Application of rfpl1s-201 in the preparation of drugs for inhibiting proliferation, invasion and/or metastasis of ovarian cancer
An RFPL1S-201, ovarian cancer technology, applied in drug combinations, pharmaceutical formulations, anti-tumor drugs, etc., to achieve the effects of increasing survival, promoting tumor chemotherapy sensitivity, and inhibiting tumor progression
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Embodiment 1
[0039] 1. Cell lines
[0040] The ovarian cancer cell line SKOV3 was purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences; the normal ovarian epithelial cell line IOSE386, and the ovarian cancer cell lines A2780, HO-8910, HO-8910PM, OVCAR3, and OVCAR8 were purchased from KGI. Cell culture conditions are as follows:
[0041] Culture medium: SKOV3: McCoy’s 5A incomplete medium (1×) + 10% fetal bovine serum (FBS)
[0042] A2780: DMEM incomplete high-glucose medium (1×) + 10% fetal bovine serum (FBS)
[0043] Digestive solution: 0.25% Trypsin-EDTA (1×)
[0044] Freezing medium: fetal bovine serum (FBS): dimethyl sulfoxide (DMSO): = 9: 1
[0045] 2. Clinical specimens
[0046] The samples in this study were collected from patients undergoing surgery in Nanjing Maternal and Child Health Hospital from 2016 to 2017. A total of 13 normal ovarian tissue samples (from cervical cancer and endometrial cancer with preventive ovary rese...
Embodiment 2
[0057] 1. Construction of RFPL1S-201 interference RNA and overexpression plasmid:
[0058]The interfering RNA sequence of RFPL1S-201 and the control interfering RNA sequence were designed and synthesized by American Thermo Fisher Company.
[0059] The RFPL1S-201 overexpression plasmid pc-RFPL1S-201 was constructed and synthesized by Shanghai Jierui Bioengineering Co., Ltd. on the basis of pcDNA3.1(+), the cloning site is: NheI / NotI, and LncRNA RFPL1S-201 (ENST00000419368. 1) The full length is connected to the cytomegalovirus (CMV) promoter of the pcDNA3.1 vector. Use the OMEGA kit (D6915-03) plasmid to measure the concentration of the extracted plasmid, and detect that the sequence is connected successfully, and use it directly for subsequent experiments or store it at -80°C.
[0060] 2. Cell Transfection
[0061] 1) When the confluence of SKOV3 and A2780 cells cultured in the 6-well plate reaches 90%-95%, the RFPL1S-201 overexpression plasmid, control pcDNA3.1 plasmid, RFP...
Embodiment 3
[0068] Example 3 Cell Proliferation Experiment
[0069] 1) Count the cells after 24 hours of transient transfection, dilute to the desired concentration with medium containing 10% FBS, and add the cell suspension to a 96-well plate. Each well contains about 1000 cells in a 100μ suspension, divided into 0 hours. , 24h, 48h, 72h four groups, 5-6 auxiliary wells for each group, placed at 37°C, 5% CO 2 Wait for the cells to adhere to the wall for 3-4 hours in the incubator;
[0070] 2) Measure the cell viability according to the grouping time after the cells adhere to the wall: replace the medium with a mixed solution with 10% CCK-8 reagent, 37°C, 5% CO 2 After reacting in the incubator for 1 hour, detect on a microplate reader, detection wavelength: 450nm.
[0071] see results figure 2 A-B and image 3 A-B; It can be seen from the figure that overexpression of RFPL1S-201 can significantly inhibit the proliferation of ovarian cancer cells, and knockdown of RFPL1S-201 can sign...
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