Interfering sequences of lncRNA and their applications

Abstract

The present invention relates to the field of gene engineering technology, and specifically discloses an interference sequence of lncRNA and an application thereof, wherein the lncRNA is lncRNA MSTRG.2684.1, and the interference sequence is lncRNA MSTRG.2684.1-sh3. The interference sequence 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 stage of cashmere goats.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly relates to interfering sequences of lncRNA and their applications. Background Art

[0002] Cashmere goats are livestock with multiple uses. The cashmere produced is well-known at home and abroad for its whiteness, softness, fineness, and good luster. The quality and output of cashmere in China rank first in the world. The cashmere produced by Inner Mongolia cashmere goats has a soft touch and good luster, and is a high-grade textile raw material with high economic value.

[0003] Hair follicles determine the output and quality of cashmere. They are tiny organs attached to the skin and can be divided into primary hair follicles that grow coarse hair and secondary hair follicles that grow fine hair. Hair follicles are developed through the interaction of epithelial cells and fibroblasts. The most remarkable characteristic is regeneration, and its morphogenesis during the fetal period involves a series of interactions between the epidermis and the dermis, and the process is relatively complex. Hair follicles are formed during the embryonic period, mainly relying on a series of signal transmissions that occur between the dermis and the epidermis, inducing the orderly proliferation and differentiation of epithelial cells and fibroblast populations. During the embryonic period, fibroblasts, under the induction of "important genetic factors", finally form structures such as dermal papillae and dermal sheaths through continuous proliferation and differentiation; the dermal papilla stimulates the proliferation and differentiation of epithelial cells, promotes epithelial cells to send feedback signals to adjacent mesenchymal cells to regulate the growth of hair follicles, thereby forming different structures of hair follicles. Research results show that the morphogenesis of primary and secondary hair follicles in cashmere goats both starts during the fetal period, and the morphogenesis of primary hair follicles is earlier than that of secondary hair follicles. At 45 days of gestation in the fetus, the epidermal structure is basically formed, but the primary hair follicles have not yet formed, and keratinized cells are neatly arranged in the basal layer of the epidermis. The primary hair follicles start to occur at 55 days of gestation and are basically developed at 135 days of gestation; the secondary hair follicles start to occur at 75 days of gestation and are basically developed 6 months after birth.

[0004] Cashmere is produced by the secondary hair follicles of cashmere goats. The traits of skin hair follicles in cashmere goats have a direct and important impact on the output and quality of fine hair. The key cells for hair follicle development are fibroblasts. The development of hair follicles is a process of continuous proliferation and differentiation of epithelial cells and fibroblasts. Fibroblasts form dermal aggregates through continuous proliferation and differentiation, and the dermal aggregates further form dermal papilla cells through continuous aggregation and differentiation. The dermal papilla is composed of dermal papilla cells, and this structure is an inductive structure for sending and receiving signals, playing a leading role in the development and periodic growth of hair follicles. Therefore, developing a product that promotes the proliferation of fibroblasts is of great significance for studying the development of secondary hair follicles in cashmere goats. Summary of the Invention

[0005] To obtain a product that promotes the proliferation of fibroblasts, the present invention provides an interfering sequence of lncRNA and its application. The interfering sequence 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.

[0006] The present invention provides an interfering sequence of lncRNA. The lncRNA is lncRNA MSTRG.2684.1, and the interfering sequence is lncRNA MSTRG.2684.1-sh3;

[0007] The sense strand of the lncRNA MSTRG.2684.1-sh3 is as shown in SEQ ID NO.9;

[0008] The antisense strand of the lncRNA MSTRG.2684.1-sh3 is as shown in SEQ ID NO.10.

[0009] The interfering sequence lncRNA MSTRG.2684.1-sh3 provided by the present invention can inhibit the expression of lncRNA MSTRG.2684.1, and effectively improve the proliferation ability of fibroblasts by increasing the proportion of cells in the S phase of the cell cycle, and promote the occurrence and development of secondary hair follicles in the embryonic period of cashmere goats.

[0010] The present invention also provides an interfering plasmid, which is obtained by ligating the interfering sequence with the pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector.

[0011] The pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector is purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.

[0012] The present invention also provides a lentivirus resuspension solution, which contains the interfering plasmid.

[0013] Furthermore, the lentivirus resuspension solution is obtained by packaging lentivirus by co-transfecting an auxiliary plasmid and the interfering plasmid into cells using a three-plasmid lentivirus system.

[0014] The present invention also provides the application of an lncRNA MSTRG.2684.1 gene inhibitor in the preparation of a fibroblast proliferation promoter. The only effective ingredient of the gene inhibitor is the interfering sequence of the lncRNA or the interfering plasmid.

[0015] Furthermore, the fibroblasts are fibroblasts in the embryonic period of cashmere goats.

[0016] The present invention also provides an application of an inhibitor of the lncRNA MSTRG.2684.1 gene in the preparation of a promoter for the development of secondary hair follicles in cashmere goats, wherein the only active ingredient of the gene inhibitor is the interfering sequence of the lncRNA or the interfering plasmid.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. The present invention identifies an lncRNA MSTRG.2684.1 related to the development of secondary hair follicles in cashmere goats during the embryonic period, and designs and obtains an interfering sequence lncRNA MSTRG.2684.1-sh3 of lncRNA MSTRG.2684.1. The interfering sequence of lncRNA MSTRG.2684.1 effectively improves the proliferation ability of fibroblasts by increasing the proportion of cells in the S phase of the cell cycle, and promotes the development of secondary hair follicles in cashmere goats during the embryonic period. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1 Screening of lncRNAs related to the development of secondary hair follicles in the present invention.

[0021] Figure 2 Fluorescence diagram of lncRNA MSTRG.2684.1-sh3 interfering with fibroblast cell line in the present invention.

[0022] Figure 3 Relative expression levels of lncRNA MSTRG.2684.1 in fibroblast cell lines interfered with different interfering sequences of lncRNA MSTRG.2684.1 in the present invention; 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.2684.1-sh1 interfering vector, sh2 is the fibroblast cell line transfected with the lncRNA MSTRG.2684.1-sh2 interfering vector, and sh3 is the fibroblast cell line transfected with the lncRNA MSTRG.2684.1-sh3 interfering vector; ** represents p < 0.01, with significant difference; **** represents p < 0.0001, with extremely significant difference.

[0023] Figure 4It shows the proliferation of fibroblasts after interference with lncRNA MSTRG.2684.1-sh3.

[0024] Figure 5 It shows the cell cycle of fibroblasts after interference with lncRNA MSTRG.2684.1-sh3;

[0025] In the figure, A shows the proportion of cells in different cell cycles in NC group fibroblasts;

[0026] B shows the proportion of cells in different cell cycles in sh-NC group fibroblasts;

[0027] C shows the proportion of cells in different cell cycles in lncRNA MSTRG.2684.1-sh3 group fibroblasts;

[0028] D shows the statistical results of the proportion of fibroblasts in different cell cycles in NC group, sh-NC group and lncRNA MSTRG.2684.1-sh3 group.

[0029] Figure 6 It is the fragment from the 0bp to 3780bp of the full-length sequence of lncRNA MSTRG.2684.1 of the present invention.

[0030] Figure 7 It is the fragment from the 3781bp to 7680bp of the full-length sequence of lncRNA MSTRG.2684.1 of the present invention.

[0031] Figure 8 It is the fragment from the 7681bp to 11700bp of the full-length sequence of lncRNA MSTRG.2684.1 of the present invention.

[0032] Figure 9 It is the fragment from the 11701bp to 15000bp of the full-length sequence of lncRNA MSTRG.2684.1 of the present invention.

[0033] Figure 10 It is the fragment from the 15001bp to 16849bp of the full-length sequence of lncRNA MSTRG.2684.1 of the present invention.

[0034] The full-length sequence of the said lncRNA MSTRG.2684.1 is Figures 6 to 10 formed by connecting the sequences in Detailed implementation mode

[0035] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the protection scope of the present invention is not limited by the specific embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention 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.

[0036] Example 1: Screening of lncRNAs during the development of secondary hair follicles in cashmere goats.

[0037] 1. Collection of cashmere goat body side skin

[0038] 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.

[0039] The experimental sheep breed was Inner Mongolia Albas cashmere goats.

[0040] 2. RNA extraction and quality inspection

[0041] 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 of 260 / 230 ≥ 1.5, 260 / 280 ≥ 1.8, and RIN ≥ 7, the RNA passed the quality inspection and could be used for subsequent sequencing.

[0042] 3. Whole-genome transcriptome sequencing of cashmere goat fetal body side skin during embryogenesis

[0043] Using Ribo-Zero TMThe rRNA Removal Kit was used to perform rRNA removal on the total RNA of 12 samples, 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. The sequencing adapters and low-quality data were removed using Cutadpter, resulting in 1,023,889,360 valid data. Subsequently, Hisat was used to align the processed valid data to the reference genome. The percentage of reads aligned to the reference genome among the valid reads was higher than 94%, the percentage of reads aligned to unique positions among the valid reads was higher than 77%, and the percentage of reads aligned to multiple positions among the valid reads was higher than 17%.

[0044] 4. lncRNA Identification and Differential Expression Analysis

[0045] According to the characteristics that lncRNAs cannot encode proteins and have long transcripts, we excluded transcripts that did not conform to the characteristics of lncRNAs. Subsequently, the coding ability of the remaining transcripts was predicted using two software programs, CNCI and CPC, and transcripts that might encode proteins were filtered out. After a series of screenings, a total of 6,630 lncRNA transcripts were finally identified.

[0046] 5. Differential Expression Analysis

[0047] To further explore the regulatory role of lncRNAs in the early development of Inner Mongolia cashmere goat hair follicles, we divided the 4 periods into 6 comparison groups, and used String Tie to calculate the FPKM value, that is, the gene expression level. This calculation method can eliminate the influence of sequencing depth, gene length, and differences between samples on gene expression levels; EdgeR was used to perform differential expression analysis on the genes identified in 4 different embryonic periods, with the screening conditions of differential fold |log2foldchange|≥1 and significant P-value≤0.05.

[0048] 6. Preliminary Screening of Genes Related to the Development of Secondary Hair Follicles

[0049] 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 used as Stage A, and the three comparison groups of d75vsd45, d75vsd55, and d75vsd65 are used as Stage B. The intersection of Stage A and Stage B is taken, and the 158 lncRNAs after removing the intersection part from Stage B are regarded as related to the development process of secondary hair follicles. For example Figure 1 。

[0050] 8. Determination of lncRNAs related to the development of secondary hair follicles

[0051] 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.2684.1 was significantly lowly expressed compared to other lncRNAs during the critical period of 75 days when secondary hair follicles began to occur. Therefore, lncRNA MSTRG.2684.1 was finally selected as the lncRNA related to the development of secondary hair follicles

[0052] The full-length sequence of lncRNA MSTRG.2684.1 is spliced in sequence from the sequences in Figures 6 - 10 with a length of 16,849 bp. The full-length sequence of lncRNA MSTRG.2684.1 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

[0053] Example 2: Construction and screening of lncRNA MSTRG.2684.1 interference cell lines

[0054] 1. Construction of lncRNA MSTRG.2684.1 - shRNA vector

[0055] According to the lncRNA MSTRG.2684.1 sequence information, the target fragment was amplified and purified by PCR and PAGE purification methods. Finally, 3 pairs of shRNAs of lncRNA MSTRG.2684.1 were obtained, which were named lncRNAMSTRG.2684.1-sh1, lncRNA MSTRG.2684.1-sh2, and lncRNA MSTRG.2684.1-sh3 respectively. The shRNA sequence information is shown in Table 1. The lncRNA MSTRG.2684.1-sh1, lncRNA MSTRG.2684.1-sh2, and lncRNA MSTRG.2684.1-sh3 fragments were respectively ligated to the pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector by T4 ligation method to complete the construction of lncRNA MSTRG.2684.1 interfering plasmids. The lncRNA MSTRG.2684.1 interfering plasmids include lncRNAMSTRG.2684.1-sh1 interfering plasmid, lncRNA MSTRG.2684.1-sh2 interfering plasmid, and lncRNA MSTRG.2684.1-sh3 interfering plasmid. Subsequently, the constructed lncRNA MSTRG.2684.1 interfering plasmids were mixed with the transformed competent DH5a, the bacterial solution was spread on the plate and cultured in the incubator for 16 h; single colonies were selected for colony verification, and 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.2684.1-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.2684.1 gene. The siRNA sequence information of lncRNA MSTRG.2684.1 is shown in Table 2.

[0056] The pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector was purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.

[0057]

[0058]

[0059] 2. Lentivirus packaging

[0060] Using a three-plasmid lentiviral system, the helper plasmids psPAX2 and pMD2G and the above-constructed lncRNA MSTRG.2684.1 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 double antibiotics to obtain a resuspended solution containing lentivirus, simply referred to as lentivirus resuspended solution, and stored at -80 °C. The fresh medium without double antibiotics was: DMEM medium + 10% FBS serum.

[0061] 3. Culture of fibroblast cells in the embryonic period of cashmere goats

[0062] 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 fibroblast cells in the embryonic period of cashmere goats. The specific steps are as follows:

[0063] 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) Use a small scissors after autoclaving to cut the tissue sample into pieces until it becomes a meat paste, inoculate it into a cell culture flask with autoclaved forceps, and invert the culture flask for about 4 min. (4) Flip the culture flask so that the side with the tissue block is facing 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 medium is changed every 4 days.

[0064] 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 poorest tolerance to trypsin, the fibroblasts can completely float in the digestive solution in 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 pellet. (6) Add 1 mL of complete culture medium to the centrifuge tube and gently pipette until no cell pellet 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 Cultivate in an incubator for about 3 days to obtain fibroblasts; the whole process is carried out on a sterilized laminar flow bench, and all the equipment used has been sterilized or autoclaved; the purified fibroblasts are spindle-shaped.

[0065] 4. Construction of lncRNA MSTRG.2684.1 interference cell line

[0066] Digest fibroblasts with trypsin and adjust the cell concentration to 2×10 4 cells / well in a 24-well plate and change the medium every 2 days. Before transfection, add 5 μg / mL of Polybrene dilution to the fibroblasts and incubate for 4 h; then, discard the original medium, add the medium with half the required volume and containing the lentivirus resuspension to culture. After 4 h of transfection, supplement the required medium, and the multiplicity of infection of each experimental group is 5. After 24 h 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 Cultivate in an incubator. After 72 h of transfection, observe the transfection situation 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.2684.1 interference cell line is obtained, including the lncRNA MSTRG.2684.1-sh1 interference cell line, abbreviated as sh1, the lncRNA MSTRG.2684.1-sh2 interference cell line, abbreviated as sh2, and the lncRNA MSTRG.2684.1-sh3 interference cell line, abbreviated as sh3.

[0067] Figure 2 The lncRNA MSTRG.2684.1-sh3 interference fibroblast cell line constructed by the present invention. In the figure, the cells grow densely and show an obvious spindle shape, indicating that the lncRNA MSTRG.2684.1-sh3 interference fibroblast cell line is successfully constructed.

[0068] 5. Screening of lncRNA MSTRG.2684.1-interfered cell lines: The total RNA of the constructed lncRNA MSTRG.2684.1-sh1 fibroblast cell line, lncRNA MSTRG.2684.1-sh2 fibroblast cell line, and lncRNA MSTRG.2684.1-sh3 fibroblast cell line was extracted by the Trizol reagent method. According to the sequence information of lncRNA MSTRG.2684.1, primers were designed, and the primer information is shown in Table 3. The expression changes of lncRNA MSTRG.2684.1 in each cell line after interference were detected by real-time fluorescence quantitative PCR.

[0069]

[0070] The specific steps of real-time fluorescence quantitative PCR were as follows: The total RNA of different interfered cell lines that had been 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 to perform 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 2-ΔΔCt method was used to calculate the relative expression level of the gene, and the primers used were the primers in Table 3. －△△Ct The results are as shown. Compared with the sh-NC group, lncRNA MSTRG.2684.1 was significantly downregulated in the lncRNA MSTRG.2684.1-sh1, lncRNA MSTRG.2684.1-sh2, and lncRNA MSTRG.2684.1-sh3 groups, and the interference efficiency of sh3 was the best. Therefore, the lncRNA MSTRG.2684.1-sh3 interfered fibroblast cell line was selected for subsequent research.

[0071] Results are as Figure 3 shown. Compared with the sh-NC group, lncRNA MSTRG.2684.1 was significantly downregulated in the lncRNA MSTRG.2684.1-sh1, lncRNA MSTRG.2684.1-sh2, and lncRNA MSTRG.2684.1-sh3 groups, and the interference efficiency of sh3 was the best. Therefore, the lncRNA MSTRG.2684.1-sh3 interfered fibroblast cell line was selected for subsequent research.

[0072] Example 3: Effects of lncRNA MSTRG.2684.1-sh3 interference on the development of secondary hair follicles.

[0073] 1. Effects of lncRNA MSTRG.2684.1-shRNA on the proliferation of fibroblasts

[0074] The cell proliferation of lncRNA MSTRG.2684.1-sh3 fibroblast cell line, blank control fibroblast cell line and interference control fibroblast cell line was detected by Cell Counting Kit-8 (CCK8) assay. Cells were seeded in 96-well plates, 10 μL of CCK8 solution was added to each well, incubated in an incubator for 1 - 4 h, and the absorbance of each well was measured at 450 nm. A two-tailed T-test was used to compare the results of different groups, and the data were expressed as mean ± standard deviation. Graphpad Prism 9.0 was used to visualize the data.

[0075] The results were as Figure 4 shown that compared with the NC group and sh-NC group, the proliferation ability of fibroblasts was significantly enhanced after lncRNA MSTRG.2684.1 interference; it was indicated that lncRNA MSTRG.2684.1 inhibited the proliferation ability of fibroblasts, and its inhibitor lncRNA MSTRG.2684.1-sh3 could effectively improve the proliferation ability of fibroblasts.

[0076] 2. Effect of lncRNA MSTRG.2684.1-shRNA on the cell cycle of fibroblasts

[0077] The cell cycle was determined by DNA content quantification method. Cells of lncRNA MSTRG.2684.1-sh3 fibroblast cell line, blank control fibroblast cell line and interference control fibroblast cell line were collected and prepared into single-cell suspensions with a cell concentration of 1×10 6 / mL. 1 mL of 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 4℃ refrigerator for fixation. After 24 h of fixation, the cells were centrifuged and the fixative was washed away with PBS; 100 μL of RNase A solution was added to the cell pellet, the cells were resuspended, and incubated in a 37℃ water bath for 30 min. Subsequently, 400 μL of propidium iodide staining solution was added and mixed well, and incubated in the dark at 4℃ for 30 min. Flow cytometry was used to detect the red fluorescence at an excitation wavelength of 488 nm.

[0078] The results were as Figure 5 shown that Figure 5 as shown in A of Figure 5 the NC group, G1 phase accounted for 66% of the cells, S phase accounted for 12.2% of the cells, and G2 / M phase accounted for 21.5% of the cells in fibroblasts; Figure 5As shown in C, in fibroblasts transfected with lncRNA MSTRG.2684.1-sh3, the G1 phase accounted for 48.9% of the cells, the S phase accounted for 23.7% of the cells, and the G2 / M phase accounted for 27.3% of the cells. It can be seen that compared with the NC group and the sh-NC group, lncRNA MSTRG.2684.1-sh3 significantly increased the number of cells in the S phase, while the proportion of cells in the G1 phase was significantly decreased; indicating that lncRNA MSTRG.2684.1-sh3 can effectively increase the proportion of cells in the S phase, thereby promoting cell proliferation.

[0079] In summary, as a class of non-coding RNAs, lncRNAs actively 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, we screened a total of 158 lncRNAs related to secondary hair follicle development, among which lncRNA MSTRG.2684.1 was significantly downregulated at 75 days during secondary hair follicle development. Therefore, an inhibitor of lncRNA MSTRG.2684.1 was synthesized and its effect on fibroblasts, the key cells for hair follicle development, was detected. The study found that lncRNA MSTRG.2684.1 significantly inhibited the proliferation ability of fibroblasts, and its inhibitor lncRNA MSTRG.2684.1-sh3 could effectively improve the proliferation ability of fibroblasts by increasing the proportion of cells in the S phase of the cell cycle, and promote the development of secondary hair follicles during the embryonic period of cashmere goats.

[0080] 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.

[0081] 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 is also intended to include these modifications and variations.

Claims

1. The interfering molecule of lncRNA is characterized by: The lncRNA is lncRNA MSTRG.2684.1, and the interfering molecule is lncRNA MSTRG.2684.1-sh3; The positive strand of the lncRNA MSTRG.2684.1-sh3 is shown in SEQ ID NO.9; The antisense strand of the lncRNA MSTRG.2684.1-sh3 is shown in SEQ ID NO.

10.

2. Interference plasmid, characterized in that, The interfering plasmid is obtained by connecting the interfering molecule described in claim 1 with the pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector.

3. A lentivirus resuspension, characterized in that: The lentiviral resuspension contains the interfering plasmid according to claim 2.

4. The lentivirus resuspension according to claim 3, 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.

5. Application of lncRNA MSTRG.2684.1 gene inhibitor in the preparation of cashmere goat fibroblast proliferation promoter, characterized in that: The only active ingredient of the gene inhibitor is the interfering molecule of the lncRNA according to claim 1 or the interfering plasmid according to claim 2.

6. Use of the lncRNAMSTRG.2684.1 gene inhibitor according to claim 5 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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