miR-26a作為胃癌預后生物標志物初探
發(fā)布時間：2018-08-27 11:36
【摘要】：背景:胃癌(gastric cancer,GC)是目前世界上發(fā)病率和死亡率分居第五位和第三位的常見惡性腫瘤。近年來,胃癌的全球發(fā)病率和死亡率都呈下降趨勢,但胃癌仍是發(fā)展中國家癌癥相關(guān)死亡的主要原因。在世界范圍內(nèi),胃癌的男性發(fā)病率是女性的兩倍,且發(fā)病率與死亡率都有顯著的地域差別。在我國,胃癌是排名第三的致死性腫瘤,僅次于肺癌和肝癌。早期胃癌并無明顯癥狀,臨床確診時多為中晚期,從而失去最佳治療時機。盡管胃癌的診斷手段和治療方案日漸先進,但是胃癌患者的預后仍然不佳。微小RNA(micro RNA,mi RNA)是在真核生物中發(fā)現(xiàn)的一類內(nèi)源性的具有調(diào)控功能的非編碼蛋白質(zhì)小RNA,參與腫瘤細胞的生物調(diào)控過程,在腫瘤的診斷和預后中扮演著重要的角色。大量研究報道m(xù)i RNAs與包括胃癌在內(nèi)的很多種惡性腫瘤的預后密切相關(guān)。選擇合適的mi RNAs作為胃癌的預后生物標志物具有巨大的臨床價值。本實驗室前期通過Taq Man低密度芯片(Taq Man low density array,TLDA)篩選出四個在胃癌血漿中表達差異明顯的mi RNAs(mi R-148a、mi R-142-3p、mi R-26a和mi R-195),本研究擬在胃癌患者的組織中檢測出特異性表達的mi RNAs,并將其作為胃癌預后的生物學標志。方法:本研究通過RT-PCR(實時定量聚合酶鏈式反應)實驗在一階段南通胃癌石蠟樣本中檢測mi R-148a、mi R-142-3p、mi R-26a和mi R-195的表達水平,篩選出與胃癌預后相關(guān)的mi RNAs,在宜興石蠟樣本中繼續(xù)驗證一階段研究結(jié)果。在Pubmed中搜索關(guān)于候選mi RNAs、gastric cancer和survival或prognosis有關(guān)的文獻,運用STATA11.0等軟件進行Meta分析后,再確定進行下一步的進行功能研究的mi RNA。在胃癌細胞株MGC 803、BGC 823、SGC 7901、MKN 28和正常細胞株GES-1檢測mi RNAs本底表達,選擇表達最低的細胞株進行功能實驗探索。經(jīng)RT-PCR證實轉(zhuǎn)染成功后,通過CCK-8(cell counting kit-8,細胞計數(shù)試劑盒)細胞增殖試驗分析細胞增殖能力的改變,運用平板克隆試驗檢測單個細胞的惡性程度,采用Transwell細胞遷移侵襲試驗檢驗細胞遷移和侵襲能力的變化,應用流式細胞儀檢測細胞周期凋亡的變化。結(jié)果:在一階段南通387例石蠟樣本結(jié)果顯示,mi R-26a和mi R-148a的表達水平與預后顯著相關(guān),P值分別為0.009和0.005;mi R-142-3p和mi R-195的表達水平于預后無關(guān),P值分別為0.320和0.119。因此在二階段宜興石蠟樣本中不在驗證mi R-142-3p和mi R-195。mi R-26a和mi R-148a的表達水平、浸潤深度、淋巴結(jié)轉(zhuǎn)移、TNM分期、分化程度與不良預后均有關(guān)系。多因素Cox回歸模型顯示mi R-26a和mi R-148a能改善胃癌的預后(mi R-26a:HR=0.76,95%CI=0.61-0.94;mi R-148a:HR=0.73,95%CI=0.58-0.91);另外,TNM分期和化療也與胃癌預后顯著相關(guān),P值均0.05。鑒于前期已有多篇有關(guān)mi R-148a與胃癌研究報道,本研究主要側(cè)重于mi R-26a與胃癌預后相關(guān)的生物學機制探討。在胃癌細胞株MGC 803中mi R-26a顯著低于正常細胞GES-1(P0.01)。在MGC 803細胞中過表達mi R-26a 48h后,增殖能力有明顯下降(P0.001);克隆形成率下降了69%(P=0.004);癌細胞通過小室的遷移率下降了48%(P=0.028);侵襲能力顯著下降(P=0.031)。流式細胞術(shù)顯示增加細胞內(nèi)mi R-26a表達,細胞周期無明顯改變,但細胞凋亡率明顯增高(P=0.004)。結(jié)論:癌組織中的mi R-26a、mi R-148a的表達水平可能與胃癌預后相關(guān)。腫瘤的TNM分期、浸潤深度、淋巴結(jié)轉(zhuǎn)移等均能影響胃癌生存時間。過表達mi R-26a可抑制胃癌的發(fā)生和發(fā)展。我們的研究提示mi R-26a的表達水平可以用來預測胃癌患者手術(shù)預后的情況,是潛在的預后預測生物標志物,為其今后的臨床治療提供重要參考。
[Abstract]:BACKGROUND: Gastric cancer (GC) is the fifth and third most common malignant tumor in the world. In recent years, the incidence and mortality of gastric cancer have been declining globally, but gastric cancer is still the main cause of cancer-related death in developing countries. In the world, the incidence of male gastric cancer is female. Gastric cancer is the third most lethal tumor in China, after lung cancer and liver cancer. Early gastric cancer has no obvious symptoms, and most of the clinical diagnosis is in the middle and advanced stages, thus losing the best opportunity for treatment. The prognosis of cancer patients is still poor. MicroRNAs (micro RNAs) are a class of endogenous non-coding protein small RNAs found in eukaryotes, which are involved in the biological regulation of tumor cells and play an important role in the diagnosis and prognosis of cancer. The prognosis of various malignant tumors is closely related. Selection of appropriate mi RNAs as prognostic biomarkers of gastric cancer is of great clinical value. MiR-195. We intend to detect the expression of specific mi RNAs in gastric cancer tissues and use them as biological markers for prognosis of gastric cancer. Methods: The expression of MI R-148a, mi R-142-3p, mi R-26a and MI R-195 in paraffin samples of one-stage Nantong gastric cancer was detected by RT-PCR (real-time quantitative polymerase chain reaction). In Pubmed, we searched for the literature about candidate mi RNAs, gastric cancer, survival or prognosis, and then used STATA 11.0 software for meta-analysis to determine the next step of functional research in the stomach. The expression of MIRNAs in cancer cell lines MGC 803, BGC 823, SGC 7901, MKN 28 and normal cell lines GES-1 were detected. The cell lines with the lowest expression were selected for functional experiment. After the transfection was confirmed by RT-PCR, the cell proliferation test of CCK-8 (cell counting kit-8, cell counting kit-8) was used to analyze the change of cell proliferation ability. Prolongation test was used to detect the degree of malignancy of single cell, Transwell cell migration and invasion test was used to examine the changes of cell migration and invasion ability, and flow cytometry was used to detect the changes of cell cycle apoptosis. The expression levels of MIR-142-3p and MIR-195 were 0.009 and 0.005, respectively. The expression levels of MIR-142-3p and MIR-195 were not correlated with prognosis, P values were 0.320 and 0.119, respectively. Cox regression model showed that MIR-26a and MIR-148a could improve the prognosis of gastric cancer (MIR-26a: HR = 0.76, 95% CI = 0.61-0.94; MIR-148a: HR = 0.73, 95% CI = 0.58-0.91); in addition, TNM staging and chemotherapy were also significantly correlated with the prognosis of gastric cancer, P values were 0.05. Biological mechanisms associated with prognosis of gastric cancer were investigated. MiR-26a in gastric cancer cell line MGC 803 was significantly lower than that in normal cell GES-1 (P 0.01). Overexpression of MIR-26a in MGC 803 cells significantly decreased the proliferation ability (P 0.001), the clone formation rate (P = 0.004), the migration rate of cancer cells through compartments (P = 0.028), and the invasion rate (P = 0.028). The expression of MIR-26a and MIR-148a in cancer tissues may be related to the prognosis of gastric cancer. TNM stage, depth of invasion and lymph node metastasis can all affect the prognosis of gastric cancer. Overexpression of MIR-26a can inhibit the occurrence and development of gastric cancer. Our study suggests that the expression of MIR-26a can be used to predict the prognosis of gastric cancer patients after surgery. It is a potential biomarker for predicting prognosis and provides an important reference for future clinical treatment.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R735.2
本文編號：2207153
[Abstract]:BACKGROUND: Gastric cancer (GC) is the fifth and third most common malignant tumor in the world. In recent years, the incidence and mortality of gastric cancer have been declining globally, but gastric cancer is still the main cause of cancer-related death in developing countries. In the world, the incidence of male gastric cancer is female. Gastric cancer is the third most lethal tumor in China, after lung cancer and liver cancer. Early gastric cancer has no obvious symptoms, and most of the clinical diagnosis is in the middle and advanced stages, thus losing the best opportunity for treatment. The prognosis of cancer patients is still poor. MicroRNAs (micro RNAs) are a class of endogenous non-coding protein small RNAs found in eukaryotes, which are involved in the biological regulation of tumor cells and play an important role in the diagnosis and prognosis of cancer. The prognosis of various malignant tumors is closely related. Selection of appropriate mi RNAs as prognostic biomarkers of gastric cancer is of great clinical value. MiR-195. We intend to detect the expression of specific mi RNAs in gastric cancer tissues and use them as biological markers for prognosis of gastric cancer. Methods: The expression of MI R-148a, mi R-142-3p, mi R-26a and MI R-195 in paraffin samples of one-stage Nantong gastric cancer was detected by RT-PCR (real-time quantitative polymerase chain reaction). In Pubmed, we searched for the literature about candidate mi RNAs, gastric cancer, survival or prognosis, and then used STATA 11.0 software for meta-analysis to determine the next step of functional research in the stomach. The expression of MIRNAs in cancer cell lines MGC 803, BGC 823, SGC 7901, MKN 28 and normal cell lines GES-1 were detected. The cell lines with the lowest expression were selected for functional experiment. After the transfection was confirmed by RT-PCR, the cell proliferation test of CCK-8 (cell counting kit-8, cell counting kit-8) was used to analyze the change of cell proliferation ability. Prolongation test was used to detect the degree of malignancy of single cell, Transwell cell migration and invasion test was used to examine the changes of cell migration and invasion ability, and flow cytometry was used to detect the changes of cell cycle apoptosis. The expression levels of MIR-142-3p and MIR-195 were 0.009 and 0.005, respectively. The expression levels of MIR-142-3p and MIR-195 were not correlated with prognosis, P values were 0.320 and 0.119, respectively. Cox regression model showed that MIR-26a and MIR-148a could improve the prognosis of gastric cancer (MIR-26a: HR = 0.76, 95% CI = 0.61-0.94; MIR-148a: HR = 0.73, 95% CI = 0.58-0.91); in addition, TNM staging and chemotherapy were also significantly correlated with the prognosis of gastric cancer, P values were 0.05. Biological mechanisms associated with prognosis of gastric cancer were investigated. MiR-26a in gastric cancer cell line MGC 803 was significantly lower than that in normal cell GES-1 (P 0.01). Overexpression of MIR-26a in MGC 803 cells significantly decreased the proliferation ability (P 0.001), the clone formation rate (P = 0.004), the migration rate of cancer cells through compartments (P = 0.028), and the invasion rate (P = 0.028). The expression of MIR-26a and MIR-148a in cancer tissues may be related to the prognosis of gastric cancer. TNM stage, depth of invasion and lymph node metastasis can all affect the prognosis of gastric cancer. Overexpression of MIR-26a can inhibit the occurrence and development of gastric cancer. Our study suggests that the expression of MIR-26a can be used to predict the prognosis of gastric cancer patients after surgery. It is a potential biomarker for predicting prognosis and provides an important reference for future clinical treatment.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R735.2
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