Team:JSNU-China/Description
Background
Although it is one of the most researched and funded fields in medicine, cancer is still a major cause of morbidity and mortality all over the world, with 14 million new cases and over 8 million deaths per year. It is also the second cause of death and is responsible for quarter of the death cases among developed countries.
People have tried many ways to keep health or prevent cancer, such as eating some healthy diet, keeping active, avoiding certain infections and so on. Anthocyanin works as a natural colorants in food with health-promoting properties, which could protect our heart and boost our cancer defense, and during the process of preventing and curing cancer, doctors always encourage patients to use anthocyanin-like foods and drugs. However, anthocyanin cannot kill cancer cells and prevent more from growing in our bodies effectively.
About anthocyanin
Anthocyanin is a type of natural flavonoid and antioxidant with powerful anti-cancer properties. Anthocyanin will be able to protect against cancer indirectly through the prevention of DNA damage. Anthocyanin can also make cancer cells not be successfully diffused, thereby protecting the healthy cells not being eroded by cancerous ones.
About KLF4
Kruppel-like-factor 4（KLF4）is a zinc-finger transcriptional factor that regulate gene expression. KLF4’s expression level is different in gastric cancer, lung cancer, bladder cancer ,breast cancer and oral squamous cell.
Experiment
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 anthocyanin. The results of fluorescence microscopy and cell counting kit 8(CCK8) suggested anthocyanin can kill SUM52 cells.
Fig2.SUM52-GFP treated with different concentrations of anthocyanin(4x)
Fig3.CCK8 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 CCK8 kit. CCK8 results showed that anthocyanin could kill HGC27 cell easily 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.CCK8 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 both as activator and repressor.
4.We tested KLF4’s expression in various cancer cells by q-PCR and western blot. The results shown 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 line (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-express 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 3days, HEK293T-KLF4 cells all dead while HEK293T cells dead after 6 days with anthocyanin treatment. The results of CCK8 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)CCK8 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 3days with anthocyanin treatment.
b)CCK8 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 ZF1, Lu J, Zheng YL, Wu DM, Hu B, Shan Q, Cheng W, Li MQ, Sun YY. 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. doi: 10.1016/j.jnutbio.2012.07.009. Epub 2012 Sep 17.
[2] LU J, WU D M, ZHENG Y L, 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 [J]. Brain Pathology, 2010, 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
Future Work
According to KLF4 structure, We construct to build another four mutant.
However, due to time constraints, we have not had time to make this part of the work, which will be our next will try to complete the work
Protocal
The experimental operation we mainly used
1.EnoGeneCell Counting Kit-8
Detection principle
◆Cell Counting Kit Acronym CCK8 kit is based WST-8 (chemical name: 2- (2-methoxy-4-nitro-phenyl) -3- (4-nitro-phenyl) -5- (2 , 4-sulfophenyl) -2H- tetrazolium monosodium salt) is widely used in cell proliferation and cytotoxicity of fast, high-sensitivity detection kit.
◆WST-8 belonging to MTT upgrade products, works as follows: In the case of electronic coupling agent is present and can be restored Dehydrogenase mitochondria produce highly water-soluble orange formazan product (formazan). Color depth and proliferation of cells is directly proportional to cell toxicity. OD value was measured using a microplate reader at a wavelength in the 450mM indirectly reflect the number of viable cells.
◆CCK8 method is widely used, such as drug screening, cell proliferation assay, cytotoxicity assays, tumor susceptibility testing and detection of biological activity and other factors.
Cell Viability Assay
1, the cell suspension seeded in 96-well plates (100μL / well). The plates were pre-incubated in an incubator (37 ℃, 5% CO2).
2, was added to each well 10 μL CCK8 solution (be careful not to bubble Kong Zhongsheng, they can affect the reading OD values).
3, the plates were incubated in the incubator for 1-4 hours.
4, then determined by comparing the absorbance at 450nm.
5, if the OD value temporarily, can be added to 10 μL 0.1M HCL solution or 1% w / v SDS solution to each well, and the cover plates stored in the dark at room temperature. Within 24 hours measured absorbance change does not occur.
Cell viability * (%) = [A (dosing) -A (Blank)] / [A (0 dosing) -A (Blank)] × 100
2.Molecular Cloning
3.Western blot
Extract proteins
1.1000r/min centrifuge for 5 minutes to remove PBS.
2.Add lysis buffer and shake it.
3.12000r/min freezing centrifuge for 15minutes
4.Add loading buffer and boil for 10 minutes
5.Put it on the ice.
SDS-PAGE
1. Plates:The sealed box with silica gel placed flat on the glass, and then the concave glass and flat glass overlap, the two glass stand up to make contact with the bottom of the desktop, two glass hand clamped into the electrophoresis tank, then insert Xiecha board to moderate degree, you can glue.
2. Polymer gel:Separating gel and stacking gel preparation: The following table sequentially and proportion solution, configuration of 10% separating gel and 4.8% stacking gel.
3.
| Separating gel | Stacking gel | |
| ddH2O | 4.95ml | 3.05ml |
| Arc-Bis(30%) | 6ml | 0.67ml |
| Tris-Hcl | 3.75ml(PH=8.8) | 1.25ml(PH=6.8) |
| 10%SDS | 150ul | 50ul |
| 10%APS | 150ul | 50ul |
| TEMED | 15ul | 10ul |
Following the above table, mix them.After separating gel formulation, add it to the gel, be careful not to produce bubbles. Then add water carefully to make surface covered with water.Put them on room temperature about 30-40min. When absorb the water completely, prepare stacking gel according to the above table. Add stacking gel and insert the comb .Finally,carefully remove the sample.
4.Loading:add sample to the sample tank
5.Electrophoresis
Transfer and block
1.transfer the protein to PVDF membrane.
2.block the membrane with 5% milk 1h.
3.add the first antibody and overnight at 4 degrees temperature.
4.wash membrane 15min 3 times.
5.add second antibody 1h at room temperature.
6.wash membrane 15min 3 times.
Expose
Expose and we can see the result clearly.
4.Cell culture
Cell culture technology refers to the number of cells after a lot of training to become a simple single cell or a little more differentiated cells, which are essential aspects of cloning technology. We can get a lot of cells or metabolites through cell culture. Because biological products are derived from the cells, it can be said cell culture technology is biotechnology core, the most basic technology.
Cell recovery
1.We put cryopreserved tubes in 37 degrees temperature water bath pot and shake it until dissolved.We can complete the thawing about one to two minutes.
2.Put cryopreservation solution and 5 ml of culture into a centrifuge tube and Pipet.
3.1000r/min centrifuge for 5 minutes
4.Add the precipitate to complete medium, pipetting, transferred to a Petri dish.
Cell culture
Add 55ml serum and 6ml Dual anti to the medium.
Collect cell
6.Add Trypsin and stop digestionafter cell culture medium plus curled .
7.Absorb the culture medium to completely blow down cells.
8.Transfer the cells to a centrifuge tube and centrifuge for 5 minutes
9.Discard the supernatant, add wash cells with PBS.
10.1000r/min centrifuge for 5 minutes
11.Discard the supernatant and add frozen -80 degrees to cryopreservate.
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Address:Jiangsu Normal University, 101 Shanghai Rd, Tongshan District, Xuzhou, China
Mail:jsnuchina@163.com
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