Use of emc8 gene inhibitor in preparation of medicine for treating gastric cancer
A gene suppression, gastric cancer technology, applied in the field of biomedicine, to achieve the effect of changing the cycle distribution, inhibiting the formation of clones, and inhibiting the proliferation rate
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Embodiment 1
[0065] Example 1 Effect of EMC8 gene on the proliferation of gastric cancer cells
[0066] 1.1 Target cells
[0067] The target cells selected in Example 1 are AGS cells (belonging to human gastric adenocarcinoma cells) as shown in Table 3, and the culture medium is a complete medium, that is, F12 medium supplemented with 10% fetal bovine serum.
[0068] Table 3 Example 1 Target cells and culture medium thereof
[0069] target cell complete medium 96-well plate cell count AGS cells (gastric adenocarcinoma cells) F12+10%FBS 1500~2000 pcs / hole
[0070] 1.2 Target virus
[0071] In order to ensure the efficiency of gene interference, in Example 1, 3 RNA interference target sequences were designed for the EMC8 gene. The 3 RNA interference target sequences are shown in Table 4, and 3 plasmid vectors carrying different target sequences were mixed in equal proportions for lentiviral packaging , so as to ensure the knockdown efficiency of the EMC8 gene af...
Embodiment 2
[0119] Example 2 Screening of EMC8 gene inhibitor shRNA and construction of interference lentivirus
[0120] 2.1 Preparation of EMC8 gene interference lentivirus:
[0121] 2.1.1 EMC8 gene target sequence:
[0122] The target sequence of the EMC8 gene is shown in Table 4.
[0123] 2.1.2 Interfering with lentiviral vectors
[0124] Vector name: GV493 (the vector is provided by Jikai Gene Company)
[0125] Element sequence: hU6-MCS-CBh-gcGFP-IRES-puromycin
[0126] Control number: CON313
[0127] Control insert sequence: TTCTCCGAACGTGTCACGT (SEQ ID NO: 4)
[0128] Carrier map such as Figure 4 shown.
[0129] 2.1.3 Design the shRNA interference sequence according to the EMC8 gene target sequence, and synthesize the oligonucleotide chain
[0130] The designed shRNA interference sequence is shown in Table 8, and the shRNA oligonucleotide chain is synthesized according to the sequence, wherein, EMC8-RNAi(70235-11)-a and EMC8-RNAi(70235-11)-b synthesize oligonucleotides Chai...
Embodiment 3
[0172] Example 3 Effects of EMC8 Gene Inhibitors on Gastric Cancer Cells
[0173] MTT experiments, cell apoptosis detection experiments, cell clone formation experiments, and cell cycle detection experiments were carried out to illustrate the effects of EMC8 gene inhibitors on gastric cancer cells, and at the same time prove the effect of the interfering lentivirus constructed in Example 2 on gastric cancer cells. Specifically, unless otherwise specified, the experiments described in Example 3 were conducted using the design in Table 15.
[0174] Table 15 Basic experimental design
[0175]
[0176] 3.1 MTT assay to detect cell viability
[0177] 3.1.1 Experimental steps
[0178] (1) According to 1.4.1-1.4.2 in Example 1, the culture of target cells and lentivirus infection are carried out, and the lentivirus is a lentivirus (NC group) containing a control gene (SEQ ID NO: 4) and Example 2 Prepare the EMC8 gene interference lentivirus LV-EMC8-RNAi (70237-1) (KD group), co...
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