siRNA for inhibiting MCM7, composition and application of siRNA
A composition and drug technology, applied in the field of biomedicine, can solve problems such as siRNA that have not yet been seen, achieve significant clinical application prospects and economic value, improve efficiency, and improve therapeutic effects
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Embodiment 1
[0071] Example 1 siRNA design
[0072] According to the basic principle of siRNA target sequence, the siRNA sequence (1 21 nucleotides) expressed by the human MCM7 gene transcript (NM_001278595.1) was designed and synthesized, that is, the sense strand and the antisense strand of siRNA, and its base sequence is:
[0073] siRNA-1 sense strand: 5'-GGACUUAAUUUGUGAGAAUdTdT-3'; (SEQ ID NO.1)
[0074] siRNA-1 antisense strand: 5'-AUUCUCACAAAUUGAGUCCdTdT-3' (SEQ ID NO.2).
[0075] or
[0076] siRNA-2 sense strand: 5'-GGAAGUGGUAAAUAAAGAUdTdT-3' (SEQ ID NO.3);
[0077] siRNA-2 antisense strand: 5'-AUCUUUAUUUACCACUUCCdTdT-3' (SEQ ID NO.4).
[0078] The base sequence of the negative control RNA (NC) is:
[0079] Sense strand: 5'-CUCUUAGCCAAUAUUCGCUdTdT-3' (SEQ ID NO.5);
[0080] Antisense strand: 5'-AGCGAAUAUUGGCUAAGAGdTdT-3' (SEQ ID NO. 6).
[0081] The siRNA sequence of the present invention and the 3' end of the sense strand and the antisense strand of the control RNA are added ...
Embodiment 2
[0084] Example 2 Transfection of siRNA in cells
[0085] The liposome Lipofectamine RNAiMax was used as the transfection reagent, and the steps were in accordance with the operating procedures of Thermo Fisher Scientific Company. The cell lines used were HepG2 and Hep3B liver cancer cell lines, SGC-7907 gastric cancer cell line, PC3 prostate cancer cell line and MCF7 breast cancer cell line.
[0086] The experimental method of the present invention not indicating specific conditions, generally according to conventional conditions such as the conditions described in "Molecular Cloning: Laboratory Guide" (New York: Cold Spring Harbor Laboratory Press, 1989) by Sambrook et al. suggested conditions.
[0087] The transfection steps are as follows: inoculate the above-mentioned different cell lines in 12-well plates, culture overnight at 37° C. and 5% CO 2 , so that the cell growth density reaches 40-50%. Referring to the operating procedure of Lipofectamine RNAiMax (Thermo Fisher...
Embodiment 3
[0088] Example 3 siRNA inhibits MCM7mRNA expression detection
[0089] Methods: Cells were collected after transfection, total RNA was extracted, and real-time fluorescent quantitative PCR was performed after reverse transcription to detect the expression of MCM7mRNA after siRNA treatment of cancer cells.
[0090] The siRNA prepared by the present invention and the negative control RNA (NC) were respectively transfected into the liver cancer cell line HepG2, and the same method was also used to transfect the liver cancer cell line Hep3B. After 24 hours of transfection, the cells were collected, and an appropriate amount of cells were re-inoculated in In 6-well plates, cells were harvested after 72 hours to extract total RNA. Reverse transcription, and real-time fluorescent quantitative PCR detection of MCM7mRNA.
[0091] 1. Total RNA Extraction
[0092] (1) Collect tumor cells into centrifuge tubes, centrifuge at 800rpm for 3min, discard supernatant, add PBS to wash once and...
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