The Influence And Mechanism Of KRAS Gene On Glycolysis In Lung Cancer Cell

BACKGROUND:Lung cancer is the most common cancer in our country and even the whole world, the incidence and mortality rate rank the top of list. With the development of medicine, for the treatment of lung cancer, in addition to the traditional treatment of surgery, chemotherapy and radiotherapy, the gradual rise of the targeted therapy, making the treatment more individual. It is necessary to detect mutations individuals. The study found that expression of KRAS oncogene occupyed 30% in the lung cancer, mainly exist in the non-small cell lung cancer.It is the one of the important proto-oncogene,and connected with cancer growth, proliferation and angiogenesis closely, and related with poor prognostic.The function of the KRAS protein has similar with G proteins, and has the ability to bind with guanylate.When it binds to GTP, play a role with a variety of effector molecules such as MAPK,STAT, PI3 K,and thereby regulating cell growth, proliferation and apoptosis. A lot of research which target to above-mentioned signal pathway, so far it has not found an effective therapeutic target.Normal cell is mainly dependent on mitochondrial oxidative phosphorylation to generate the required energy, and most of the tumor cells, not by aerobic oxidation, aerobic glycolysis is its main production mode, a phenomenon known as "Warburg effect ", that is, under aerobic conditions, cancer cells exhibited efficient glucose uptake, and produce large amounts of lactic acid.2011 Douglas Hanahan et al published a review in the journal Cell, take the energy metabolism as one of the signal of cancer cells. This feature can promote cancer cell proliferation, invasion and mediating drug resistance. However, the precise molecular mechanisms of Warburg effect is not clear, more and more evidence shown glycolysis has largely connected with oncogenes, such as ras, src, myc, p53 etc.In recent years, colon cancer cells and pancreatic cancer cells were found that KRAS can regulate the glycolytic pathway to promote the growth and maintain tumor.Studies have shown that KRAS can regulates glycolytic by upregulating HIF-1, mTOR, Pak1 and other pathways. In the field of lung cancer, the relationship with KRAS and glycolysis not research a lot, and the mechanism of the KRAS regulate glycolysis pathway is not fully clear yet.Purpose:This experiment attempts to understand the impact of KRAS gene on the glycolysis pathway in lung cancer cells. The expression of KRAS, HK-2,PKM2 in non-small cell lung cancer cells and the amount of lactate concentration.After knocking down and upregulating the expression of KRAS,determined the expressions of HK2, PKM2 and. To prove KRAS can influence glycolysis pathway in lung cancer through the above research.Methods and Results:The first part: The expression of KRAS and glycolytic key enzyme in three lung cancer cell linesThe study collected RNA,total protein and the supernatant in culture medium of H1299, A549, SPC-A1 cells. The expressions of KRAS, HK2, PKM2 in 3 lung cancer cells H1299, A549 and SPC-A 1 were determined with q RT-PCR and Western blot, and assessed lactate concentration at the same time.1. The expression of KRAS H1299 cells were the highest among above 3 lung cancer cell lines determined with qRT-PCR and Western blot. The difference was statistically significant(P <0.05).2. The expression of HK2,PKM2 H1299 cells were the highest among above 3 lung cancer cell lines determined with qRT-PCR and Western blot. The difference was statistically significant(P <0.05).3. The lactate secretionin H1299 cells were the highest among above 3 lung cancer cell lines. The difference was statistically significant(P <0.05).The second part: The influence glycolytic key enzyme HK2, PKM2 in lung cancer cellAfter transfected with H1299 cells which have the highest expressionof KRAS, detect its interference efficiency. The cells are grouped into: control group(CON group), negative sequence control group(NC group), the effective sequence interference group(KD group). The expressions of HK2, PKM2 were assessed with q RT-PCR and Western blot in KD group significantly reduced,as well as the lactate secretionin. Treatment with TNF-αin H1299 cells and transfecte with negative control and KRAS-shRNA,After that,the expression levels of KRAS,HK2, PKM2, as well as the lactate concentration were rised.1. The three sequences, and a negative control sequence to interference KRAS gene in H1299 cells. Detected that KRAS-RNAi(10300-1) is the most effective sequence,and the difference was statistically significance(P <0.05), Use this interference sequence with lung cell to observed stable cell lines for subsequent experiments.2. After knocking down KRAS, q RT-PCR and Western blot detect the expression n of HK2, PKM2 in KD group, compared with CON and NC group,the levels were significantly lower than before, the difference was statistically significance(P <0.05).3. After knocking down KRAS, the lactate secretionin detected reduced that compared with CON and NC group, the difference was statistically significant(P <0.05).4. The effect of different concentrations of TNF-α in H1299 cells, Western blot showed that the highest expression of KRAS with concentration of 20 ng / ml TNF-α.5. Treatment with 20 ng / ml TNF-αin NC and KD group,the expression levels of KRAS,HK2, PKM2 were rised,the difference was statistically significant(P <0.05).6. Treatment with 20 ng / ml TNF-αin NC and KD group, lactate concentration were rised, the difference was statistically significant(P <0.05).Conclusions:1. In three lung cancer cells, the expression of KRAS correlated with HK2 and PKM2.2. The expression of KRAS influence lactic acid secretion, suggesting that KRAS involved in the glycolytic pathway in lung cancer cells.3. KRAS influence glycolytic pathway.by regulating HK2 and PKM2.