CN104498497A - miRNA related to milk goat mammary epithelial cell apoptosis and application thereof in breeding of milk goats - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, and provides miRNA related to milk goat mammary epithelial cell apoptosis and application thereof in breeding of milk goats. The small RNA molecule miR-143 is capable of inhibiting proliferation of mammary epithelial cells by delaying a cell cycle progress; by utilizing the characteristic, the inventor finds that miR-143 or precursor expression thereof can be inhibited through the genetic engineering technology; mammary epithelial cell apoptosis can be indirectly inhibited; therefore, the purposes of delaying mammary gland degradation, prolonging the lactation period and increasing the lactation amount of milk livestock can be achieved, and thus, the miRNA disclosed by the invention has high application value.

Description

A miRNA related to apoptosis of dairy goat mammary epithelial cells and its application in dairy goat breeding

technical field

The invention relates to the technical field of genetic engineering, and provides a miRNA related to apoptosis of dairy goat mammary gland epithelial cells and its application in dairy goat breeding.

Background technique

The process of mammary gland development, lactation and degeneration of dairy goats has a certain regularity, which involves mammary gland cell growth and development, proliferation and apoptosis, and death. Molecular means are now used to regulate the apoptosis process of mammary gland cells and delay the mammary gland degeneration process to prolong the lactation period of dairy goats, increase lactation volume, improve the genetic performance of dairy goat lactation, and provide new ideas and useful clues for the molecular improvement of Laoshan dairy goats , is the main direction of current technological development.

miRNA is a kind of regulatory small RNA molecule with a length of about 22nt (nucleotide), single-stranded, non-protein-coding, and highly conserved in evolution, which is widely involved in neurogenesis by regulating the expression of target genes. Development, cell differentiation, proliferation and apoptosis, fat metabolism (Xu et al, 2003), breast development and other physiological processes, and play an important role as tumor suppressor gene or oncogene.

The study of miRNA in animals was first discovered in C. elegans, which is called Lin-4, a heterochronous switch gene. Although this gene does not encode a protein, it can delay the developmental events of C. elegans. Later, researchers such as Reinhart discovered a second same heterochronous switch gene, Let-7, which can regulate the transition of nematode developmental stages. Later, researchers discovered that many miRNAs of the Let-7 family, such as miR-148, miR-84, and miR-241, were also involved in the regulation of the transition of nematode developmental stages.

With the continuous development of sequencing technology, especially with the development of second-generation sequencing technology in recent years, researchers can identify and discover some new miRNAs in large quantities, so that the development of miRNAs is increasing day by day, and the identified new miRNA has risen exponentially, and miRNA has rapidly become a research hotspot in the field of biology due to its unique regulation mode and expression characteristics.

Hormones, growth factors and some proteins play key roles in mammary gland development, lactation and regression. After the discovery of microRNA, its role in organ development has gradually attracted the attention of researchers. In 2010, scientists from Göttingen, Frankfurt and Hannover found that hormones and proteins are not the complete determinants of mammary gland development, and some small RNA molecules also play a key role in this process. The mice used in the experiment have All the hormones, growth factors and proteins necessary for mammary gland development, but the lack of miR-122 and miR-132 led to the complete failure of mouse mammary ducts to develop. microRNAs play an important role in organ development.

As a brand-new small RNA molecule, miR-143 has been reported to be able to inhibit the proliferation of cancer cells, which in turn can be used to inhibit the growth of tumors. However, due to the differences between cancer cells and normal cells, this effect Whether it can be applied to normal cells is still unknown, so far there is no report on the role of miR-143 in breast cell apoptosis and its breeding application.

Contents of the invention

The inventors of the present invention have provided a miRNA related to the apoptosis of dairy goat mammary gland epithelial cells and its application in dairy goat breeding, aiming at the situation of the above-mentioned prior art, the small RNA molecule miR-143 can delay cell cycle The process inhibits the proliferation of mammary epithelial cells. The inventors used this characteristic to find that inhibiting the expression of miR-143 or its precursors through genetic engineering technology can indirectly inhibit the apoptosis of mammary epithelial cells, so as to delay mammary gland degeneration, prolong lactation period, and improve milk production. It can be used to measure the milk production of livestock and has extremely high application value.

The inventor first provided a miRNA related to the apoptosis of dairy goat mammary gland epithelial cells, the miRNA is miR-143, its gene sequence is shown in SEQ ID NO.1, and its precursor gene sequence is shown in SEQ ID NO.2 Show.

In order to verify the relevant role of this miRNA, the inventors used MTT method, fluorescent Hoechst/PI double staining technique and flow cytometry to study the effect of miR-143 on the proliferation and apoptosis of breast epithelial cells. Cell cycle progression can inhibit cell proliferation and promote apoptosis; the specific process is as follows:

The inventors first transiently transfected miR-143 into primary mammary epithelial cells cultured in vitro, and used the MTT method to detect the results of cell proliferation. It was found that at 48h and 72h after transfection, the cell viability was significantly higher than the colorimetric value of the transfection group. At 48h, the difference between the two groups reached a very significant level (P<0.01), indicating that miR-143 inhibits the proliferation of mammary epithelial cells;

On the basis of this work, the inventors further verified the effect of miR-143 on breast epithelial cell apoptosis by fluorescent Hoechst/PI fluorescent double staining technique and flow cytometry, and detected the expression of apoptosis marker genes by qRT-PCR :

The fluorescent double-staining technique showed that the Hoechst33342-stained nuclei in the blank group were in a normal state, and the Hoechst33342-stained cells in the control group were also in a normal state, while the cell nuclei in the transfection group stained by Hoechst33342 were incomplete in shape under a fluorescent microscope and showed fragmented apoptosis. small body, showing obvious apoptosis;

Flow cytometry analysis found that miR-143 delayed the progress of the cell cycle by arresting the cell cycle in the G1 phase. In the control group, only the normal diploid cell DNA peak (G1 peak) was detected, and the G1 peak in the transfection group A small peak, the so-called apoptotic peak (Ap peak), appeared before, indicating that miR-143 promotes the apoptosis of mammary epithelial cells;

The results of qRT-PCR detection showed that compared with the control group, the expression level of the apoptosis gene BAX in the transfection group was significantly increased (P<0.01), and the expression level of BCL2 in the transfection group was lower, but the difference was not significant, further illustrating miR-143 promotes apoptosis of mammary epithelial cells.

Based on the above findings, the inventors further suppressed the expression of miR-143 in dairy goats through genetic engineering technology, which can indirectly inhibit the apoptosis of mammary epithelial cells, so as to delay mammary gland degeneration, prolong the lactation period, and increase the milk production of dairy livestock. High application value. In addition, the expression of the precursor sequence shown in SEQ ID NO.2 in dairy goats can also be suppressed to achieve the above-mentioned purpose.

In summary, the inventors of the present invention provided a miRNA related to dairy goat mammary epithelial cell apoptosis and its application in dairy goat breeding on the basis of sufficient experiments. The small RNA molecule miR-143 can be passed through Delaying the cell cycle progress inhibits the proliferation of mammary epithelial cells. The inventors used this characteristic to find that inhibiting the expression of miR-143 or its precursor in dairy goats through genetic engineering technology can indirectly inhibit the apoptosis of mammary epithelial cells, so as to delay mammary gland degeneration , prolong the lactation period, and increase the milk production of dairy animals, which has extremely high application value.

Description of drawings

Figure 1 is the grayscale image of the immunofluorescence identification results of primary mammary epithelial cells cultured in vitro,

The dark area in the lower right corner of the figure is the immunofluorescence identification of the marker protein keratin 18 of mammary gland epithelial cells; it can be seen that what we cultured in vitro is indeed mammary gland epithelial cells;

Figure 2 is a schematic diagram of the MTT method detection results of the effect of miR-143 on cell viability,

In the figure, A is the cell growth curve of the control group and the transfection group; B is the MTT detection result of the control group and the transfection group;

Figure A is the cell growth curve between the control group and the transfection group obtained by cell counting. The results show that after miR-143 transfection, the cell count is significantly reduced and the growth is inhibited; in the figure B is the growth curve between the control group and the transfection group measured by the MTT method. The results of the absorbance value of the cells in the transfection group showed that the absorbance value of the cells in the transfection group was significantly reduced when the cells were cultured for 48 hours; both parts of the results indicated that miR-143 had a certain inhibitory effect on the growth of breast epithelial cells cultured in vitro;

Figure 3 is a grayscale schematic diagram of the fluorescent Hoechst33342/PI double-staining detection results of the effect of miR-143 on cell apoptosis,

Group Ⅰ (Ⅰ-1, Ⅰ-2, Ⅰ-3) in the figure is the positive control; Group Ⅱ (Ⅱ-1, Ⅱ-2, Ⅱ-3) is the transfection group; Group Ⅲ (Ⅲ-1, Ⅲ- 2,Ⅲ-3) is negative control; 1 column (Ⅰ-1,Ⅱ-1,Ⅲ-1) is normal cells; 2 columns (Ⅰ-2,Ⅱ-2,Ⅲ-2) are PI staining; 3 columns (Ⅰ-3,Ⅱ-3,Ⅲ-3) are stained for Hoechst33342;

After PI staining, dead cells can be observed under the microscope, and after Hoechst33342 staining, apoptotic cells can be observed and present apoptotic granules; the experimental results show that after PI staining of all experimental cells, only a small amount of cell death was found, Most of the cells were normal growth cells, but after Hoechst33342 staining, the cells in the transfection group showed obvious apoptotic state. The results showed that miR-143 had obvious pro-apoptotic effect on mammary epithelial cells cultured in vitro;

Figure 4 is a schematic diagram of the flow cytometry detection results of the effect of miR-143 on the cell cycle,

In the figure, A is the cell cycle distribution of the positive control; B is the cell cycle distribution of the transfection group; C is the statistical result of the cell cycle distribution among different groups, showing that after miR-143 treatment, the G1 phase becomes longer and the S phase becomes shorter, and both The difference between the treatment groups reached a significant level, and the results indicated that miR-143 could arrest the growth cycle of cells cultured in vitro in G1 phase, thus delaying the progress of the cell cycle;

Figure 5 is a schematic diagram of the flow cytometry detection results of the effect of miR-143 on breast epithelial cell apoptosis,

In the figure, A is the positive control group; B is the transfection group,

The results showed that only the normal diploid cell DNA peak (G1 peak) was detected in the control group, while a small peak appeared before the G1 peak in the transfection group, the so-called apoptotic peak (Ap peak). The results showed that miR-143 Mammary epithelial cells cultured in vitro can promote apoptosis;

Figure 6. Schematic diagram of the qRT-PCR detection results of the expression of apoptosis-related genes BAX and BCL2 after miR-143 overexpression. The results showed that the mRNA expression of the apoptosis gene BAX in the transfection group was significantly higher than that of the positive control. Reached extremely significant (P<0.01), anti-apoptotic gene BCL2 in the control group, compared with the transfection group, its mRNA expression was significantly increased, the difference reached significance (P<0.05), the results showed that miR-143 had a significant effect on in vitro culture mammary gland cells are apoptotic.

Detailed ways

The present invention is further defined in the following examples. According to the above description and these examples, those skilled in the art can determine the essential features of the present invention, and can make various changes to the present invention without departing from the spirit and scope of the present invention. and modified to suit various uses and conditions. Unless otherwise specified, what the present invention adopts is the prior art in this field;

The preparation of the cell culture medium: a solution with a volume ratio of FBS:DMEM-F12 of 1:9, containing 5 mg/L bovine insulin, 5 mg/L hydrocortisone, 10 μg/L epidermal growth factor and 100,000 IU/L green chain Mycin double antibody;

The digestion solution is an EDTA solution of PBS without calcium and magnesium ions and 0.25wt% trypsin mixed in equal volumes, the digestion condition is room temperature 25°C, and the digestion time of fibroblasts is 2-3 minutes;

The EDTA solution of FBS, DMEM-F12 and 0.25wt% trypsin used was purchased from gibco company, bovine insulin, hydrocortisone and penicillin streptomycin double antibody were purchased from Sigma company, and epidermal growth factor was purchased from Invitrogen company;

Example 1. Primary Mammary Epithelial Cell Culture

1. Breast tissue collection and processing

Obtain dairy goat mammary gland tissue by conventional means. When sampling, obtain about 30g of mammary gland tissue with abundant acinus and less connective tissue and fat, wash it in sterile PBS containing 3% double antibody, and place it in low temperature for 2 hours. Take it back to the lab. Before culturing, the mammary gland tissue was washed 3-5 times in a culture dish with PBS containing 1% double antibody until the color of the tissue turned pale. Cut the tissue into pieces with surgical scissors (into small pieces of chyle of about 1 mm ³ ), and then wash twice with PBS containing 1% double antibody until the tissue pieces turn white.

2. Tissue Block Inoculation and Culture

(1) Use a straw or ophthalmic tweezers to inoculate small pieces of chylo-like tissue in a culture bottle: place about ten pieces in each 25cm ² plate evenly, and the distance between each small piece is about 0.5cm;

(2) Gently turn the culture bottle over so that the tissue block faces down, and place it in a 37°C incubator for about 2-3 hours, so that the tissue block is firmly attached to the surface of the culture bottle;

(3) Add 3-4ml of full medium containing 1% double antibody (medium components include: DMEM/F12 basal medium, 10% fetal bovine serum FBS, insulin, hydrocortisone and epidermal growth factor), gently Rotate the culture bottle so that the culture medium slowly infiltrates the tissue block;

(4) Put the culture bottle into an incubator at 37° C. and 5% CO ₂ and cultivate it under the condition of changing the liquid once every 3 days;

(5) After 10 days, observe the cells crawling out under a microscope, and change the medium every 2 days after the cells grow out.

3. Cell purification:

The cultured mammary epithelial cells are mixed with fibroblasts, and the time required for digestion and adhesion of the two types of cells is different for purification. After the cells grow well, the cells are digested and passaged with 0.25% trypsin, and finally obtained For purer breast epithelial cells, the specific operation is as follows:

(1) Take out the culture bottle and absorb the old culture medium, rinse the cells with 1-2mL PBS, add 1mL of 0.25% trypsin to digest;

(2) When observed under a microscope, fibroblasts will detach from the flask wall earlier than epithelial cells. Therefore, when cells detach, discard the digestion solution and the detached cells, and add 1 mL of 0.25% trypsin to continue digestion. When the cells are all detached, add the whole culture medium to stop the digestion;

(3) Repeated purification for 3-4 generations can eliminate fibroblasts to obtain purer mammary epithelial cells;

The inventor carried out the immunofluorescence identification of the primary mammary epithelial cells cultured in vitro, and the results are shown in Figure 1, wherein the dark area in the lower right corner is the immunofluorescence identification of the mammary epithelial cell marker protein keratin 18; it can be seen that we cultured in vitro What was obtained was indeed mammary epithelial cells.

Example 2. Transient transfection of cells

The transfection assay was divided into 3 groups: transfection group, negative control and positive control

Firstly, miR-143 was connected to the pcDNA3.1 vector according to the conventional method to obtain the transfection plasmid for the transfection group;

The pcDNA3.1 empty vector was used as the transfection plasmid of the positive control group;

Negative control is not using any transfection plasmid;

The transfection step was carried out according to the instructions of the Lipofectamine ^TM LTX and PLUS ^TM Reagents (invitrogen) transfection kit;

(1) For culturing well-adhered cells in the exponential growth phase, digest with trypsin to make a single-cell suspension, and evenly inoculate the same amount in a 24-well culture plate;

(2) After culturing for 24 hours, cell transfection was performed when the cell confluency reached 70-80%;

(3) Dilute each group of plasmids and internal reference vector plasmid pGL4.74 (transfection plasmid and internal reference plasmid at a mass ratio of 30:1) to 100 μl Opti-MEM I at a final plasmid concentration of 0.8 μg per well, plasmid DNA (μg) Add Lipofectamine ^TM PLUS (μl) at a volume ratio of 1:1 and mix by blowing. After 5 minutes ^, add an appropriate amount of Lipofectamine ^TM LTX transfection reagent and gently blow and mix. Continue to incubate at room temperature for 30 minutes;

(4) Add 100 μL of the mixed solution to each cell culture well to be transfected, and add the double-antibody-containing culture solution (a solution with a volume ratio of FBS:DMEM-F12 of 1:9, containing 5 mg/L bovine insulin, 5 mg/L L hydrocortisone, 10 μg/L epidermal growth factor, and 100,000 IU/L penicillin-streptomycin double antibody;) replace with the above-mentioned culture medium without antibiotics, add 400 μL to each well, and set 4 duplicate wells in each group;

(5) Put the culture plate into a 37°C 5% CO ₂ incubator to continue culturing, replace the transfection solution 6 hours after transfection, continue culturing with normal culture solution, and detect the fluorescence intensity 24-48 hours after transfection.

Example 3. MTT method to detect the effect of miR-143 on cell proliferation

(1) Cell inoculation: use DMEM/F12 culture medium containing 10% fetal bovine serum to make a single cell suspension, after repeated exploration, finally inoculate a 96-well plate at a density of 3×10 ⁴ cells per well, culture each well Liquid volume 200μl;

(2) Cell culture: put the 96-well culture plate into an incubator with 5% CO ₂ and saturated humidity at 37°C for culture;

(3) Cell transfection: After culturing for 24 hours, when the cells grew to 70-80% confluence, each 96-well culture plate was transfected simultaneously, and each plate was divided into 3 groups (miR-143 transfection group, control group (positive), blank group (negative)), each group was provided with 6 duplicate wells, medium without antibiotics was used during transfection, and normal medium was replaced 12 hours after transfection;

(4) Cell coloration: Fluorescence values were detected at 0h, 48h, and 72h of transfection, and 20 μl of MTT solution with a final concentration of 5 mg/ml was added to each well, and the culture was terminated after continued incubation for 4 hours. Carefully discard all the supernatant in the wells with a syringe, and at the same time add 150 μl dimethyl sulfoxide (DMSO) to each well, and shake slightly for 10 minutes to fully dissolve the crystals;

(5) Colorimetry: After mixing, use a microplate reader to measure the absorbance value of each well at a wavelength of 490nm, and perform statistical analysis on the obtained data; determine the number of viable cells by comparing the absorbance values of the overexpression group and the control group (the results are shown in Fig. 2).

Example 4. Fluorescent Hoechst33342/PI double staining to detect the effect of miR-143 on cell apoptosis

(1) Fixed cultured cells were digested and suspended cells were collected by centrifugation at 1000rpm/min, and 10 ⁵ -10 ⁶ cells were suspended in 1 mL of culture medium, then 10 μL of Hoechst 33342 dye solution was added, mixed well, and incubated at 37°C 5～15min;

(2) Cells were centrifuged at 4°C, 500-1000rpm/min for 5min, and the supernatant was discarded;

(3) Add 1.0mL Buffer A working solution (dilute 10×Buffer A 10 times with double distilled water) to suspend the cells, add 5μL PI staining solution, place in the dark at room temperature for 5-15min, and mix well;

The blank group, the transfection group and the control group all carried out the above operations;

(4) Fluorescence microscope observation: Heochst 33342 will produce fluorescence (Ⅰ-3, Ⅱ-3, Ⅲ-3) with ultraviolet light excited by krypton laser, and the apoptotic cells will appear brighter (Ⅱ-3); PI excited fluorescence with argon ion laser, and dead cells produced fluorescence (I-2, II-2, III-2); the results are shown in Figure 3.

Example 5. Effect of miR-143 on cell apoptosis detected by flow cytometry

1. Cell sample preparation:

(1) Digest the cells with trypsin and collect them in a 5mL centrifuge tube;

(2) centrifuge at 1000rpm/min for 5min, discard the supernatant;

(3) Add 1 mL of pre-cooled PBS to suspend the cells, then centrifuge, discard and leave a little supernatant to suspend the cells;

2. Cell fixation:

Add 1mL ice bath to pre-cool 70% ethanol, gently pipette the cells to mix, transition at 4°C;

3. PI staining:

(1) Centrifuge the fixed cells at 1000rpm/min for 5min, discard the supernatant;

(2) Add ice-bath pre-cooled PBS and wash twice;

(3) Add PI dye to the centrifuge tube, mix gently, and incubate at 37°C for 30 minutes in the dark;

4. Flow detection and analysis:

A flow cytometer was used to detect red fluorescence at an excitation wavelength of 488 nm, and simultaneously detect light scattering. Cellular DNA content analysis and light scattering analysis were performed using appropriate analysis software, and the results are shown in Figures 4 and 5.

Example 6. qRT-PCR detection of apoptosis-related marker gene expression

(1) Total RNA extraction and reverse transcription: use TIANGEN’s RNAsimple Total RNA Kit total RNA extraction kit (Cat.#DP419) to extract total RNA from cells and tissues, and refer to the kit instructions for specific operations; the extracted RNA was extracted using TaKaRa The company's RrimeScript ^TM RT reagent Kit (Cat. #RR037A) was reverse-transcribed to synthesize the first strand of cDNA, and stored at -20°C for future use.

(2) Instructions for primer design: primers were designed according to the sequences of sheep anti-apoptosis gene BCL2 (NCBI accession number: JN036559.1) and bovine apoptosis gene BAX (NCBI accession number: NM_173894.1). The primer information is shown in the following table:

(3) qRT-PCR reaction: with GAPDH as the internal reference gene, according to the TaKaRa company's Premix EX Taq ^TM Ⅱ (DRR081A) instructions were added, and the reaction system was 15 μl.

(4) Analysis and statistics of the results: the reaction results were initially analyzed for the obtained Ct values by the supporting software of MX3000P. The relative value of each gene expression was calculated according to the standard curve using the ^2-△△CT method, and the expression level of the gene was expressed as ^2-△△CT ± SE, and the difference was significant using the one-way ANOVA method in the SAS9.2 software The relative expression levels of the apoptosis gene BAX in the transfection group and the control group were 0.9506 and 0.6416, as shown in Figure 6, and the expression level of BAX in the miR-143 transfection group was extremely significantly increased and reached a very significant level (P<0.01); the relative expression levels of BCL2 in the transfection group and the control group reached 0.4653 and 0.5967, respectively, and the difference was significant (P<0.05)), indicating that miR-143 has an apoptotic effect on breast cells cultured in vitro.

Test case

After the inventor obtained the above-mentioned miRNA related to the apoptosis of dairy goat mammary gland epithelial cells, whose gene sequence is shown in SEQ ID NO.1, and its precursor sequence shown in SEQ ID NO.2, the inventor used conventional genetic engineering The technology inhibits the expression of miR-143 or its precursor sequence in dairy goats, and the results confirm that the mammary gland degeneration time of the dairy goats tested was delayed by one week at the shortest and three weeks at the longest, and the milk production was increased on average compared with the unsuppressed control group 8%, it can be seen that by inhibiting the expression of miR-143 or its precursor, it can indirectly inhibit the apoptosis of mammary epithelial cells, so as to delay mammary gland degeneration, prolong the lactation period, and increase the milk production of dairy livestock, which has extremely high application value.

Claims (4)

1. A miRNA relevant to the apoptosis of dairy goat mammary gland epithelial cells, characterized in that: the miRNA is miR-143, and its gene sequence is as shown in SEQ ID NO.1. 2. miRNA according to claim 1 is characterized in that: its precursor gene sequence is as shown in SEQ ID NO.2. 3. the application of miRNA described in claim 1 in dairy goat breeding is characterized in that, suppresses the expression of gene sequence such as sequence shown in SEQ ID NO.1 in dairy goat body. 4. application according to claim 3, is characterized in that, suppresses the expression of gene sequence such as the precursor sequence shown in SEQ ID NO.2 in dairy goat body.