], A is the expression level diagram of the target gene after interfering with lncRNA TCONS-00237214 COL11A1 In [description of the figure], B is the expression level diagram of the target gene after interfering with lncRNA TCONS-00237214 COL1A1 of the target gene.

[0032] Figure 5 This is the cell proliferation detection diagram of the present invention by the CCK8 method.

[0033] Figure 6 This is the cell migration detection diagram of the present invention by the cell scratch test.

[0034] Figure 6 In [description of the figure], A is the result diagram of the cell scratch experiment; B is the result diagram of the bright field cell migration rate of the dermal papilla cell line; C is the result diagram of the fluorescence cell migration rate of the dermal papilla cell line.

[0035] Figure 7 This is the result diagram of the detection of the cell cycle of dermal papilla cells by flow cytometry of the present invention.

[0036] Figure 7 In [description of the figure], A is the result diagram of the cell cycle analysis by flow cytometry of the NC group; B is the result diagram of the cell cycle analysis by flow cytometry of the MT + interfering lncRNA TCONS-00237214 group; C is the result diagram of the cell cycle analysis by flow cytometry of the interfering lncRNA TCONS-00237214 group; D is the statistical analysis diagram of the cell cycle of dermal papilla cells.

[0037] Figure 8 This is the result diagram of the detection of apoptosis of dermal papilla cells by flow cytometry of the present invention.

[0038] Figure 8 In [description of the figure], A is the quadrant diagram of cell apoptosis of the NC group; B is the quadrant diagram of cell apoptosis of the MT + interfering lncRNA TCONS-00237214 group; C is the quadrant diagram of cell apoptosis of the interfering lncRNA TCONS-00237214 group; D is the result diagram of the detection of apoptosis rate by qRT-PCR of the NC, MT + interfering lncRNA TCONS-00237214 group and the interfering lncRNA TCONS-00237214 group. Detailed implementation methods

[0039] For the convenience of understanding the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are given. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. 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.

[0040] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those of ordinary skill in the technical field to which the present invention pertains. The terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments, and are not intended to limit the present invention.

[0041] The beneficial effects of the present invention are illustrated by the following specific embodiments:

[0042] The main instruments and equipment used in the present invention are as follows:

[0043] Carbon dioxide incubator, purchased from Eppendorf; ultracentrifuge, purchased from Eppendorf; fluorescence microscope, purchased from Nikon; microplate reader, purchased from Bio-Tek; Nano Drop 2000 ultraviolet spectrophotometer, purchased from Thermo; ultra-low temperature refrigerator, purchased from Thermo Fisher Scientific; agarose gel electrophoresis tank; inverted fluorescence microscope; mini centrifuge; fully automatic ultrapure water instrument; pressure cooker, autoclave; -80 °C ultra-low temperature refrigerator; flow cytometer, purchased from Beckman CytoFLEX.

[0044] The main reagents used in the present invention are all purchased from Bio-Rad Biotechnology Distribution Department and are as follows:

[0045] DMEM medium, purchased from Tecono; fetal bovine serum, purchased from Tecono; triple antibiotic penicillin + streptomycin + amphotericin, purchased from gibco; 0.25% (mass concentration) trypsin containing EDTA; PBS buffer, purchased from gibco; 0.25% (mass concentration) trypsin without EDTA; DMSO; CCK8 kit; melatonin, M5250, purchased from Sigma; FITC Annexin V Apoptosis, purchased from BD Pharmingen TM; PI staining solution, purchased from MCE.

[0046]

[0047] Example 1

[0048] 1.1 Test method

[0049] 1.1.1 Culture of dermal papilla cells

[0050] (1)After shearing the collected tissue in PBS buffer containing triple antibiotics, cut it into tissue blocks the size of match heads and place them in PBS buffer for later use.

[0051] (2)Digest the tissue blocks in 0.25% trypsin (mass concentration) for 45 minutes.

[0052] (3)Terminate the digestion with a medium supplemented with 20% fetal bovine serum (volume fraction), then spread it in a culture dish and place it in an inverted position in a carbon dioxide incubator for 6 hours.

[0053] (4)After the tissue adheres to the wall, slowly add the medium and continue culturing.

[0054] (5)During the period when primary cells crawl out, replace the medium irregularly and observe the cell growth situation.

[0055] (6)After the primary cells cover the culture dish, aspirate the medium and wash it twice with PBS buffer containing triple antibiotics.

[0056] (7)Add 1 mL of 0.25% trypsin (mass concentration) and place it in a carbon dioxide incubator at 30 °C for digestion for 5 minutes.

[0057] (8)Add serum-containing medium to terminate the digestion. This step separates epithelial cells and a small amount of fibroblasts.

[0058] (9)Wash it twice again with PBS buffer containing triple antibiotics.

[0059] (10)Add 0.25% trypsin (mass concentration) and place it in a carbon dioxide incubator at 37 °C for 5 minutes, then add serum-containing medium to terminate the digestion.

[0060] (11)Transfer the cell suspension in the medium to a 15 mL centrifuge tube and then place it in a centrifuge at 1200 rpm for 5 minutes.

[0061] (12)The obtained precipitate is dermal papilla cells. Aspirate the supernatant, add a medium containing 10% fetal bovine serum (volume fraction) and culture it in a carbon dioxide incubator at 37 °C.

[0062] 1.1.2 Construction of lentiviral interference vector

[0063] The lentiviral vector was constructed by Shanghai Genechem Co., Ltd. First, the target gene was determined, and the interfering vector was constructed by selecting the siRNA target site. It relied on RNA polymerase III promoters including U6 and Cbh, and expressed in cells by manipulating a small hairpin RNA, abbreviated as shRNA for short hairpinRNA in English. Then, a negative control was designed. The siRNA used as the negative control should have the same composition as the selected siRNA sequence. Usually, the method is to scramble the base sequence in the selected siRNA. Subsequently, the DNA single strand of the siRNA sequence was annealed and cloned downstream of the promoter of the corresponding vector. Starting from the transcriptional 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. siRNAs with a GC content of about 40% were more effective. Secondly, the potential sequences were compared with the corresponding genomic databases, such as those of humans, mice, rats, etc., to exclude those sequences homologous to other coding sequences / ESTs.

[0064] The sequence of the shRNA is shown in SEQ ID NO.2 as: ccggGCGCAATGAAGACAGGTTAGTctcgagACTAACCTGTCTTCATTGCGCtttttg.

[0065] 1.1.3 Lentiviral transfection of dermal papilla cells

[0066] The true dermal papilla cells cultured by the 6-well plate method were cultured in a 37 °C incubator with 5% carbon dioxide by volume fraction until the confluence rate reached 20% - 30%; then continued to be cultured with serum-free DMEM, and HitransG-P virus enhancer was added; based on the preliminary experiment, the dosage of the virus was calculated according to the method of MOI = 10 and cultured under the conditions of 37 °C and 5% carbon dioxide by volume fraction; after 12 hours, the serum-free DMEM was aspirated and replaced with a complete medium containing serum and triple antibiotics, and then cultured for about 72 hours; in the experimental group with better transfection effect, it was cultured for 24 hours with a final dosage of 5 μg / mL under the action of puromycin.

[0067] Note: The lentiviral vector was constructed and packaged by Shanghai Genechem Co., Ltd. An interfering vector of lncRNA TCONS-00237214 and an shRNA negative control were constructed.

[0068] 1.1.4 Extraction of total RNA from dermal papilla cells

[0069] Extract the total RNA of the dermal papilla cell line when the cell confluence reaches 80%; aspirate and discard the complete DMEM medium, add 1 mL of pre-warmed PBS buffer and wash three times; add 1 mL of Trizol to lyse the cells; then add chloroform and mix well, and aspirate the aqueous phase after centrifugation; add an equal volume of isopropanol to the aqueous phase and mix well, discard the supernatant after centrifugation, add 75% ethanol by volume to wash the precipitate; discard the supernatant after centrifugation, air-dry at room temperature, add RNase-free water to fully dissolve the RNA; store the obtained RNA at -70 °C. Note: The dermal papilla cells for total RNA extraction are cultured in a six-well plate.

[0070] 1.1.5 qRT-PCR detection

[0071] Reverse transcription using the Kangwei reagent HiFiScript gDNA Removal RT MasterMix kit, as shown in Table 1, to obtain the cDNA template; prepare the cDNA template and primers according to Table 2, use GAPDH as an internal standard to quantify lncRNA and target genes, as shown in Table 3; use the 2 -△△CT CT relative expression calculation formula to calculate the quantitative data of lncRNA and target genes. The Pearson correlation analysis method is used to calculate the relative expression levels of lncRNAs and target genes, and Graphpad prisim9.0 is used for visualization. The primers used were designed by Sangon Biotech (Shanghai) Co., Ltd., and the primer sequence information for lncRNA and mRNA is shown in Table 4.

[0072] Table 1 Reverse transcription reaction system

[0073]

[0074] Table 2 qRT-PCR reaction system

[0075]

[0076] Table 3 qRT-PCR reaction program

[0077]

[0078] Table 4 lncRNA and mRNA primer sequence information

[0079]

[0080] 1.1.6 Detection of dermal papilla cell proliferation by CCK8 method

[0081] Three groups were set up, namely the negative control group NC, the group interfering with lncRNA TCONS-00237214, and the MT + interfering lncRNA TCONS-00237214 group. MT is melatonin. Cells were collected using the enzyme digestion method. 100 μL of cell suspension was inoculated into a 96-well plate and cultured in a constant temperature carbon dioxide incubator for 24 hours. At the same time every day, the corresponding 96-well culture plates at different times were taken out. 10 μL of CCK8 reagent was added to each well. After 2 hours, the absorbance value at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The measured OD values were sorted out and calculated, and plotted using GraphPad 9.0.

[0082] 1.1.7 Scratch test to detect the migration of dermal papilla cells

[0083] Before the scratch experiment, several marker lines with a spacing of 0.5 cm were drawn horizontally on the back of a six-well plate using a marker pen. Cells were inoculated into the six-well plate with the spaced lines and cultured at 37 °C in an atmosphere of 5% carbon dioxide by volume until 100% confluence was reached. Then, a scratch was made perpendicular to the marker line using a 200 μL pipette tip. After the scratch was completed, it was washed 3 times with pre-warmed PBS buffer. It was cultured with serum-free DMEM and placed in an incubator at 37 °C in an atmosphere of 5% carbon dioxide by volume. Photos were taken and observed at the same position at 0 h and 24 h, and the scratched area was statistically analyzed using Imagej software. The formula for calculating the migration rate: Migration rate = (Blank area at 0 h - Blank area at 24 h) / Blank area at 0 h.

[0084] 1.1.8 Flow cytometry to detect the cell cycle of dermal papilla cells

[0085] Single cell suspensions were prepared and centrifuged at 4 °C and 1200 rpm for 5 min. Pre-cooled ethanol at a volume fraction of 70% was added to the cell pellet collected above, and it was pipetted evenly and incubated overnight at 4 °C. After removing the supernatant, it was suspended with 1 mL of pre-cooled PBS buffer, centrifuged at 4 °C and 1200 rpm for 5 minutes, and then the supernatant was removed and saved as a pellet. 1 mL of pre-cooled ethanol at a volume fraction of 70% was added to the cell pellet collected above, gently pipetted to make it uniform, and fixed at 4 °C for more than 2 h. After centrifuging at 4 °C and 1200 rpm for 5 minutes, the supernatant was removed, and 1 mL of pre-cooled PBS buffer was added to re-suspend it. After centrifuging at 4 °C and 1200 rpm for 5 minutes, the supernatant was removed, and the cell pellet was retained. A suitable PI staining working solution was prepared according to the dosage of the test substance and placed at 4 °C, which was valid within 24 h. 0.5 mL of PI staining solution was added to the suspended cells and cultured in the dark at 37 °C for 40 minutes. Red fluorescence was detected using a flow cytometer at a laser wavelength of 488 nm, and light scattering was also detected to analyze DNA content and light scattering.

[0086] 1.1.9 Detection of dermal papilla cell apoptosis by flow cytometry

[0087] Prepare a single cell suspension, centrifuge at 4°C and 1200 rpm for 5 min; 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 and 1200 rpm for 5 min, discard the supernatant; wash the cells once with PBS buffer, discard the supernatant after centrifugation, add 100 μL of diluted 1x Annexin V Binding Buffer to resuspend the cells; add 5.0 μL of Annexin V-APC Reagent and 5.0 μL of PI Reagent to the cell suspension; gently vortex and mix well, incubate at room temperature in the dark for 15 min; add 400 μL of diluted 1x Annexin V Binding Buffer to mix the sample; immediately perform on-machine detection. If detection cannot be performed in a timely manner, place it on ice in the dark and complete the detection within 1 hour.

[0088] 1.2 Results and analysis

[0089] 1.2.1 Morphological observation of dermal papilla cells

[0090] After 10 days, as shown in A of Figure 1 , some cells can be seen growing from the surrounding edges around the tissue block. These cells are short spindle-shaped or irregular square-shaped and aggregate together. At 15 days, as shown in B of Figure 1 , these cells will start to enter the logarithmic growth phase, and a large amount of cytoplasm will appear during this process. Soon, it gradually loses its original morphology. As shown in the figure, during the initial growth process, more easily growing epidermal cells will climb out. Most of them are irregular quadrilateral and gather in groups, and it can be determined that they are a relatively primitive type of dermal papilla cells. The morphology of dermal papilla cells after passage is as shown in C of Figure 1 , and the cell growth rate accelerates after passage. The morphology of dermal papilla cells after adding melatonin is as shown in D of Figure 1 . After adding melatonin, the proliferation rate of dermal papilla cells is faster, and the cell morphology is more obvious, which can be used for subsequent experiments.

[0091] 1.2.2 Identification of lentiviral interference vector

[0092] Determine the siRNA sequences of the target gene and the negative control. The vector element of the negative control NC is hU6-MCS-CMV-puro, and the vector element for interfering with lncRNA TCONS-00237214 is hU6-MCS-CBh-gcGFP-IRES-puromycin, as shown in Figure 2As shown, their GC content is 47.62% as shown in Table 5. After sequence alignment, it was found that there was homology of 16 consecutive bases only with two genes, and there was no homology of 16 consecutive bases with any sequence in the genome except this. This target construction is effective and has good results, indicating that the vector construction is successful and subsequent experiments can be carried out.

[0093] Table 5 Target sequence

[0094]

[0095] 1.2.3 Construction of stably transfected virus dermal papilla cell line

[0096] The FITC protein fluorescence results of each experimental group showed that the cells in the experimental groups all emitted green fluorescence and had normal morphology, indicating good transfection effect. After successfully constructing the lncRNA TCONS-00237214 interference vector, the third-generation dermal papilla cells were transfected. After 72 hours of transfection, the transfection efficiency was observed through an inverted fluorescence microscope. The transfection efficiency was the highest and the cell morphology was the best, as shown in Figure 3 A - F. The transfection efficiency was up to more than 80% and the cell death was less, so further research could be carried out. In addition, the qRT-PCR results showed that, as shown in Figure 3 G, in dermal papilla cells, the relative expression levels of interfering lncRNA TCONS-00237214 and melatonin MT + interfering lncRNA TCONS-00237214 were both extremely significantly less than those of the NC group, * indicates P<0.05 , indicating that the construction of the stably transfected dermal papilla cell line interfering with lncRNA TCONS-00237214 was successful.

[0097] 1.2.4 qRT-PCR detection COL11A1 / COL1A1 Expression at the dermal papilla cell level

[0098] By using qRT-PCR to measure the expression in the stably transfected dermal papilla cell line, the results were as shown in COL11A1 / COL1A1 Figure 4 . In the dermal papilla cell line interfering with lncRNA TCONS-00237214, the relative expression level of Figure 4 was extremely significantly different from that of the corresponding NC group, ** indicates COL11A1 / COL1A1 , but after adding melatonin, the expression level of the target gene showed a significant increase, * indicates P<0.01 . It was found that melatonin could increase the relative expression level of the target gene by regulating the expression of lncRNA, indicating that lncRNA TCONS-00237214 was positively correlated with P<0.05 COL11A1 / COL1A1 COL11A1 / COL1A1 in dermal papilla cells. Melatonin promoted the expression of lncRNA and had a regulatory effect at the mRNA level.

[0099] 1.2.5 Detection of dermal papilla cell proliferation by CCK8 method

[0100] The results of the CCK8 method test are shown as Figure 5 shown. Compared with the NC group, after interfering with the dermal papilla cells of lncRNA TCONS - 00237214, the cell proliferation level decreased extremely significantly, ** indicating P<0.01 ; after adding melatonin, the proliferation level of dermal papilla cells with interference of lncRNA TCONS - 00237214 also decreased extremely significantly, ** indicating P<0.01 , but compared with the cell proliferation ability of interfering with lncRNA TCONS - 00237214, the cell proliferation ability of interfering with lncRNA TCONS - 00237214 after adding melatonin increased significantly, * indicating P<0.05 , indicating that interfering with lncRNA TCONS - 00237214 can inhibit the proliferation of dermal papilla cells, but adding melatonin can promote the proliferation of dermal papilla cells.

[0101] 1.2.6 Detection of dermal papilla cell migration by scratch test

[0102] The results of the cell scratch test are shown as Figure 6 shown. In dermal papilla cells, the migration rate of the NC group was greater than that of the group interfering with lncRNA TCONS - 00237214, and the difference between the two groups was extremely significant, P<0.01 ; after adding melatonin, the migration rate of dermal papilla cells with interference of lncRNA TCONS - 00237214 was also extremely significantly lower than that of the NC group, * indicating P<0.05 , but compared with the migration rate of dermal papilla cells interfering with lncRNA TCONS - 00237214, the migration rate of dermal papilla cells interfering with lncRNA TCONS - 00237214 after adding melatonin increased significantly, * indicating P<0.05 , indicating that melatonin can promote the migration rate of dermal papilla cells. The above research shows that interfering with lncRNA TCONS - 00237214 inhibits the migration of dermal papilla cells.

[0103] 1.2.7 Detection of dermal papilla cell cycle by flow cytometry

[0104] The results of flow cytometry analysis of the dermal papilla cell cycle are as Figure 7 shown in A - C of. It was found that most cells with interference of lncRNA TCONS - 00237214 stayed in the G0 / G1 phase, and at the same time, the transition from the G0 / G1 phase to the S phase and the G2 / M phase was inhibited, and the cell proliferation index decreased extremely significantly. As Figure 7As shown in D in [reference], the proportion of dermal papilla cells interfering with lncRNA TCONS-00237214 in the S phase and G2 phase after adding melatonin was also extremely significantly lower than that of the NC group, ** indicating P<0.01 , but compared with the cell cycle of dermal papilla cells interfering with lncRNATCONS-00237214, the proportion of G0 / G1 phase of dermal papilla cells interfering with lncRNA TCONS-00237214 decreased relatively after adding melatonin, and at the same time, the degree of inhibition of the transition from G0 / G1 phase to S phase and G2 / M phase was alleviated, indicating that melatonin can accelerate the process of the dermal papilla cell cycle. The results obtained from the above research are consistent with the experimental results of CCK8 to detect cell proliferation, indicating that interfering with lncRNA TCONS-00237214 can inhibit the cell cycle process and thus inhibit cell proliferation by inhibiting the transition from G0 / G1 phase to S phase and G2 / M phase.

[0105] 1.2.8 Detection of dermal papilla cell apoptosis by flow cytometry

[0106] After collecting dermal papilla cells transfected for 72 hours, apoptosis was measured by flow cytometry using AnnexinV-APC / DAPI double staining method. The results are as Figure 8 shown. Compared with the NC group, interfering with lncRNA TCONS-00237214 extremely significantly promoted dermal papilla cell apoptosis, ** indicating P<0.01 ; after adding melatonin, interfering with lncRNA TCONS-00237214 also extremely significantly inhibited dermal papilla cell apoptosis, * indicating P<0.05 ; compared with the group interfering with lncRNA TCONS-00237214, the group adding melatonin + interfering with lncRNA TCONS-00237214 reduced the proportion of early and late apoptosis and inhibited cell apoptosis, indicating that adding melatonin in dermal papilla cells can inhibit cell apoptosis. The above research shows that interfering with lncRNA TCONS-00237214 plays a role in promoting dermal papilla cell apoptosis.

[0107] In summary, the present invention established a stable transfected dermal papilla cell line interfering with lncRNA TCONS-00237214. The qRT-PCR detection results showed that in dermal papilla cells, lncRNA TCONS-00237214 and COL11A1 / COL1A1There was a positive correlation. Melatonin promoted the expression of lncRNA and had a regulatory effect at the mRNA level. The results of CCK8 assay, cell migration and flow cytometry experiments showed that interfering with lncRNA TCONS-00237214 could inhibit cell proliferation and cell migration and promote cell apoptosis by regulating the cell cycle process. Melatonin could regulate the expression of lncRNA TCONS-00237214, participate in cell cycle transformation, promote the proliferation and migration of dermal papilla cells, and inhibit the apoptosis of dermal papilla cells, thereby affecting the hair follicle development.

[0108] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, 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, it should be considered as the scope recorded in this specification.

[0109] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended 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; 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 .

4. The use according to claim 3, characterized in that: The hair papilla cells are real fur papilla cells of cashmere goats.

5. The use according to claim 3, characterized in that: The interference carrier is used for preparing a product for inhibiting proliferation or migration of hair papilla cells.

6. The use according to claim 3, characterized in that: The interference vector is used for preparing a product that promotes apoptosis of hair papilla cells.

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