肝癌組織miR- 148a表達變化及其與患者臨床病理參數(shù)和術(shù)后早期復(fù)發(fā)的關(guān)系

周興芹，陳鐘，常仁安

(南通大學(xué)附屬醫(yī)院,江蘇南通226001)

肝癌組織miR- 148a表達變化及其與患者臨床病理參數(shù)和術(shù)后早期復(fù)發(fā)的關(guān)系

周興芹，陳鐘，常仁安

(南通大學(xué)附屬醫(yī)院,江蘇南通226001)

目的觀察原發(fā)性肝癌(HCC)組織微小RNA- 148a(miR- 148a) mRNA表達變化，并分析其與患者臨床病理參數(shù)和術(shù)后早期復(fù)發(fā)的關(guān)系。方法選擇HCC組織及其配對的癌旁正常組織各142例份，采用qRT- PCR法檢測miR- 148a mRNA表達。分析HCC組織miR- 148a mRNA表達與患者臨床病理參數(shù)及術(shù)后早期復(fù)發(fā)的關(guān)系。結(jié)果HCC組織miR- 148a mRNA相對表達量明顯低于癌旁正常組織(P<0.05)。以142例HCC患者miR- 148a mRNA相對表達量的均數(shù)為截斷值，將患者分為miR- 148a mRNA高表達者65例和miR- 148a mRNA低表達者77例。miR- 148a mRNA表達與腫瘤大小、微血管侵犯及TNM分期有關(guān)(P均<0.05)，而與年齡、性別、肝硬化、血清AFP水平、HBV感染、組織分化程度及腫瘤數(shù)目無關(guān)(P均>0.05)。142例HCC患者中，早期復(fù)發(fā)88例、未復(fù)發(fā)54例。早期復(fù)發(fā)者miR- 148a mRNA相對表達量明顯低于未復(fù)發(fā)者(P<0.05)。88例早期復(fù)發(fā)者中，miR- 148a mRNA低表達者55例、miR- 148a mRNA高表達者33例，miR- 148a mRNA低表達者早期復(fù)發(fā)率明顯高于miR- 148a mRNA高表達者(P<0.05)。采用受試者工作特征(ROC)曲線評價miR- 148a mRNA預(yù)測HCC術(shù)后早期復(fù)發(fā)的價值，結(jié)果顯示，miR- 148a mRNA預(yù)測HCC術(shù)后早期復(fù)發(fā)的ROC曲線下面積為0.710(95%CI：0.660～0.769，P<0.01)，其cut off值為0.31，此時miR- 148a mRNA預(yù)測HCC術(shù)后早期復(fù)發(fā)的敏感性、特異性分別77.1%、71.8%。結(jié)論HCC組織中miR- 148a mRNA低表達，其表達變化與腫瘤進展有關(guān)。miR- 148a mRNA可作為HCC患者術(shù)后早期復(fù)發(fā)的預(yù)測因子之一。

原發(fā)性肝癌；微小RNA- 148a；臨床病理參數(shù)；復(fù)發(fā)

全球范圍內(nèi)每年因肝癌死亡的患者約70萬，其中我國約占50%[1]。目前，原發(fā)性肝癌(HCC)的治療方法有多種。手術(shù)治療仍是HCC的首選治療方法，但術(shù)后易復(fù)發(fā)，術(shù)后2年內(nèi)復(fù)發(fā)率為62.4%～77.8%，5年生存率較低[2～5]。因此，探討HCC術(shù)后早期復(fù)發(fā)的分子機制，尋找準確預(yù)測HCC術(shù)后早期復(fù)發(fā)的腫瘤分子生物學(xué)標志物意義重大。微小RNA(miRNA)是一類由19～25個核苷酸組成的、高度保守的內(nèi)源性非編碼小分子RNA，可通過與目標基因mRNA的3′非翻譯區(qū)結(jié)合，調(diào)控下游靶基因表達，繼而發(fā)揮相應(yīng)的生物學(xué)功能[6,7]。近年研究發(fā)現(xiàn)，miRNA與腫瘤的發(fā)生、發(fā)展密切相關(guān)[8,9]。miR- 148a是miRNA家族的成員之一，研究發(fā)現(xiàn)，其在胃癌、結(jié)腸癌及非小細胞肺癌等多種惡性腫瘤組織中低表達，具有抑癌基因作用[10～12]。但目前關(guān)于miR- 148a mRNA在HCC組織中表達的報道甚少，且其與患者臨床病理參數(shù)及術(shù)后早期復(fù)發(fā)的關(guān)系尚不完全明確。2011年1月～2014年7月，本研究觀察了HCC組織中miR- 148a mRNA表達變化，現(xiàn)分析其與患者臨床病理參數(shù)及術(shù)后早期復(fù)發(fā)的關(guān)系。

1 臨床資料

1.2 HCC組織及其配對癌旁正常組織miR- 148a mRNA表達比較 將手術(shù)切除的HCC組織及其配對的癌旁正常組織(距癌組織邊緣≥2 cm)離體30 min內(nèi)置于液氮中，再轉(zhuǎn)入- 80 ℃低溫冰箱中凍存。取部分HCC組織及癌旁正常組織，采用TRIzol法提取組織總RNA，紫外分光光度計檢測總RNA純度，OD260/OD280為1.8～2.0，瓊脂糖凝膠電泳鑒定總RNA完整，符合實驗要求。取總RNA 1 μg，按RNA逆轉(zhuǎn)錄試劑盒說明逆轉(zhuǎn)錄為cDNA，然后按qRT- PCR試劑盒說明進行PCR擴增。引物序列：miR- 148a上游引物：5′- ACACTCCAGCTGGGACAAA-GTTCTG- 3′，下游引物： 5′- CTCAACTGGTGTCGTGG-AGTCGGCAATTCAGTTGAGTCAGTGCAC- 3′；內(nèi)參U6上游引物：5′- CTCGCTTCGGCAGCACA- 3′，下游引物：5′- AACGCTTCACGAATTTGCGT- 3′。PCR反應(yīng)體系共25 μL：SYBR? Premix Ex TaqTMⅡ(2×)12.5 μL，cDNA 2 μL，上下游引物各1 μL，無酶水8.5 μL；反應(yīng)條件：95 ℃預(yù)變性10 min，95 ℃ 15 s、60 ℃ 60 s共40個循環(huán)。以U6為內(nèi)參，采用2-ΔΔCt法計算miR- 148a mRNA相對表達量。實驗重復(fù)3次，取平均值。結(jié)果顯示，HCC組織miR- 148a mRNA相對表達量為0.35±0.04，癌旁正常組織為1.00±0.02，二者比較P<0.05。以142例患者miR- 148a mRNA相對表達量的均數(shù)為截斷值，miR- 148a mRNA高表達者65例，miR- 148a mRNA低表達者77例。

1.2 miR- 148a mRNA表達與HCC患者臨床病理參數(shù)的關(guān)系 miR- 148a mRNA表達與腫瘤大小、微血管侵犯及TNM分期有關(guān)(P均<0.05)，與年齡、性別、肝硬化、血清AFP水平、HBV感染、組織分化程度及腫瘤數(shù)目無關(guān)(P均>0.05)。見表1。

表1 miR- 148a mRNA表達與HCC患者 臨床病理參數(shù)的關(guān)系[例(%)]

1.3 miR- 148a mRNA表達與HCC患者術(shù)后早期復(fù)發(fā)的關(guān)系 所有患者術(shù)后隨訪2年。術(shù)后1年內(nèi)每2個月門診或住院復(fù)查1次，術(shù)后1～2年每2～3個月復(fù)查1次。復(fù)查AFP、胸部X線、腹部B超，必要時進行CT、MRI或PET- CT檢查。以術(shù)后2年內(nèi)復(fù)發(fā)定義為早期復(fù)發(fā)。142例HCC患者中，早期復(fù)發(fā)88例、未復(fù)發(fā)54例。早期復(fù)發(fā)者和未復(fù)發(fā)者miR- 148a mRNA相對表達量分別為0.20±0.03、0.59±0.06，二者比較P<0.05。88例早期復(fù)發(fā)者中，miR- 148a mRNA低表達者55例、高表達者33例，miR- 148a低表達者早期復(fù)發(fā)率明顯高于miR- 148a高表達者(P<0.05)。

1.4 miR- 148a mRNA表達預(yù)測HCC術(shù)后早期復(fù)發(fā)的價值 ROC曲線分析顯示，miR- 148a mRNA表達預(yù)測HCC術(shù)后早期復(fù)發(fā)的曲線下面積為0.710(95%CI：0.660～0.769，P<0.01)，其cut off值為0.31，此時miR- 148a mRNA預(yù)測HCC術(shù)后早期復(fù)發(fā)的敏感性、特異性分別為77.1%、71.8%。見圖1。

2 討論

圖1 miR- 148a mRNA表達預(yù)測HCC患者術(shù)后早期復(fù)發(fā)的ROC曲線

目前關(guān)于HCC術(shù)后早期復(fù)發(fā)的分子生物學(xué)機制尚不完全明確，臨床上缺乏特異性強、敏感性高地預(yù)測HCC術(shù)后早期復(fù)發(fā)指標。Budhu等[13]研究發(fā)現(xiàn)，Th2- dominant細胞因子與HCC術(shù)后早期復(fù)發(fā)有關(guān)；Woo等[14]利用基因芯片技術(shù)篩選出了12種與HCC術(shù)后早期復(fù)發(fā)相關(guān)的分子生物學(xué)標志物，但其預(yù)測效果均不令人滿意。

miR- 148a是miR- 148a/152家族成員之一，定位于染色體7p15.2，其成熟體由22個核苷酸組成，含有一個包含8個核苷酸區(qū)域的種子序列。研究證實，miR- 148a在多種惡性腫瘤組織中表達下調(diào)，并可影響腫瘤細胞增殖、侵襲及遷移等生物學(xué)行為[10～12]；其在胃癌組織中低表達，且在Ⅲ、Ⅳ期胃癌組織中的表達較Ⅰ、Ⅱ期明顯降低，提示miR- 148a mRNA低表達與胃癌的惡性進展有關(guān)[15]。Szafranska等[16]研究發(fā)現(xiàn)，miR- 148a mRNA在胰腺癌組織中表達下調(diào)，miR- 148a mRNA低表達可造成其靶基因DNMT1表達升高，從而增加DNMT1對抑癌基因的過度甲基化，繼而促進胰腺癌的惡性進展。Tsai等[17]研究發(fā)現(xiàn)，復(fù)發(fā)性結(jié)腸癌患者血清miR- 148a mRNA水平降低，并可作為結(jié)腸癌根治性切除術(shù)后早期復(fù)發(fā)的分子生物學(xué)標志物。

既往研究發(fā)現(xiàn)，miR- 148a mRNA在HCC組織中表達下調(diào)[18]，過表達miR- 148a mRNA能夠阻滯細胞周期，抑制HCC細胞增殖[19]。Heo等[20]研究發(fā)現(xiàn)，miR- 148a mRNA在HCC組織中表達下調(diào)，并與患者預(yù)后不良有關(guān)。本研究結(jié)果顯示，HCC組織中miR- 148a mRNA表達降低，且早期復(fù)發(fā)者miR- 148a mRNA表達較未復(fù)發(fā)者顯著降低，提示miR- 148a mRNA表達降低可能與HCC術(shù)后早期復(fù)發(fā)有關(guān)。本研究還發(fā)現(xiàn)，miR- 148a mRNA低表達與腫瘤大小、微血管侵犯、TNM分期及淋巴結(jié)轉(zhuǎn)移有關(guān)，而與年齡、性別、肝硬化、血清AFP水平、HBV感染、組織分化程度及腫瘤數(shù)目無關(guān)。提示miR- 148a mRNA低表達與HCC患者病情及惡性進展有關(guān)。

目前，關(guān)于HCC術(shù)后早期復(fù)發(fā)的判定尚無明確標準。有學(xué)者將HCC術(shù)后2年內(nèi)復(fù)發(fā)定義為早期復(fù)發(fā)[21，22]，也有學(xué)者選擇1年[23]甚至6個月[24]作為術(shù)后早期復(fù)發(fā)的界線。本研究將術(shù)后2年內(nèi)復(fù)發(fā)定義為早期復(fù)發(fā)，術(shù)后隨訪發(fā)現(xiàn)，142例HCC患者中早期復(fù)發(fā)88例，且miR- 148a mRNA低表達者早期復(fù)發(fā)率明顯高于miR- 148a mRNA高表達者。ROC曲線分析顯示，miR- 148a mRNA表達預(yù)測HCC術(shù)后早期復(fù)發(fā)的曲線下面積為0.710，其cut off值為0.31，此時miR- 148a mRNA預(yù)測HCC術(shù)后早期復(fù)發(fā)的敏感性、特異性分別為77.1%、71.8%，提示miR- 148a mRNA對預(yù)測HCC術(shù)后早期復(fù)發(fā)有一定臨床價值。

綜上所述，HCC組織中miR- 148a mRNA低表達，其表達變化與腫瘤進展有關(guān)；miR- 148a mRNA可作為HCC患者術(shù)后早期復(fù)發(fā)的預(yù)測因子之一。

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ExpressionchangesofmiR- 148ainhepaticcarcinomatissuesanditsrelationshipswithclinicopathologicalparametersandearlypost-operativerecurrence

ZHOUXingqin,CHENZhong,CHANGRen′an

(AffiliatedHospitalofNantongUniversity,Nantong226001,China)

To observe the expression changes of microRNA- 148a (miR- 148a) in the primary hepatic carcinoma (HCC) tissues, and further to analyze its relationships with the clinicopathological parameters of patients and their early post- operative recurrence.MethodsQuantitative RT- PCR (qRT- PCR) were used to detect the relative expression of miR- 148a in 142 cases of HCC tissues and 142 cases of paired adjacent normal tissues. The relationships between miR- 148a expression and the clinicopathological parameters as well as early post- operative recurrence in HCC patients were analyzed.ResultsThe relative expression of miR- 148a was significantly lower in HCC tissues than in the adjacent normal tissues (P<0.05). The mean of the relative expression of miR- 148a in HCC tissues from 142 patients was set as cut- off value, thereafter, patients were categorized into the high- expression group (n=65) and low- expression group (n=77). The expression of miR- 148a was significantly associated with tumor size, microvascular invasion, and TNM stages (P<0.05), while was not statistically associated with age, gender, cirrhosis, serum level of AFP, HBV infection, histological differentiation, or tumor numbers (P>0.05). In the 142 HCC patients, 88 of them suffered from early post- operative recurrence, while the other 54 patients did not. The expression of miR- 148a was significantly lower in patients with early post- operative recurrence than that of those without recurrence (P<0.05). In the 88 patients with early post- operative recurrence, there were 55 cases of patients with low expression of miR- 148a and 33 patients with high expression of miR- 148a. The early recurrence rate was significantly higher in patients with low expression of miR- 148a than that of those with high expression of miR- 148a (P<0.05). Receiver operating characteristic (ROC) curve was used to evaluate the predictive value of miR- 148a in early post- operative recurrence. Consequently, area under the curve was 0.710 (95%CI:0.660- 0.769,P<0.01), and the cut- off value of relative mRNA expression of miR- 148a was 0.31. Therefore, the sensitivity and specificity of miR- 148a expression in predicting early post- operative recurrence of HCC was 77.1% and 71.8%, respectively.ConclusionThe miR- 148a is low expressed in HCC tissues, and the expression change is associated with tumor progression. Moreover, miR- 148a can be taken as an effective predictive indicator for early post- operative recurrence of HCC.

primary hepatic carcinoma; micro RNA- 148a; clinicopathological parameters; recurrence

江蘇省臨床醫(yī)學(xué)科技專項基金資助項目(BL2014060)；江蘇省“科教興衛(wèi)工程”醫(yī)學(xué)領(lǐng)軍人才和創(chuàng)新團隊基金資助項目(LJ201134)；南通市科技計劃項目(MS22016041)。

周興芹(1978- )，女，主治醫(yī)師，主要研究方向為惡性腫瘤的分子生物學(xué)機制。E- mail： zhouxingqin666@126.com

常仁安(1975- )，男，副主任醫(yī)師，主要研究方向為肝臟惡性腫瘤的診治。E- mail： xuluzlk@yeah.net

10.3969/j.issn.1002- 266X.2017.36.003

R735.7

A

1002- 266X(2017)36- 0009- 04

2017- 02- 21)