Interference sequence of lncRNA and application thereof

Abstract

The invention relates to the technical field of gene engineering, and particularly discloses an interference sequence of lncRNA and application of the interference sequence, the lncRNA is lncRNA MSTRG.2684.1, and the interference sequence is lncRNA MSTRG.2684.1-sh3. According to the interference sequence provided by the invention, the proliferation capacity of fibroblasts is effectively improved by increasing the proportion of cells in the S period of a cell cycle, and generation and development of secondary hair follicles in the embryonic period of the down producing goat are promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to an interference sequence of lncRNA and an application thereof. Background Art

[0002] Cashmere goats are livestock with multiple uses. The cashmere they produce is famous at home and abroad for its whiteness, softness, fineness and good luster. The quality and output of cashmere in my country are both the highest in the world. The cashmere produced by cashmere goats in Inner Mongolia Autonomous Region is soft to the touch and has a good luster. It is a high-grade textile raw material with high economic value.

[0003] Hair follicles determine the production and quality of cashmere. They are tiny organs attached to the skin and can be divided into primary follicles for growing coarse hair and secondary follicles for growing down hair. Hair follicles develop from the interaction between epithelial cells and fibroblasts. The most notable characteristic is regeneration. The occurrence of their morphology 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 in the embryonic period, mainly relying on a series of signal transductions between the dermis and the epidermis, inducing the orderly proliferation and differentiation of epithelial cells and fibroblast groups. During the embryonic period, fibroblasts, under the induction of "important genetic factors", continue to proliferate and differentiate, and eventually form structures such as hair papilla and dermal sheath; hair papilla continuously stimulates the proliferation and differentiation of epithelial cells, promoting epithelial cells to send feedback signals to adjacent mesenchymal cells to regulate the growth of hair follicles, thereby forming different structures of hair follicles. The results of the study showed that the morphogenesis of primary and secondary follicles of cashmere goats is initiated in the fetal period, and the morphogenesis of primary follicles is earlier than that of secondary follicles. At 45 days of gestation, the epidermal structure is basically formed, but the primary hair follicles have not yet formed, and the keratinized cells are neatly arranged in the basal layer of the epidermis. The primary hair follicles begin to form at 55 days of gestational age, and the development is basically completed at 135 days of gestational age; the secondary hair follicles begin to form at 75 days of gestational age, and the development is basically completed 6 months after birth.

[0004] Cashmere is produced by the secondary hair follicles of cashmere goats. The characteristics of cashmere goat skin hair follicles have a direct and important impact on the yield and quality of cashmere. 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 dermal aggregates further form hair papilla cells through continuous aggregation and differentiation. The hair papilla is composed of hair papilla cells. This structure is an inductive structure that sends and receives signals and plays a leading role in the development and cyclical growth of hair follicles. Therefore, the development of a product that promotes the proliferation of fibroblasts is of great significance to the study of the development of secondary hair follicles in cashmere goats. Summary of the invention

[0005] In order to obtain a product that promotes the proliferation of fibroblasts, the present invention provides an interference sequence of lncRNA and an application thereof. 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, thereby promoting the occurrence and development of secondary hair follicles in the embryonic stage of cashmere goats.

[0006] The present invention provides an interference sequence of lncRNA, wherein the lncRNA is lncRNA MSTRG.2684.1, and the interference sequence 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.

[0007] The interference 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, thereby promoting the occurrence and development of secondary hair follicles in the embryonic stage of cashmere goats.

[0008] The present invention also provides an interference plasmid, which is obtained by connecting the interference sequence with a pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector.

[0009] The pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector was purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.

[0010] The present invention also provides a lentiviral resuspension, wherein the lentiviral resuspension comprises the interfering plasmid.

[0011] Furthermore, 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.

[0012] The present invention also provides the use of a lncRNA MSTRG.2684.1 gene inhibitor in the preparation of a fibroblast proliferation promoter, wherein the only active ingredient of the gene inhibitor is the interference sequence of the lncRNA or the interference plasmid.

[0013] Furthermore, the fibroblasts are fibroblasts from the embryonic stage of cashmere goats.

[0014] The present invention also provides the use of a lncRNA MSTRG.2684.1 gene inhibitor in the preparation of a cashmere goat secondary hair follicle development promoter, wherein the only active ingredient of the gene inhibitor is the interference sequence of the lncRNA or the interference plasmid.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention identifies a lncRNA MSTRG.2684.1 that is related to the occurrence and development of secondary hair follicles in the embryonic stage of cashmere goats, and designs an interference sequence of lncRNA MSTRG.2684.1, lncRNA MSTRG.2684.1-sh3. The interference 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, thereby promoting the occurrence and development of secondary hair follicles in the embryonic stage of cashmere goats. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is the screening of lncRNA related to the development of secondary hair follicles in the present invention.

[0018] Figure 2 This is a fluorescence image of the fibroblast cell line interfering with lncRNA MSTRG.2684.1-sh3 in the present invention.

[0019] Figure 3 is the relative expression amount of lncRNAMSTRG.2684.1 in the fibroblast cell lines interfered with by different lncRNA MSTRG.2684.1 interference sequences in the present invention; wherein, sh-NC is a fibroblast cell line transfected with an empty vector, sh1 is a fibroblast cell line transfected with a lncRNAMSTRG.2684.1-sh1 interference vector, sh2 is a fibroblast cell line transfected with a lncRNA MSTRG.2684.1-sh2 interference vector, and sh3 is a fibroblast cell line transfected with a lncRNA MSTRG.2684.1-sh3 interference vector; ** represents p < 0.01, and the difference is significant; **** represents p < 0.0001, and the difference is extremely significant.

[0020] Figure 4This is the proliferation of fibroblasts after lncRNA MSTRG.2684.1-sh3 interference.

[0021] Figure 5 This is the cell cycle status of fibroblasts after lncRNA MSTRG.2684.1-sh3 interference; In the figure, A is the percentage of cells in different cell cycles in NC group fibroblasts; B is the percentage of cells in different cell cycles in sh-NC group fibroblasts; C is the percentage of cells in different cell cycles in the fibroblasts composed of lncRNA MSTRG.2684.1-sh3; D is the statistical percentage of fibroblasts in different cell cycles in the NC group, sh-NC group and lncRNA MSTRG.2684.1-sh3 group.

[0022] Figure 6 It is the fragment of 0 bp to 3780 bp in the full-length sequence of lncRNA MSTRG.2684.1 of the present invention.

[0023] Figure 7 It is the fragment of 3781 bp to 7680 bp in the full-length sequence of lncRNA MSTRG.2684.1 of the present invention.

[0024] Figure 8 It is the fragment of 7681 bp to 11700 bp in the full-length sequence of lncRNA MSTRG.2684.1 of the present invention.

[0025] Fig. 9 It is the fragment of 11701 bp to 15000 bp in the full-length sequence of lncRNA MSTRG.2684.1 of the present invention.

[0026] Fig.10 It is the fragment of 15001 bp to 16849 bp in the full-length sequence of lncRNA MSTRG.2684.1 of the present invention.

[0027] The full-length sequence of lncRNA MSTRG.2684.1 is Figure 6 to Figure 10 The sequences in are connected in sequence. DETAILED DESCRIPTION

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0029] Example 1: Screening of lncRNAs in the development of secondary hair follicles in cashmere goats.

[0030] 1. Collection of skin from the side of the cashmere goat Twelve 3-year-old ewes with good production performance and the same growth environment were selected for estrus synchronization, and skin tissue samples from the fetus were collected at 45, 55, 65 and 75 days of embryo, 3 in each period, a total of 12 fetal skin samples. After collection, they were numbered and quickly placed in liquid nitrogen for quick freezing.

[0031] The experimental sheep breed is Inner Mongolia Albas cashmere goat.

[0032] 2. RNA extraction and quality inspection Trizol reagent method was used to extract total RNA from the lateral skin samples of cashmere goat embryos at 45, 55, 65 and 75 days. NanoDrop ND-1000 and Agilent 2100 were used to detect RNA purity and integrity. When OD values ​​260 / 230 ≥ 1.5, 260 / 280 ≥ 1.8 and RIN ≥ 7, the RNA quality inspection was qualified and subsequent sequencing could be carried out.

[0033] 3. Whole genome transcriptome sequencing of the lateral skin of cashmere goats during embryonic stage Utilizing Ribo-Zero TM The total RNA of 12 samples was treated with rRNA Removal Kit to remove rRNA, and the remaining RNA was reverse transcribed to form the final product cDNA. Finally, paired-end sequencing was performed on Illumina Hiseq 4000. A total of 1 063 299 566 raw data were obtained from 12 sequencing databases. Cutadpter was used to remove sequencing adapters and low-quality data, and 1 023 889 360 valid data were obtained. Subsequently, Hisat was used to align the processed valid data with the reference genome. The percentage of reads aligned to the reference genome accounted for more than 94% of the valid reads, the percentage of reads aligned to a unique position accounted for more than 77% of the valid reads, and the percentage of reads aligned to multiple positions accounted for more than 17% of the valid reads.

[0034] 4. lncRNA identification and differential expression analysis Based on the characteristics of lncRNA that it cannot encode proteins and has long transcripts, we eliminated transcripts that did not meet the characteristics of lncRNA. Subsequently, we used CNCI and CPC software to predict the coding ability of the remaining transcripts and filter out transcripts that may encode proteins. After a series of screening, a total of 6,630 lncRNA transcripts were finally identified.

[0035] 5. Differential Expression Analysis To further explore the regulatory role of lncRNA in the early development of hair follicles in Inner Mongolia cashmere goats, we divided the four periods into six comparison groups and used String Tie to calculate the FPKM value, i.e., the expression level of the gene. This calculation method can eliminate the effects of sequencing depth, gene length, and differences between samples on gene expression. EdgeR was used to perform differential expression analysis on the genes identified in the four different embryonic stages, with the difference multiple |log2foldchange|≥1 and significant P-value≤0.05 as screening conditions.

[0036] 6. Preliminary screening of genes related to the development of secondary hair follicles According to the characteristics of the development period of primary and secondary hair follicles in cashmere goats: 45 days: primary and secondary hair follicles did not occur; 55 days, primary hair follicles began to form; 65 days, primary hair follicles continued to grow downward; 75 days, the hair buds of primary hair follicles penetrated into the dermis, and secondary hair follicles began to occur. The three comparison groups of d55vsd45, d65vsd45, and d65vsd55 in the sequencing results were used as Stage A, and the three comparison groups of d75vsd45, d75vsd55, and d75vsd65 were used as Stage B. The intersection of Stage A and Stage B was taken, and the 158 lncRNAs after the intersection was removed from Stage B were considered to be related to the development process of secondary hair follicles. Figure 1 .

[0037] 8. Determination of lncRNAs in the development of secondary hair follicles Combined with the morphological changes of secondary hair follicles in cashmere goats at different embryonic stages, the present invention screened out 158 ​​lncRNAs related to the development of secondary hair follicles in cashmere goats by using differential expression analysis. Among them, lncRNA MSTRG.2684.1 was significantly less expressed than other lncRNAs in the critical period of 75 days when secondary hair follicles began to develop. Therefore, lncRNA MSTRG.2684.1 was finally selected as the lncRNA related to the development of secondary hair follicles.

[0038] The full-length sequence of lncRNA MSTRG.2684.1 was Figure 6-Figure 10 The sequences in the sequence are spliced ​​in sequence, with a length of 16849bp. The full-length sequence of lncRNA MSTRG.2684.1 comes from the SRA database, which is obtained by performing data quality assessment on the basis of the original transcriptome sequencing data, aligning with the reference genome, and then splicing and assembling the transcripts. The accession numbers in the SRA database are SRR13306938-SRR13306949.

[0039] Example 2: Construction and screening of lncRNA MSTRG.2684.1 interfering cell lines.

[0040] 1. Construction of lncRNA MSTRG.2684.1-shRNA vector According to the sequence information of lncRNA MSTRG.2684.1, the target fragment was amplified and purified by PCR and PAGE purification, and finally three pairs of lncRNA MSTRG.2684.1 shRNA were obtained, which were named lncRNA MSTRG.2684.1-sh1, lncRNA MSTRG.2684.1-sh2 and lncRNA MSTRG.2684.1-sh3, and their 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 connected to the pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector by T4 ligation method to complete the construction of lncRNA MSTRG.2684.1 interference plasmid. The lncRNA MSTRG.2684.1 interference plasmid includes lncRNA MSTRG.2684.1-sh1 interference plasmid, lncRNA MSTRG.2684.1-sh2 interference plasmid and lncRNA MSTRG.2684.1-sh3 interference plasmid. Subsequently, the constructed lncRNA MSTRG.2684.1 interference plasmid was mixed with the transformation competent DH5a, the bacterial solution was coated and placed in an incubator for 16 hours; single clones were selected for colony verification, and positive clones were sequenced for verification; if the sequencing results were consistent with the target sequence, plasmid extraction and purification were performed. Among them, the lncRNA MSTRG.2684.1-shRNA synthesized by PCR amplification is a hairpin structure, which can be cut in cells to form siRNA, which 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.

[0041] The pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector was purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.

[0042]

[0043]

[0044] 2. Lentivirus packaging The three-plasmid lentiviral system was used to co-transfect the auxiliary plasmids psPAX2 and pMD2G and the above-constructed lncRNAMSTRG.2684.1 interference plasmid into 293T cells, respectively. The virus supernatant was collected at 48h and 72h after transfection, and the supernatant was taken 48h after transfection and added with new culture medium. After collection, it was filtered into a 40mL centrifuge tube with a 0.45μm filter and centrifuged at 4°C and 7200rpm for 120min. The virus precipitate was resuspended in 500μL of fresh culture medium without double antibodies to obtain a resuspension containing lentivirus, referred to as lentivirus resuspension, which was stored at -80°C. The fresh culture medium without double antibodies is: DMEM culture medium + 10% FBS serum.

[0045] 3. Culture of fibroblasts from cashmere goat embryos The embryonic cashmere goat skin tissue was cultured using tissue block culture method, and the primary cells were purified and cultured using enzyme digestion method, and finally the embryonic cashmere goat fibroblasts were obtained. The details are as follows:

[0046] Tissue block culture experiment: (1) Collect skin tissue from the embryonic stage of cashmere goats and store it in PBS liquid at low temperature for subsequent cell culture. (2) The environment around the test bench is disinfected by spraying with alcohol. The collected samples are disinfected with 75% alcohol for about 30 seconds and washed with PBS three times. (3) Use small scissors after high pressure to cut the tissue sample into pieces until it is like meat paste. Use forceps after high pressure sterilization to inoculate it into a cell culture bottle, and invert the culture bottle for about 4 minutes. (4) Turn the culture bottle over so that the side with the tissue block faces up and place it in an incubator at 37°C and 5% CO2 for about 30 minutes. (5) Take out the inverted cell culture bottle and add complete culture medium. (6) Replace the cell culture liquid according to the crawling of cells from the skin tissue in the bottle. Usually, it only needs to be replaced once in the first week, and then every 4 days.

[0047] Enzyme digestion experiment: (1) When the primary cells grow to confluence, remove the tissue blocks and cell culture medium in the culture flask and wash with PBS three times. (2) Add 2mL of trypsin containing EDTA and phenol red to the cell culture flask and digest for about 3 minutes in an incubator at 37℃ and 5% CO2; gradually separate and purify different cell lines according to the different tolerance of different cells to trypsin. Since fibroblasts have the worst tolerance to trypsin, fibroblasts can completely float in the digestion solution in about 3 minutes. (3) Add an equal amount of complete culture medium to the culture flask to stop digestion. (4) Aspirate the liquid containing fibroblasts in the culture flask and transfer it to a 15mL centrifuge tube and centrifuge at 1500rpm for 3 minutes. (5) Discard the supernatant and retain the fibroblast pellet. (6) Add 1mL of complete culture medium to the centrifuge tube and gently blow until the cell pellet at the bottom is no longer visible to the naked eye. (7) Aspirate the culture medium containing fibroblasts in the centrifuge tube into a new culture flask and add complete culture medium to 3mL in the culture flask. (8) Place the culture flask containing fibroblasts in an incubator at 37°C and 5% CO2 for cultivation. Fibroblasts can be obtained after about 3 days of cultivation. The entire process is performed on a sterilized clean bench, and all equipment used has been sterilized or autoclaved. The purified fibroblasts are spindle-shaped.

[0048] 4. Construction of lncRNA MSTRG.2684.1 interference cell line Digest the fibroblasts with trypsin and adjust the cell concentration to 2 × 10 4 Cells were cultured in 24-well plates, and the medium was changed every 2 days. Before transfection, 5 μg / mL of Polybrene dilution was added to the fibroblasts for 4 h; then, the original culture medium was discarded, and the culture medium containing 1 / 2 of the required volume and lentivirus resuspension was added for culture. After 4 h of transfection, the required culture medium was replenished, and the multiplicity of infection of each experimental group was 5. After 24 h of transfection, the culture medium containing lentivirus resuspension was discarded, replaced with fresh culture medium, and continued to be cultured in a 37°C, 5% CO2 incubator. 72 hours after transfection, the transfection situation was observed under a fluorescence microscope, and Puromycin was added to each test group for resistance screening at a final concentration of 5 μg / mL, thereby obtaining lncRNA MSTRG.2684.1 interference cell lines, including lncRNA MSTRG.2684.1-sh1 interference cell line, abbreviated as sh1, lncRNA MSTRG.2684.1-sh2 interference cell line, abbreviated as sh2, and lncRNA MSTRG.2684.1-sh3 interference cell line, abbreviated as sh3.

[0049] Figure 2This is 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-shaped morphology, indicating that the lncRNA MSTRG.2684.1-sh3 interference fibroblast cell line was successfully constructed.

[0050] 5. Screening of lncRNA MSTRG.2684.1 interference cell lines: Trizol reagent method was used to extract 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. Primers were designed according to the sequence information of lncRNA MSTRG.2684.1. The primer information is shown in Table 3. Real-time fluorescence quantitative PCR was used to detect the expression changes of lncRNA MSTRG.2684.1 after interference in each cell line.

[0051]

[0052] The specific steps of real-time fluorescence quantitative PCR are as follows: the total RNA extracted from different interference cell lines was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit with gDNAEraser kit; then, the real-time fluorescence quantitative PCR was performed on the LightCycler®96 Real-Time PCR platform using the TBGreen Premix Ex Taq Ⅱ kit. The experimental conditions were 95°C pre-denaturation for 30 seconds, 1 cycle, then 95°C denaturation for 10 seconds, 60°C annealing for 30 seconds, and 72°C extension for 10 seconds, for 40 cycles; the internal reference gene was β-actin; all experiments were repeated 3 times, using 2 －△△Ct Methods The relative expression of genes was calculated using the primers listed in Table 3.

[0053] The results are as follows Figure 3 As shown in the figure, compared with the sh-NC group, lncRNA MSTRG.2684.1 was significantly down-expressed in the lncRNA MSTRG.2684.1-sh1, lncRNA MSTRG.2684.1-sh2, and lncRNA MSTRG.2684.1-sh3 groups, among which sh3 had the best interference efficiency. Therefore, lncRNA MSTRG.2684.1-sh3 was selected to interfere with the fibroblast cell line for subsequent studies.

[0054] Example 3: Effect of lncRNA MSTRG.2684.1-sh3 interference on the development of secondary hair follicles.

[0055] 1. Effect of lncRNA MSTRG.2684.1-shRNA on fibroblast proliferation 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 CCK8 assay. The cells were seeded in 96-well plates, 10 μL CCK8 solution was added to each well, incubated in an incubator for 1-4h and the absorbance of each well was measured at 450nm. The results of different groups were compared using a two-tailed T test, and the data were expressed as mean ± standard deviation. Graphpad Prism 9.0 was used to visualize the data.

[0056] The results are as follows Figure 4 As shown, compared with the NC group and sh-NC group, the proliferation ability of fibroblasts was significantly enhanced after lncRNA MSTRG.2684.1 interference, indicating that lncRNA MSTRG.2684.1 inhibits the proliferation ability of fibroblasts, and its inhibitor lncRNA MSTRG.2684.1-sh3 can effectively improve the proliferation ability of fibroblasts.

[0057] 2. Effect of lncRNA MSTRG.2684.1-shRNA on fibroblast cell cycle The cell cycle was determined by DNA content quantification. 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 with a cell concentration of 1×10 6 / mL single cell suspension, take 1mL of single cell suspension for centrifugation and discard the supernatant. Add 70% pre-cooled ethanol to the cells, resuspend the cells and place them in a 4℃ refrigerator for fixation. After fixation for 24 hours, centrifuge and wash the fixative with PBS; add 100 μL RNase A solution to the cell pellet, resuspend the cells, and bathe at 37℃ for 30 minutes. Subsequently, add 400 μL of propidium iodide staining solution and mix thoroughly, and incubate at 4℃ in the dark for 30 minutes. Flow cytometry detects red fluorescence at an excitation wavelength of 488nm.

[0058] The results are as follows Figure 5 As shown, Figure 5 As shown in A, in NC group fibroblasts, G1 phase accounted for 66% of cells, S phase accounted for 12.2% of cells, and G2 / M phase accounted for 21.5% of cells; Figure 5 As shown in B, in the sh-NC group of fibroblasts, the G1 phase accounted for 62.3% of the cells, the S phase accounted for 14.7% of the cells, and the G2 / M phase accounted for 24% of the cells; Figure 5Figure C shows that in the lncRNA MSTRG.2684.1-sh3 group of fibroblasts, 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 S phase cells, while the proportion of G1 phase cells was significantly reduced; indicating that lncRNA MSTRG.2684.1-sh3 can effectively increase the proportion of S phase cells, thereby promoting cell proliferation.

[0059] In summary, lncRNA, as a type of non-coding RNA, actively regulates the occurrence and development of secondary hair follicles in the embryonic stage of cashmere goats. Through high-throughput sequencing and morphological analysis of the occurrence and development of secondary hair follicles in different embryonic stages, we screened a total of 158 lncRNAs related to the occurrence and development of secondary hair follicles, among which lncRNA MSTRG.2684.1 was significantly low-expressed at 75 days when secondary hair follicles occurred. Therefore, the lncRNA MSTRG.2684.1 inhibitor 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 of fibroblasts, and its inhibitor lncRNA MSTRG.2684.1-sh3 could effectively improve the proliferation of fibroblasts by increasing the proportion of cells in the S phase of the cell cycle, thereby promoting the occurrence and development of secondary hair follicles in the embryonic stage of cashmere goats.

[0060] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0061] 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 equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. The interference sequence of lncRNA is characterized by: The lncRNA is lncRNA MSTRG.2684.1, and the interference sequence 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 sequence 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. Use of a lncRNA MSTRG.2684.1 gene inhibitor in the preparation of a fibroblast proliferation promoter, characterized in that: The only active ingredient of the gene inhibitor is the interference sequence of the lncRNA according to claim 1 or the interference plasmid according to claim 2.

6. Use of the lncRNA MSTRG.2684.1 gene inhibitor according to claim 5 in the preparation of a fibroblast proliferation promoter, characterized in that: The fibroblasts are fibroblasts from the embryonic stage of cashmere goats.

7. Application of lncRNA MSTRG.2684.1 gene inhibitor in the preparation of a promoter for the development of secondary hair follicles in cashmere goats, characterized in that: The only active ingredient of the gene inhibitor is the interference sequence of the lncRNA according to claim 1 or the interference plasmid according to claim 2.

Images