Part:BBa_K2796029:Experience

We used lentiviral vector to express miR135b-3p in gastric cancer cells and detected its function.Including the use of fluorescence imaging to detect transfection efficiency.Using cck-8 detected the activity of gastric cancer cells after expression of miR-135b-3p.Scratch test and transwell test were used to detect migration and invasion of gastric cancer cells.We also use flow cytometry to analyze cell cycles.

The transfection effect of miR overexpressing plasmid was detected by fluorescence

mir135b-3p overexpression vector was transfected into human embryonic renal epithelial cell line HEK293T, gastric cancer cell line MKN45 and SGC7901 through lentivirus. The cells were transfected by puromycin screening. Figure A-C shows the transfection efficiency through the mRFP reporting system under an inverted fluorescence microscope at 40 times objective lens. The results showed that mir135b-3p expression plasmid was successfully transfected into cells and expressed. Figure D-F is the observation result under 100 times objective lens.

Using cell wound scratch assay to analyze cell migration

We examined the effects of mir-135b-3p on gastric cancer cell lines MKN45 and SGC7901 through scratch test. It can be observed from the experimental results that the control gastric cancer cells almost healed after 24 hours of scratch, while mir-135b-3p significantly inhibited the migration ability of gastric cancer cell lines MKN45 and SGC7901.

Using CCK8 (Cell Counting Kit-8) experiment to detect cell proliferation.

a.Because the cck-8 reagent can be reduced by dehydrogenase in the cell's mitochondria to form a highly water-soluble yellow formazan. The amount of formazan is directly proportional to the number of living cells . Therefore, we used cck-8 to detect the number of living cells . Because 450 nm is specific absorption peak of formazan, we used Abs 450 to detect formazan. The results showed that overexpression of miR-135b-3p could significantly reduce the number of gastric cancer cell MKN45.

b.As shown in the previous experiment, overexpression of miR-135b-3p significantly reduced the number of gastric cancer cell SGC7901. The decline in the number of cells on the fifth day may be due to the depletion of nutrients in the cell culture medium.

Using transwell experiment to analyze cell invasion ability.

a.The tanswell chamber is divided into two parts by polycarbonate film and matrix glue. Tumor cells were added to the upper compartment and culture medium was added to the lower compartment. Due to the high nutrient content of the lower compartment, tumor cells will pass through the matrix glue and migrate down the chamber. The results showed that overexpression of mir-135b-3p inhibited the invasion of gastric cancer cell MKN45.

b.Cancer cells need to secrete protease to digest the stromal glue before they can pass through the compartment. We measured the number of cells in the lower chamber using the cck-8 reagent. The results show that overexpression of mir-135b-3p inhibited the invasion of gastric cancer cell SGC7901.

Flow cytometry（FCM） shows cell cycle was inhibited by miR-135b-3p

The effect of overexpression of miR-135b-3p on the cell cycle of gastric cancer cell line MKN45 was analyzed by using flow cytometry. A complete cell cycle includes the M phase (cell division phase) and the interphase. Interphase is divided into phase G1 (early DNA synthesis), S (DNA synthesis) and G2 (late DNA synthesis). As we can be observed from this figure, the number of cells in the G1 phase of MKN45 cells overexpressing miR-135b-3p increased, while the number of cells in the G2 phase decreased. This indicated that mir-135b-3p affected the DNA synthesis of gastric cancer cell MKN45, i.e. S phase.

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