CN107354157B - siRNA for specifically inhibiting LAMB1 gene expression, recombinant vector and application thereof - Google Patents
siRNA for specifically inhibiting LAMB1 gene expression, recombinant vector and application thereof Download PDFInfo
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Abstract
The invention discloses siRNA for specifically inhibiting LAMB1 gene expression, a recombinant vector thereof and application thereof in reversing ovarian cancer taxol resistance, belonging to the technical field of molecular biology and biomedicine. The siRNA comprises a sense strand and an antisense strand, wherein the sense strand: 5'-UGUUUGAAAGCCGAAUCUGCG-3', respectively; antisense strand: 5'-CAGAUUCGGCUUUCAAACAAA-3' are provided. The siRNA provided by the invention can specifically and efficiently inhibit the mRNA and protein expression of LAMB1 gene in tumor cells, reduce the proliferation of tumor cells, increase the apoptosis, reduce the migration and invasion capacity of tumor cells, and effectively reverse the drug resistance of ovarian cancer cells to paclitaxel. The invention also provides application of the siRNA and the recombinant vector thereof in preparing medicines for treating ovarian cancer, hepatocellular carcinoma, colorectal cancer, malignant glioma, prostatic cancer or gastric cancer and reversing drug resistance of ovarian cancer.
Description
Technical Field
The invention relates to the technical field of molecular biology and biological medicine, in particular to siRNA for specifically inhibiting LAMB1 gene expression, a recombinant vector and application thereof.
Background
RNA interference (RNAi) is a widely occurring sequence-specific post-transcriptional gene silencing mechanism in animals and plants. In 1998, Andrew Fire, an American scientist, first found that a mixture of sense and antisense strands (i.e., dsRNA) produced at least 10 times the effect of gene silencing over antisense nucleotides in C.elegans, and induced the same phenomenon in progeny. The mechanism research of RNAi phenomenon shows that the micro siRNA can silence a large amount of target RNA through post-transcriptional gene silencing, and the key molecule for efficiently and specifically degrading homologous RNA to cause sequence-specific gene silencing is small double-stranded oligonucleotide with 21-23 bases, also called small interfering RNA (siRNA). Further research shows that the double-stranded RNA shorter than 21bp or longer than 25bp can not effectively start RNAi, and the gene silencing effect is obviously reduced or even eliminated as long as one base is mismatched, so that the specificity of the siRNA effect is fully embodied.
siRNA showing strong gene silencing efficacy has attracted attention as a significant finding in recent biotechnology because it has specificity and high efficiency in blocking gene suppression function of homologous genes and is a powerful tool for gene function studies. siRNA has many incomparable advantages and features over traditional methods. Although there are several methods for inhibiting the expression of specific genes, such as antisense RNA, knock-out (knock-out), etc., siRNA has shown significant advantages over these techniques: it has higher specificity and persistence compared to antisense RNA; compared with complex and time-consuming gene knockout, siRNA is a simpler and more effective means. At present, the method of using siRNA as gene silencing is widely used for the research of malignant tumor mechanisms, the key point of the research is mainly focused on how to improve the gene silencing efficacy, including target gene locus selection, optimization of an introduction system, influence on host cell functions and the like, and the research and development process of tumor specific high-efficiency targeted therapy strategies based on the technology is promoted.
Ovarian cancer is one of the most common gynecological malignant tumors, and the mortality rate of ovarian cancer is the first of all gynecological tumors. The biggest obstacle to ovarian cancer treatment is the development of tumor cell resistance, especially the emergence of multidrug resistance. With the implementation of the platinum/taxol medicament combined chemotherapy, the primary chemotherapy response rate of the ovarian cancer can reach 80%. But most patients relapse within 2-3 years. Even if the ovarian cancer is treated by chemotherapeutics with completely different action mechanisms, the ovarian cancer is resistant, so that the survival rate of the treated tumor cannot be obviously improved. The five-year survival rate of the advanced ovarian cancer always ranges from 20% to 30%. High recurrence rate and high drug resistance rate after recurrence are one of the leading causes of high mortality of ovarian cancer, and the search for a solution to drug resistance in ovarian cancer chemotherapy is an important subject of current tumor research.
the ECM forms a complex network structure, supports and connects tissue structures, regulates physiological activities of tissues and cells, can combine signal molecules such as growth factors and the like to start various signal transduction pathways so as to regulate functions of cell morphology, migration movement, proliferation, differentiation and the like, the occurrence, development, invasion and metastasis of malignant tumors are frequently accompanied by changes of ECM expression, laminin beta 1(laminin β, LAMB1), chromosomes are positioned at 7q31.1, are one of large families of extracellular basal lamina heterotrimeric glycoproteins, play an important role in a plurality of basic biological processes, including cell adhesion and migration, embryonic development, angiogenesis, tissue, wound healing and the like, and the fact that some documents reported in recent years show that the malignant tumor contains high-grade glioma gene expression in the colorectal cancer, tissue, wound healing and the like, and the malignant tumor tissue contains high-grade glioma gene expression of LAMB 18, and the glioma gene expression of LAMB 2 in the malignant tumor tissue contains high-grade glioma gene expression of LAMB 18, and the glioma gene expression of LAMB 19 is obviously detected on the malignant tumor tissue, and the malignant tumor is obviously expressed on the prostate tissue.
Although the specific mechanism of action of LAMB1 in tumors is not clear so far, the involvement of LAMB1 in the pathogenesis and progression of tumors has been increasingly recognized. Our previous studies showed that LAMB1 is abnormally elevated in the paclitaxel-resistant cell line A2780/Taxol for ovarian cancer, but whether the gene is related to the onset and drug resistance of ovarian cancer is still to be further studied. Therefore, the interference of the RNAi technology on the LAMB1 gene expression in the ovarian cancer drug-resistant cell strain is an important supplement to the pathogenesis of ovarian cancer and an important exploration and application to the ovarian cancer and the drug-resistant treatment thereof.
Disclosure of Invention
The invention aims to provide siRNA for specifically inhibiting LAMB1 gene expression, which is used for researching the pathogenesis of ovarian cancer. The invention also aims to provide application of the siRNA in preparing medicaments for treating ovarian cancer and reversing paclitaxel drug resistance.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention designs and synthesizes 3 pairs of siRNA for specifically inhibiting LAMB1 gene expression, and the siRNA is transfected into an ovarian cancer Taxol resistant cell strain A2780/Taxol, and the result shows that the S1 has the most obvious interference effect for inhibiting LAMB1 gene expression.
The invention provides siRNA (S1) for specifically inhibiting LAMB1 gene expression, which comprises a sense strand and an antisense strand,
the sense strand: 5'-UGUUUGAAAGCCGAAUCUGCG-3' (SEQ ID NO. 1);
the antisense strand: 5'-CAGAUUCGGCUUUCAAACAAA-3' (SEQ ID NO. 2).
Preferably, the sense and antisense strands are 2 ' -methoxy modified at the 5 ' and 3 ' ends of the 3 bases. The research of the invention proves that the stability of the siRNA (S1) modified by the 2' -methoxy group is increased, the capability of resisting the hydrolysis of ribozyme in vivo can be improved, the immune stimulation reaction is reduced, the action time of siRNA interfering the expression of gene to be reduced is prolonged, and the action has high efficiency and specificity.
The invention provides an RNA interference kit comprising a DNA sequence encoding the siRNA. The kit comprises a DNA plasmid vector cloned with the siRNA, and when the kit is applied, the plasmid vector transcribes and expresses the siRNA in eukaryotic cells, so that the expression of the LAMB1 gene is silenced.
The invention provides a recombinant vector containing a DNA sequence for encoding the siRNA. Preferably, the original vector used is the lentiviral vector plko.1puro.
The invention also provides a construction method of the recombinant vector, which comprises the following steps:
(1) synthesizing a LAMB1-S1 fragment, selecting two restriction enzyme sites of Age I and EcoR I, and designing an shRNA sequence according to the sequence of S1, wherein the sequence is as follows:
sense strand:
5’-CCGGTTTGTTTGAAAGCCGAATCTGCGTTCAAGAGACGCAGATTCGGCTTTCAAACAAATTTTTTGGTACC-3’(SEQ ID NO.7)；
antisense strand:
5’-AATTGGTACCAAAAAATTTGTTTGAAAGCCGAATCTGCGTCTCTTGAACGCAGATTCGGCTTTCAAACAAA-3’(SEQ ID NO.8)；
(2) annealing to obtain a DNA fragment of LAMB 1-S1;
(3) pLKO.1-LAMB1-sh1 recombinant vector was constructed using lentiviral vector pLKO.1puro.
The siRNA provided by the invention can efficiently and specifically inhibit the expression of the LAMB1 gene of ovarian cancer cells, reduce cell proliferation, increase apoptosis and reduce cell migration and invasion capacity, so that the siRNA and the recombinant vector serving as an LAMB1 gene expression inhibitor can be applied to the research of the pathogenesis of tumor diseases.
The invention provides application of the siRNA and the recombinant vector in preparation of a LAMB1 gene expression inhibitor.
The invention provides application of the siRNA and the recombinant vector in preparing medicaments for treating ovarian cancer, hepatocellular carcinoma, colorectal cancer, malignant glioma, prostatic cancer or gastric cancer.
The research of the invention shows that after the siRNA is transfected by the paclitaxel resistant strain A2780/Taxol, the sensitivity of the cell strain to paclitaxel is obviously improved, and the reversal index is 15.28, which shows that the siRNA provided by the invention has very obvious reversal effect on the drug resistance of the paclitaxel resistant strain A2780/Taxol, therefore, the siRNA has potential application value on the treatment of reversing the drug resistance of ovarian cancer paclitaxel.
The invention provides application of the siRNA and the recombinant vector in preparation of a medicine for reversing drug resistance of ovarian cancer taxol.
The invention has the following beneficial effects:
the siRNA provided by the invention can specifically and efficiently inhibit the mRNA and protein expression of LAMB1 gene in tumor cells, reduce the proliferation of tumor cells, increase the apoptosis, reduce the migration and invasion capacity of tumor cells, and effectively reverse the drug resistance of ovarian cancer cells to paclitaxel, and has important significance in the study of tumor pathogenesis and the preparation of drugs for tumor treatment and ovarian cancer drug resistance treatment.
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FIG. 1 shows the qRT-PCR detection of LAMB1mRNA expression in A2780 cells and A2780/Taxol cells, wherein A is A2780 cells and AR is A2780/Taxol cells.
FIG. 2 shows Western Blotting to detect LAMB1 protein expression of A2780 cells and A2780/Taxol cells, wherein A is A2780 cells and AR is A2780/Taxol cells.
FIG. 3 shows the expression of LAMB1mRNA in A2780/Taxol cells 48h after transfection with S1, S2, S3 by qRT-PCR.
FIG. 4 shows Western Blotting to detect the expression of LAMB1 protein in A2780/Taxol cells 72h after transfection with S1, S2 and S3.
FIG. 5 is a schematic diagram of pLKO.1-LAMB1-sh1 recombinant plasmid and inserted restriction enzyme sites.
FIG. 6 is a schematic diagram of a small hairpin shRNA. The U6 promoter directs the transcription of downstream small hairpin shRNA; comprises 23S 1 sense strand bases and 23S 1 antisense strand bases.
FIG. 7 shows the protein expression of LAMB1 in A2780/Taxol cells after pLKO.1-LAMB1-sh1 transfection detected by Western Blotting.
FIG. 8 shows the number and morphology of A2780/Taxol cells after pLKO.1-LAMB1-sh1 transfection under phase contrast microscope observation.
FIG. 9 shows the proliferation of A2780/Taxol cells detected by bromine labeling method after transfection of pLKO.1-LAMB1-sh 1.
FIG. 10 shows the detection of Caspase3 activity on apoptosis of A2780/Taxol cells after pLKO.1-LAMB1-sh1 transfection.
FIG. 11 shows the migration ability of A2780/Taxol cells after pLKO.1-LAMB1-sh1 transfection in cell scratch assay.
FIG. 12 shows the migration ability (A) and invasion ability (B) of A2780/Taxol cells after transfection of pLKO.1-LAMB1-sh1 by Transwell assay.
FIG. 13 shows the reversal of paclitaxel resistance in A2780/Taxol cells after transfection with pLKO.1-LAMB1-sh 1.
Detailed Description
The present invention will be further described with reference to the following examples. The methods used in the following examples are intended to better understand the invention but are not intended to limit it. Unless otherwise specified, the experimental methods mentioned in the examples are all conventional methods, and the experimental materials used are all purchased from conventional reagents companies.
The SPSS 18.0 statistical analysis software is adopted, and the data of each sample are averaged to be +/-standard deviation
As shown, the differences between the two groups were measured by T Test (Independent-Sample T Test), and the differences between the groups were measured by One-Way analysis of variance (One-Way ANOVA), IC50Using probit regression analysis, P < 0.05 was statistically different.
Ovarian cancer cell strain A2780 and ovarian cancer Taxol resistant cell strain A2780/Taxol are preserved in a cell bank of a key laboratory of female reproductive health research in Zhejiang province;
rabbit anti-human LAMB1 primary antibody (Cat.23498-1-AP), mouse anti-human GAPDH primary antibody (Cat.60004-1-Ig), horseradish peroxidase-labeled goat anti-mouse IgG (H + L) secondary antibody (Cat.SA00001-1), and horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) secondary antibody (Cat.SA00001-2) were purchased from Proteitech corporation;
western Blotting Luminol Reagent kit (Cat. sc-2048) was purchased from Santa Cruz;
cDNA reverse transcription kit PrimeScriptTMRT Master Mix (Cat. RR036A), fluorescent quantitative PCR detection kit SYBR Premix Ex Taq (perfect Real time, Cat. DRR041A) purchased from TaKaRa company; lipofectamine3000 transfection kit (cat. l3000008) was purchased from Invitrogen;
the eukaryotic expression vector selects an RNAi vector pLKO.1puro, which is derived from a global scientist plasmid sharing non-profit tissue Addgene;
restriction enzymes Age I (Cat. R0552S), EcoR I (Cat. R0101S), Kpn I (Cat. R0142S) were purchased from NEB; t4 ligase (Cat.2011A), a DNA fragment purification kit (Cat.9761), a DNA gel recovery kit (Cat.9762) and a plasmid DNA small purification kit (Cat.9760) are all purchased from TaKaRa company;
siRNA was synthesized by TaKaRa; PCR primers and DNA for cloning were synthesized by Shanghai Bioengineering Co., Ltd;
prestained protein Marker (cat.26616) available from Fermentas corporation;
cell Proliferation ELISA, BrdU (colorimetric, Cat.11647229001) was purchased from Roche;
CaspACE Assay System (colorimetric, Cat. G7351) available from Promega;
cell migration, invasion model Transwell Permeable Supports (Cat.3428) was purchased from Corning;
Lab-Tek II Chamber Slide System-Lab-Tek Chamber Slide System was purchased from Nunc corporation (Cat.154526);
BD MatrigelTMbasic Membrane matrix membranes (cat.356234) available from BD;
siRNA Negative Control AllStars Negative Control SiRNA (Cat.1027281) was purchased from QIAGEN;
SDS-PAGE gel preparation kit (cat. cw0022m) purchased from kazai century corporation;
0.45um PVDF membrane (Cat. IPVH00010) was purchased from Millipore;
paclitaxel (cat. P106868) was purchased from Aladdin.
Example 1 study of LAMB1 expression differences in ovarian cancer cell line A2780 and paclitaxel-resistant cell line A2780/Taxol thereof
First, real-time fluorescent quantitative RT-PCR (qRT-PCR) is used for detecting LAMB1 gene mRNA expression
After 48h incubation, the medium in the 6-well plate was aspirated, washed twice with PBS, and total RNA was extracted with Trizol, and the RNA concentration was determined with a ThermoNano Drop2000 spectrophotometer, according to the SYBR Premix Ex Taq (perfect reaction time) kit instructions. First step RNA denaturation. Reaction system: RNA0.5ug, adding DEPC water to 6.8 ul; reaction conditions are as follows: incubate at 70 ℃ for 10min and place on ice. And a second step of reverse transcription. Reaction system: reverse transcription was performed according to PrimeScript RTMaster Mix kit instructions; reaction conditions are as follows: incubating at 42 deg.C for 60min, inactivating at 85 deg.C for 5min, and storing at-20 deg.C.
Taking 1ul of reverse transcription product to perform fluorescent quantitative PCR reaction. PCR primer sequences:
5’-ATCGCAGATTCGGCTTTCAA-3’；
5'-TCTTCCCGTCTTCCTTTCCG-3', product length: 236 bp;
reaction conditions are as follows: 10s at 95 ℃, 5s at 95 ℃ and 30s at 6 ℃ for 40 cycles.
the expression level of LAMB1mRNA in each group of samples was calculated by the 2- △ CT method.
As a result: as shown in FIG. 1, the expression of LAMB1mRNA was 68.35% higher in A2780/Taxol cells compared to its parental cell A2780 (P < 0.05), indicating that LAMB1mRNA was significantly higher in the paclitaxel-resistant cell line.
Second, Western Blotting detection of LAMB1 protein expression
After culturing for 72h, absorbing and removing the culture medium in the 6-well plate, washing with PBS for 3 times, adding RIPA protein lysate (100 ul/well), blowing and beating for several times, incubating on ice for 5min to fully crack, centrifuging at 4 ℃, 12000 rpm for 5min, collecting supernatant, and subpackaging at-20 ℃ for storage; denaturing each sample at 95 deg.c for 5min, taking 10ul sample, 8% SDS-PAGE electrophoresis at 200V for 10 min; 100V for 100 min; turning to a PVDF membrane: 110V for 120 min; sealing with TBS sealing solution containing 5% skimmed milk powder for 60 min; primary antibody incubation: LAMB1 primary antibody (1: 2000), GAPDH primary antibody (1: 5000) were incubated at room temperature for 2 h; washing membrane with TBS for 10min × 3 times; and (3) secondary antibody incubation: incubating a goat anti-mouse IgG (H + L) secondary antibody (1: 10000) marked by horseradish peroxidase and a goat anti-rabbit IgG (H + L) secondary antibody (1: 10000) marked by horseradish peroxidase for 1H; washing membranes with TBST for 10min × 3 times and TBS for 10min × 1 time; after ECL development, the images were scanned with ImageQuant LAS4000mini (GE Healthcare).
As a result: as shown in FIG. 2, the expression of LAMB1 protein was also significantly increased in A2780/Taxol cells compared to its parent cell, A2780 (P < 0.05).
Example 2 LAMB1siRNA design Synthesis
The LAMB1 gene mRNA sequence (NM-002291.2) was searched in Genebank, and 3 pairs of siRNA sequences (shown in SEQ ID NO.1-SEQ ID NO.6) were obtained by on-line design using siDirect Ver2.0 software (http:// siDirect2.rnai. jp /). In the design process, a sequence simultaneously meeting three algorithms (Ui-Tei x Reynolds x Amarzguioui) reported in the literature is selected, a 23nt length segment with the highest siRNA action specificity is selected, the design can avoid the generation of an interferon-like immune response in future in-vivo experiments, 100nt after an initiation codon is selected, 5 'and 3' end UTR regions are avoided, and the GC content is controlled to be 30-70%. 3 pairs of siRNA with the length of 23nt are selected as experimental screening interference fragments, the structural characteristics are that the 3' ends of the sense strand and the antisense strand are respectively externally hung with two basic groups, and the structural characteristics are as follows
Followed by BLASTN(https://blast.ncbi.nlm.nih.gov/Blast.cgi)Homology search is carried out on line, sequences with homology are excluded, and the influence of nonspecific fragments on the specific action effect of siRNA is avoided as much as possible.
And finally, carrying out 2 '-OMe (2' -methoxyl) modification on 3 continuous purine (pyrimidine) bases at the 5 'end and the 3' end of the sense strand and the antisense strand during chemical synthesis, so that the chemical stability of the siRNA molecule in cells is increased, and the time and effect of siRNA interfering gene expression down regulation are prolonged. The final sequences and modifications are shown in Table 1 and formula (I) below:
TABLE 1
Example 3 detection and screening of interference Effect of three pairs of LAMB1 siRNAs on LAMB1 Gene in Taxol-resistant Strain A2780/Taxol for ovarian cancer
Firstly, experimental grouping:
A2780/Taxol normal group (no siRNA transfection), hereinafter referred to as AR;
A2780/Taxol negative control group (transfection negative control siRNA), hereinafter referred to as AR-N;
A2780/Taxol panel (transfection S1), hereinafter referred to as AR-S1;
A2780/Taxol panel (transfection S2), hereinafter referred to as AR-S2;
A2780/Taxol panel (transfection S3), hereinafter referred to as AR-S3.
Two, grouped transfection
To ensure transfection efficiency and reduce cytotoxicity, we used Lipofectamine3000 transfection reagent for siRNA transfection. One day before transfection, cells were trypsinized and counted, and cells were plated in six-well plates at a density of 0.5X 10 on the day of transfection6Per ml, cells were fused to 70-90%. Diluting 5ul Lipofectamine3000 reagent with 125 ul serum-free OPTI-MEM medium per well and mixing well; preparing siRNA premix, diluting siRNA with 125 mul of serum-free OPTI-MEM culture medium to a final concentration of 50nM, and fully mixing; adding siRNA premix (1:1) into diluted Lipofectamine3000 reagent, and incubating for 5min at room temperature; finally, the siRNA-liposome complex was added to the cells at 37 ℃ with 5% CO2The culture is continued. LAMB1mRNA expression was measured after 48h, and LAMB1 protein expression was measured after 72 h.
Thirdly, detecting the mRNA expression of the LAMB1 gene by real-time fluorescent quantitative RT-PCR (qRT-PCR)
the detection steps are the same as the previous steps, and the expression quantity of LAMB1mRNA in each group of samples is calculated by adopting a 2- △ CT method.
As a result: as shown in fig. 3, after the a2780/Taxol cells were transfected with S1, S2, and S3, respectively, the expression of LAMB1mRNA was significantly reduced, wherein the S1 interference effect was 85.09% lower than that of the negative control group, and was significantly different (P < 0.05) than that of S2 (65.02%) and S3 (21.786%). The results show that S1 has the best interference effect on LAMB 1.
Fourthly, detecting LAMB1 protein expression by Western Blotting
The detection procedure was as before, and after ECL development, the Image was scanned with Image Quant LAS4000mini (GE Healthcare).
As a result: as shown in FIG. 4, LAMB1 protein expression was significantly reduced in A2780/Taxol cells after transfection with S1 (P < 0.05) compared to the negative control, and was significantly different from S2 and S3 (P < 0.05). The results show that S1 has the best interference effect on protein expression of LAMB1 in the paclitaxel resistant strain A2780/Taxol. Therefore, S1 was selected as siRNA for subsequent studies.
Example 4 construction of eukaryotic vector pLKO.1-LAMB1-sh1 and detection of interference effect on LAMB1 Gene expression
Firstly, experimental grouping:
A2780/Taxol normal group (without transfection of any vector), hereinafter referred to as AR;
A2780/Taxol negative control group (pLKO.1puro empty vector transfected), hereinafter referred to as AR-N;
A2780/Taxol panel 1 (pLKO.1-LAMB 1-sh1 transfection), hereinafter referred to as AR-sh 1;
II, synthesis of LAMB1-S1 fragment
Two enzyme cutting sites of Age I and EcoR I are selected, and shRNA sequence is designed according to the sequence of S1 and is constructed into a eukaryotic expression vector pLKO.1puro. The sequence is as follows:
sense strand:
5’-CCGGTTTGTTTGAAAGCCGAATCTGCGTTCAAGAGACGCAGATTCGGCTTTCAAACAAATTTTTTGGTACC-3’(SEQ ID NO.7)；
antisense strand:
5’-AATTGGTACCAAAAAATTTGTTTGAAAGCCGAATCTGCGTCTCTTGAACGCAGATTCGGCTTTCAAACAAA-3’(SEQ ID NO.8)；
thirdly, construction of eukaryotic vector pLKO.1-LAMB1-sh1
pLKO.1-LAMB1-sh1 recombinant expression vector was constructed using eukaryotic expression vector pLKO.1puro (FIGS. 5 and 6), and the detailed method was described in molecular cloning laboratory Manual of Cold spring harbor Press, USA.
The sense and antisense strands of LAMB1-S1 were annealed (denaturation at 95 ℃ C. for 2 min; annealing by slow cooling to 25 ℃ C.) and stored at 4 ℃. Completely digesting the vector pLKO.1puro by Age I and EcoR I, and standing overnight at 37 ℃; and recovering DNA from the enzyme digestion product by using a DNA gel recovery kit. The annealing product and the carrier enzyme digestion recovery fragment are subjected to a connection reaction, and the reaction system (10 ul): 1ul of T4 ligase, 1ul of T4 ligase buffer solution, and carrying out enzyme digestion on the annealing product and a carrier to recover a fragment mixture (the molar ratio is 3: 1); supplementing deionized water to 10ul, and connecting overnight at 16 ℃; placing 5ul of the ligation product in 100ul of JM109 competent bacteria, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 90s, carrying out ice bath for 5min, adding 1000ul of LB culture medium, carrying out shake culture at 37 ℃ for 30min, centrifuging at 5000rpm for 5min, discarding the supernatant, uniformly coating the bacteria on an LB plate (containing 50ug/ml ampicillin), and carrying out inverted culture at 37 ℃ for overnight; selecting a plurality of independent colonies, inoculating the colonies into an LB culture medium containing corresponding resistance, and oscillating the colonies overnight at 37 ℃ for strain expansion; collecting bacteria, extracting by using a plasmid DNA purification kit to obtain plasmid DNA, carrying out enzyme digestion identification on KpnI to obtain pLKO.1-LAMB1-sh1 recombinant plasmid.
Fourth, pLKO.1-LAMB1-sh1 recombinant plasmid transfected cells is observed to interfere effect
pLKO.1-LAMB1-sh1 was transfected into A2780/Taxol cells in log phase growth. Transfection 5ul Lipofectamine3000 reagent was diluted in 125. mu.l serum-free OPTI-MEM medium per well and mixed well according to Lipofectamine3000 protocol; adding 5ug of recombinant plasmid DNA into 125 mul of serum-free OPTI-MEM culture medium, adding 10ul of P3000 reagent, and mixing to obtain recombinant plasmid premix; the diluted Lipofectamine3000 reagent was added to the recombinant plasmid premix (1:1) and incubated at room temperature for 5 mm; finally, 250ul of the recombinant plasmid-liposome complex was added to the cells at 37 ℃ with 5% CO2The culture is continued. LAMB1 protein expression was detected after 72 h.
As shown in FIG. 7, after transfection of pLKO.1-LAMB1-sh1 recombinant plasmid, compared with negative control (transfection of empty plasmid), LAMB1 protein expression in A2780/Taxol cell is significantly reduced (P < 0.05), and the result shows that the protein expression of LAMB1 can be effectively interfered by the transfection of pLKO.1-LAMB1-sh1 recombinant plasmid.
Example 5 Effect of pLKO.1-LAMB1-sh1 transfection on tumor cell proliferation, apoptosis, migration and invasion after specific blockade of LAMB1 expression
Firstly, experimental grouping:
A2780/Taxol normal group (without transfection of any vector), hereinafter referred to as AR;
A2780/Taxol negative control group (pLKO.1puro empty vector transfected), hereinafter referred to as AR-N;
A2780/Taxol panel (pLKO.1-LAMB 1-sh1 transfection), hereinafter referred to as AR-sh 1.
Two, grouped transfection
The transfection procedure was as before, and cells were cultured after transfection for detection of proliferation, apoptosis, migration and invasion of cells.
Third, cell proliferation assay
Cells are transfected into a 96-well plate pLKO.1-LAMB1-sh1 and then cultured for 72h, 10ul BrdU labeling solution is added into each well until the final BrdU concentration is 10uM, and the cells are incubated for 2h at 37 ℃; absorbing BrdU labeling solution, adding 200ul FixDenat into each well, and incubating at 20 deg.C for 30 min; removing FixDenat by suction, adding 100 ula-BrdU-POD into each well, and incubating at 20 deg.C for 90 min; washing 3 times with 200ul Washingsolution per well; adding 100 ul/well substrate solution, incubating at 20 deg.C for 20min, detecting absorbance (A) at wavelength of 370nm (reference wavelength of 492nm), and measuring cell proliferation ability with AExperimental group/AControl groupAnd (4) showing.
As shown in FIGS. 8 and 9, in A2780/Taxol cells, pLKO.1-LAMB1-sh1 is transfected, and the observation under a phase contrast microscope shows that the number of cells in an experimental group is obviously reduced, the number of suspension cells is increased, and cell debris is increased; the cell proliferation results tested by the bromine standard method show that: compared with a negative control, the cell proliferation capacity of the experimental group is reduced by 33.21%, and the difference is significant (P is less than 0.05). Thus, the expression of LAMB1 can be specifically blocked, and the tumor cell proliferation can be inhibited.
Fourthly, detecting apoptosis by Caspase3 activity
Collecting cells after transfection for 72h, adjusting cell density to 1 × 10 with lysate8-/ml, split on ice for 15min, 15000g × 20min, collect supernatant. Positive and negative control samples were prepared simultaneously according to CaspACE Assay System (colorimetric) instructions, and the concentrations of each histone were determined and adjusted to be the same. Adding Caspace AssayBuffer32ul, DMSO2ul, 100nM DTT 10ul, deionized water to adjust the volume to 98ul, adding 2ul DEVD-pNA substrate, incubating at 37 ℃ for 4h, detecting the absorbance at 405nM, and measuring the absorbance by deltaCaspase3 activity was calculated for each group of samples by method A.
The result is shown in figure 10, after pLKO.1-LAMB1-sh1 is transfected by A2780/Taxol cells, Caspase3 activity is increased by 2.92 times, and compared with a negative control, the significant difference (P is less than 0.05) is generated, which indicates that tumor cell apoptosis can be promoted after the expression of LAMB1 is specifically blocked.
Fifth, cell migration ability test
And uniformly marking transverse lines at the back of the six-hole plate by using a straight ruler, wherein one transverse line is marked by about 0.5cm and penetrates through the holes, and each hole is provided with at least 6 transverse lines. After the cells are transfected with pLKO.1-LAMB1-sh1, when the cells are continuously cultured for 24 hours and fused into a single-layer state, a 200ul gun head is used for vertically scratching the selected area in a six-hole plate, the scratched cells are removed by washing with PBS for 3 times, and serum-free culture medium is added for continuous culture. Photographing is carried out at the time points of 0h, 24h and 48h, 6 horizontal lines are randomly selected, and the average value of the distances between cells is calculated.
The result is shown in fig. 11, after cells are transfected for 24h and 48h by pLKO.1-LAMB1-sh1, the distance between A2780 and Taxol cells is obviously greater than that of a negative control group, the healing capacity of the cells after scratching is obviously reduced, and the result shows that the cells can inhibit the migration of tumor cells after the expression of LAMB1 is specifically blocked.
Sixth, Transwell test for detecting cell migration ability
48h after cell transfection, the cells were collected by trypsinization, resuspended in serum-free medium and adjusted to a cell density of 5X 105Adding 2ml of cell suspension into an upper chamber, adding 2ml of 10% FBS complete culture medium into a lower chamber, continuously culturing for 24h, taking out a small chamber, washing with PBS for 3 times, carefully removing cells on the surface of the upper layer of the upper chamber by using a cotton swab, inverting and airing, fixing with 95% ethanol for 25min, staining with hematoxylin, observing under a microscope, counting and taking a picture. 10 fields were counted per chamber, averaged and analyzed for changes in cell migration capacity.
The results of the cell migration experiments are shown in FIG. 12A, after pLKO.1-LAMB1-sh1 is transfected, the cell numbers of the A2780/Taxol experimental group and the negative control group penetrating the chamber are respectively 48 +/-12 and 277 +/-42, and the two groups have significant statistical difference (P is less than 0.05); the results show that the tumor cell migration can be obviously inhibited after the expression of the LAMB1 is specifically blocked.
Seventhly, cell invasion capacity detection by Transwell test
Liquefying matrigel stored at-20 deg.C at 4 deg.C, mixing matrigel with OPTI-MEM at a ratio of 1:6 ice, diluting, coating the upper surface of the bottom membrane of the chamber, solidifying at 37 deg.C for 30min, and removing the liquid separated out from the chamber. The remaining steps after matrigel coating were as above, 10 fields were counted per chamber, and changes in cell invasiveness were counted and analyzed by averaging.
The results of the cell invasion assay are shown in FIG. 12B, the numbers of cells penetrating the chamber of the A2780/Taxol test group and the negative control group are 39 + -10 and 175 + -39, respectively, and the two groups have statistical difference (P < 0.05); the results show that tumor cell infiltration can be significantly inhibited after the expression of the LAMB1 is specifically blocked.
Example 6 pLKO.1-LAMB1-sh1 specifically blocks the reversal of drug resistance in ovarian cancer after LAMB1 expression
Firstly, experimental grouping:
a2780 normal group (without transfection of any vector), hereinafter referred to as a;
A2780/Taxol normal group (without transfection of any vector), hereinafter referred to as AR;
A2780/Taxol negative control group (pLKO.1puro empty vector transfected), hereinafter referred to as AR-N;
A2780/Taxol panel (pLKO.1-LAMB 1-sh1 transfection), hereinafter referred to as AR-sh 1.
Two, grouped transfection
The transfection procedure was as before, and cells were cultured for 24h after transfection.
Thirdly, detecting the sensitivity of the cells to the paclitaxel after pLKO.1-LAMB1-sh1 transfection
Taking cells in logarithmic growth phase from each group, re-suspending the cells after trypsinization, counting the cells and adjusting the density of the cell suspension to 1 × 105And/ml, inoculating into a 96-well plate and continuing culturing for 24 hours. The next day, adding paclitaxel into each group, setting concentration gradient at 200ug/ml, 100ug/ml, 50ug/ml, 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.125ug/ml, 0ug/ml, after 24h, measuring absorbance (A) at wavelength 370nm (reference wavelength 492nm) by bromine standard method, and calculating the inhibition rate of paclitaxel on each group of cells, wherein the inhibition rate is AExperimental group/ANegative control group.3 replicate wells were set for each concentration and the average was taken.
The drug concentration at 50% inhibition was half the Inhibitory Concentration (IC)50)；
IC of drug-resistant strain A2780/Taxol50IC with its parent cell line A278050The ratio of (A) to (B) is the Resistance Fold (RF);
IC of drug-resistant strain A2780/Taxol50IC after transfection with pLKO.1-LAMB1-sh1 (reversal agent)50The ratio of (A) to (B) is the resistance Reversal Index (RI).
The results are shown in Table 2, Table 3 and FIG. 13, IC of A2780/Taxol vs. paclitaxel50(37.17 +/-5.47 ug/ml) is significantly higher than the IC of parent A2780 for paclitaxel50(1.27 +/-0.28 ug/ml), the drug resistance times are as high as 29.27, which indicates that the sensitivity of A2780/Taxol to paclitaxel is obviously lower than that of a parent cell A2780, and the drug resistance is high. And after the A2780/Taxol is transfected with pLKO.1-LAMB1-sh1, the sensitivity to the paclitaxel is obviously improved (2.36 +/-0.77 ug/ml), and the reversion effect of the transfected pLKO.1-LAMB1-sh1 to the drug resistance of the A2780/Taxol is very obvious, and the reversion index is 15.28.
TABLE 2 drug sensitivity of A2780 and A2780/Taxol to paclitaxel
TABLE 3 reversion of sensitivity of A2780/Taxol to paclitaxel drug after pLKO.1-LAMB1-sh1 transfection
SEQUENCE LISTING
<110> Zhejiang university
<120> siRNA for specifically inhibiting LAMB1 gene expression, recombinant vector and application thereof
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Claims (3)
1. The application of the siRNA for specifically inhibiting the LAMB1 gene expression in the preparation of the medicine for reversing the drug resistance of the ovarian cancer paclitaxel is characterized in that the siRNA comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 2.
2. The use of claim 1, wherein 3 bases at the 5 ' and 3 ' ends of the sense and antisense strands are 2 ' -methoxy modified.
3. The application of a recombinant vector containing a DNA sequence of siRNA for specifically inhibiting LAMB1 gene expression in the preparation of drugs for reversing drug resistance of ovarian cancer paclitaxel is characterized in that the siRNA comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 2.
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