本文選題：KRAS + 非小細(xì)胞肺癌��； 參考：《第三軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：背景:肺癌是我國(guó)乃至全球最常見(jiàn)的惡性腫瘤,其發(fā)病率及死亡率均居榜首[1]。隨著醫(yī)學(xué)事業(yè)的發(fā)展,對(duì)于肺癌的治療,除了手術(shù)、化療、放療等傳統(tǒng)治療手段外,逐漸興起了靶向治療,使治療更加個(gè)體化,檢測(cè)個(gè)體的突變基因成為了必要[2]。研究發(fā)現(xiàn)30%的肺癌中存在致癌基因KRAS的表達(dá),而且主要存在于非小細(xì)胞肺癌(non-small cell cancer,NSCLC)中,是重要的原癌基因之一,與腫瘤的生長(zhǎng)、增殖及血管生成密切相關(guān),且是不良的預(yù)后因素[3-6]。KRAS基因編碼的蛋白,與G蛋白的功能相似,具有與鳥(niǎo)苷酸結(jié)合的能力,當(dāng)其與三磷酸鳥(niǎo)苷(Guanosine triphosphate,GTP)結(jié)合后,可與多種效應(yīng)分子如促有絲分裂原活化蛋白激酶(Mitogen-actived Protein Kinases,MAPK),信號(hào)傳導(dǎo)與轉(zhuǎn)錄激活因子(Signal transducers and act ivators of transcription,STAT)和磷酸肌醇3激酶(Phosphotylinosital 3 kinase,PI3K)等發(fā)生作用,從而調(diào)節(jié)細(xì)胞的生長(zhǎng)、增殖與凋亡[7,8]。以上述信號(hào)通路為靶點(diǎn)的研究很多,但目前為止,并未發(fā)現(xiàn)有效的治療靶點(diǎn)。正常分化的細(xì)胞主要依賴于線粒體的氧化磷酸化生成所需的能量,而大部分腫瘤細(xì)胞,不是靠有氧氧化,是以有氧糖酵解為其主要產(chǎn)能方式,這種現(xiàn)象稱(chēng)為“Warburg效應(yīng)”,即在有氧條件下,惡性腫瘤細(xì)胞表現(xiàn)出高效攝取葡萄糖,并產(chǎn)生大量乳酸[9,10]。2011年Douglas Hanahan等在Cell雜志上發(fā)表綜述,將能量代謝異常作為腫瘤細(xì)胞的標(biāo)志之一[11]。該特性能夠促進(jìn)腫瘤細(xì)胞的增殖、侵襲及參與介導(dǎo)耐藥。然而Warburg效應(yīng)的確切分子機(jī)制尚不清楚,越來(lái)越多的證據(jù)表明,該效應(yīng)與癌基因有很大的關(guān)系,如Ras、src、myc、p53等[12,13]。近年來(lái),在結(jié)腸癌細(xì)胞、胰腺癌細(xì)胞等其他腫瘤性疾病中發(fā)現(xiàn)KRAS通過(guò)調(diào)控糖酵解途徑促使腫瘤的生長(zhǎng)以及增殖[14-17],研究表明KRAS可通過(guò)調(diào)節(jié)缺氧誘導(dǎo)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)等通路調(diào)節(jié)糖酵解途徑[18-20]。在肺癌領(lǐng)域,KRAS與糖酵解的研究甚少,且KRAS調(diào)控糖酵解途徑的機(jī)制尚未完全明了。目的:本實(shí)驗(yàn)?zāi)康氖橇私鈑ras基因?qū)Ψ伟┘?xì)胞中糖酵解途徑的影響。首先觀察非小細(xì)胞肺癌細(xì)胞中kras、己糖激酶-2(hexokinase-2,hk-2)、丙酮酸激酶m2型(pyruvatekinasem2)的表達(dá)情況及乳酸生成量,通過(guò)敲降或上調(diào)kras表達(dá)水平,檢測(cè)hk2、pkm2的表達(dá)情況及乳酸生成量。通過(guò)以上研究,以證明kras對(duì)肺癌細(xì)胞糖酵解途徑的影響及其機(jī)制。方法和結(jié)果:第一部分:kras以及糖酵解關(guān)鍵酶在三種肺癌細(xì)胞株中的表達(dá)水平本研究收集h1299、a549、spc-a1細(xì)胞的rna、總蛋白以及培養(yǎng)基中的上清液,通過(guò)qrt-pcr、westernblot方法測(cè)定三種肺癌細(xì)胞的kras、hk2、pkm2,同時(shí)檢測(cè)乳酸生成情況。1.在三種肺細(xì)胞中,qrt-pcr、westernblot結(jié)果顯示h1299細(xì)胞的kras表達(dá)量最高,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。2.在三種肺癌細(xì)胞中,qrt-pcr、westernblot結(jié)果顯示h1299細(xì)胞的hk2、pkm2表達(dá)量最高,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。3.在三種肺癌細(xì)胞中,乳酸濃度檢測(cè)顯示h1299細(xì)胞上清液中乳酸濃度最高,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。第二部分:kras表達(dá)對(duì)肺癌細(xì)胞糖酵解關(guān)鍵酶hk2、pkm2的影響將上述三種肺癌細(xì)胞中kras表達(dá)量最高的細(xì)胞株h1299予以慢病毒干擾,并檢測(cè)其干擾效率。將細(xì)胞分組為:空白對(duì)照組(con組)、陰性序列對(duì)照組(nc組)、有效序列干擾組(kd組),采用qrt-pcr、westernblot檢測(cè)kd組細(xì)胞中hk2、pkm2的表達(dá)量較前明顯降低,乳酸生成量也較前明顯降低。tnf-α作用于nc組及kd組細(xì)胞,再次用qrt-pcr、westernblot檢測(cè)細(xì)胞中kras、hk2、pkm2的表達(dá)水平以及乳酸生成量,與nc組比較,tnf-α作用后,kras、hk2、pkm2表達(dá)量較前升高,乳酸生成量也較前升高。1.將三種針對(duì)kras基因的干擾序列以及陰性對(duì)照序列分別感染h1299細(xì)胞,檢測(cè)發(fā)現(xiàn)kras-rnai(10300-1)該干擾序列較其余兩組序列及陰性對(duì)照組有顯著下降,差異有統(tǒng)計(jì)學(xué)意義(p0.05),并將該組干擾序列成功構(gòu)建穩(wěn)定細(xì)胞株以備后續(xù)實(shí)驗(yàn)。2.敲降KRAS后,qRT-PCR、Western blot檢測(cè)KD組細(xì)胞中的HK2、PKM2表達(dá),與CON及NC組比較,敲降后H1299細(xì)胞中HK2、PKM2表達(dá)水平較前明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。3.敲降KRAS后,乳酸濃度檢測(cè)顯示,與CON及NC組比較,敲降后H1299細(xì)胞的乳酸生成量減少,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。4.不同濃度TNF-α作用于H1299細(xì)胞,Western blot結(jié)果顯示以濃度20ng/mlTNF-α作用H1299細(xì)胞后,KRAS蛋白表達(dá)最高。5.以20ng/ml TNF-α作用NC組及KD組細(xì)胞后,采用qRT-PCR、Western blot檢測(cè)顯示細(xì)胞中KRAS、HK2、PKM2均升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。6.以20ng/ml TNF-α作用NC組及KD組細(xì)胞后,乳酸濃度檢測(cè)顯示作用后細(xì)胞上清液中乳酸濃度較前升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1.在三種肺癌細(xì)胞中,KRAS表達(dá)量與糖酵解關(guān)鍵酶HK2、PKM2表達(dá)量相關(guān)。2.KRAS表達(dá)水平對(duì)乳酸分泌有影響,提示KRAS參與了肺癌細(xì)胞中糖酵解途徑。3.KRAS通過(guò)調(diào)節(jié)糖酵解關(guān)鍵酶HK2、PKM2,影響糖酵解途徑。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R734.2

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本文編號(hào)：2022924