Difference between revisions of "Team:JSNU-China/Description"

·Home

·Project (Background | Experiment | Future work | Protocal)

· Parts

·Team (Members | Attributions | PI&Advisor | Acknowledgements)

·Human Partice (Silver | Quick-flashing | Collaboration)

·Notebook

·Safety

1.We constructed GFP-plasmid and transfected it into SUM52 cells. After 72h, we could detect green fluorescent protein in almost 90% of SUM52 cells.

Fig1.Transfect GFP-plasmid into SUM52 with liposome

2.We set up one SUM52-GFP stable cell line and treated it with different concentrations of anthocyanin. The results of fluorescence microscopy and cell counting kit-8(CCK-8) suggested anthocyanin can kill SUM52 cells.

Fig2.SUM52-GFP treated with different concentrations of anthocyanin(4x)

Fig3.CCK-8 kit to test the cell viability of SUM52-GFP cells

3.We treated normal human gastric epithelial cell line- GES1 and gastric cancer cell line-HGC27 with different concentrations of anthocyanin and tested the cells' viability with CCK-8 kit.The result of CCK-8 showed that anthocyanin had the effect of killing HGC27 cell and had a little effect on GES1 cell lines. We also found normal cells can't endure high concentration of anthocyanin for a long time.

Fig4.CCK-8 kit to test the cell viability of cell lines

(a)GES1 cell (b)HGC27

These experiments showed anthocyanin was a kind of powerful anti-cancer substance. There are many opportunities for humanity to intake anthocyanin for a long time in the course of their entire life. But no research has data to prove the cancer incidence is different between inadequate or excessive intake anthocyanin. Why humanity can't prevent and destroy cancers with it? Maybe, we need push it. We try to find a gene which is special for cancer cell. Is there a hidden traitor in cancer cells? KLF4 is a transcription factor acting as both activator and repressor.

4.We tested KLF4’s expression in various cancer cells by q-PCR and western blot. The results showed that KLF4’s expression is different in a bunch of cancer cells cultured in our lab, mostly downregulation compared with normal human gastric epithelial cell line- GES1.

Fig5. KLF4’s expression in different cancer cell lines

(a)q-PCR (b)Western Blot

5.We constructed KLF4 over-expression and 5 mutant plasmids.

1)Design 6 templates with specific restriction sites for cloning

Fig6. The schematic diagram of five mutants

2)

3)Cloning of "master" into vector

Fig7.BBa_K2000006

After 3 weeks, we selected one KLF4 over-expression cell line from HEK293T cell with G418. WB’s results make sure KLF4 over-expressed in our HEK293-KLF4 cell.

Fig8.KLF4 over expressed in HEK293T-KLF4 cell.

6.Then, we treated HEK293T-KLF4 and HEK293T cells with different concentrations of anthocyanin. After 3 days, HEK293T-KLF4 cells all dead while HEK293T cells dead after 6 days with anthocyanin treatment. The results of CCK-8 kit and light microscopy are consistent which showed upregulation of KLF4 will make HEK293T cell more sensitive to anthocyanin.

Fig9.HEK293T cells treated with different concentrations of anthocyanin

(a)Light microscope showed HEK293T cells dead after 6 days with anthocyanin treatment.

(b)CCK-8 also detected HEK293T cells dead with anthocyanin treatment.

Fig10.HEK293T-KLF4 cells treated with different concentrations of anthocyanin

(a)Light microscope showed HEK293T-KLF4 cells dead after 3 days with anthocyanin treatment.

(b)CCK-8 also detected HEK293T cells dead with anthocyanin treatment.

Our results are so funny, just as the Chinese old saying goes, A mix of Jacks and Jills makes a tough job a breeze. KLF4 maybe is Ms. anthocyanin's Mr. right. Upregulation of KLF4's expression maybe help increase cancer cells' sensitivity to anthocyanin. Another truth is , cancer cells will tolerate it after a long time of taking in anthocyanin. KLF4 downregulated in some cancer cells to help its cancer stem cell to escape from immune system, including lost sensibility to anti-cancer drug. That's why we can't use anthocyanin in clinical cancer therapy. Maybe, our work throw a little light in this dilemma, we can find a plant inside the cancer cells to cooperate with anthocyanin and destroy cancer cell.

In the future, we'll do more jobs about the molecular mechanism of anthocyanin and KLF4. We also finished constructing 5 mutants to explore the relation of KLF4 and anthocyanin.

References:

[1] Zhang ZF, Lu J, Zheng YL, et al. Purple sweet potato color attenuates hepatic insulin resistance via blocking oxidative stress and endoplasmic reticulum stress in high-fat-diet-treated mice.J Nutr Biochem. 2013,Jun 24(6):1008-18.

[2] Lu J, Wu DM, Zheng YL et al. Purple Sweet Potato Color Alleviates D‐galactose‐induced Brain Aging in Old Mice by Promoting Survival of Neurons via PI3K Pathway and Inhibiting Cytochrome C‐mediated Apoptosis . Brain Pathol. 2010 May;20(3):598-612.

[3] Capra hircus Kruppel-like factor 4 (KLF4) mRNA, complete cds.https://www.ncbi.nlm.nih.gov/nucleotide/1021691550?report=genbank&log$=nuclalign&blast_rank=10&RID=Z74WM35U015

Compared with traditional chemotherapy, combining eating foods rich in anthocyanin with gene therapy has many advantages, such as no harm and lower cost in treatment. Since everybody's genes are different, we can treat patients one-to-one in gene therapy. In this way, we will get a more efficient result and we don't need to take medicine any more. We can take in anthocyanin from grapes, cherries, strawberries and some food that we can purchase easily.