CN111944812A - siRNA molecule of targeting Fascin gene and application thereof - Google Patents
siRNA molecule of targeting Fascin gene and application thereof Download PDFInfo
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Abstract
The invention discloses a group of siRNA molecules targeting Fascin genes and application thereof, belonging to the technical field of biological medicines. The siRNA molecule consists of a sense strand and an antisense strand, and in vitro experiments prove that the antisense strand of the siRNA molecule can be specifically combined with mRNA for inhibiting a Fascin gene to degrade the mRNA, so that the translation process after transcription is interfered, tumor cell apoptosis is induced, tumor cell metastasis and invasion are inhibited, and the purpose of treating tumors is achieved.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a group of siRNA molecules targeting Fascin genes and application thereof.
Background
Primary liver cancer, especially Hepatocellular carcinoma (HCC), is the most common primary tumor of the liver, and the incidence and mortality of the primary cancer tend to increase. Its global incidence is currently the fifth of all malignancies, and the third highest in the rate of cancer mortality, with approximately 80 million people dying from hepatocellular carcinoma each year. About half of new liver cancer cases occur in China every year in China, and the second cause of cancer death in China is listed, so that the health of the China is seriously harmed. Liver cancer is high in malignancy degree, hidden in morbidity, difficult in early diagnosis and fast in progress, and most patients have reached middle and late stages with extensive infiltration or distant metastasis and cannot be resected by surgery. One important reason for poor prognosis of liver cancer is the lack of effective therapeutic means. Therefore, besides emphasizing and paying attention to early detection and early treatment, the exploration of an effective treatment method has important significance for improving the prognosis of the liver cancer patient.
The human Fascin gene belongs to the Fascin family, and the protein coding product is a cytoskeletal protein with the molecular weight of 55kDa, which is combined with F actin and positioned in the core actin bundles of male pseudopoda and microspine at the edge of cytoplasmic tension fiber and cell membrane fold. Fascin plays an important role in cell migration, cell adhesion, intercellular information exchange and other processes. The existing research shows that the expression of the Fascin protein is obviously increased in various tumors, and the Fascin protein participates in malignant processes of proliferation, growth, invasion and the like of the tumors and is closely related to the prognosis of the tumors.
RNA interference (RNAi) is a form of post-transcriptional gene silencing in which small interfering RNA (siRNA) initiates degradation of its cognate messenger RNA (mRNA) (Nature 1998,391: 806-. Has proved to have great potential in the treatment of various virus infectious diseases and tumors, and is an ideal therapeutic means for blocking gene expression. RNAi technology opens up a brand new therapeutic field, and currently, dozens of siRNA drugs enter the clinical stage internationally.
Disclosure of Invention
The invention aims to provide a group of siRNA molecules targeting a Fascin gene and application thereof, wherein an antisense chain of the siRNA molecules can be specifically combined with mRNA for inhibiting the Fascin gene to degrade the mRNA, so that the translation process after transcription is interfered, tumor cell apoptosis is induced, and tumor cell metastasis and invasion are inhibited, thereby achieving the purpose of treating tumors.
In order to achieve the purpose, the invention adopts the following technical scheme:
the siRNA molecule of targeting Fascin gene consists of a sense strand and an antisense strand, and the sequence of the siRNA molecule is as follows:
sense strand: 5 '-CGUUCGGUUCAAGGUGAADTT-3' (SEQ ID NO:1),
antisense strand: 5 '-UUCACCUGAACCCGAACGdTdT-3' (SEQ ID NO: 2).
The siRNA molecule is applied to the preparation of drugs for inhibiting the function of Fascin genes in cells.
The siRNA molecule is applied to the preparation of a medicament for preventing and/or treating liver cancer.
Further, the siRNA molecule can induce apoptosis of liver cancer cells.
Further, the siRNA molecule can inhibit the metastasis and invasion of liver cancer cells.
In vitro experiments prove that the antisense strand of the siRNA molecule can be specifically combined with mRNA for inhibiting a Fascin gene to degrade the mRNA, so that the translation process after transcription is interfered, tumor cell apoptosis is induced, tumor cell metastasis and invasion are inhibited, and the purpose of treating tumors is achieved.
The siRNA molecule can be applied to the preparation of drugs for inhibiting the function of the Fascin gene in the regulatory cell to play the role of RNA interference, induce the apoptosis of tumor cells and inhibit the metastasis and invasion of the tumor cells, thereby achieving the purpose of treating tumors.
Drawings
FIG. 1 shows the real-time quantitative PCR detection of mRNA expression levels of Fascin in liver cancer cells (HepG2 and Huh7) and normal liver cells LO2 in example 1.
FIG. 2 is a graph showing that Western Blot in example 1 detects the protein expression levels of Fascin in liver cancer cells (HepG2 and Huh7) and normal liver cells LO 2.
FIG. 3 shows that the siRNA reduces the mRNA expression level of Fascin in HepG2 of liver cancer cells by the real-time quantitative PCR detection in example 1.
FIG. 4 shows that the siRNA reduces the protein expression level of Fascin in HepG2 of liver cancer cells by the real-time quantitative PCR detection in example 1.
FIG. 5 shows that the Western Blot detection of siRNA in example 1 down-regulates the mRNA expression level of Fascin in Huh7 of hepatoma cells.
FIG. 6 shows that the siRNA can regulate the protein expression level of Fascin in the liver cancer cell Huh7 by the real-time quantitative PCR detection in example 1.
FIG. 7 is a graph showing the effect of the MTT method in example 1 on cell proliferation ability after siRNA down-regulates the expression level of Fascin in hepatoma cells HepG 2.
FIG. 8 is a graph showing the effect of MTT method in example 1 on cell proliferation ability after siRNA down-regulates the expression level of Fascin in Huh7 of hepatoma cells.
FIG. 9 is a cell scratching experiment in example 1 to examine the effect of siRNA on cell migration ability after down-regulating the expression level of Fascin in hepatoma cells HepG 2.
FIG. 10 is a graph of the cell scratch test in example 1 to examine the effect of siRNA on cell migration ability after down-regulating the expression level of Fascin in Huh7 of hepatoma cells.
FIG. 11 is a diagram showing the effect of siRNA on cell invasion ability after down-regulating the expression level of Fascin in hepatoma cells HepG2, as detected by the Transwell cell invasion assay in example 1.
FIG. 12 is a diagram showing the effect of siRNA on cell invasion ability after down-regulating the expression level of Fascin in hepatoma cells Huh7, as detected by the Transwell cell invasion assay in example 1.
Detailed Description
For convenience, in the following, the terms "siRNA", "siRNA sequence" or "siRNA molecule" are interchangeable and mean the same and range.
Wherein, the siRNA is a double-stranded structure formed by annealing a sense strand and an antisense strand.
The siRNA molecules of the present invention are designed from a functionally conserved region of the open reading frame of the Fascin gene.
siRNA can be prepared by a variety of methods, such as: the method comprises the steps of chemical synthesis, in-vitro transcription, enzyme digestion of long-chain dsRNA, vector expression of siRNA, synthesis of siRNA expression elements by PCR and the like, provides a selectable space for researchers, and can better obtain the gene silencing efficiency.
The siRNA molecule can be used as an effective component for preparing drugs for regulating the function of the Fascin gene in cells, in particular to an effective component for anti-tumor drugs.
For application purposes, the siRNA molecule may be administered as a drug directly to a specific site on the recipient, such as a tumor tissue.
The dosage form of the drug of the present invention may take various forms as long as it is suitable for administration to the corresponding disease and properly maintains the activity of the siRNA molecule. For example, for an injectable delivery system, the dosage form may be a lyophilized powder.
Optionally, any pharmaceutically acceptable carrier and adjuvant may be included in the above pharmaceutical dosage form, as long as it is suitable for the corresponding administration system and properly maintains the activity of the siRNA molecule.
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.
The following examples are given for the purpose of illustration only and are not intended to be limiting.
Example l
First, experiment method
Step 1, cell culture
Hepatoma cell lines HepG2 and Huh7 were purchased from Shanghai institute of cell culture, and cultured in DMEM medium (Thermo Fisher) containing 10% FBS (Thermo Co.) to which penicillin and streptomycin (Thermo Co.) were added at final concentrations of 100U/mL and 100. mu.g/mL, respectively, in a carbon dioxide incubator at 37 ℃.
Step 2, siRNA in vitro transfection
siRNA sequences, Fascin siRNAs, were designed that target the Fascin (NCBI number: NM-003088) gene. Negative control sequences (NC) were designed as controls. The sequences are shown in Table 1.
TABLE 1 siRNA sequences and control sequences
Taking the cells cultured in the step 1 and in the logarithmic growth phase, and inoculating the cells in a 96-well plate at a density of 5 multiplied by 104Per well, 24 well plate by 1.5X 105Hole/hole6 the pore plate is 1 multiplied by 106Cells were inoculated per well and divided into a Fascin siRNA transfected group and an untreated group that was not transfected with siRNA, and NC transfected group as a negative control. Liposomes for each experimental group were used with the corresponding siRNA as indicated
2000(Thermo Fisher Co., Ltd.) was transfected into the cells to give a final siRNA concentration of 50nM, and after culturing at 37 ℃ for 4 hours, the culture medium was changed to DMEM medium containing 10% FBS.
All experiments were repeated 3 times, and the results are expressed as mean ± SD and statistically analyzed using SPSS 19.0. Statistical differences were analyzed using one-way anova and two-sided t-test. P <0.05 indicates significant difference. In all graphs, a significant difference compared to the untreated group is indicated.
Secondly, real-time quantitative PCR (RT-qPCR) detection of mRNA expression level
Cell culture and siRNA in vitro transfection were performed as described above. The in vitro transfection was performed using 96-well plates.
48h after transfection, total cellular RNA was used
RNA extraction reagent (Thermo Fisher Co., Ltd.) was extracted and detected by RT-qPCR kit (Biomics) according to the protocol. The primer sequences are shown in Table 2, and the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, 45 cycles of denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30 s.
TABLE 2 primers for RT-QPCR detection
As shown in fig. 1, mRNA expression levels of Fascin in the hepatoma cell lines HepG2 and Huh7 were significantly higher than that of normal hepatocyte LO2(P < 0.05); as shown in fig. 3 and 4, Fascin siRNA effectively inhibited the mRNA expression of the Fascin gene in HepG2 and Huh7 cells. Compared with the untreated group, the inhibition rate of Fascin siRNA reaches 65% and 75% (P <0.05), respectively.
Third, Western Blot detects the protein expression level of the gene
Cell culture and siRNA in vitro transfection were performed as described above.
Cells were seeded in 6-well plates at 37 ℃/5% CO2Culturing in an incubator for 24h, and transfecting siRNA when the cell fusion degree reaches 70-80%; after 24h, the culture solution is discarded, and the cells are washed for 2 times by PBS; the operation is carried out on ice, 50 mu L of SDS protein lysate is added into each hole to lyse cells, the cells are fully and uniformly mixed to be viscous, the cells are scraped off by a scraper and transferred into a centrifugal tube with the volume of 1.5mL, the centrifugal tube is placed on the ice after being heated in boiling water bath for 10min, the temperature is 4 ℃, the rpm is 12000 multiplied by 15min, and supernatant fluid is extracted; after SDS-PAGE (5% lamination gel and 8% separation gel) electrophoresis, a wet transfer printer is adopted to transfer the protein in the gel to a PVDF membrane at a constant current of 200mA for 2 h; immersing the PVDF membrane in a confining liquid (5% skimmed milk) and slowly shaking the PVDF membrane on a shaking table for 2 hours; adding primary antibody (1:1000), and incubating at 37 deg.C for 2 h; rinsing the membrane with TBST three times for 10min each time; adding secondary antibody (goat anti-mouse IgG-HRP, 1:1000), and incubating at 37 deg.C for 2 h; after the secondary antibody incubation is finished, rinsing the membrane for three times by using TBST, wherein each time lasts for 5-10 min; carrying out ECL luminescence development on the washed film; and (4) analyzing results: using the internal reference gene as an internal control, the gray value of the target band was analyzed using Image J software, and the relative expression level of the target gene was calculated as the gray value of the target band/the gray value of the internal reference in the same sample.
As shown in fig. 2, the protein expression level of Fascin in the hepatoma cell lines HepG2 and Huh7 was significantly higher than that of normal hepatocyte LO2(P < 0.05); as shown in fig. 5 and 6, Fascin siRNA effectively inhibited protein expression of the Fascin gene in HepG2 and Huh7 cells. Inhibition rates of Fascin siRNA reached 68% and 75%, respectively, compared to the untreated group (P < 0.05).
Fourth, MTT method for detecting cell proliferation
Cell culture and siRNA in vitro transfection were performed using 96-well plates using the methods described above. Before transfection, when the confluency of cells reached about 75%, cells in logarithmic growth phase were plated in 96-well cell culture plates at a seeding density of 5X 104Per well, repeat 3 wells.
OD values of samples of the Fascin siRNA transfection group, NC transfection group and untreated group at 24h, 48h, 72h and 96h of transfection, and samples of the untransfected group were measured by the following method: adding 10 mul MTT into each hole, and placing the culture box at 37 ℃ for 4h in a dark place; adding 150 μ l DMSO into each well, and standing in 37 deg.C incubator for 10 min; after the mixture is blown and uniformly mixed, 120 mul of the mixture is put into another clean 96-well plate, 120 mul of DMSO is taken as a blank control for zero adjustment, and OD is measured on an enzyme-linked immunosorbent assay (Bio-Rad company) with the wavelength of 490 nm; and (5) carrying out data processing, and drawing a cell growth curve.
As shown in fig. 7 and 8, Fascin siRNA treated HepG2 and Huh7 cells 48h, 72h and 96h had significant cell growth inhibitory effect (P <0.05) compared to untreated and NC groups.
Fifth, cell migration detection by cell scratch test
Cell culture and siRNA in vitro transfection were performed as described above.
With a density of 1.5X 105HepG2 and Huh7 cells per well were plated in 24-well plates and transfected. After 48h, the confluent cells were streaked with a pipette tip, washed with PBS buffer at pH 7.4, and serum-free DMEM medium (Thermo Fisher Co.) was added. Cell migration was observed by taking pictures 24h and 48h after scratching and experiments were done in 3 groups in parallel with 4 fields per plate.
As shown in fig. 9 and 10, migration of HepG2 and Huh7 was effectively inhibited when the Fascin siRNA treated HepG2 and Huh7 cells 24 and 48h (P <0.05) compared to the untreated group and the negative control group.
Sixth, Transwell cell invasion experiment
Cell culture and siRNA in vitro transfection were performed as described above. The in vitro transfection was performed using 24-well plates.
Cell migration was measured 72h after transfection using 24-well membrane filters (Corning Bioscience, USA). 1.5X 105The individual cells were plated in the upper chamber to migrate into serum-containing DMEM medium (Gibco Co.) for 24 h. The cells remaining in the upper chamber were removed with a cotton swab and the cells migrated into the lower chamber were fixed with 10% formaldehyde for 30 s. Finally, cells were stained with 0.1% crystal violet for 4min, followed by 3 washes with PBS buffer at pH 7.4. Cells were counted in 200-fold magnification fields, and 5 fields were counted for each condition.
As shown in fig. 11 and 12, Fascin siRNA significantly inhibited HepG2 and Huh7 cell invasion (P <0.05) compared to untreated and NC groups.
Sequence listing
<110> university of southeast Tong
<120> siRNA molecule targeting Fascin gene and application thereof
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Claims (5)
1. A siRNA molecule targeting a Fascin gene, characterized by: consists of a sense strand and an antisense strand, and the sequence of the antisense strand is as follows:
sense strand: 5 '-CGUUCGGUUCAAGGGUGAAdTdT-3',
antisense strand: 5 '-UUCACCUGAACCCGAACGdTdT-3'.
2. Use of the siRNA molecule of claim 1 for the preparation of a medicament for inhibiting the function of a Fascin gene in a cell.
3. The use of the siRNA molecule of claim 1 in the preparation of a medicament for the prevention and/or treatment of liver cancer.
4. Use according to claim 3, characterized in that: the siRNA molecule can induce the apoptosis of the liver cancer cell.
5. Use according to claim 3, characterized in that: the siRNA molecule can inhibit the metastasis and invasion of liver cancer cells.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100297109A1 (en) * | 2007-11-21 | 2010-11-25 | Cornell University | Methods for inhibiting fascin |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100297109A1 (en) * | 2007-11-21 | 2010-11-25 | Cornell University | Methods for inhibiting fascin |
Non-Patent Citations (4)
| Title |
|---|
| J SURG ONCOL: "Fascin expression in progression and prognosis of hepatocellular carcinoma" * |
| 周亮;王德盛;: "Fascin-1与消化系恶性肿瘤" * |
| 孔静萍;顾栋桦;: "RNAi沉默Fascin基因表达对HEp2细胞株生物学特性的影响" * |
| 林跃丰等: "肌成束蛋白-1表达对肝癌细胞增殖及细胞骨架的影响" * |
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