Application of inhibiting IncRNA expression in treatment of stomach cancer
A gastric cancer, 00211901 lncRNA technology, applied in the field of biopharmaceuticals, can solve the problems affecting the invasion and metastasis of gastric cancer cells
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Embodiment 1
[0031] Example 1 Overexpression and inhibition of expression of 00211901 lncRNA
[0032] Gastric cancer MKN74 cells, product number BS-C61866280, were purchased from Shanghai Binsui Biotechnology Co., Ltd., and were cultured and subcultured for 3 generations according to the instructions to prepare for transfection.
[0033] Adopt liposome 2000 reagent (U.S. Invitro}en company) to transfect siRNA-00211901 (5'-ggatggatctctctgagtga-3') and eukaryotic expression vector pcDNA3.0-00211901 (constructed by the conventional construction method in this field; Wherein 00211901 The sequence is shown in SEQ ID NO: 1), before the specific transfection, trypsinize the MKN74 cells grown to the logarithmic phase, and adjust the cell concentration to 1×10 6cells / ml, conventionally cultured for 24 hours, transfected cells with siRNA, and conventionally cultured the transfected gastric cancer cells, and detected 00211901 gene knockout and overexpression after 48 hours.
Embodiment 2
[0034] Example 2 RNA extraction and real-time fluorescent quantitative polymerase chain reaction detection
[0035] Total RNA of gastric cancer cells before and after transfection was extracted by TRIzoI reagent. According to the preparation process, Bestar reverse transcription system fluorescent quantitative polymerase chain reaction (FQ-PCR) kit was used. Use 7500 real-time FQ-PCR system (ABI Company, USA) for real-time quantitative polymerase chain reaction (RT-qPCR) detection (the double-stranded DNA is denatured at 90-95°C, then rapidly cooled to 40-60°C, the primers are annealed and combined to the target sequence, then rapidly increase the temperature to 70-75°C, and cycle 30-40 times). Each detection method was a triple method, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control gene. The primer sequence used: 00211901 5'-agactggcttcctctctcct-3'(forward), 5'-atgggatccaaatatcttcc-3'(reverse); GAPDH5'-TGTTC-GTCATGGGTGTGAAC-3'(forward...
Embodiment 3
[0036] Example 3 Cell Apoptosis Detection
[0037] Collect cells transfected for 48 hours, take 1ml of cells, wash with pre-cooled PBS and resuspend in 1×Bindingbuffer, add AnnexinV-FITC and PI respectively, 5μl and 10μl respectively, keep away from light for 15-20min at room temperature, add 300μl of 1×Bindingbuffer, 1h Internal flow cytometry detection. The experiment was repeated three times. Statistical method SPSS21.0 software was used for analysis, and the measurement data was expressed as x±s. Differences among multiple groups were compared using one-way analysis of variance, and pairwise comparisons were performed using SNK-q test. P<0.05 was considered statistically significant.
[0038] siRNA transfection induced apoptosis of MKN74 cells in each group The results of apoptosis rate detection are as follows: figure 2 The apoptosis rate of transfected gastric cancer cells detected by flow cytometry showed that the apoptosis rate of siRNA group was significantly highe...
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