KRAS基因表达对肺癌细胞糖酵解途径的影响及机制

发布时间：2018-06-15 17:44

本文选题：KRAS + 非小细胞肺癌　；参考：《第三军医大学》2015年硕士论文

【摘要】：背景:肺癌是我国乃至全球最常见的恶性肿瘤,其发病率及死亡率均居榜首[1]。随着医学事业的发展,对于肺癌的治疗,除了手术、化疗、放疗等传统治疗手段外,逐渐兴起了靶向治疗,使治疗更加个体化,检测个体的突变基因成为了必要[2]。研究发现30%的肺癌中存在致癌基因KRAS的表达,而且主要存在于非小细胞肺癌(non-small cell cancer,NSCLC)中,是重要的原癌基因之一,与肿瘤的生长、增殖及血管生成密切相关,且是不良的预后因素[3-6]。KRAS基因编码的蛋白,与G蛋白的功能相似,具有与鸟苷酸结合的能力,当其与三磷酸鸟苷(Guanosine triphosphate,GTP)结合后,可与多种效应分子如促有丝分裂原活化蛋白激酶(Mitogen-actived Protein Kinases,MAPK),信号传导与转录激活因子(Signal transducers and act ivators of transcription,STAT)和磷酸肌醇3激酶(Phosphotylinosital 3 kinase,PI3K)等发生作用,从而调节细胞的生长、增殖与凋亡[7,8]。以上述信号通路为靶点的研究很多,但目前为止,并未发现有效的治疗靶点。正常分化的细胞主要依赖于线粒体的氧化磷酸化生成所需的能量,而大部分肿瘤细胞,不是靠有氧氧化,是以有氧糖酵解为其主要产能方式,这种现象称为“Warburg效应”,即在有氧条件下,恶性肿瘤细胞表现出高效摄取葡萄糖,并产生大量乳酸[9,10]。2011年Douglas Hanahan等在Cell杂志上发表综述,将能量代谢异常作为肿瘤细胞的标志之一[11]。该特性能够促进肿瘤细胞的增殖、侵袭及参与介导耐药。然而Warburg效应的确切分子机制尚不清楚,越来越多的证据表明,该效应与癌基因有很大的关系,如Ras、src、myc、p53等[12,13]。近年来,在结肠癌细胞、胰腺癌细胞等其他肿瘤性疾病中发现KRAS通过调控糖酵解途径促使肿瘤的生长以及增殖[14-17],研究表明KRAS可通过调节缺氧诱导因子-1(Hypoxia inducible factor-1,HIF-1)、雷帕霉素靶蛋白(Mammalian target of rapamycin,m TOR)、p21活化激酶1(P21-activated kinase1,Pak1)等通路调节糖酵解途径[18-20]。在肺癌领域,KRAS与糖酵解的研究甚少,且KRAS调控糖酵解途径的机制尚未完全明了。目的:本实验目的是了解kras基因对肺癌细胞中糖酵解途径的影响。首先观察非小细胞肺癌细胞中kras、己糖激酶-2(hexokinase-2,hk-2)、丙酮酸激酶m2型(pyruvatekinasem2)的表达情况及乳酸生成量,通过敲降或上调kras表达水平,检测hk2、pkm2的表达情况及乳酸生成量。通过以上研究,以证明kras对肺癌细胞糖酵解途径的影响及其机制。方法和结果:第一部分:kras以及糖酵解关键酶在三种肺癌细胞株中的表达水平本研究收集h1299、a549、spc-a1细胞的rna、总蛋白以及培养基中的上清液,通过qrt-pcr、westernblot方法测定三种肺癌细胞的kras、hk2、pkm2,同时检测乳酸生成情况。1.在三种肺细胞中,qrt-pcr、westernblot结果显示h1299细胞的kras表达量最高,差异有统计学意义(p0.05)。2.在三种肺癌细胞中,qrt-pcr、westernblot结果显示h1299细胞的hk2、pkm2表达量最高,差异有统计学意义(p0.05)。3.在三种肺癌细胞中,乳酸浓度检测显示h1299细胞上清液中乳酸浓度最高,差异有统计学意义(p0.05)。第二部分:kras表达对肺癌细胞糖酵解关键酶hk2、pkm2的影响将上述三种肺癌细胞中kras表达量最高的细胞株h1299予以慢病毒干扰,并检测其干扰效率。将细胞分组为:空白对照组(con组)、阴性序列对照组(nc组)、有效序列干扰组(kd组),采用qrt-pcr、westernblot检测kd组细胞中hk2、pkm2的表达量较前明显降低,乳酸生成量也较前明显降低。tnf-α作用于nc组及kd组细胞,再次用qrt-pcr、westernblot检测细胞中kras、hk2、pkm2的表达水平以及乳酸生成量,与nc组比较,tnf-α作用后,kras、hk2、pkm2表达量较前升高,乳酸生成量也较前升高。1.将三种针对kras基因的干扰序列以及阴性对照序列分别感染h1299细胞,检测发现kras-rnai(10300-1)该干扰序列较其余两组序列及阴性对照组有显著下降,差异有统计学意义(p0.05),并将该组干扰序列成功构建稳定细胞株以备后续实验。2.敲降KRAS后,qRT-PCR、Western blot检测KD组细胞中的HK2、PKM2表达,与CON及NC组比较,敲降后H1299细胞中HK2、PKM2表达水平较前明显降低,差异有统计学意义(P0.05)。3.敲降KRAS后,乳酸浓度检测显示,与CON及NC组比较,敲降后H1299细胞的乳酸生成量减少,差异有统计学意义(P0.05)。4.不同浓度TNF-α作用于H1299细胞,Western blot结果显示以浓度20ng/mlTNF-α作用H1299细胞后,KRAS蛋白表达最高。5.以20ng/ml TNF-α作用NC组及KD组细胞后,采用qRT-PCR、Western blot检测显示细胞中KRAS、HK2、PKM2均升高,差异有统计学意义(P0.05)。6.以20ng/ml TNF-α作用NC组及KD组细胞后,乳酸浓度检测显示作用后细胞上清液中乳酸浓度较前升高,差异有统计学意义(P0.05)。结论:1.在三种肺癌细胞中,KRAS表达量与糖酵解关键酶HK2、PKM2表达量相关。2.KRAS表达水平对乳酸分泌有影响,提示KRAS参与了肺癌细胞中糖酵解途径。3.KRAS通过调节糖酵解关键酶HK2、PKM2,影响糖酵解途径。
[Abstract]:Background: lung cancer is the most common malignant tumor in China and even in the world. Its incidence and mortality rate are at the top of [1]. with the development of medical cause. For the treatment of lung cancer, besides the traditional treatment methods such as surgery, chemotherapy and radiotherapy, the target therapy has been gradually developed to make the treatment more individualized, and the detection of individual mutant genes has become the necessary [2].. The study found that 30% of lung cancer has the expression of oncogene KRAS, which is mainly in non-small cell cancer (NSCLC), one of the important proto oncogenes, closely related to the growth, proliferation and angiogenesis of the tumor, and is a poor prognostic factor encoded by the [3-6].KRAS gene, similar to the function of the G protein. Having the ability to bind with guanosine, when it is combined with Guanosine triphosphate, GTP, can be used with a variety of effector molecules such as Mitogen-actived Protein Kinases kinase (MAPK), signal conduction and transcription activator (Signal transducers and act ivators) and phosphoric acid. Inositol 3 kinase (Phosphotylinosital 3 kinase, PI3K) and so on, thus regulating cell growth, proliferation and apoptosis of [7,8]. with the above signal pathway as the target of a lot of research, but so far, no effective therapeutic targets have been found. Part of the tumor cells, not by aerobic oxidation, are mainly produced by aerobic glycolysis, which is called the "Warburg effect", that is, in the oxygen condition, the malignant tumor cells show high glucose uptake and produce a large amount of lactic acid [9,10].2011 Douglas Hanahan in Cell magazine, and the energy metabolism is abnormal. As one of the marker of tumor cells [11]., this characteristic can promote the proliferation, invasion and involvement of tumor cells. However, the exact molecular mechanism of the Warburg effect is not clear. More and more evidence suggests that this effect has a great relationship with the oncogene, such as Ras, SRC, Myc, p53 and other [12,13]. in recent years, in colon cancer cells, pancreatic cancer. It is found that KRAS promotes the growth of the tumor and the proliferation of [14-17] by regulating glycolysis pathway. The study shows that KRAS can regulate the hypoxia inducible factor -1 (Hypoxia inducible factor-1, HIF-1), the target protein of the Mammalian target (Mammalian target of rapamycin). In the field of lung cancer, KRAS and glycolysis are poorly studied by the pathway of glycolysis, and the mechanism of glycolysis by KRAS is not fully understood. Objective: the purpose of this study was to understand the effect of KRAS gene on glycolysis in lung cancer cells. First, to observe KRAS, hexokinase -2 (hexokinase-2) in non-small cell lung cancer cells. HK-2) the expression of pyruvate kinase M2 (pyruvatekinasem2) and the production of lactic acid, by knocking down or up-regulated KRAS expression, detecting the expression of hK2, pkm2 and lactic acid production. Through the above study, the effect and mechanism of KRAS on the glycolysis pathway of lung cancer cells and its mechanism. Methods and results: the first part: KRAS and glycolysis The expression level of key enzyme in three lung cancer cell lines, the study collected the RNA of H1299, A549, SPC-A1 cells, the total protein and the supernatant in the medium. The KRAS, hK2, pkm2 of three lung cancer cells were measured by qRT-PCR and Westernblot method, and.1. was detected in three lung cells, qRT-PCR, Westernblot results showed fine. The expression of KRAS was the highest, and the difference was statistically significant (P0.05).2. in three lung cancer cells. QRT-PCR and Westernblot showed the highest expression of hK2 and pkm2 in H1299 cells, and the difference was statistically significant (P0.05).3. in three lung cancer cells, the concentration of lactic acid was the highest in the H1299 cell supernatant, and the difference was statistically significant Meaning (P0.05). The second part: the effect of KRAS expression on the key enzyme of glycolytic glycolysis of lung cancer cells, hK2, pkm2, and the interference efficiency of the cell line of the three lung cancer cells with the highest KRAS expression, and detecting the interference efficiency. The cells were divided into blank control group (CON group), negative sequence control group (NC group), effective sequence interference group (Group KD group). Using qRT-PCR and Westernblot to detect hK2 in the cells of group KD, the expression of pkm2 was significantly lower than that before, and the production of lactic acid was significantly reduced by.Tnf- alpha in NC group and KD group cells. Again, qRT-PCR, Westernblot were used to detect the KRAS, hK2, expression level and lactic acid production. The amount of.1. was higher than before, and the production of lactic acid was also increased by.1.. H1299 cells were infected by KRAS gene interference sequence and negative control sequence, and kras-rnai (10300-1) was found to be significantly lower than that of the other two groups and negative control groups, and the difference was statistically significant (P0.05), and the group interference sequence was formed. After a stable cell line was constructed to prepare the cell line for subsequent.2. knockdown KRAS, qRT-PCR and Western blot were used to detect HK2, PKM2 expression in the KD group, compared with the CON and NC groups. The amount of lactic acid in H1299 cells decreased, and the difference was statistically significant (P0.05), and the different concentrations of.4. TNF- alpha acted on H1299 cells. The results of Western blot showed that after the concentration of 20ng/mlTNF- a in H1299 cells, the expression of KRAS protein was the highest. KRAS, HK2, PKM2 were all increased, the difference was statistically significant (P0.05).6. with 20ng/ml TNF- alpha in NC group and KD group cells, lactic acid concentration detection showed that the lactic acid concentration in the cell supernatant was higher than before, the difference was statistically significant (P0.05). Conclusion: 1. in three lung cancer cells, KRAS expression and glycolysis key enzyme HK2, expression amount The expression level of related.2.KRAS has an effect on lactic acid secretion, suggesting that KRAS participates in the glycolytic pathway.3.KRAS in lung cancer cells by regulating glycolysis key enzyme HK2, PKM2, and affecting glycolysis pathway.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

【二级参考文献】