Designed by: Shuangshuang Pu   Group: iGEM18_LZU-CHINA   (2018-10-04)


  • microRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. miRNAs are transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts (pri-miRNAs) that can be either protein-coding or non-coding. The primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce an approximately 70-nt stem-loop precursor miRNA (pre-miRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA and antisense miRNA star (miRNA*) products. The mature miRNA is incorporated into a RNA-induced silencing complex (RISC) , which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in translational inhibition or destabilization of the target mRNA.
  • The expression of hsa-mir-135b-3p can be cut into mir-135b-3p, mir-135b-3p was screened from TCGA database and its survival index in gastric cancer is -0.294 (anything below -0.15 indicate inhibiting effect on digestive tumors) using the method of bioinformatics. We have verified its effect through a series of cellular function experiments such as CCK8, flow cytometry, transwell and scratch assay.

  • 2018 LZU-CHINA 29design1.png

    • Our design is show in above figure. Three microRNA were transcript by three different inducible promoters. We plan to use biotin-induced system to regulate miR135b-3p expression, tetracycline induction system to regulate miR942-5p expression, and galactose induction system was applied to regulate miR769-5p expression. So far, we have verified the function of mir135p-3p and biotin-inducing system, respectively.

      Biological information analysis

      2018 LZU-CHINA 41 bio1.png

      a.According to bioinformatics prediction analysis, miR-135b-3p can inhibit 50 genes expression. The redder the color, the higher the gene expression, and the bluer the color, the lower the gene expression. As you can see from the diagram. When miR-135b-3p expression was at a high level, most genes were at a low level. As the expression level of miR-135b-3p gradually decreased, most genes showed high expression level. However, some gene expression levels are not linearly correlated, which may be due to the interaction between genes.

      2018 LZU-CHINA 41 bio2.png

      b.The gene pathways that miR-135b-3p may affected and the interactions between these gene pathways.

      2018 LZU-CHINA 41 bio3.png

      c.The interaction network between the same functional proteins affected by miR-135b-3p.

      2018 LZU-CHINA 41 bio4.png

      d.GO (Gene Ontology) analysis. The molecular function affected by miR-135b-3p were analyzed by GO database, and there was a total of 20 molecular function related to miR-135b-3p. The color and size of the histogram represent the P value. The darker the color, the smaller the P value, the stronger the correlation between this function and miR-135b-3p.

      Experimental results

    • The transfection effect of miR overexpressing plasmid was detected by fluorescence
      2018 LZU-CHINA 29 experimental result1.png

      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
      2018 LZU-CHINA 29 experimental result2.png

      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.
      2018 LZU-CHINA 29 experimental result3.png

      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.

      2018 LZU-CHINA 29 experimental result4.png

      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.
      2018 LZU-CHINA 29 experimental result5.png

      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.

      2018 LZU-CHINA 29 experimental result6.png

      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
      2018 LZU-CHINA 29 experimental result7.png

      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.

      Sequence and Features

      Assembly Compatibility:
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