Primer mixture for quantitative detection of long-chain non-coding gene FAM91A2, shRNA for knocking down expression of FAM91A2 and preparation method of plasmid

Abstract

The invention discloses a primer mixture for quantitative detection of a long-chain non-coding gene FAM91A2, which is characterized by comprising single-chain DNA (deoxyribonucleic acid) as shown in SEQ ID NO: 1-4, single-chain DNA as shown in SEQ ID NO: 2-4 and single-chain DNA as shown in SEQ ID NO: 3-4, the invention further discloses a detection kit, the detection kit is used for detecting the long-chain non-coding gene FAM91A2, and the detection kit comprises the primer mixture, nuclease-free sterilized water and a sample DNA (Deoxyribonucleic Acid); the invention further discloses shRNA for knocking down expression of the long-chain non-coding gene FAM91A2, the shRNA comprises a positive chain and a negative chain, the base sequence of the positive chain is shown as SEQ ID NO: 5, and the base sequence of the negative chain is shown as SEQ ID NO: 6.

Description

Technical Field

[0001] The present invention relates to the field of gene detection. Background Art

[0002] The development of RNA sequencing technology and expression transcript mapping has shown that although the human genome is universally transcribed, only a small portion of RNA (2%) encodes proteins, and most expressed transcripts do not encode proteins. This subclass of non-coding transcripts with variable lengths and functions is collectively referred to as non-coding RNA (ncRNA), among which non-coding RNAs with a length of more than 200nt are called long non-coding RNA (lncRNA).

[0003] Recent studies have revealed the biological roles of lncRNA in various normal and pathological physiological processes, such as development, metastasis, apoptosis, stem cell pluripotency, proliferation, DNA damage, differentiation and cancer. Since the abnormal expression levels of some lncRNAs can be detected in tumor tissues or body fluids, their specific expression in certain types of cancer can be used as markers for diagnosing tumors or as targets for treating cancer, such as ovarian cancer (OC), hepatocellular carcinoma, endometrial cancer, lung cancer, breast cancer and prostate cancer. Abnormal expression or mutation of lncRNA is often associated with proliferation, tumor microenvironment remodeling, metastasis and resistance to tumor cell death at different stages of tumor development.

[0004] So far, a large number of lncRNAs related to ovarian cancer have been found to be abnormally upregulated in ovarian cancer and are involved in the occurrence, progression and chemotherapy resistance of OC. In OC cells and tumor tissues, except for a few special cases, the expression of most lncRNAs shows an upregulated trend, which suggests that downregulating their expression may have potential benefits for patient treatment. With the increase in the content of lncRNA research, the changes in the expression profile, tissue specificity and regulatory effects of lncRNA in OC have an important role in promoting the development of OC diagnosis and treatment. Therefore, it is crucial to obtain the development of ovarian cancer as early and as conveniently as possible for disease diagnosis. In the early stage, lncRNA chips and bioinformatics analysis found that lncRNA FAM91A2 (also known as LINC00869) is highly expressed in ovarian cancer. Summary of the invention

[0005] The purpose of the present invention is to solve the problems existing in the prior art and disclose a primer mixture for quantitative detection of long non-coding gene FAM91A2, comprising:

[0006] The single-stranded DNA composition is as described in SEQ ID NO: 1-4.

[0007] The present application also discloses a detection kit for detecting the long non-coding gene FAM91A2 as described above, the detection kit comprising:

[0008] the primer mixture;

[0009] Nuclease-free sterile water;

[0010] Sample DNA.

[0011] Specifically, the kit includes:

[0012] Forward primer: 0.2 μl;

[0013] Reverse primer: 0.2 μl;

[0014] Sample DNA: 0.1 μl;

[0015] Nuclease-free sterile water: 3.6 μl.

[0016] The present application also discloses an application of a primer mixture for quantitative detection of long non-coding gene FAM91A2 in the preparation of a detection kit.

[0017] The present application also discloses a shRNA for knocking down the expression of the long non-coding gene FAM91A2, wherein the shRNA comprises a positive strand and a reverse strand, the base sequence of the positive strand is shown in SEQ ID NO: 5, and the base sequence of the reverse strand is shown in SEQ ID NO: 6.

[0018] The shRNA is completed by reacting the positive strand and the reverse strand in an annealing reaction solution.

[0019] The present application also discloses a method for preparing a plasmid, the steps of which are as follows:

[0020] S1: preparing the shRNA according to claim 5;

[0021] S2: digest the pLKO.1-puro vector to obtain the digestion product;

[0022] S3: recover the enzyme cleavage product by gel recovery to obtain the vector product;

[0023] S4: connecting the shRNA prepared in S1 to the vector product to obtain a connection product;

[0024] S5: Transform the ligation product into competent cells, then culture and screen positive clones. Name the correct plasmid pLKO.1-shFAM91A2, then amplify and preserve.

[0025] The present application also discloses a plasmid, wherein the plasmid connects the shRNA to the pLKO.1-puro vector, and is prepared by the plasmid preparation method described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the melting curve of FAM91A2 gene qPCR amplification.

[0027] Figure 2 is the expression level of FAM91A2 in ovarian cancer tissues.

[0028] Figure 3 is the expression of FAM91A2 in different ovarian cancer cell lines and normal ovarian epithelial cell lines.

[0029] Figure 4 qPCR identification of ovarian cancer cell lines with stable knockdown of FAM91A2.

[0030] Figure 5 Schematic diagram of the ability of knocking down FAM91A2 to inhibit the proliferation of ovarian cancer cells.

[0031] Figure 6 Schematic diagram of knocking down FAM91A2 to inhibit the migration and invasion ability of ovarian cancer cells. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0033] Embodiment 1:

[0034] 1. Cell Culture

[0035] 1. Cell Recovery

[0036] Take out the required cells from liquid nitrogen or -80℃ freezer, quickly place them in a 37-40℃ water bath, quickly rotate the cryotube, and after the cells in the tube melt into liquid, transfer the cell suspension to a 15ml centrifuge tube, centrifuge at 4℃, 1000rpm for 5min; then prepare a 6cm cell culture dish and add 2ml culture medium to it; take out the centrifuge tube and carefully discard the supernatant; add 1ml culture medium to the cell pellet and gently blow to resuspend the cells; finally, plant the resuspended cell suspension into the culture dish and shake it gently to evenly distribute the cells throughout the culture dish, then place the culture dish in a cell culture incubator for culture.

[0037] 2. Cell Culture

[0038] Human ovarian cancer cell line A2780 and human ovarian epithelial cell line Hosepic use 1640 medium containing 10% serum and 1× penicillin-streptomycin; human ovarian cancer cell lines SKOV3, OVCAR3, HO8910 and human embryonic kidney cells HEK-293T use high-glucose DMEM medium containing 10% serum and 1× penicillin-streptomycin. Culture conditions: 37°C, 5% CO2.

[0039] 3. Cell Passaging

[0040] Take out the cells that need to be passaged from the incubator, discard the old culture medium, and wash the cells twice with sterilized 1×PBS solution to remove the residual old culture medium; add an appropriate amount of trypsin to the culture dish for digestion for 1-5 minutes, and then add an equal amount of culture medium containing serum to stop digestion; then gently blow the cells off the wall of the culture dish, collect them in a 15ml centrifuge tube, and centrifuge them at 4℃, 1000rpm for 5 minutes; after centrifugation, carefully discard the supernatant, and then add 1ml of fresh culture medium to the centrifuge tube and gently blow to resuspend the cells, then aspirate an appropriate amount of cells for cell counting, and finally aspirate the required amount of cells according to the experimental requirements and plant them in the culture dish, add an appropriate volume of culture medium, and gently shake the culture dish to make the cells evenly distributed in the culture dish.

[0041] 4. Cell Cryopreservation

[0042] Take out the cells to be frozen from the incubator, digest and centrifuge the cells according to the cell passage method in Part 3 above, discard the supernatant after centrifugation, count the cells, resuspend the cells in 500-1000 μl of serum-free cell freezing solution, and gently pipette to mix. The concentration is 1×10 6 / ml, add to the cell cryopreservation tube; finally, cover the mouth of the cryopreservation tube with sealing film. The cell sample should be placed in a -80℃ refrigerator for initial cryopreservation. If long-term cryopreservation is required, the cells should be placed in a -80℃ refrigerator for one to two days, and after they are fully cooled, they should be transferred to a liquid nitrogen tank to ensure their long-term stability and safety.

[0043] 2. Cell RNA Extraction

[0044] 1. Prepare pre-cooled PBS buffer, isopropanol, 75% ethanol, and RNase-free ddH2O before the experiment;

[0045] 2. Remove the cells, discard the culture medium, and wash the cells 1-2 times with pre-cooled PBS. Try to absorb all the PBS liquid in the last wash;

[0046] 3. Add 500 μl RNA-easy Isolation Reagent (Novozyme, Cat. No. R701) to each well of the six-well plate, making sure that the solution completely covers the cell surface, and then blow the cells off with a pipette; then transfer the lysate in the well to an EP tube, and blow repeatedly with a pipette to fully lyse the cells;

[0047] 4. Add 200 μl of RNase-free ddH2O to the lysate in the EP tube, mix by inverting, and let stand at room temperature for 5 min; then centrifuge at 12000g for 15 min at room temperature;

[0048] 5. After centrifugation, the lysate will be clearly separated into layers, with the upper layer being the aqueous phase and the lower layer being the precipitate. Carefully pipette 500 μl of the upper aqueous phase and transfer it to a new EP tube;

[0049] 6. Then add an equal volume of 500 μl of isopropanol, mix well, and then let stand at room temperature for 10 minutes; then centrifuge again at 12000g for 10 minutes;

[0050] 7. After centrifugation, carefully discard the supernatant (be careful not to suck away the white precipitate at the bottom);

[0051] 8. Then add 500 μl of 75% ethanol, flick the bottom of the tube to suspend the precipitate, and turn it upside down several times; then centrifuge at 9000g for 5 minutes, and finally discard the supernatant completely;

[0052] 9. Open the EP tube cap, place it in a clean bench and let it dry at room temperature for several minutes, then add 20-50 μl RNase-free ddH2O to dissolve the precipitate, and pipette repeatedly to fully dissolve the RNA precipitate;

[0053] 10. Check the concentration and purity of the extracted RNA and store it at -80℃.

[0054] 3. Tissue RNA Extraction

[0055] 1. Prepare mortar, isopropanol, 75% ethanol, and RNase-free ddH2O before the experiment;

[0056] 2. Place the mortar on ice to pre-cool, take about 100 mg of ovarian tissue specimen, add 1 ml RNAiso Plus (Takara, Cat. No. 9109), and grind carefully until the tissue is lysed into a liquid paste; then collect the lysate in a 1.5 ml EP tube and place it on ice for 5 minutes;

[0057] 3. Add 200 μl of chloroform, invert several times to mix, and place on ice for 5 min;

[0058] 4. Centrifuge at 12000g, 4°C for 15 min;

[0059] 5. After centrifugation, the liquid in the EP tube is divided into three layers. Carefully pipette the upper transparent liquid into a new EP tube;

[0060] 6. Add an equal volume of isopropanol, invert several times to mix, and then let stand on ice for 10 minutes;

[0061] 7. Then centrifuge at 12000g and 4℃ for 10min. After centrifugation, white RNA precipitate can be seen at the bottom of the tube.

[0062] 8. Discard the supernatant, keep the precipitate at the bottom of the tube, and add 500 μl of 75% ethanol (prepared with RNase-free ddH2O), invert and mix several times, and then centrifuge at 7500g, 4℃ for 5 minutes;

[0063] 9. After centrifugation, discard the supernatant, open the EP tube, and place the EP tube in a clean bench to dry at room temperature for several minutes to remove residual ethanol. Then add 20-50μl RNase-free ddH2O to dissolve the precipitate, and repeatedly pipette until the precipitate is completely dissolved;

[0064] 10. Check the concentration and purity of the extracted RNA and store it at -80℃.

[0065] 4. Reverse Transcription PCR

[0066] Use the reverse transcription kit TransScript Uni One-Step gDNA Removaland cDNA Synthesis SuperMix (Quanshijin Company, catalog number AT311) to prepare the reverse transcription PCR reaction solution according to the system in Table 1 and mix gently.

[0067] Table 1 Reverse transcription PCR system

[0068]

[0069] 2. PCR reaction procedure: incubate at 50℃ for 15min, heat at 85℃ for 5s, and store the obtained cDNA product at -20℃. 5. Real-time fluorescence quantitative PCR (qPCR)

[0070] 1. Prepare the reaction system according to Table 2:

[0071] Table 2 qPCR reaction system

[0072]

[0073] 2. qPCR reaction program: 95℃, 30s; 95℃, 5s; 60℃, 15s; 72℃, 30; 35 cycles; finally, use the instrument default program to draw the melting curve, such as Figure 1 As shown, it was shown that the amplification of FAM91A2 gene was specific amplification.

[0074] 3. qPCR primer sequences are shown in Table 3.

[0075] Table 3 qPCR primer sequences

[0076]

[0077] Six results

[0078] The results showed that the expression of FAM91A2 in ovarian cancer tissues was higher than that in non-ovarian cancer tissues ( Figure 2 A). Subsequently, ovarian cancer patients were divided into early (stage I and II) and late (stage III and IV) according to clinical characteristics to analyze the expression of FAM91A2. After qPCR detection, it was found that the expression level of FAM91A2 in the tissues of patients with late ovarian cancer showed a significant upward trend compared with that in the tissues of patients with early stage ( Figure 2 B). These results demonstrate that FAM91A2 expression is elevated in ovarian cancer, and the increase in FAM91A2 is more significant as the disease progresses. qPCR was used to detect the expression level of FAM91A2 in four ovarian cancer cell lines (SKOV3, OVCAR3, HO8910, and A2780) and a normal ovarian epithelial cell line (Hosepic). The results showed that compared with normal ovarian epithelial cell lines, the expression level of FAM91A2 in ovarian cancer cell lines was significantly increased, and the expression increase was particularly significant in SKOV3 and A2780 cells ( Figure 3 ).

[0079] Example 2

[0080] Preparation of ovarian cancer cell lines with stable knockdown of FAM91A2

[0081] 1. Preparation of FAM91A2 knockdown plasmid

[0082] 1. Send the sequences of SEQ ID NO: 5-6 to the company to synthesize dry powder primers;

[0083] 2. Dilute the dry powder primer to a 20 μM suspension with ddH2O, prepare an annealing reaction solution according to the system in Table 4, perform an annealing reaction, and obtain an annealing reaction product;

[0084] Table 4 Annealing reaction system

[0085]

[0086] 3. pLKO.1-puro vector digestion;

[0087] (1) AgeⅠ digestion system is shown in Table 5. The digestion conditions are digestion in a water bath at 37°C for 2 h;

[0088] Table 5 pLKO.1-puro vector restriction enzyme digestion system

[0089]

[0090] (2) Purification of products after enzyme cleavage:

[0091] 1) Add 250 μl of Buffer B3 to the product after AgeⅠ digestion and mix thoroughly with a pipette; then transfer it to the purification column, centrifuge at 8000g for 30 seconds, and discard the liquid in the collection tube;

[0092] 2) Next, add 500 μl of Wash Solution to the purification column and centrifuge at 9000 g for 30 seconds. Discard the liquid in the collection tube. Repeat this operation once to ensure sufficient washing. Finally, centrifuge the purification column empty for 1 minute.

[0093] 3) Transfer the purification column to a new 1.5 ml EP tube, add 35 μl Elution Buffer, let stand for 1 min, centrifuge at 9000 g for 1 min, and collect the purified product in the EP tube.

[0094] (3) The purified product obtained in the previous step was digested with EcoRI. The system is shown in Table 6. The digestion conditions are digestion in a water bath at 37°C for 2 h;

[0095] Table 6 pLKO.1-puro vector restriction enzyme digestion system

[0096]

[0097] 4. Gel recovery of the product after enzyme cleavage:

[0098] (1) First, prepare a 1% agarose gel; then add 5 μl of 10× Loading Buffer to the above digested product, mix thoroughly, add it together with DNA Maeker into the agarose gel well for electrophoresis at 110 V for 40 min;

[0099] (2) After electrophoresis, remove the gel, soak it in EB dye solution for 1 min, and then rinse it with running water for 5 min; then place the gel under ultraviolet light to observe the bands, and accurately cut out the band containing the target fragment for gel recovery;

[0100] (3) Set the water bath to 50°C and calculate the gel weight of the cut target band; add 300 μl of Buffer B2 for every 100 mg of gel; after adding Buffer B2, place in a 50°C water bath for 5-10 min until the gel block is completely dissolved;

[0101] (4) Transfer the dissolved gel mixture to a dedicated adsorption column, centrifuge at 8000 g for 30 seconds, and discard the waste liquid in the collection tube;

[0102] (5) Then add 500 μl of Wash Solution to the adsorption column, centrifuge at 9000 g for 30 seconds, and discard the waste liquid in the collection tube; repeat this step once; finally, centrifuge the empty adsorption column for 1 minute;

[0103] (6) Then, transfer the adsorption column to a new EP tube, add 20 μl of Elution Buffer to the adsorption column, let it stand at room temperature for 1 min, and then centrifuge at 9000 g for 1 min; collect the product in the EP tube and store it at -20°C;

[0104] 5. After the enzyme digestion is completed, the obtained product is subjected to a ligation reaction with the annealing product prepared in step 2; the reaction conditions are 16° C. overnight to obtain a ligation product, and the reaction system is shown in Table 7;

[0105] Table 7T4 DNA ligase ligation system

[0106]

[0107] 6. Transform competent cells with ligation product: First, take 10 μl of ligation product, add 100 μl DH5α competent cells to it, and place it on ice for 30 minutes; then quickly put it in a 42°C water bath for heat shock treatment for 90 seconds, take it out immediately after heat shock and put it back on ice, and let it stand for 2 minutes; then add 300 μl liquid LB culture medium to it, and shake it on a shaker at 37°C for 1 hour; then take it out and evenly spread it on an LB solid agar plate containing ampicillin, and finally turn the plate upside down and place it in a 37°C incubator for overnight culture;

[0108] 7. The next day, take out the LB plate from the incubator, pick 5 colonies from it and inoculate them into 3 ml of liquid LB culture medium containing ampicillin, and culture them on a shaker at 37°C overnight;

[0109] 8. Take out the overnight culture solution and use the EasyPure Plasmid MiniPrep Kit to extract the plasmid (Quanshijin Company, product number EM101-02). The specific steps are as follows:

[0110] (1) Transfer the liquid bacterial solution in the LB tube to the EP tube, centrifuge at 10,000 g for 1 min, and remove the supernatant (absorb as much as possible);

[0111] (2) Add 250 μl of colorless solution RB (add RNase A in the kit to RB in advance) and oscillate to suspend the bacterial precipitate; then add 250 μl of blue solution LB and mix by inverting the tube until the color of the bacterial solution changes from semi-translucent blue to translucent blue;

[0112] (3) Then add the yellow solution NB and mix well. When the bacterial solution turns yellow and a precipitate forms, let it stand at room temperature for 2 min.

[0113] (4) Centrifuge at 16,000 g for 10 min, then transfer the supernatant to a centrifuge column; centrifuge at 12,000 g for 1 min, and discard the flow-through;

[0114] (5) Add 650 μl of WB solution to the centrifuge column and centrifuge at 12,000 g for 1 min. Discard the flow-through and centrifuge again for 2 min to ensure that the residual WB is completely removed.

[0115] (6) Transfer the centrifuge column to a new EP tube, add 30-50 μl of EB solution to the center of the column, and then let it stand at room temperature for 1 min, then centrifuge at 10,000 g for 1 min to elute the plasmid DNA;

[0116] (7) Detect the purity and concentration of the extracted plasmid and store it at -20°C.

[0117] 9. The proposed plasmid was sent for sequencing identification; the correctly identified plasmid was named pLKO.1-shFAM91A2, and after transformation and amplification, it was stored at -20°C.

[0118] 2. Lentivirus Preparation

[0119] 1. Prepare HEK-293T cells with good growth status, and prepare for transfection after the confluence reaches 70% in a 6 cm culture dish; 2. The transfection system is as follows: add 3 μg of target plasmid (pLKO.1-shFAM91A2), control plasmid (pLKO.1-Control), 2 μg of psPAX2 and 1 μg of pMD2.G plasmid to a sterile EP tube, make up to 250 μl with DMEM without double antibody and serum, and mix thoroughly; then add 18 μl of liposome nucleic acid transfection reagent to another sterile EP tube, make up to 250 μl with DMEM without double antibody and serum, mix gently, and let stand at room temperature for 5 minutes; mix the liquid in the plasmid tube and the liposome tube and let stand at room temperature for 20 minutes;

[0120] 3. Take out the HEK-293T cells to be used, discard the original culture medium, gently wash them with PBS, add 2.5 ml of DMEM without double antibody and serum, gently add the mixture of plasmid and liposome to the cell culture dish, gently mix, and culture in the incubator. After 8 hours of transfection, replace the liquid in the culture dish with 3 ml of DMEM containing 5% serum and continue to culture for 24 hours; 4. 24 hours after transfection, filter and collect the virus liquid with a syringe and a 0.45 μm filter, and add 3 ml of DMEM containing 5% serum to the culture dish and continue to culture for 24 hours; 48 hours after transfection, filter and collect the virus liquid with a 0.22 μm filter; The collected virus liquid was packaged and stored at -80°C.

[0121] 3. Cell infection and cell screening and identification

[0122] 1. Take the cells to be infected in the logarithmic growth phase (select SKOV3 cells and A2780 cells with relatively high FAM91A2 expression), inoculate an appropriate amount into a 24-well plate, wait for them to adhere to the wall, discard the old culture medium, wash twice with PBS, add 250 μl of virus solution and 250 μl of DMEM containing 5% serum and 0.5 μl of polybrene, culture for 24 hours, and repeat the above operation for secondary infection;

[0123] 2. After the secondary infection, discard the liquid in the well plate, wash twice with PBS, add DMEM medium containing 10% serum and a final concentration of 1 μg / ml puromycin to select cells; observe the cells every day and change the medium until the cells no longer die;

[0124] 3. Cell identification: Extract RNA from the selected stable cell lines and detect the expression of FAM91A2 by qPCR.

[0125] IV. Experimental Methods

[0126] (I) CCK8 cell proliferation assay

[0127] 1. First, take out the cells of the control group and the experimental group in the logarithmic growth phase, then digest and centrifuge these cells, and resuspend the cells in 1 ml of culture medium; dilute the cells to an appropriate multiple and count them; prepare the required cell suspension, with 1000 cells in each 100 μl of culture medium;

[0128] 2. In a 96-well plate, add 100 μl of cells to each well, that is, plant 1000 cells in each well, set up 4-5 replicate wells for each cell type, and plant 5 plates in total; and add cell-free culture medium to each plate as a blank control;

[0129] 3. After the cells in the 96-well plate adhere to the wall, take out the first plate, add 10 μl of CCK8 reagent (Yeasen, Cat. No. 40203) to each well, and place it in an incubator in the dark for 2 hours;

[0130] 4. After the incubation time is over, take out the first 96-well plate, measure the absorbance value of each well at a wavelength of 450 nm using an enzyme reader, and record the data at this time as the data at 0 h;

[0131] 5. Repeat the above steps at the same time point every day for the next 4 days, and record the absorbance values ​​as 24h, 48h, 72h, and 96h data in sequence;

[0132] 6. Subtract the absorbance value of the blank group from the absorbance value of the control group and the target group of cells, and make a line graph of the absorbance values ​​at 0h, 24h, 48h, 72h, and 96h for statistical analysis.

[0133] (II) Plate clone formation experiment

[0134] 1. Take out the cells of the control group and the experimental group in the logarithmic growth phase, digest and count them, and then prepare the cell suspension of the required concentration so that there are 1000 cells in each 200 μl suspension;

[0135] 2. Add 2 ml of culture medium containing 10% FBS to the six-well plate, and add 200 μl of the prepared cell suspension, and mix gently; 3. Replace the culture medium for the cells every 3 days, and culture for about 10 days (the specific number of days is adjusted according to the cell growth status);

[0136] 4. When obvious monoclonal cell clusters were observed, the 6-well plate was removed, washed twice with PBS, and 1 ml of methanol was added to each well for fixation for 30 min.

[0137] 5. Discard the methanol, wash away the residual methanol with PBS, then add 1 ml of crystal violet staining solution and stain for 15 minutes in the dark;

[0138] 6. Discard the crystal violet stain, wash away the residual stain with PBS, wait for the 6-well plate to dry, and take a picture of each well;

[0139] 7. Count the number of monoclonal cell clusters in each well and perform statistical analysis.

[0140] (III) Cell scratch assay

[0141] 1. Draw a straight line every 1 cm on the back of the 6-well plate as a mark;

[0142] 2. Digest the cells in the control group and the experimental group in the logarithmic growth phase, count, resuspend and evenly inoculate into a 6-well plate (adjust the amount of inoculated cells according to the cell growth rate so that the cell confluence reaches about 95% before scratching the next day);

[0143] 3. After the cells have grown, use a sterile 200μl Tip to draw a line perpendicular to the back mark. Use the same Tip, speed and force to draw a line in each well.

[0144] 4. Discard the culture medium in the 6-well plate, wash the scratched cells with 1×PBS, and then add 2 ml of serum-free cell culture medium;

[0145] 5. Use a camera to take a photo of the scratch at 0h, and select the intersection of the back mark line and the scratch line as the shooting field; then place the cells in an incubator for culture;

[0146] 6. After 24 or 36 h of incubation, remove the 6-well plate and take a photo of the scratch at the same position as the photo taken at 0 h;

[0147] 7. Finally, calculate the relative migration rate of the cells: relative migration rate = (0h distance - 24h distance) ÷ 0h distance × 100%.

[0148] (IV) Transwell cell migration assay

[0149] 1. First, take the cells of the control group and the experimental group in the logarithmic growth phase, digest and centrifuge them, and resuspend the cells with 1 ml of serum-free DMEM culture medium; count the cells and prepare the cell suspension according to the cell concentration to ensure that there are 30,000 cells in every 300 μl of suspension (the number of cells can be increased or decreased appropriately for different cells); add 600 μl of culture medium containing 10% serum to the 24-well plate, and place the small chamber on it, add 300 μl of the prepared cell suspension to the upper chamber, and finally put the 24-well plate into the incubator for 12-24 hours;

[0150] 2. After culturing for an appropriate time, gently remove the chamber, discard the cell suspension in the upper chamber, and gently wash the upper chamber with PBS, repeating 2-3 times; then add 800 μl of methanol to a new well of the 24-well plate, add 200 μl of PBS to the upper chamber, and place the chamber in methanol for 30 minutes;

[0151] 3. Add 800 μl of crystal violet staining solution to the new well, place the fixed chamber into it, and stain for 15 minutes in the dark;

[0152] 4. Take out the chamber, immerse it in PBS to wash off the excess dye, and then use a cotton swab to carefully wipe off the water and residual cells in the upper chamber. Finally, after the chamber is naturally dried, use a microscope to observe the cells migrating at the bottom of the chamber and take pictures;

[0153] 5. Count the number of cells in each field of view and perform statistical analysis.

[0154] (V) Transwell cell invasion assay

[0155] 1. Place the matrix gel in a 4°C refrigerator the night before the experiment to slowly dissolve;

[0156] 2. Mix the matrix gel and serum-free culture medium at a ratio of 3:97, then add 100 μl of serum-free culture medium containing matrix gel to each chamber and place it in the incubator until it solidifies for later use;

[0157] 3. The remaining steps are the same as (IV).

[0158] Five results

[0159] 1. Successfully constructed an ovarian cancer cell line with knockdown of FAM91A2

[0160] First, we selected SKOV3 and A2780 cell lines with relatively high FAM91A2 expression, and used lentiviral plasmid vector transfection technology to encapsulate and collect the virus solution and then infect the cells; then we used the puromycin resistance gene carried on the vector to select cells and construct a stable cell line with stable knockdown of FAM91A2. The knockdown efficiency of FAM91A2 was detected by qPCR. The results showed that the expression levels of SKOV3 and A2780 cells after transfection with pLKO.1-shFAM91A2 were significantly reduced compared with those of the control group transfected with pLKO.1-shNC, proving that the ovarian cancer cell line with knockdown of FAM91A2 was successfully constructed.

[0161] 2. Knockdown of FAM91A2 inhibits the proliferation of ovarian cancer cells

[0162] In order to study the effect of FAM91A2 on the proliferation ability of tumor cells, the above-mentioned stable transfected cell lines with knockdown of FAM91A2 that were transfected with lentiviral vectors and stably screened were used for proliferation-related cell experiments. pLKO.1-shFAM91A2 was the experimental group, and pLKO.1-shNC was the control group. The role of FAM91A2 in cell proliferation was detected by plate cloning experiments and CCK8. In the CCK8 experiment, the number of cells in the control group and the experimental group was consistent at the initial measurement. The absorbance values ​​of the cells after adding CCK8 reagent were detected for 4-5 consecutive days. It was found that after knocking down FAM91A2, the proliferation ability of the experimental groups of SKOV3 and A2780 cell lines was significantly inhibited compared with the control group ( Figure 5 A). In order to further verify the effect of FAM91A2 on cell proliferation, a cell clone formation experiment was conducted. The results showed that the number of cloned cell clusters formed on the cell plate in the experimental group decreased significantly ( Figure 5 B) confirmed again that knocking down FAM91A2 can significantly inhibit the proliferation of cells.

[0163] 3. Knockdown of FAM91A2 inhibits migration and invasion of ovarian cancer cells

[0164] The results of the scratch assay showed that after knocking down FAM91A2 in SKOV3 and A2780 cells, the migration rate of cells was calculated for 24 h. The migration distance of cells in the pLKO.1-shFAM91A2 group was significantly shorter than that of cells in the pLKO.1-shNC group ( Figure 6 A). Transwell migration assay confirmed that after the same number of cells were inoculated in the chamber and cultured under the same conditions, the number of cells in the pLKO.1-shFAM91A2 group that passed through the membrane of the chamber and entered the lower part of the membrane was significantly lower than that in the pLKO.1-shNC group ( Figure 6 B). After the matrix gel was laid in the Transwell chamber, cells were inoculated and the invasion ability of the cells was observed. The results showed that the number of cells in the pLKO.1-shFAM91A2 group that penetrated the matrix gel and entered the bottom of the chamber membrane was less than that in the pLKO.1-shNC group ( Figure 6 C) These results indicate that knocking down FAM91A2 expression significantly inhibited the migration and invasion abilities of ovarian cancer cells.

Claims

1. A primer mixture for quantitative detection of long non-coding gene FAM91A2, characterized in that: include: The single-stranded DNA sequences are as described in SEQ ID NOs: 1-4.

2. Use of the primer mixture for quantitative detection of the long non-coding gene FAM91A2 as claimed in claim 1 in the preparation of a detection kit.

3. A detection kit, characterized in that: For detecting the long non-coding gene FAM91A2 according to claim 1, the detection kit comprises: The primer mixture of claim 1; Nuclease-free sterile water; Sample DNA.

4. The detection kit according to claim 2, characterized in that include: Forward primer: 0.2 μl; Reverse primer: 0.2 μl; Sample DNA: 0.1 μl; Nuclease-free sterile water: 3.6 μl.

5. A shRNA for knocking down the expression of the long non-coding gene FAM91A2, characterized in that: The shRNA comprises a forward strand and a reverse strand, the base sequence of the forward strand is shown in SEQ ID NO:5, and the base sequence of the reverse strand is shown in SEQ ID NO:

6.

6. The shRNA for knocking down the expression of the long non-coding gene FAM91A2 according to claim 5, characterized in that: The method is completed by reacting the positive strand and the reverse strand as claimed in claim 5 in an annealing reaction solution.

7. A method for preparing a plasmid, characterized in that: Here are the steps: S1: preparing the shRNA according to claim 5; S2: digest the pLKO.1-puro vector to obtain the digestion product; S3: gel recovery of the enzyme cleavage product to obtain the vector product; S4: connecting the shRNA prepared in S1 to the vector product to obtain a connection product; S5: Transform the ligation product into competent cells, then culture and screen positive clones. Name the correct plasmid pLKO.1-shFAM91A2, then amplify and preserve it.

8. A plasmid, characterized in that The plasmid connects the shRNA described in claim 5 to the pLKO.1-puro vector.

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