Application of lncRNA inhibitor in preparing a fibroblast proliferation promoter

Abstract

The present invention relates to the field of genetic engineering technology, and specifically discloses the application of an lncRNA inhibitor in the preparation of a fibroblast proliferation promoter. The lncRNA is lncRNA MSTRG.12349.1, and the only active ingredient in the lncRNA inhibitor is the interfering sequence of the lncRNA, and the interfering sequence is lncRNA MSTRG.12349.1-sh1. The interfering sequence lncRNA MSTRG.12349.1-sh1 provided by the present invention effectively improves the proliferation ability of fibroblasts by increasing the proportion of cells in the S phase of the cell cycle, and promotes the occurrence and development of secondary hair follicles in the embryonic period of cashmere goats.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to the application of lncRNA inhibitors in the preparation of fibroblast proliferation promoters. Background Art

[0002] Cashmere goats are a special type of livestock with an important position in the global livestock industry. They are distributed between latitudes 25°N and 55°N and longitudes 40°E and 125°E. In China, they are mainly distributed in four regions: southwest, northwest, north China, and northeast. Inner Mongolia cashmere goats are an excellent local breed mainly for cashmere production. The cashmere produced by them is slender and soft, and is an extremely precious natural fiber. Textiles made of cashmere are light, thin, soft, and have remarkable warmth retention effects, and are highly sought after by the public. Cashmere grows from the secondary hair follicles of cashmere goats, and its output is relatively low. According to statistics, the annual global cashmere output only accounts for about 0.2% of the total animal fiber output, which makes the market price of cashmere remain high. High-quality cashmere products are even more expensive, which also brings considerable economic benefits to farmers.

[0003] Hair follicles are unique and relatively complex skin appendages in mammals, and play an important role in functions such as maintaining skin homeostasis, regulating body temperature, and metabolism. In addition, it controls hair growth, determines the quality and output of animal hair, and is an important economic trait of hair-using animals. The formation of hair follicles is particularly crucial during the embryonic period, and the development at this stage will directly determine the quality and output of cashmere. Therefore, in-depth exploration of the development mechanism of cashmere goat hair follicles is of great significance for improving cashmere quality, increasing output, and even promoting the prosperous development of the entire cashmere industry. The skin of cashmere goats contains primary hair follicles and secondary hair follicles. Among them, secondary hair follicles are the key organs for growing cashmere. Research shows that during the development process of cashmere goat hair follicles, the development of primary hair follicles always precedes that of secondary hair follicles. Starting from the 45th day of embryonic development, the fetal skin has initially formed a complete epidermal structure. On the 55th day of embryo, the hair bud structure of primary hair follicles begins to appear. With the continuous development, by the 65th day, these hair bud structures continue to grow downward. On the 75th day, the hair bud structure of secondary hair follicles appears for the first time.

[0004] The development of hair follicles is an intricate physiological process, which depends on the precise signal transduction between the dermis and the epidermis to form a complete hair follicle structure. Among them, dermal papillae are formed by the proliferation and differentiation of dermal fibroblasts. This structure is the signal center in the entire hair follicle structure and is the key to ensuring the normal growth of hair follicles. Therefore, the proliferation of fibroblasts is of great significance for promoting the development of secondary hair follicles in cashmere goats. It is necessary to explore new ways to promote the proliferation of cashmere goat fibroblasts. Summary of the Invention

[0005] To explore new ways to promote the proliferation of fibroblast cells in cashmere goats, the present invention provides the application of lncRNA inhibitors in the preparation of fibroblast proliferation promoters. By interfering with the sequence to inhibit the expression of lncRNA MSTRG.12349.1, the proportion of cells in the S phase of the cell cycle is increased, thereby effectively improving the proliferation ability of fibroblast cells and further promoting the occurrence and development of secondary hair follicles in the embryonic stage of cashmere goats.

[0006] The present invention provides the application of lncRNA inhibitors in the preparation of fibroblast proliferation promoters, wherein the lncRNA is lncRNA MSTRG.12349.1, and the lncRNA inhibitor takes the interference sequence of lncRNA as the only active ingredient, and the interference sequence is lncRNA MSTRG.12349.1-sh1;

[0007] The sense strand of the lncRNA MSTRG.12349.1-sh1 is as shown in SEQ ID NO.5;

[0008] The antisense strand of the lncRNA MSTRG.12349.1-sh1 is as shown in SEQ ID NO.6.

[0009] The lncRNA inhibitor provided by the present invention takes the interference sequence of lncRNA as the only active ingredient. The interference sequence lncRNA MSTRG.12349.1-sh1 can inhibit the expression of lncRNA MSTRG.12349.1, effectively improve the proliferation ability of fibroblast cells by increasing the proportion of cells in the S phase of the cell cycle, and thus promote the occurrence and development of secondary hair follicles in the embryonic stage of cashmere goats.

[0010] The present invention also provides the interference sequence of lncRNA, and the interference sequence is lncRNA MSTRG.12349.1-sh1.

[0011] The present invention also provides an interference plasmid, which is obtained by ligating the interference sequence with the pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector.

[0012] The pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector is purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.

[0013] The present invention also provides a lentiviral resuspension solution, which contains the interference plasmid.

[0014] Furthermore, the lentiviral resuspension solution is obtained by packaging lentivirus by co-transfecting the helper plasmid and the interference plasmid into cells using a three-plasmid lentiviral system.

[0015] The present invention also provides an application of an lncRNA MSTRG.12349.1 gene inhibitor in preparing a fibroblast proliferation promoter, wherein the only active ingredient of the gene inhibitor is the interfering sequence of the lncRNA, the interfering plasmid or the lentivirus resuspension solution.

[0016] Furthermore, the fibroblast is a fibroblast in the embryonic period of cashmere goats.

[0017] The present invention also provides an application of an lncRNA MSTRG.12349.1 gene inhibitor in preparing a promoter for the occurrence and development of secondary hair follicles in cashmere goats, wherein the only active ingredient of the gene inhibitor is the interfering sequence of the lncRNA, the interfering plasmid or the lentivirus resuspension solution.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. The present invention identifies an lncRNA MSTRG.12349.1 related to the occurrence and development of secondary hair follicles in the embryonic period of cashmere goats, and designs and obtains an interfering sequence of lncRNA MSTRG.12349.1. Among them, lncRNA MSTRG.12349.1-sh1 effectively improves the proliferation ability of fibroblasts by increasing the proportion of cells in the S phase of the cell cycle, and promotes the occurrence and development of secondary hair follicles in the embryonic period of cashmere goats. Description of the Drawings

[0020] 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.

[0021] Figure 1 Screening of lncRNA related to the occurrence and development of secondary hair follicles in the present invention.

[0022] Figure 2 Fluorescence image of lncRNA MSTRG.12349.1-sh1 interfering with fibroblast cell line in the present invention.

[0023] Figure 3Relative expression levels of different lncRNA MSTRG.12349.1 interfering sequences in interfering with lncRNA MSTRG.12349.1 in fibroblast cell lines; among them, sh-NC is the fibroblast cell line transfected with the empty vector, sh1 is the fibroblast cell line transfected with the lncRNA MSTRG.12349.1-sh1 interfering vector, sh2 is the fibroblast cell line transfected with the lncRNA MSTRG.12349.1-sh2 interfering vector, and sh3 is the fibroblast cell line transfected with the lncRNA MSTRG.12349.1-sh3 interfering vector; *** represents p < 0.001, with extremely significant differences; **** represents p < 0.0001, with extremely extremely significant differences; ns indicates no significant differences.

[0024] Figure 4 Proliferation of fibroblasts after interference with lncRNA MSTRG.12349.1-sh1.

[0025] Figure 5 Cell cycle of fibroblasts after interference with lncRNA MSTRG.12349.1-sh1;

[0026] In the figure, A is the proportion of cells in different cell cycles in NC group fibroblasts;

[0027] B is the proportion of cells in different cell cycles in sh-NC group fibroblasts;

[0028] C is the proportion of cells in different cell cycles in lncRNA MSTRG.12349.1-sh1 group fibroblasts;

[0029] D is the statistical chart of the proportion of fibroblasts in different cell cycles in NC group, sh-NC group and lncRNA MSTRG.12349.1-sh1 group.

[0030] Figure 6 The fragment from the 1st bp to the 3000th bp in the full-length sequence of lncRNA MSTRG.12349.1 of the present invention.

[0031] Figure 7 The fragment from the 3001st bp to the 8700th bp in the full-length sequence of lncRNA MSTRG.12349.1 of the present invention.

[0032] Figure 8 The fragment from the 87001st bp to the 11111bp in the full-length sequence of lncRNA MSTRG.12349.1 of the present invention.

[0033] The full-length sequence of the said lncRNA MSTRG.12349.1 isFigures 6 - 8 The sequences in Specific embodiments

[0034] The specific embodiments of the present invention will be described in detail below. However, it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0035] Example 1: Screening of lncRNAs during the development of secondary hair follicles in cashmere goats.

[0036] 1. Collection of cashmere goat body side skin

[0037] Twelve 3-year-old female goats with good production performance and the same growth environment were selected for estrus synchronization treatment, and fetal body side skin tissue samples were collected at 45 days, 55 days, 65 days, and 75 days of gestation. Three samples were collected at each stage, for a total of 12 fetal body side skin samples. After collection, they were numbered and quickly placed in liquid nitrogen for cryopreservation.

[0038] The experimental sheep breed was the Albas type cashmere goat in Inner Mongolia Autonomous Region.

[0039] 2. RNA extraction and quality control

[0040] Total RNA was extracted from the fetal body side skin samples of cashmere goats at 45 days, 55 days, 65 days, and 75 days of gestation using the Trizol reagent method. The purity and integrity of the RNA were detected using NanoDrop ND-1000 and Agilent 2100 respectively. When the OD values 260 / 230 ≥ 1.5, 260 / 280 ≥ 1.8, and RIN ≥ 7, the RNA passed the quality control and could be used for subsequent sequencing.

[0041] 3. Whole-genome transcriptome sequencing of cashmere goat fetal body side skin

[0042] Using Ribo-Zero TMThe rRNA of 12 samples was removed using the rRNA Removal Kit, and the remaining RNA was reverse transcribed to form the final product cDNA. Finally, paired-end sequencing was performed on the Illumina Hiseq 4000. A total of 1,063,299,566 raw data were obtained from 12 sequencing databases. After removing sequencing adapters and low-quality data using Cutadpter, 1,023,889,360 valid data were obtained. Subsequently, the processed valid data were aligned to the reference genome using Hisat. The percentage of reads aligned to the reference genome was higher than 94% of the valid reads, the percentage of reads aligned to a unique location was higher than 77% of the valid reads, and the percentage of reads aligned to multiple locations was higher than 17% of the valid reads.

[0043] 4. Identification and differential expression analysis of lncRNAs

[0044] According to the characteristics that lncRNAs cannot encode proteins and have long transcripts, the present invention excluded transcripts that did not conform to the characteristics of lncRNAs. Subsequently, the coding ability of the remaining transcripts was predicted using two software, CNCI and CPC, and transcripts that might encode proteins were filtered. After a series of screenings, a total of 6,630 lncRNA transcripts were finally identified.

[0045] 5. Differential expression analysis

[0046] To further explore the regulatory role of lncRNAs in the early development of Inner Mongolia cashmere goat hair follicles, the present invention divided the 4 periods into 6 comparison groups and used String Tie to calculate the FPKM value, i.e., the gene expression level. This calculation method can eliminate the influence of sequencing depth, gene length, and differences between samples on the gene expression level. EdgeR was used to perform differential expression analysis on the genes identified in 4 different embryonic periods, with the screening conditions of a differential multiple |log2foldchange|≥1 and a significant P-value≤0.05.

[0047] 6. Preliminary screening of genes related to secondary hair follicle development

[0048] According to the characteristics of the development stages of primary and secondary hair follicles in cashmere goats: at 45 days, primary and secondary hair follicles have not occurred; at 55 days, primary hair follicles begin to form; at 65 days, primary hair follicles continue to grow downward; at 75 days, the hair buds of primary hair follicles penetrate into the dermis layer, and at the same time, secondary hair follicles begin to occur. The three comparison groups of d55vsd45, d65vsd45, and d65vsd55 in the sequencing results are regarded as Stage A, and the three comparison groups of d75vsd45, d75vsd55, and d75vsd65 are regarded as Stage B. Take the intersection of Stage A and Stage B, and the 158 lncRNAs after removing the intersection part from Stage B are regarded as related to the development process of secondary hair follicles, such as Figure 1 。

[0049] 8. Determination of lncRNAs related to the development of secondary hair follicles

[0050] Combined with the morphological changes of secondary hair follicles in different embryonic stages of cashmere goats, and using differential expression analysis, the present invention finally screened out 158 lncRNAs related to the development of secondary hair follicles in cashmere goats. Among them, lncRNAMSTRG.12349.1 was significantly lowly expressed compared with other lncRNAs during the critical period of 75 days when secondary hair follicles began to occur. Therefore, lncRNA MSTRG.12349.1 was finally selected as the lncRNA related to the development of secondary hair follicles.

[0051] The full-length sequence of lncRNA MSTRG.12349.1 is successively spliced from the sequences in Figures 6 - 8 with a length of 11111bp. The full-length sequence of lncRNA MSTRG.12349.1 is derived from the SRA database and is obtained by performing data quality assessment, reference genome alignment, and then transcript splicing and assembly on the basis of the original transcriptome sequencing data. The accession numbers in the SRA database are SRR13306938~SRR13306949.

[0052] Example 2: Construction and screening of lncRNA MSTRG.12349.1 interference cell lines.

[0053] 1. Construction of lncRNA MSTRG.12349.1-shRNA vector

[0054] According to the lncRNA MSTRG.12349.1 sequence information, the target fragment was amplified and purified by PCR method and PAGE purification method. Finally, 3 pairs of shRNAs of lncRNA MSTRG.12349.1 were obtained, which were named lncRNAMSTRG.12349.1-sh1, lncRNA MSTRG.12349.1-sh2 and lncRNA MSTRG.12349.1-sh3 respectively. The shRNA sequence information is shown in Table 1.

[0055] The lncRNA MSTRG.12349.1-sh1, lncRNA MSTRG.12349.1-sh2 and lncRNA MSTRG.12349.1-sh3 fragments were respectively ligated with the pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector by T4 ligation method to complete the construction of the lncRNA MSTRG.12349.1 interfering plasmid. The lncRNA MSTRG.12349.1 interfering plasmid includes lncRNA MSTRG.12349.1-sh1 interfering plasmid, lncRNA MSTRG.12349.1-sh2 interfering plasmid and lncRNAMSTRG. 12349.1-sh3 interfering plasmid.

[0056] Subsequently, the constructed lncRNA MSTRG.12349.1 interfering plasmid was mixed with the transformed competent DH5a, and the bacterial solution was spread on the plate and cultured in the incubator for 16 h; single colonies were selected for colony verification, and the positive clones were verified by sequencing; if the sequencing results were consistent with the target sequence, plasmid extraction and purification were carried out. Among them, the lncRNAMSTRG.12349.1-shRNA synthesized by PCR amplification is a hairpin structure, which can be cleaved in cells to form siRNA. The siRNA binds to the RNA-induced silencing complex, thereby inhibiting the expression of the lncRNA MSTRG.12349.1 gene. The siRNA sequence information of lncRNA MSTRG.12349.1 is shown in Table 2.

[0057] The pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector was purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.

[0058]

[0059]

[0060] 2. Lentivirus packaging

[0061] Using a three-plasmid lentiviral system, the helper plasmids psPAX2 and pMD2G and the above-constructed lncRNA MSTRG.12349.1 interfering plasmid were co-transfected into 293T cells respectively. After transfection, the viral supernatants were collected at 48 h and 72 h respectively. After collecting the supernatant at 48 h of transfection, fresh medium was added. After collection, it was filtered through a 0.45 μm filter into a 40 mL centrifuge tube and centrifuged at 4 °C and 7200 rpm for 120 min. The viral pellet was resuspended in 500 μL of fresh medium without antibiotics to obtain a resuspended solution containing lentivirus, simply referred to as the lentivirus resuspended solution, and stored at -80 °C. The fresh medium without antibiotics is: DMEM medium + 10% FBS serum.

[0062] 3. Cultivation of fibroblasts in the embryonic period of cashmere goats

[0063] The primary culture of embryonic cashmere goat skin tissue was carried out by the tissue block culture method, and the primary cells were purified and cultured by the enzyme digestion method to finally obtain fibroblasts in the embryonic period of cashmere goats. The specific steps are as follows:

[0064] Tissue block culture experiment: (1) Collect the skin tissue of cashmere goats in the embryonic period and store it at low temperature in PBS solution for subsequent cell culture. (2) Spray the surrounding environment of the test bench with alcohol for disinfection. Disinfect the collected samples with 75% alcohol for about 30 s and wash them 3 times with PBS. (3) Cut the tissue samples into minced meat with a small scissors after autoclaving, inoculate them into a cell culture flask with autoclaved forceps, and invert the culture flask for about 5 min. (4) Turn the culture flask over so that the side with tissue blocks faces up and place it in an incubator at 37 °C and 5% CO 2 for about 30 min. (5) Take out the inverted cell culture flask and add complete culture medium. (6) Replace the cell culture liquid according to the emergence of cells from the skin tissue in the flask. Usually, it only needs to be replaced once in the first week, and then the liquid is changed every 4 days.

[0065] Enzyme digestion method experiment: (1) When the primary cells grow to confluence, remove the tissue blocks and cell culture medium in the culture flask and wash them 3 times with PBS. (2) Add 2 mL of trypsin containing EDTA and phenol red to the cell culture flask and place it at 37 °C and 5% CO 2Digest in an incubator for about 3 minutes; gradually isolate and purify different cell lines according to the different tolerances of different cells to trypsin. Since fibroblasts have the worst tolerance to trypsin, fibroblasts can completely float in the digestive solution after about 3 minutes. (3) Add complete medium equal to the amount of trypsin to the culture flask to terminate digestion. (4) Aspirate the liquid containing fibroblasts in the culture flask and transfer it to a 15 mL centrifuge tube, and centrifuge at 1500 rpm for 3 minutes. (5) Discard the supernatant and retain the fibroblast precipitate. (6) Add 1 mL of complete culture medium to the centrifuge tube and gently pipette until no cell precipitate can be seen at the bottom with the naked eye. (7) Aspirate the culture medium containing fibroblasts in the centrifuge tube into a new culture flask, and supplement the complete culture medium in the culture flask to 3 mL. (8) Place the culture flask containing fibroblasts in an incubator at 37 °C and 5% CO 2 for culture. Fibroblasts can be obtained after culturing for about 3 days; the whole process is operated on a sterilized super clean bench, and all the equipment used has been sterilized or autoclaved; the purified fibroblasts are spindle-shaped.

[0066] 4. Construction of lncRNA MSTRG.12349.1 interference cell line

[0067] Digest fibroblasts with trypsin and adjust the cell concentration to 2×10 4 cells per well in a 24-well plate and change the medium every 2 days. Add 5 μg / mL of Polybrene dilution to fibroblasts for incubation for 4 hours before transfection; then, discard the original medium, add the medium with half of the required volume and containing the lentivirus resuspension for culture. After 4 hours of transfection, supplement the required medium, and the multiplicity of infection of each experimental group is 5. After 24 hours of transfection, discard the medium containing the lentivirus resuspension and replace it with fresh medium, and continue to culture in an incubator at 37 °C and 5% CO 2 . After 72 hours of transfection, observe the transfection efficiency under a fluorescence microscope. Add Puromycin to each experimental group for resistance screening, and the final concentration is 5 μg / mL, that is, the lncRNA MSTRG.12349.1 interference cell line is obtained, including the lncRNA MSTRG.12349.1-sh1 interference cell line, abbreviated as sh1, the lncRNA MSTRG.12349.1-sh2 interference cell line, abbreviated as sh2, and the lncRNA MSTRG.12349.1-sh3 interference cell line, abbreviated as sh3.

[0068] Figure 2 The lncRNA MSTRG.12349.1-sh1 interference fibroblast cell line constructed in the present invention. The cells in the figure grow densely and show an obvious spindle shape, indicating that the lncRNA MSTRG.12349.1-sh1 interference fibroblast cell line is successfully constructed.

[0069] 5. Screening of lncRNA MSTRG.12349.1 interfering cell lines: The total RNA of the constructed lncRNA MSTRG.12349.1-sh1 fibroblast cell line, lncRNA MSTRG.12349.1-sh2 fibroblast cell line, and lncRNA MSTRG.12349.1-sh3 fibroblast cell line was extracted by the Trizol reagent method. According to the lncRNA MSTRG.12349.1 sequence information, primers were designed, and the primer information is shown in Table 3. The expression changes of lncRNA MSTRG.12349.1 in each cell line after interference were detected by real-time fluorescence quantitative PCR.

[0070]

[0071] The specific steps of real-time fluorescence quantitative PCR were as follows: The total RNA of different interfering cell lines extracted was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser kit; subsequently, the TBGreen Premix Ex Taq Ⅱ kit was used for real-time fluorescence quantitative PCR on the LightCycler® 96 Real-Time PCR platform. The experimental conditions were pre-denaturation at 95°C for 30 s for 1 cycle, then denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 10 s for 40 cycles; the internal reference gene selected was β-actin; all experiments were performed with 3 technical replicates, and the relative expression levels of the genes were calculated using the 2 －△△Ct method, and the primers used were the primers in Table 3.

[0072] The results were as Figure 3 shown. Compared with the sh-NC group, lncRNA MSTRG.12349.1 was significantly down-regulated in the lncRNA MSTRG.12349.1-sh1 and lncRNA MSTRG.12349.1-sh2 groups, and the interference efficiency of sh1 was the best. Therefore, the lncRNA MSTRG.12349.1-sh1 interfering fibroblast cell line was selected for subsequent research.

[0073] Example 3: Effects of lncRNA MSTRG.12349.1-sh1 interference on the development of secondary hair follicles.

[0074] 1. Effects of lncRNA MSTRG.12349.1-shRNA on the proliferation of fibroblasts

[0075] The cell proliferation of the lncRNA MSTRG.12349.1-sh1 fibroblast cell line, the blank control fibroblast cell line, and the interference control fibroblast cell line was detected by the Cell Counting Kit-8 (CCK8) assay. The cells were seeded in 96-well plates, 10 μL of CCK8 solution was added to each well, and the plates were incubated in an incubator for 3 h. The absorbance of each well was measured at 450 nm. The results of different groups were compared using a two-tailed T-test, and the data were expressed as the mean ± standard deviation. Graphpad Prism 9.0 was used to visualize the data.

[0076] The results are as Figure 4 shown. Compared with the NC group and the sh-NC group, the proliferation ability of fibroblasts was significantly enhanced after the interference of lncRNA MSTRG.12349.1, indicating that lncRNA MSTRG.12349.1 inhibited the proliferation ability of fibroblasts, and its inhibitor lncRNA MSTRG.12349.1-sh1 could effectively improve the proliferation ability of fibroblasts.

[0077] 2. Effect of lncRNA MSTRG.12349.1-shRNA on the cell cycle of fibroblasts

[0078] The cell cycle was determined by DNA content quantification. The cells of the lncRNA MSTRG.12349.1-sh1 fibroblast cell line, the blank control fibroblast cell line, and the interference control fibroblast cell line were collected and a single-cell suspension with a cell concentration of 1×10 6 / mL was prepared. 1 mL of the single-cell suspension was taken for centrifugation, and the supernatant was discarded. 70% pre-cooled ethanol was added to the cells, the cells were resuspended and placed in a refrigerator at 4°C for fixation. After 24 h of fixation, the cells were centrifuged and the fixative was washed off with PBS. 100 μL of RNase A solution was added to the cell pellet, the cells were resuspended, and incubated in a water bath at 37°C for 30 min. Subsequently, 400 μL of propidium iodide staining solution was added and mixed well, and incubated in the dark at 4°C for 30 min. Flow cytometry was used to detect the red fluorescence at an excitation wavelength of 488 nm.

[0079] The results are as Figure 5 shown. Figure 5 As shown in A of Figure 5 it, in the NC group fibroblasts, the G1 phase accounted for 69% of the cells, the S phase accounted for 13.3% of the cells, and the G2 / M phase accounted for 16.7% of the cells; Figure 5As shown in C, in fibroblasts transfected with lncRNA MSTRG.2684.1-sh1, cells in G1 phase accounted for 43.9% of the total cells, cells in S phase accounted for 27.8%, and cells in G2 / M phase accounted for 27%. It can be seen that compared with the sh-NC group, lncRNA MSTRG.12349.1-sh1 significantly increased the number of cells in S phase, while the proportion of cells in G1 phase was significantly decreased, indicating that lncRNA MSTRG.12349.1-sh1 can effectively increase the proportion of cells in S phase, thereby promoting cell proliferation.

[0080] In summary, as a class of non-coding RNAs, lncRNAs positively regulate the development of secondary hair follicles during the embryonic period of cashmere goats. Through high-throughput sequencing and morphological analysis of secondary hair follicle development at different embryonic stages, 158 lncRNAs related to secondary hair follicle development were screened in this invention. Among them, lncRNA MSTRG.12349.1 was significantly down-regulated at 75 days during secondary hair follicle development. Therefore, an inhibitor of lncRNA MSTRG.12349.1 was synthesized and its effect on fibroblasts, the key cells for hair follicle development, was detected. The study found that lncRNA MSTRG.12349.1 significantly affected the proliferation ability of fibroblasts, and its inhibitor lncRNA MSTRG.12349.1-sh1 could effectively improve the proliferation ability of fibroblasts by increasing the proportion of cells in S phase of the cell cycle, and promote the development of secondary hair follicles during the embryonic period of cashmere goats.

[0081] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0082] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. Application of lncRNA inhibitor in the preparation of cashmere goat fibroblast proliferation promoter, characterized in that: The lncRNA is lncRNA MSTRG.12349.1, and the lncRNA inhibitor has the interference sequence of lncRNA as the only effective ingredient, and the interference sequence is lncRNA MSTRG.12349.1-sh1; The positive strand of the lncRNA MSTRG.12349.1-sh1 is shown in SEQ ID NO.5; The antisense strand of the lncRNA MSTRG.12349.1-sh1 is shown in SEQ ID NO.

6.

2. The interfering molecule of lncRNA is characterized by: The interfering molecule is the lncRNAMSTRG.12349.1-sh1 described in claim 1.

3. Interference plasmid, characterized in that, The interfering plasmid is obtained by connecting the interfering sequence described in claim 2 with the pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector.

4. A lentiviral resuspension, characterized in that: The lentiviral resuspension contains the interfering plasmid according to claim 3.

5. The lentiviral resuspension according to claim 4, characterized in that The lentiviral resuspension is obtained by co-transfecting the auxiliary plasmid and the interference plasmid into cells using a three-plasmid lentiviral system to achieve lentiviral packaging.

6. Application of lncRNA MSTRG.12349.1 gene inhibitor in the preparation of cashmere goat fibroblast proliferation promoter, characterized in that: The only active ingredient of the gene inhibitor is the interference sequence of the lncRNA according to claim 2, the interference plasmid according to claim 3 or the lentivirus resuspension according to claim 4.

7. Use of the lncRNA MSTRG.12349.1 gene inhibitor according to claim 6 in the preparation of a cashmere goat fibroblast proliferation promoter, characterized in that: The cashmere goat fibroblasts are fibroblasts from the embryonic stage of cashmere goats.

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