Application of a long noncoding RNA and its encoded polypeptide in liver cancer risk / progression assessment and treatment

Abstract

The present invention belongs to the field of biomedicine technology, and discloses the use of a reagent for detecting a biomarker in preparing a product for liver cancer risk or progression assessment, wherein the biomarker is lncRNAAC016727.1 and / or a polypeptide encoded therein; further discloses the use of a substance for inhibiting the expression of lncRNAAC016727.1 and / or a polypeptide encoded therein in preparing a liver cancer treatment drug, and a drug for treating liver cancer, wherein the drug comprises a substance for inhibiting the expression of lncRNAAC016727.1 and / or a polypeptide encoded therein. The present invention provides a new biomarker and a new treatment strategy for liver cancer risk or progression assessment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a long non-coding RNA and its encoded polypeptide in liver cancer risk / progression assessment and treatment. Background Art

[0002] The progression of liver cancer is closely related to the hardness of liver tissue. As liver cancer progresses, the hardness of liver tissue gradually increases, and the change in the hardness of the extracellular matrix (ECM) is the main reason for the change in the hardness of liver tissue. Changes in ECM hardness cause changes in the mechanical microenvironment of liver cancer cells, leading to the proliferation, migration and drug resistance of liver cancer cells, thereby promoting the progression of liver cancer.

[0003] Long non-coding RNA (lncRNA) plays an important role in various types of tumors, including liver cancer, and regulates the occurrence and development of tumors. Recent studies have shown that in addition to participating in classical ways such as transcriptional regulation, chromatin structure and epigenetic regulation, and RNA indirect regulation, lncRNA can also translate a polypeptide to promote or inhibit tumor progression in various tumors. Although lncRNA-encoded peptides have important functions in various types of tumors, the specific role and molecular mechanism of lncRNA-encoded peptides in the response of liver cancer cells to matrix hardness still need further study. Summary of the invention

[0004] The present invention takes liver cancer as the research background, screens out lncRNAs with coding potential from lncRNAs differentially expressed on substrates of different hardness, studies the effects of the small peptides encoded by them on the biological behaviors of liver cancer cells, and determines the polypeptide SAP encoded by lncRNAAC016727.1 and its effects on liver cancer. Based on this, the present invention provides the following technical solutions:

[0005] The first aspect of the present invention provides a use of a reagent for detecting a biomarker in preparing a product for assessing the risk or progression of liver cancer, wherein the biomarker is lncRNAAC016727.1 and / or a polypeptide encoded thereby.

[0006] In the above-mentioned technical scheme of use, the nucleotide sequence of the transcript of the lncRNAAC016727.1 is shown as SEQ ID NO.15, and the amino acid sequence of the polypeptide encoded thereby is shown as SEQ ID NO.16.

[0007] In the above-mentioned technical scheme of use, high expression of lncRNAAC016727.1 and / or its encoded polypeptide indicates a high risk of liver cancer or progression of liver cancer in the patient.

[0008] The second aspect of the present invention provides a product, which contains a detection reagent for lncRNAAC016727.1 and / or a polypeptide encoded by it.

[0009] The above-mentioned product, the reagent for detecting lncRNAAC016727.1 includes primers for specific amplification of lncRNAAC016727.1, and the sequence of the primers is:

[0010] Forward primer: 5′-GCCTCCTCGGTCCAAGCCATTCT-3′;

[0011] Reverse primer: 5′-ATGTCGCAGATACGGGTAAATAA-3′;

[0012] The products include chips, kits or test strips for assessing patients' liver cancer risk or progression.

[0013] The third aspect of the present invention provides the use of a substance that inhibits the expression of lncRNAAC016727.1 and / or a polypeptide encoded thereby in the preparation of a drug for treating liver cancer.

[0014] In the above-mentioned application technology solution, the substances include compounds, proteins, and nucleotide molecules.

[0015] In the above-mentioned application technology solution, the nucleotide molecule includes siRNA and shRNA, and the shRNA is selected from shRNA#1 and shRNA#2, and the nucleotide sequence is as follows:

[0016]

[0017] In the above-mentioned application technology scheme, the substance inhibits the proliferation, migration and / or invasion of liver cancer cells, or inhibits the progression of liver cancer.

[0018] The fourth aspect of the present invention provides a drug for treating liver cancer, wherein the drug comprises a substance that inhibits the expression of lncRNAAC016727.1 and / or a polypeptide encoded thereby; preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0019] The beneficial effects of the present invention are:

[0020] RNAseq and qRT-PCR were used to screen lncRNAs that were differentially expressed in HepG2 cells on different substrate hardnesses, and the polypeptide SAP encoded by lncRNAAC016727.1 was identified by constructing an ORF-GFP fusion protein vector and performing immunofluorescence experiments. Then, in order to explore the effect of polypeptide SAP on the proliferation of liver cancer cells, MHCC97H and HepG2 liver cancer stable cell lines were constructed to overexpress polypeptide SAP and knock down the transcript USP34-DT-201 where polypeptide SAP was located, respectively. These cells were then used as research objects to detect the effect of polypeptide SAP on the proliferation ability of liver cancer cells at the cellular level through CCK-8 experiments and clone formation experiments, and to verify the effect of polypeptide SAP on the proliferation of liver cancer cells at the molecular level through Western blot detection of the expression of tumor cell proliferation-related marker proteins. Finally, in order to explore the effect of polypeptide SAP on the migration and invasion of liver cancer cells, the above two types of cell lines were used as research objects, and the effect of polypeptide SAP on the migration and invasion of liver cancer cells was detected by Transwell experiment at the cellular level, and the expression of tumor cell migration and invasion-related marker proteins was detected by Western blot at the molecular level to verify the effect of polypeptide SAP on the migration and invasion of liver cancer cells. The present invention provides a new biomarker and a new treatment strategy for the risk or progression assessment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The lncRNAs differentially expressed in HepG2 cells on different substrate hardnesses in RNAseq data are shown (Harder: harder substrate; Softer: softer substrate).

[0022] Figure 2 The results of qRT-PCR detection of the mRNA expression of lncRNAs in HepG2 liver cancer cells on substrates of different hardness are shown (n=6, *P<0.05, **P<0.01, ***P<0.001).

[0023] Figure 3 This is a schematic diagram of the construction of the ORF-GFP fusion protein vector.

[0024] Figure 4 This is the expression of green fluorescent protein in 293T cells after transfection of ORF-GFPmut plasmid.

[0025] Figure 5 This is the expression of green fluorescent protein in 293T cells after transfection of AC016727.1-ORF7 related plasmid (scale bar: 20 μm).

[0026] Figure 6The results of immunofluorescence detection of the expression of polypeptide SAP in 293T, MHCC97H and HepG2 cells (scale bar: 50 μm).

[0027] Figure 7 Transwell assay for migration ability of MHCC97H and HepG2 cells overexpressing polypeptide SAP (L-SAP): (a) representative image of MHCC97H migration; (b) statistical graph of the number of MHCC97H migrated cells; (c) representative image of HepG2 migration; (d) statistical graph of the number of HepG2 migrated cells; scale bar: 100 μm; n=3, *P<0.05, ***P<0.001).

[0028] Figure 8 Transwell assay shows the migration ability of MHCC97H cells with knockdown of the expression of USP34-DT-201 transcript containing the polypeptide SAP (shRNA#1-SAP and shRNA#2-SAP): (a) representative image of MHCC97H migration; (b) statistical graph of the number of MHCC97H migrated cells; (c) representative image of HepG2 migration; (d) statistical graph of the number of HepG2 migrated cells; scale bar: 100 μm; n=3, *P<0.05).

[0029] Fig. 9 The invasive ability of MHCC97H cells overexpressing the SAP polypeptide (L-SAP) was detected by Transwell assay: (a) representative image of MHCC97H invasion; (b) statistical image of the number of MHCC97H invading cells; scale bar: 100 μm; n=3, **P<0.01).

[0030] Fig.10 Western blot was used to detect the expression of migration- and invasion-related proteins in MHCC97H cells overexpressing the SAP polypeptide (L-SAP): (a) representative electrophoresis image of MHCC97H protein; (b) statistical graph of grayscale value of MHCC97H; (c) representative electrophoresis image of HepG2 protein; (d) statistical graph of grayscale value of HepG2 (n=3, ns, P>0.05, *P<0.05, **P<0.01). DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0032] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0033] Example 1

[0034] 1 Experimental Materials and Methods

[0035] 1.1 Screening of lncRNAs with differential expression on substrates of different hardness in RNAseq data

[0036] (1) Cell culture

[0037] Human hepatoma cell HepG2 was revived and cultured, and subsequent experiments were carried out when the cells grew well and the growth density reached about 90%.

[0038] (2) Preparation of PDMS 3D microcolumn base glue

[0039] The polydimethylsiloxane SYLGARDTM 184Silicone ElastomerKit (PDMS, USA) material consists of two parts: silicone elastic colloid and silicone elastomer curing agent. We prepared base glues of different hardness by adjusting the ratio of PDMS colloid and curing agent, among which the "harder base glue" simulates the hardness of liver cancer pathological state, and the "softer base glue" simulates the hardness of liver in normal physiological state. The applicant's research team previously tested the Young's modulus of the two hardness base glues by atomic force microscopy, among which the Young's modulus of the "harder base glue" was about 5000kpa, and the Young's modulus of the "softer base glue" was about 300kpa.

[0040] ① For base glue with higher hardness: weigh 6g of PDMS colloid and add 0.6g of PDMS curing agent; for base glue with softer hardness: weigh 6g of PDMS colloid and add 0.2g of PDMS curing agent.

[0041] ② In order to prepare PDMS base glue with higher hardness and softer hardness, we will weigh the mixed silicone elastomer and curing agent and stir them with a 1mL pipette tip to mix them thoroughly and evenly.

[0042] ③ Carefully pour the mixture onto the 3D microcolumn model, flatten it, and use a vacuum pump to remove the bubbles in the mixture.

[0043] ④ Take out the 3D micro-column model covered with PDMS from the vacuum pump and place it on a horizontal table for 20-30 minutes to allow the PDMS glue to flatten the entire 3D topological micro-column model.

[0044] ⑤ Place on a constant temperature heater and cure at 80°C for 12 hours.

[0045] ⑥After heating and curing, gently demold the PDMS glue from the 3D microcolumn model, cut the PDMS glue into the size of a six-well cell culture plate with a blade, and place it in the six-well plate.

[0046] ⑦ Place the six-well plate covered with PDMS base glue of different hardness in the clean bench and sterilize it by UV irradiation overnight.

[0047] (3) Cultivation of liver cancer cells on PDMS substrate

[0048] ① Add 2-3 mL of pre-prepared Fn (fibronectin) solution (Fn:PBS=1:100) to the six-well plate irradiated under ultraviolet light overnight, and then incubate at 37°C for 3 hours or 4°C overnight to promote subsequent cell adhesion.

[0049] ② When the HepG2 cells are growing well and the growth density is about 90%, digestion is performed and the single cell suspension of HepG2 cells is counted using a hemocytometer.

[0050] ③ After counting the cells, prepare the cell suspension to be inoculated according to the concentration, and then inoculate HepG2 cells on the PDMS base glue with different hardness.

[0051] ④HepG2 cells were cultured at 37°C and 5% CO2 for 48 hours.

[0052] ⑤ When the HepG2 cell density reaches about 80%-90%, the HepG2 cells are harvested.

[0053] ⑥Use Trizol lysis buffer provided in the RNA extraction kit to lyse HepG2 cells and prepare for RNA extraction.

[0054] (4) RNAseq data analysis

[0055] In the early stage, HepG2 liver cancer cells were cultured on substrates of different hardness and RNAseq analysis was performed on them. According to the Log2FC(Softer / Harder) values ​​of lncRNAs differentially expressed on different substrate hardness in the sequencing data, they were divided into lncRNAs upregulated on harder substrates (Log2FC(Softer / Harder)>1) and lncRNAs downregulated on harder substrates (Log2FC(Softer / Harder)<-1). Many of these lncRNAs are new transcripts, so we selected lncRNAs reported in the literature as the objects of subsequent analysis and prediction.

[0056] 1.2. qRT-PCR verification of differential expression of lncRNA on substrates of different hardness

[0057] HepG2 liver cancer cells cultured on substrates of different hardness were collected, RNA was extracted, and qRT-PCR experimental steps were performed to detect the expression differences of lncRNA. The primers used for detection are shown in Table 1.

[0058] Table 1 qRT-PCR primer sequences

[0059]

[0060] 1.3 ORFfinder and GWIPS-viz Browser predict ORFs with coding potential

[0061] (1)Use GWIPS-viz Browser

[0062] ①Open the URL in the browser https: / / gwips.ucc.ie / .

[0063] ② On the genome page of GWIPS-viz, select the organism of interest (human in this case) in the Genome drop-down menu and enter the gene ID in the Gene Search box.

[0064] ③ Scroll down to the following track sections: "Ribosome Profile - Start Ribosome", "Ribosome Profile - Elongation Ribosome", and "mRNA Sequence Coverage", and set the corresponding "Global Aggregation Track" to "Full". In addition, set "Basic Position" and "Refseq Gene Track" in the "Annotation Track" group to "Full".

[0065] ④ Zoom to the area of ​​interest (such as an exon on a transcript): Click and hold the left mouse button on the edge of the exon in the "Basic Position Track", drag the mouse to the other edge of the exon, and then release the mouse button.

[0066] (2)ORFfinder use

[0067] ①Open the URL in the browser https: / / www.ncbi.nlm.nih.gov / orffinder / .

[0068] ② On the ORFfinder page, enter the gene accession number or the gene sequence in FASTA format in the first column, and you can also enter the coordinates of the query sequence in the attached column.

[0069] ③ In the second column, select the following information: minimum ORF length, classical genetic code, and ORF start codon. Submit. ④ After submission, you can see the number of ORFs contained in the gene, the location, direction, length, and translated amino acid sequence of each ORF.

[0070] (3) Prediction of ORFs with coding potential

[0071] ① First, open the Ensembl database in your browser ( http: / / asia.ensembl.org / index.html ) and select the Human GRCh38.p13 version.

[0072] ② Retrieve the transcript information of lncRNA, which includes the number of transcripts and specific data in each transcript, such as the location, direction and number of exons of a transcript in the chromosome.

[0073] ③Based on the search information, record the location of each exon of each transcript of each lncRNA on the chromosome.

[0074] ④Open GWIPS-viz Genome Browser, enter the chromosomal distribution location of a certain exon in the search bar, check the corresponding "track information", you can see the specific segments of the translation start signal and translation extension signal on the exon, and record the chromosomal location with translation information.

[0075] ⑤Query the ORF distribution information of the corresponding transcript of the lncRNA where the exon is located in ORFfinder.

[0076] ⑥ Match the fragments with translation start signals and translation extension signals in GWIPS-viz Genome Browser with the ORFs retrieved in ORFfinder one by one. Those that can be matched are preliminarily identified as ORFs with coding potential, and their specific information is recorded.

[0077] 1.4 ORF-GFP fusion protein vector verifies the coding potential of ORF

[0078] (1) Preparation of LB solid (liquid) medium

[0079] Prepare a sterile 250mL conical flask, weigh 2.00g NaCl, 1.00g yeast extract, 2.00g tryptone and 3.00g agar (liquid does not need agar) and add them, then use a measuring cylinder to measure 200mL 3d H2O and pour it into the conical flask, stir with a glass rod to make the solid components completely mixed, seal the conical flask mouth with a breathable sealing film, and sterilize with high temperature and high pressure steam. The conical flask with antibiotics is added with 200μL kanamycin or ampicillin (if you want to make a blank culture plate, do not add antibiotics), the concentration is 0.05g / mL.

[0080] (2) Bacteria activation: conventional method.

[0081] (3) Preparation of competent cells: conventional method.

[0082] (4) Overview of plasmid construction process

[0083] ① Construction of pEGFP-N1-GFPmut plasmid

[0084] a. Using pEGFP-N1 plasmid as template, design synthetic primers, mutate the ATGGTG start codon of the wild-type GFP fragment to ATTGTT, and then amplify the target gene DNA fragment GFPmut using a high-fidelity enzyme. (When designing primers, add BamH I and Not I restriction sites at the 5' end of the front and back primers respectively)

[0085] b. The PCR product was subjected to agarose gel electrophoresis and then recovered and purified using the "Omega Gel Recovery Kit".

[0086] c. Double-digest the PCR product, and then double-digest the purified fragment with BamH I and Not I, and then purify it after digestion.

[0087] d. pEGFP-N1 was double-digested with BamH I and Not I, and then purified by gel recovery.

[0088] e. Use T4 ligase to connect the purified GFPmut fragment to the pEGFP-N1 vector after double enzyme digestion and purification. (Use Takara's DNALigation Kit Ver.2.1 kit)

[0089] f. The obtained ligation product was transformed into DH5α E. coli competent cells and plated on solid LB medium containing kanamycin.

[0090] g. On the second day, pick a single colony and expand the culture in liquid LB medium containing kanamycin.

[0091] h. Perform colony PCR test when the bacterial solution is turbid.

[0092] i. Screen out the colony PCR-positive strains and send them to Shanghai Sangon Biotechnology Co., Ltd. for DNA sequencing.

[0093] j. Expand the culture of the strain with correct sequencing and extract the plasmid.

[0094] ② Construction of ORF-GFPmut plasmid

[0095] a. Using the cDNA obtained by reverse transcription as a template, amplify the ORF of the target gene DNA fragment by PCR using a high-fidelity enzyme. (When designing primers, add seamless cloning sites of HindIII and BamH I to the 5' end of the front and rear primers respectively)

[0096] b. The PCR product was recovered and purified after agarose gel electrophoresis.

[0097] c. pEGFP-N1-GFPmut was cut with restriction endonucleases HindIII and BamH I, and then recovered and purified after enzyme digestion.

[0098] d. Recombinant clone the purified ORF fragment and the pEGFP-N1-GFPmut vector purified by double restriction digestion using a seamless cloning kit. The subsequent steps are the same as steps fj of "pEGFP-N1-GFPmut plasmid construction".

[0099] ③ Construction of ORFmut-GFPmut plasmid

[0100] a. Using the cDNA obtained by reverse transcription as a template, design and synthesize primers that mutate the start codon ATG of the ORF fragment to ATT, and amplify the target gene DNA fragment ORFmut using a high-fidelity enzyme. (When designing primers, add seamless cloning sites of Nhe I and Kpn I to the 5' end of the front and back primers respectively)

[0101] b. Recover and purify the PCR product.

[0102] c. pEGFP-N1-GFPmut was cut with restriction endonucleases Nhe I and Kpn I, and then purified by gel recovery after enzyme digestion.

[0103] d. Recombinant clone the purified ORFmut fragment and the pEGFP-N1-GFPmut vector purified by double restriction digestion using a seamless cloning kit. The subsequent steps are the same as steps fj of "pEGFP-N1-GFPmut plasmid construction".

[0104] (5) Amplify the target DNA gene fragment using high-fidelity enzyme PCR

[0105] PrimeSTAR Max DNA Polymerase high-fidelity enzyme kit from Takara (Japan) was used. The PCR reaction system was: PrimeSTAR Max Premix (2×) 25 μL, 1 μL each of forward and reverse primers, 1 μL of cDNA, and ddH2O supplemented to 50 μL. The PCR reaction conditions were: 98.0℃30sec; 98.0℃10sec, 58.0℃15sec, 72.0℃5sec, 35 cycles; 72.0℃5min.

[0106] (6) PCR product purification: Purification was performed using the Omega gel recovery kit.

[0107] (7) Double enzyme digestion reaction: vector pEGFP-N15 μL, 10× cut buffer 5 μL, BamHI 1 μL, Not I 1 μL, and ddH2O to 50 μL.

[0108] The above system was placed in a water bath at 37°C for 30 min. The other double enzyme digestion reaction systems were the same as above.

[0109] (8) T4 ligation (using Takara's DNA Ligation Kit Ver. 2.1)

[0110]

[0111] The above system was placed at 16°C, incubated for 30 min, and then placed in a 4°C refrigerator overnight.

[0112] (9) Seamless cloning (using Bio-Tech (China) Seamless Cloning Kit)

[0113]

[0114] The reaction system was placed at 50°C and incubated for 15 min.

[0115] (10) Conversion

[0116] ① Take a 100 μL tube of DH5α competent cell, place it on ice, wait for it to melt, add 10 μL of ligation product, flick the bottom of the centrifuge tube with your finger to mix it evenly, and let it stand in an ice box for 30 minutes.

[0117] ②After the centrifuge tube is heat-shocked in a 42°C water bath for 60-90 seconds, it is quickly placed in an ice box for 2-5 minutes.

[0118] ③ Add 1 mL of LB blank liquid culture medium without antibiotics to the product after ice bath, and incubate in a shaker at 37°C at 150 rpm for one hour.

[0119] ④ Centrifuge the bacterial solution at 2500xg for 3 minutes at room temperature. Then discard about 800μL of the supernatant medium, keep about 200μL of the supernatant, and use a pipette to blow the bacterial solution several times until it is evenly resuspended.

[0120] ⑤ Add the mixed bacterial liquid to the LB solid culture plate containing ampicillin, use pre-sterilized glass beads with high temperature and high pressure steam, spread it evenly, and culture it upside down in a 37°C incubator for 14-16 hours.

[0121] (11) Positive clone detection and expansion culture

[0122] ① On the second day, take out the culture plate, pick a single colony in the clean bench, place it in a 1.5mL centrifuge tube, place 800μL of LB liquid culture medium containing ampicillin in the centrifuge tube, pipette and mix evenly, and mark it.

[0123] ②Incubate the bacterial suspension of a single colony at 37°C and 200 rpm for 3-5 hours.

[0124] ③ The turbid bacterial solution was tested by colony PCR, and the reaction system was:

[0125]

[0126] The PCR reaction conditions were: 95.0°C for 5 min; 95.0°C for 30 sec, 55.0°C for 30 sec, 72.0°C for 30 sec, 35 cycles; 72.0°C for 5 min.

[0127] ④After the PCR reaction is completed, 6 μL of the reaction product is used for agarose gel electrophoresis, and the position of the target band is observed with a UV lamp to determine whether the obtained product meets the size of the target fragment. (This PCR Taq enzyme does not require additional DNA loading buffer)

[0128] ⑤ Add the bacterial solution with the correct band size to LB liquid culture medium containing 10 mL of ampicillin, culture it at 37°C overnight, and run the shaker at 220 rpm.

[0129] ⑥ Take 1 mL of bacterial solution and send it to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and store the rest of the bacterial solution at 4°C.

[0130] ⑦ Add the correctly sequenced bacterial solution into 20 mL of LB liquid culture medium containing ampicillin, and expand the culture at 37°C at a speed of 220 rpm.

[0131] (12) Plasmid extraction: The plasmid was extracted using the plasmid mini-prep kit from Tiangen Biochemical Technology Co., Ltd. (China).

[0132] (13) Cell transfection

[0133] ①Inoculate 293T cells in a 24-well cell culture plate and perform a plasmid transfection experiment when the 293T cell density is 40%-60%. (Use QIAGEN's Transfection Regent transfection kit)

[0134] ② Take a 1.5mL centrifuge tube and add 60μL of EC Buffer provided in the kit, then add the constructed transient transfection plasmid according to the volume of 0.2μg, and then add 1.6μL of Enhancer solution provided in the kit, use the pipette tip to blow gently to stir evenly, and let it stand at room temperature for 5 minutes.

[0135] ③ Add 5 μL of Effectene solution provided in the kit, tap the bottom of the centrifuge tube with your finger to mix it thoroughly, and let it stand at room temperature for 8-10 minutes.

[0136] ④ Add 500 μL of DMEM culture medium, gently blow and mix with the pipette tip, add the transfection complex to the culture plate, and gently shake back and forth to mix them evenly, then make the plasmid markers corresponding to each well, and then put the culture plate back into the incubator.

[0137] ⑤ After 24-48 hours, observe the expression of GFP green fluorescence under an inverted fluorescence microscope.

[0138] 1.5 Overexpression and knockdown of peptide SAP

[0139] 1.5.1 Overexpression of peptide SAP

[0140] Primers for overexpressing polypeptide SAP were designed and commissioned to be synthesized by Shanghai Sangon Biotechnology Co., Ltd. The specific primer sequences are shown in Table 2.

[0141] Table 2 Primers for constructing SAP overexpression recombinant plasmid

[0142]

[0143] 1.5.2 Construction of recombinant plasmid for knocking down the transcript USP34-DT-201 containing the polypeptide SAP

[0144] (1) BLOCK-iT on the Life Technologies website TM The RNAi Designer online tool (https: / / rnaidesigner.thermofisher.com / rnaiexpress / insert.do) was used to design two shRNA primers targeting the SAP transcript USP34-DT-201, and Qingke Biotechnology Co., Ltd. (China) was commissioned to synthesize the primers. The specific primer sequences are shown in Table 3.

[0145] Table 3 shRNA sequence primers targeting peptide SAP transcript USP34-DT-201

[0146]

[0147]

[0148] (2) High-fidelity PCR was used to amplify the target gene DNA fragment using Takara's PrimeSTARMaxDNA Polymerase high-fidelity enzyme kit.

[0149] (3) PCR product purification (Omega gel recovery kit).

[0150] (4) Double enzyme digestion reaction (double enzyme digestion with Xba I and Sal I).

[0151] (5) Seamless cloning (using the Seamless Cloning Kit from Bio-Time).

[0152] (6) Transformation into competent Escherichia coli Stbl3.

[0153] (7) Detection and expansion of positive clones.

[0154] (8) Plasmid extraction (using Tiangen Plasmid Mini-Extraction Midi Kit).

[0155] 1.5.3 Construction of MHCC97H and HepG2 cell lines with stable overexpression and knockdown of peptide SAP (1) Virus packaging

[0156] ①Inoculate 293T cells in a 60mm cell culture dish, and perform plasmid transfection experiments after the growth density of 293T cells reaches 50%-70%.

[0157] ② Take a 1.5mL centrifuge tube and add 150μL of EC Buffer provided in the kit. Then add the plasmids pCMV-dR8.2dvpr, pCMV-VSV-G and pLenti-CMV-EGFP / pLenti-CMV-SAP in a ratio of 1:3:4, a total of 5μg of the system to EC Buffer, and finally add 8μL of Enhancer solution provided in the kit. Use a pipette tip to gently pipette to mix evenly, and let it stand at room temperature for 5 minutes.

[0158] ③Finally, add 25 μL of the Effectene solution provided in the kit, gently stir the bottom of the centrifuge tube with your fingers to mix the ingredients thoroughly, and incubate at room temperature for 10-15 minutes for the next experiment.

[0159] ④ Add 1000 μL of DMEM culture medium, gently blow and mix with the pipette tip, add the transfection complex to the culture dish, and gently shake back and forth to mix them evenly, make the corresponding plasmid mark on each culture dish, and then put the culture dish back into the incubator.

[0160] ⑤On the second day, after the cells have been cultured for 12 hours, discard the waste culture medium, replace it with fresh DMEM culture medium and start timing.

[0161] ⑥ Start collecting viruses 24 hours after the first fluid change at the end of transfection. Carefully collect the virus supernatant into a new sterile 10mL centrifuge tube in the clean bench. Continue to add 4mL of fresh DMEM complete medium to the culture dish. ⑦ Collect viruses again 48 hours after the first fluid change at the end of transfection. Carefully collect the virus supernatant into the 10mL centrifuge tube from the previous day in the clean bench. The culture dish should be disinfected with 75% alcohol and discarded.

[0162] ⑧ Centrifuge the virus supernatant collected twice at 4000 rpm for 5 min, filter the virus supernatant using a 0.22 μm pore size filter membrane, and then dispense into 5 mL centrifuge tubes. If long-term storage is desired, freeze the supernatant in a -80°C refrigerator. (2) Virus infection of MHCC97H and HepG2 cells

[0163] ① After the MHCC97H and HepG2 cells grew to about 90% in the original culture flask, they were digested with trypsin and inoculated into 60 mm culture dishes for growth.

[0164] ② When the cell growth density reaches 40%-50%, wash the cells twice with sterile PBS buffer, and then add DMEM / MEM complete medium and virus supernatant in a 1:1 ratio to the culture dish. At the same time, add 4 μL polybrene to the culture dish to improve the efficiency of virus infection.

[0165] ③ Place the cell culture dish in a cell culture incubator and incubate for more than 48 hours.

[0166] ④Observe the cells under a microscope after 48 hours. When the growth density of the infected cells reaches about 80%, wash the cells twice with sterile PBS buffer.

[0167] ⑤Then add DMEM / MEM culture medium containing 2 μg / mL Puro drug into the cell culture dish to screen stable cell lines that overexpress the corresponding target fragment.

[0168] ⑥After that, change the medium every 24 hours and add DMEM / MEM culture medium containing 2μg / mL Puro drug. It takes about 2-3 weeks to screen cell lines.

[0169] ⑦Continue to observe the cell status until there is no obvious apoptosis in the cells, and then use qRT-PCR experiments to detect whether the SAP-overexpressing MHCC97H and HepG2 stable cell lines are successfully constructed.

[0170] 1.6 Immunofluorescence detection of endogenous expression of peptide SAP

[0171] (1) Human 293T, MHCC97H and HepG2 cells were inoculated into 24-well cell culture plates respectively and allowed to adhere to the plate and grow to the optimal state.

[0172] (2) When the experimental time point arrives, the culture medium in the 24-well plate is aspirated and discarded, washed twice with PBS, and 1 mL of 4% PFA (paraformaldehyde) is added, and fixed at room temperature for 30 min.

[0173] (3) Discard 4% PFA and wash with PBS three times, 5 min each time.

[0174] (4) Add 1 mL of blocking solution (blocking solution: 5% BSA and 0.4% TritonX-100 dissolved in PBS) and block for 60 min at room temperature.

[0175] (5) Discard the blocking solution in the 24-well plate, add 200 μL of primary antibody solution diluted at a ratio of 1:500 (the primary antibody solution is diluted with the blocking solution), and place in a 4°C refrigerator overnight.

[0176] (6) On the next day, the primary antibody solution was recovered and the cells were washed three times with PBS buffer, each time for 10 min.

[0177] (7) Use a pipette to aspirate the remaining PBS buffer in the 24-well plate, add 200 μL of red fluorescent labeled Cy3 secondary antibody dilution solution (the fluorescent secondary antibody solution is diluted with blocking buffer at a dilution ratio of 1:500), and incubate at room temperature in the dark for one hour.

[0178] (8) Discard the secondary antibody solution and wash the cells three times with PBS buffer, each time for 10 min.

[0179] (9) Use a pipette to remove the remaining PBS buffer in the 24-well plate, add 200 μL of DAPI blue fluorescent marker dilution solution for staining cell nuclei (the dilution ratio of DAPI with blocking solution is 1:1000), and incubate at room temperature in the dark for 5 minutes. (10) Remove DAPI and wash the cells three times with PBS buffer, each time for 10 minutes. Add 200 μL PBS buffer to each well and prepare for photography.

[0180] 1.7 Transwell assay to detect the effect of SAP on the migration of hepatoma cells

[0181] (1) Observe the overexpressed or knocked-down MHCC97H and HepG2 stable cell lines with good growth status under a microscope. When the cell growth density reaches about 90%, use trypsin to digest and then add DMEM / MEM culture medium to make a single cell suspension. Transfer the suspension into a sterile 1.5 mL centrifuge tube, centrifuge it using a handheld centrifuge, and carefully discard the supernatant with a pipette.

[0182] (2) Resuspend the cells at the bottom of the centrifuge tube with DMEM / MEM medium containing 1% FBS and dilute them to a single cell suspension of appropriate concentration.

[0183] (3) Spray and clean the blood cell counting chamber and the matching cover glass with 75% alcohol, and then place them in the clean bench to air dry naturally.

[0184] (4) Cover the middle counting area of ​​the hemocytometer with a coverslip and slowly add the diluted cell suspension to the edges of the upper and lower counting areas until it covers all the counting areas. Count the cells under an inverted microscope and record the concentration of the corresponding cells in each group of samples.

[0185] (5) Dilute the single cell suspension of each group of samples after counting to a total of 10,000 single cells per 200 μL.

[0186] (6) Place a Transwell chamber in a 24-well plate, add 500 μL of DMEM / MEM medium containing 10% FBS to the lower chamber, and add 200 μL of diluted cell suspension to the upper chamber. Repeat 3 times for each group of samples. (Note that no bubbles should be generated in either the lower or upper chamber, otherwise the cell migration process will be affected.) After inoculation, place the 24-well plate in a cell culture incubator at 37°C and culture for 48 hours.

[0187] (7) After the culture is completed, remove the 24-well plate containing the Transwell chamber. Pour the PBS buffer into a 50 mL beaker, take out the Transwell chamber and put it into the PBS buffer. Gently shake it back and forth to wash off the residual culture medium on the inner surface of the chamber as much as possible. Take another 24-well plate and add 500 μL of pre-cooled methanol to each well. Use tweezers to carefully pick up the chamber so that its chamber membrane is completely immersed in the pre-cooled methanol. Place the chamber and the 24-well plate in a 4°C refrigerator for 30 minutes to fix the cells.

[0188] (8) Gently shake the chamber back and forth in PBS buffer to thoroughly wash away the residual methanol, place it in a 24-well plate containing 500 μL of 1X crystal violet solution in each well, so that the chamber membrane is completely immersed in the crystal violet solution, and let it stand at room temperature for 1 hour or longer to stain the cells.

[0189] (9) Soak a medical cotton swab in PBS buffer, and then carefully wipe the inner surface of the chamber membrane with the medical cotton swab to wipe off the non-migrated cells. Then use kitchen paper to gently absorb the PBS buffer on the edge of the chamber, and finally dry the water on the chamber membrane in a ventilated place, and then take pictures under an inverted microscope (take 3 pictures randomly for each chamber), and finally count and save them using ImageJ.

[0190] (10) Clean and recycle the chamber. The chamber membrane was soaked in trypsin at 37°C for 10 min, then soaked in glacial acetic acid for 1 h, and then soaked in 75% alcohol overnight. Finally, it was washed with ddH2O and placed in a clean bench and irradiated with ultraviolet light overnight.

[0191] 1.8 Transwell assay to detect the effect of SAP on the invasion of liver cancer cells

[0192] (1) Place Matrigel in an ice box one day in advance, and then place the entire ice box in a 4°C refrigerator to allow the Matrigel to fully dissolve. Take a sterile 1.5 mL centrifuge tube and pre-cool it on ice. Add 40 μL of the Matrigel mixture to each chamber and the number of Transwell chambers used. Mix the Matrigel and DMEM blank culture medium in a volume ratio of 1:9 to prepare an appropriate amount of gel solution. Place the chamber on a 24-well plate, add 40 μL of the Matrigel mixture to each chamber, and then tap the 24-well plate to evenly cover the chamber membrane with the Matrigel mixture. Incubate in a 37°C cell culture incubator for 4-6 hours to facilitate the solidification of the Matrigel.

[0193] (2) Observe the overexpressed or knocked-down MHCC97H and HepG2 stable cell lines with good growth under a microscope. When the cell growth density reaches about 90%, use trypsin to digest and then add DMEM / MEM medium to make a single cell suspension. Transfer it into a sterile 1.5 mL centrifuge tube, centrifuge it with a handheld centrifuge, and carefully discard the supernatant with a pipette. Then resuspend the cells at the bottom of the centrifuge tube with DMEM / MEM medium containing 1% FBS and dilute it to a single cell suspension of appropriate concentration.

[0194] (3) Spray the blood cell counting plate and the matching cover glass with 75% alcohol and place them in a clean bench to air dry. Cover the middle counting area of ​​the blood cell counting plate with a cover glass and slowly add the diluted cell suspension to the edges of the upper and lower counting areas until it covers all the counting areas. Count the cells under an inverted microscope and record the concentration of the corresponding cells in each group of samples.

[0195] (4) Dilute the single-cell suspension of each group of samples after counting to a total of 10,000 single cells per 200 μL.

[0196] (5) Place a Transwell chamber with solidified Matrigel in a 24-well plate, add 500 μL of DMEM / MEM medium containing 10% FBS to the lower chamber, and add 200 μL of diluted cell suspension to the upper chamber. Repeat 3 times for each group of samples. (Note that no bubbles should be generated in either the lower or upper chamber, otherwise the cell migration process will be affected.) After inoculation, place the 24-well plate in a cell culture incubator at 37°C and culture for 48 hours.

[0197] (6) After the culture is completed, remove the 24-well plate containing the Transwell chamber. Pour the PBS buffer into a 50 mL beaker, take out the Transwell chamber and put it into the PBS buffer. Gently shake it back and forth to wash off the residual culture medium on the inner surface of the chamber as much as possible. Take another 24-well plate, add 500 μL of pre-cooled methanol to each well, use tweezers to carefully pick up the chamber so that its chamber membrane is completely immersed in the pre-cooled methanol, and place the chamber and the 24-well plate in a 4°C refrigerator for 30 minutes to fix the cells.

[0198] (7) Gently shake the chamber back and forth in PBS buffer to thoroughly wash away the residual methanol, place it in a 24-well plate containing 500 μL of 1X crystal violet solution in each well, so that the chamber membrane is completely immersed in the crystal violet solution, and let it stand at room temperature for 1 hour or longer to stain the cells.

[0199] (8) Soak a medical cotton swab in PBS buffer, and then carefully wipe the inner surface of the chamber membrane with the medical cotton swab to wipe off the non-migrated cells.

[0200] (9) Then, use kitchen paper to gently absorb the PBS buffer at the edge of the chamber, and finally dry the moisture on the chamber membrane in a ventilated place. Then, take pictures under an inverted microscope (take 3 pictures randomly for each chamber), and finally use Image J to count and save.

[0201] (10) Clean and recycle the chamber. The chamber membrane was soaked in trypsin at 37°C for 10 min, then soaked in glacial acetic acid for 1 h, and then soaked in 75% alcohol overnight. Finally, it was washed with ddH2O and placed in a clean bench and irradiated with ultraviolet light overnight.

[0202] 1.9 Western blot detection of the effect of SAP on the expression of proteins related to migration and invasion of liver cancer cells

[0203] Western blot was performed using conventional methods.

[0204] 2 Experimental results

[0205] 2.1 Screening of lncRNAs with differential expression on substrates of different hardness in RNAseq data

[0206] The results are as follows Figure 1 As shown, among the 11 lncRNAs, 8 lncRNAs were upregulated on harder substrate hardness, and 3 lncRNAs were downregulated on harder substrate hardness.

[0207] 2.2 qRT-PCR verification of differential expression of lncRNA on substrates of different hardness

[0208] The results are as follows Figure 2 As shown, in HepG2 liver cancer cells, lncRNA SSBP3-AS1 was downregulated on stiffer substrates, while lncRNA MAFA-AS1 and AC016727.1 were upregulated on stiffer substrates.

[0209] 2.3 ORF-GFP fusion protein vector verifies the coding potential of ORF

[0210] Schematic diagram of the ORF-GFP fusion protein vector structure Figure 3 shown.

[0211] The expression of GFP green fluorescent protein can be observed in SSBP3-AS1-ORF1, SSBP3-AS1-ORF3, AC016727.1-ORF3, and AC016727.1-ORF7 ( Figure 4 ), indicating that the start codons of these four ORFs are functional and have certain coding potentials, and may encode a polypeptide.

[0212] The expression of GFP green fluorescent protein can be observed in 293T cells transfected with GFPwt and SAP-ORF-GFPmut plasmids ( Figure 5 ), while no green fluorescent protein expression was observed in 293T cells transfected with GFPmut and SAP-ORFmut-GFPmut plasmids. This further verified that the start codon of AC016727.1-ORF7 was functional and had certain coding potential, possibly encoding a polypeptide SAP.

[0213] 2.4 Endogenous expression of peptide SAP

[0214] Through immunofluorescence experiments, anti-SAP antibodies were used to detect the expression of natural endogenous SAP in 293T, MHCC97H and HepG2 cells. It can be seen that the polypeptide SAP can be expressed in these three human cells and is mainly located in the cytoplasm ( Figure 6 ).

[0215] The above experimental results show that lncRNAAC016727.1, which responds to different substrate hardness, can encode a polypeptide SAP.

[0216] The nucleotide sequence of the transcript of lncRNAAC016727.1 is shown in SEQ ID NO.15:

[0217] GGCTCTGAGGCCCGGGGAGCGCCAGTTCCTTGAAGGTCCTCCGTCCTGGAGCAGTAAGATCCTTGACGTAACTAGAAATGGCAGGACGGAGTCTCGCTCTGTCACCCAAGCTGGAATGCCGTGGCGCAATCTTAGTTCACTGCAACCTCTGCCTCCTCGGTCCAAGCCATTCTCCTGCCTCAGCCTCCCGAATAGGCAAATGAATAAAAG AACATCTTTTATTTACCCGTATCTGCGACATTCCTCATAGTATGTTTGAAAGCCCAAATGATGGAATCCAAAACAAGTTTAAACTGTGTAGGTGGAATAGCAAGGAATGCTGGGAAACAATGAGAATTGACAGCCTGAAGTAGTAAGAAAAAGTTCGTTCTATGTTCAGGATATTCTTCAAAGTCCTAAAAATAAGTGGTGGAAACAAAA.

[0218] The amino acid sequence of the polypeptide SAP encoded by lncRNA AC016727.1 is (SEQ ID NO.16): MSQIRVNKRCSFIHLPIREAEAGEWLGPRRQRLQ.

[0219] 2.5 Effect of peptide SAP on migration of hepatoma cells

[0220] The results are as follows Figure 7 As shown, compared with the control group (L-Ctrl), the number of migrating MHCC97H and HepG2 cells overexpressing SAP (L-SAP) increased, indicating that overexpression of polypeptide SAP enhanced the migration ability of MHCC97H and HepG2 cells.

[0221] 2.6 Effect of low SAP expression on hepatoma cell migration

[0222] Compared with the control group (shRNA#NC), the number of migrating MHCC97H and HepG2 cells in which the expression of USP34-DT-201, the SAP transcript, was knocked down (shRNA#1-SAP and shRNA#2-SAP) was significantly reduced ( Figure 8 ), indicating that knocking down the expression of USP34-DT-201, the transcript where the polypeptide SAP is located, can inhibit the migration ability of MHCC97H and HepG2 cells.

[0223] 2.7 Effect of high SAP on invasion of hepatocellular carcinoma cells

[0224] Compared with the control group (L-Ctrl), the invasion ability of MHCC97H cells overexpressing SAP (L-SAP) was significantly enhanced ( Fig. 9 ), indicating that overexpression of polypeptide SAP promotes the invasion of MHCC97H liver cancer cells.

[0225] Effect of SAP on the expression of proteins related to migration and invasion of hepatocellular carcinoma cells

[0226] The results are as follows Fig.10 As shown, in MHCC97H cells, compared with the control group (L-Ctrl), when the polypeptide SAP (L-SAP) was overexpressed, the expression level of E-cad, a protein negatively correlated with cell migration and invasion, was downregulated by about 50%, but there was no statistical difference; N-cad and VIM, which were positively correlated with cell migration and invasion, were significantly upregulated by about 50%; N-cad, which was positively correlated with cell migration and invasion, was upregulated by about 130%, and VIM was upregulated by about 30%, and there was no statistical difference between the two. In HepG2 cells, compared with the control group (L-Ctrl), when the polypeptide SAP (L-SAP) was overexpressed, the expression level of E-cad, a protein negatively correlated with cell migration and invasion, was downregulated by about 40%; N-cad, VIM and MMP9, which were positively correlated with cell migration and invasion, were upregulated by about 20%, 50% and 90%, respectively, but there was no statistical difference; MMP2, which was positively correlated with cell migration and invasion, was significantly upregulated by about 40%. These results show that polypeptide SAP promotes the migration and invasion of liver cancer cells.

[0227] 3 Analysis

[0228] This study took liver cancer as the research background, and used RNAseq and qRT-PCR to screen lncRNAs that were differentially expressed in HepG2 cells on different substrate hardnesses. ORFs with coding potential were screened through bioinformatics websites such as LNCipedia, GWIPS-viz, and ORFfinder, and then the polypeptide SAP encoded by the ORF on the lncRNA was verified by ORF-GFP fusion protein vector construction and immunofluorescence experiments. Then, in order to explore the effect of polypeptide SAP on the proliferation of liver cancer cells, MHCC97H and HepG2 liver cancer stable cell lines that overexpressed polypeptide SAP and knocked down the expression of the transcript USP34-DT-201 where polypeptide SAP was located were constructed, respectively. Then, they were used as research objects, and the effect of polypeptide SAP on the proliferation ability of liver cancer cells was detected by CCK-8 experiments and clone formation experiments at the cellular level. At the molecular level, Western blot was used to detect the expression of tumor cell proliferation-related marker proteins to verify the effect of polypeptide SAP on the proliferation of liver cancer cells. Finally, in order to explore the effect of peptide SAP on the migration and invasion of liver cancer cells, the above two types of cell lines were used as research objects. The effect of peptide SAP on the migration and invasion of liver cancer cells was detected by Transwell experiment at the cellular level, and the expression of tumor cell migration and invasion-related marker proteins was detected by Western blot at the molecular level to verify the effect of peptide SAP on the migration and invasion of liver cancer cells. The main conclusions are as follows:

[0229] (1) The lncRNA AC016727.1, which is differentially expressed on substrates of different hardness, encodes a polypeptide SAP: RNAseq found that HepG2 cells had a series of differentially expressed lncRNAs on substrates of different hardness; the prediction results of the LNCipedia database showed that lncRNA SSBP3-AS1, MAFA-AS1 and AC016727.1 had certain coding potential; qRT-PCR further verified that these three lncRNAs were differentially expressed on substrates of different hardness; then, GWIPS-viz and ORFfinder were used to screen out 10 ORFs with potential coding ability from the above three lncRNAs; and then the results of cells transfected with ORF-GFP fusion protein vectors showed that SSBP3-AS1-ORF1, SSBP3-AS1-ORF3, AC016727.1-ORF3, and AC016727.1-ORF7 may encode a polypeptide. Among them, AC016727.1-ORF7 has the strongest coding ability; finally, immunofluorescence was used to verify that AC016727.1-ORF7 can encode a naturally endogenously expressed polypeptide SAP.

[0230] (2) Peptide SAP promotes liver cancer cell proliferation: First, we constructed MHCC97H and HepG2 liver cancer stable cell lines that overexpressed peptide SAP (L-SAP); at the same time, we constructed MHCC97H and HepG2 liver cancer cell lines that knocked down the expression of the transcript USP34-DT-201 where the peptide SAP is located (shRNA#1-SAP and shRNA#2-SAP); then, through CCK-8 experiments, we found that overexpression of peptide SAP in the above two liver cancer cells promoted cell proliferation, while knocking down the expression of the transcript USP34-DT-201 where the peptide SAP is located in liver cancer cells inhibited cell proliferation; clone formation experiments also showed that when the peptide SAP is overexpressed in liver cancer cells, the cell clone formation ability is enhanced and cell proliferation is promoted, while when the transcript USP34-DT-201 where the peptide SAP is located is knocked down in liver cancer cells, the cell clone formation ability is weakened and cell proliferation is inhibited; finally, through Western blotting, we found that overexpression of peptide SAP in liver cancer cells can enhance cell clone formation ability and promote cell proliferation, while knocking down the transcript USP34-DT-201 where the peptide SAP is located in liver cancer cells can weaken cell clone formation ability and inhibit cell proliferation. Blot analysis showed that in liver cancer cell lines overexpressing polypeptide SAP, PCNA and MCM2 proteins, which are positively correlated with cell proliferation, were upregulated, while p16, a protein negatively correlated with cell proliferation, was downregulated, further verifying that polypeptide SAP promoted the proliferation of liver cancer cells.

[0231] (3) Peptide SAP promotes liver cancer cell migration and invasion: Transwell experiments showed that overexpression of peptide SAP enhanced the migration ability of liver cancer cells; knockdown of the transcript USP34-DT-201 where peptide SAP was located could inhibit the migration ability of liver cancer cells; in addition, it was found that overexpression of peptide SAP promoted the invasion of MHCC97H cells; finally, Western blot showed that in the two liver cancer cell lines overexpressing peptide SAP, the expression levels of N-cad, VIM, MMP2, and MMP9 proteins, which were positively correlated with cell migration and invasion, were upregulated, while the expression level of E-cad, a protein negatively correlated with cell migration and invasion, was downregulated, further verifying that peptide SAP promoted the migration and invasion of liver cancer cells.

Claims

1. Inhibition of lncRNA AC016727.1 and / or a substance expressed by a polypeptide encoded by the same in the preparation of a drug for inhibiting the migration of liver cancer cells, wherein the substance is shRNA, and the shRNA is shRNA#1 or shRNA#2, The primer sequence of the shRNA#1 is: shRNA#1-F: 5'-CCGGGGAGCAGTAAGATCCTTGACGCTCGAGCGTCAAGGATCTTACTGCTCCTTTTTG-3', shRNA#1-R: 5'-AATTCAAAAAGGAGCAGTAAGATCCTTGACGCTCGAGCGTCAAGGATCTTACTGCTCC-3'; The primer sequence of the shRNA#2 is: shRNA#2-F: 5'-CCGGGCAGTAAGATCCTTGACGTAACTCGAGTTACGTCAAGGATCTTACTGCTTTTTG-3', shRNA#2-R: 5'-AATTCAAAAAGCAGTAAGATCCTTGACGTAACTCGAGTTACGTCAAGGATCTTACTGC-3'.

Images