靶向沉默SSBP1基因?qū)Ω伟〩epG2細(xì)胞增殖、侵襲轉(zhuǎn)移的影響

馬榮芬 陳秋英 劉艷芬

·論著·

靶向沉默SSBP1基因?qū)Ω伟〩epG2細(xì)胞增殖、侵襲轉(zhuǎn)移的影響

馬榮芬 陳秋英 劉艷芬

目的 觀察靶向沉默線粒體單鏈DNA結(jié)合蛋白(SSBP1)基因?qū)Ω伟〩epG2細(xì)胞增殖、侵襲轉(zhuǎn)移的影響。方法 設(shè)計(jì)并構(gòu)建靶向SSBP1 基因的特異性siRNA，采用脂質(zhì)體介導(dǎo)瞬時轉(zhuǎn)染肝癌HepG2細(xì)胞，細(xì)胞分3組：對照組、空白轉(zhuǎn)染組、轉(zhuǎn)染組。Real time-PCR和Western-blot檢測靶向干擾后SSBP1 mRNA和蛋白表達(dá)變化。CCK-8法檢測細(xì)胞增殖。流式細(xì)胞儀檢測細(xì)胞周期、凋亡率及線粒體膜電位。劃痕實(shí)驗(yàn)及Transwell 侵襲實(shí)驗(yàn)檢測細(xì)胞侵襲轉(zhuǎn)移能力。Western-blot檢測增殖、侵襲轉(zhuǎn)移相關(guān)基因蛋白表達(dá)狀況。結(jié)果 SSBP1 siRNA能夠顯著抑制SSBP1 mRNA和蛋白表達(dá)。與對照組和空白轉(zhuǎn)染組比較，轉(zhuǎn)染SSBP1 siRNA后HepG2細(xì)胞增殖能力、G2期和S期細(xì)胞比例、線粒體膜電位明顯降低，G1期細(xì)胞比例、細(xì)胞凋亡率明顯升高(P<0.05)；同時細(xì)胞增殖相關(guān)基因PCNA、凋亡抑制基因Bcl-2、轉(zhuǎn)移相關(guān)基因MMP-9蛋白表達(dá)顯著下調(diào)，凋亡誘導(dǎo)基因Bax蛋白表達(dá)顯著上調(diào)(P<0.05)。結(jié)論 靶向沉默SSBP1基因能夠通過線粒體途徑抑制肝癌HepG2細(xì)胞增殖、侵襲轉(zhuǎn)移，并誘導(dǎo)其凋亡。

肝癌；線粒體單鏈DNA結(jié)合蛋白；細(xì)胞增殖；侵襲轉(zhuǎn)移

近年來，我國肝癌發(fā)病率和病死率逐年增加，且呈年輕化趨勢，肝癌發(fā)病是多因素、多步驟的復(fù)雜過程[1,2]。肝癌手術(shù)切除后易復(fù)發(fā)，且對放化療不敏感，患者預(yù)后差[3]。肝癌細(xì)胞增殖活躍、凋亡減緩及侵襲轉(zhuǎn)移是導(dǎo)致治療失敗、患者病死率高的主要原因[4-6]。因此，闡明肝癌發(fā)生發(fā)展的機(jī)制，研究抑制腫瘤細(xì)胞增殖、侵襲轉(zhuǎn)移并誘導(dǎo)其凋亡的基因已經(jīng)成為治療惡性腫瘤的有效措施[7]。線粒體單鏈DNA結(jié)合蛋白(SSBP1)在真核生物進(jìn)化過程中相對保守，主要通過參與線粒體DNA(mtDNA)的復(fù)制、轉(zhuǎn)錄、損傷后修復(fù)等作用與線粒體功能密切相關(guān)[8]。有研究發(fā)現(xiàn)，SSBP1在乳腺癌、卵巢癌、結(jié)直腸癌等多種惡性腫瘤組織表達(dá)上調(diào)[9-12]。但是，目前有關(guān)SSBP1是否參與調(diào)控肝癌細(xì)胞生物學(xué)行為的研究筆者尚未見報道。本研究通過RNA干擾技術(shù)探討靶向沉默SSBP1基因?qū)Ω伟〩epG2細(xì)胞增殖、侵襲轉(zhuǎn)移的影響，旨在為尋找防治肝癌的有效靶點(diǎn)提供理論依據(jù)。

1 材料與方法

1.1 細(xì)胞、試劑與儀器 人肝癌細(xì)胞HepG2購自上海吉凱基因化學(xué)技術(shù)有限公司；RPMI 1640培養(yǎng)基、SSBP1 siRNA、LipofectamineTM 2000購自美國Invitrogen公司；Trizol、實(shí)時熒光定量試劑盒購自美國Promega公司；兔抗人PCNA、Bcl-2、Bax、MMP-9多克隆抗體購自美國Santa Cruz公司；細(xì)胞裂解液、辣根過氧化物酶標(biāo)記的抗鼠IgG抗體、BCA蛋白濃度測定試劑盒、高靈敏度化學(xué)發(fā)光檢測試劑盒購自日本Takara公司；Chemi DocTM XRS+化學(xué)發(fā)光凝膠成像系統(tǒng)購自美國Biorad公司；全自動7300型熒光定量PCR擴(kuò)增儀購自美國ABI公司；FACS Calibur型流式細(xì)胞儀購自美國BD公司。

1.2 方法

1.2.1 細(xì)胞培養(yǎng)及轉(zhuǎn)染:人肝癌細(xì)胞HepG2用含10% FBS、1%青/鏈霉素的DMEM培養(yǎng)基在37℃、5% CO2、飽和濕度條件下常規(guī)培養(yǎng)。待細(xì)胞達(dá)到90%融合時利用脂質(zhì)體LipofectamineTM 2000試劑盒進(jìn)行轉(zhuǎn)染，具體操作步驟嚴(yán)格按照試劑盒說明書進(jìn)行。細(xì)胞隨機(jī)分為3組：對照組(不進(jìn)行轉(zhuǎn)染處理)、空白轉(zhuǎn)染組(轉(zhuǎn)染空質(zhì)粒)、轉(zhuǎn)染組(轉(zhuǎn)染siRNA SSBP1)。

1.2.2 Real time-PCR:Trizol提取細(xì)胞總RNA，按照試劑盒說明書加樣進(jìn)行逆轉(zhuǎn)錄反應(yīng)，ABI 7300型熒光定量PCR儀擴(kuò)增。用儀器自帶的分析軟件得到各樣本、各基因擴(kuò)增的Ct值，以β-actin為內(nèi)參照基因，目的基因表達(dá)相對水平RQ=2-ΔΔCt。

1.2.3 Western-blot:細(xì)胞裂解后提取總蛋白，BCA法測定蛋白濃度。取30 μg 總蛋白上樣，SDS-PAGE分離后半干法電泳轉(zhuǎn)移至PVDF膜，10%脫脂奶粉封閉2 h。加入兔抗人PCNA、Bcl-2、Bax、MMP-9多克隆抗體，4℃過夜，TBST洗3次。加入辣根過氧化物酶標(biāo)記的羊抗鼠IgG，室溫孵育2 h，化學(xué)發(fā)光法顯色、定影。β-actin作為內(nèi)參照，以目的蛋白與β-actin吸光度值的比值表示目的蛋白相對表達(dá)水平。

1.2.4 CCK-8實(shí)驗(yàn):各組細(xì)胞接種于96孔板中，調(diào)整細(xì)胞接種為5×103個/孔。培養(yǎng)1 d、2 d、3 d、4 d后每孔加入10 μl CCK-8溶液，繼續(xù)培養(yǎng)2 h，于酶標(biāo)儀450 nm 波長處測定吸光度(A450)值，重復(fù)3次。

1.2.5 Annexin V-FITC/PI雙染法檢測細(xì)胞周期和凋亡率:選取對數(shù)生長期的細(xì)胞接種于6孔板，胰酶消化細(xì)胞，PBS洗2次，重懸細(xì)胞，調(diào)整細(xì)胞密度為1×109/L，加入FITC的Annexin V和PI染液，各10 μl混勻，室溫下避光反應(yīng)15 min，流式細(xì)胞儀檢測細(xì)胞周期和凋亡率。

1.2.6 劃痕實(shí)驗(yàn):用10 μl槍頭在6孔板底部做一劃痕，加入無血清DMEM培養(yǎng)基培養(yǎng)24 h，測量劃痕長度，計(jì)算細(xì)胞遷移抑制率，取均值。

1.2.7 Transwell 侵襲實(shí)驗(yàn):胰酶消化細(xì)胞，調(diào)整細(xì)胞密度為1×105/L。Transwell 上室加入400 μl細(xì)胞懸液，下室加入600 μl 10%FBS的DMEM培養(yǎng)基，培養(yǎng) 24 h。 結(jié)晶紫染色30 min，光學(xué)顯微鏡拍照，隨機(jī)選取5個視野，計(jì)數(shù)遷移至濾膜下表面的細(xì)胞數(shù)，即代表HepG2細(xì)胞侵襲力。

1.2.8 線粒體膜電位檢測:選取對數(shù)生長期的細(xì)胞接種于6孔板，調(diào)整細(xì)胞密度為1×105個/孔，加入 0.5 ml JC-1染液，避光孵育20 min，PBS洗2次，流式細(xì)胞儀檢測細(xì)胞線粒體膜電位。

2 結(jié)果

2.1 siRNA干擾后SSBP1在HepG2細(xì)胞的表達(dá) Real time-PCR和Western blot結(jié)果顯示，HepG2細(xì)胞轉(zhuǎn)染SSBP1 siRNA后SSBP1 mRNA和蛋白表達(dá)顯著低于對照組、空白轉(zhuǎn)染組(P<0.05)；對照組、空白轉(zhuǎn)染組比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表1。

組別SSBP1表達(dá)水平mRNA蛋白SSBP1siRNA組0.33±0.050.29±0.04空白轉(zhuǎn)染組2.11±0.27*1.99±0.25*對照組2.16±0.30*2.05±0.24*

注：與SSBP1 siRNA組比較,*P<0.05

2.2 SSBP1 siRNA對HepG2細(xì)胞增殖的影響 CCK-8實(shí)驗(yàn)結(jié)果顯示，HepG2細(xì)胞轉(zhuǎn)染SSBP1 siRNA后細(xì)胞A450值顯著低于對照組、空白轉(zhuǎn)染組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；對照組、空白轉(zhuǎn)染組比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表2。

組別A4501d2d3d4dSSBP1siRNA組0.29±0.040.34±0.030.49±0.070.60±0.09空白轉(zhuǎn)染組0.30±0.050.41±0.05*0.69±0.09*0.93±0.11*對照組0.28±0.040.42±0.06*0.71±0.10*0.95±0.12*

注：與SSBP1 siRNA組比較,*P<0.05

2.3 SSBP1 siRNA對HepG2細(xì)胞周期的影響 流式細(xì)胞術(shù)結(jié)果顯示，HepG2細(xì)胞轉(zhuǎn)染SSBP1 siRNA后G1期細(xì)胞比例顯著高于對照組、空白轉(zhuǎn)染組，G2期、S期細(xì)胞比例顯著低于對照組、空白轉(zhuǎn)染組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；對照組、空白轉(zhuǎn)染組比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表3。

組別G1期G2期S期SSBP1siRNA組85.34±9.678.13±0.699.39±1.10空白轉(zhuǎn)染組66.79±6.32*12.99±1.35*13.88±1.46*對照組67.12±6.34*13.65±1.46*14.24±1.62*

注：與SSBP1 siRNA組比較,*P<0.05

2.4 SSBP1 siRNA對HepG2細(xì)胞凋亡的影響 流式細(xì)胞術(shù)結(jié)果顯示，HepG2細(xì)胞轉(zhuǎn)染SSBP1 siRNA后細(xì)胞凋亡率顯著高于對照組、空白轉(zhuǎn)染組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；對照組、空白轉(zhuǎn)染組比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表4。

組別凋亡率1d2d3d4dSSBP1siRNA組2.03±0.134.90±0.637.02±0.119.26±0.12空白轉(zhuǎn)染組2.01±0.123.92±0.43*4.96±0.68*6.02±0.08*對照組2.04±0.153.89±0.40*4.95±0.71*6.00±0.09*

注：與SSBP1 siRNA組比較,*P<0.05

2.5 SSBP1 siRNA對HepG2細(xì)胞遷移的影響 劃痕實(shí)驗(yàn)結(jié)果顯示，HepG2細(xì)胞轉(zhuǎn)染SSBP1 siRNA后細(xì)胞劃痕遷移抑制率顯著高于對照組、空白轉(zhuǎn)染組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；對照組、空白轉(zhuǎn)染組比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表5。

組別遷移抑制率SSBP1siRNA組35.06±4.14空白轉(zhuǎn)染組12.21±1.11*對照組11.72±1.02*

注：與SSBP1 siRNA組比較,*P<0.05

2.6 SSBP1 siRNA對HepG2細(xì)胞侵襲力的影響 劃痕實(shí)驗(yàn)結(jié)果顯示，HepG2細(xì)胞轉(zhuǎn)染SSBP1 siRNA后細(xì)胞劃痕遷移抑制率顯著高于對照組、空白轉(zhuǎn)染組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；對照組、空白轉(zhuǎn)染組比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表6。

表6 SSBP1沉默對HepG2細(xì)胞侵襲力的影響 個,

注：與SSBP1 siRNA組比較,*P<0.05

2.7 SSBP1 siRNA對HepG2細(xì)胞線粒體膜電位的影響 流式細(xì)胞術(shù)檢測結(jié)果顯示，HepG2細(xì)胞轉(zhuǎn)染SSBP1 siRNA后細(xì)胞線粒體膜電位顯著低于對照組和空白轉(zhuǎn)染組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)；對照組和空白轉(zhuǎn)染組之間比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表7。

組別MMP(FI)SSBP1siRNA組40.12±5.02空白轉(zhuǎn)染組93.97±10.24*對照組95.56±8.53*

注：與SSBP1 siRNA組比較,*P<0.05

2.8 SSBP1 siRNA對HepG2細(xì)胞增殖、侵襲轉(zhuǎn)移相關(guān)基因蛋白表達(dá)的影響 Western blot結(jié)果顯示，HepG2細(xì)胞轉(zhuǎn)染SSBP1 siRNA后細(xì)胞增殖相關(guān)基因PCNA、凋亡抑制基因Bcl-2蛋白表達(dá)顯著低于對照組、空白轉(zhuǎn)染組，凋亡誘導(dǎo)基因Bax蛋白表達(dá)顯著高于對照組、空白轉(zhuǎn)染組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；對照組、空白轉(zhuǎn)染組比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表8。

表8 SSBP1沉默對HepG2細(xì)胞增殖、侵襲轉(zhuǎn)移相關(guān)基因蛋白表達(dá)的影響 ±s

注：與SSBP1siRNA組比較,*P<0.05

3 討論

研究發(fā)現(xiàn)，線粒體基因突變和功能障礙是惡性腫瘤發(fā)生發(fā)展的重要因素[13]。而單鏈DNA結(jié)合蛋白(SSBP1)是調(diào)控線粒體生成和功能的關(guān)鍵因子，主要參與線粒體DNA(mtDNA)的復(fù)制、轉(zhuǎn)錄、損傷后修復(fù)過程[14-16]。SSBP1表達(dá)失調(diào)與mtDNA復(fù)制速度、穩(wěn)定性及線粒體功能障礙密切相關(guān)。有研究證實(shí)，乳腺癌、大腸癌、卵巢癌、骨肉瘤、腎癌等惡性腫瘤組織異常高表達(dá)SSBP1，SSBP1可能作為潛在的腫瘤標(biāo)志物，這對于腫瘤診斷、療效及預(yù)后評估具有重要臨床價值[17,18]。

研究發(fā)現(xiàn)，線粒體途徑是誘導(dǎo)細(xì)胞凋亡的主要途徑。Wong等[19]研究表明SSBP1基因通過影響線粒體凋亡途徑參與惡性腫瘤的發(fā)生發(fā)展。已知多種蛋白參與和調(diào)控了線粒體凋亡過程，其中Bcl-2家族蛋白發(fā)揮了重要作用。Bax是主要的促凋亡蛋白，活化后從細(xì)胞核轉(zhuǎn)位進(jìn)入線粒體，從而介導(dǎo)線粒體凋亡；而Bcl-2是主要的抗凋亡蛋白，能夠抑制caspase-3活化發(fā)揮抗凋亡作用[20-22]。PCNA是細(xì)胞增殖過程中特異性表達(dá)的一種核蛋白，可作為反映細(xì)胞增殖活性的敏感指標(biāo)[23]。MMP-9屬于MMPs家族，其水平越高表示腫瘤侵襲轉(zhuǎn)移能力越強(qiáng)[24]。

為探討SSBP1參與肝癌發(fā)生發(fā)展的具體機(jī)制，本研究觀察靶向沉默SSBP1對肝癌HepG2細(xì)胞增殖、侵襲轉(zhuǎn)移的影響。結(jié)果表明，SSBP1 siRNA轉(zhuǎn)染后HepG2細(xì)胞SSBP1 mRNA和蛋白表達(dá)顯著降低，表明利用siRNA干擾SSBP1基因成功，SSBP1表達(dá)被顯著抑制。與對照組和空白轉(zhuǎn)染組比較，轉(zhuǎn)染SSBP1 siRNA后HepG2細(xì)胞增殖能力、G2期和S期細(xì)胞比例、線粒體膜電位明顯降低，G1期細(xì)胞比例、細(xì)胞凋亡率明顯升高(P<0.05)；同時細(xì)胞增殖相關(guān)基因PCNA、凋亡抑制基因Bcl-2、轉(zhuǎn)移相關(guān)基因MMP-9蛋白表達(dá)顯著下調(diào)，凋亡誘導(dǎo)基因Bax蛋白表達(dá)顯著上調(diào)(P<0.05)，提示SSBP1基因參與肝癌的發(fā)生發(fā)展，下調(diào)SSBP1表達(dá)能夠有效調(diào)控肝癌細(xì)胞周期、增殖及侵襲轉(zhuǎn)移過程。

綜上所述，本實(shí)驗(yàn)表明下調(diào)SSBP1表達(dá)能夠明顯抑制肝癌細(xì)胞增殖、侵襲轉(zhuǎn)移，降低線粒體膜電位，并誘導(dǎo)肝癌細(xì)胞凋亡。本實(shí)驗(yàn)首次證實(shí)肝癌細(xì)胞過表達(dá)SSBP1基因與細(xì)胞增殖、侵襲轉(zhuǎn)移有關(guān)。因此，SSBP1有可能成為防治肝癌的潛在靶點(diǎn)，利用特異性拮抗劑或siRNA造成SSBP1基因沉默有可能成為治療肝癌的有效措施。本研究為肝癌的靶向治療提供了新思路和新策略。但是SSBP1調(diào)控肝癌細(xì)胞增殖、侵襲轉(zhuǎn)移的具體分子機(jī)制仍需進(jìn)一步研究。

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Effects of RNAi-mediated SSBP1 gene silencing on proliferation,invasion and metastasis of human hepatocellular carcinoma HepG2 cells in vitro

MARongfen,CHENQiuying,LIUYanfen.

DepartmentofClinicalLaboratory,People’sHospitalofTanshanCity,Hebei,Tangshan063001,China

Objective To investigate the effects of RNAi-mediated SSBP1 gene silencing on proliferation,invasion and metastasis of human hepatocellular carcinoma HepG2 cells in vitro.Methods The high effective targeting SSBP1 siRNA was obtained by construction and screening,which was transfected into HepG2 cells by liposomes mediated transient transfection.The cells were divided into 3 groups: negative control group,blank transfection group (blank control group) and transfection group.The expression levels of SSBP1 mRNA and protein were detected by Real time-PCR and Western-Blot after the transfection.CCK-8 method was used to detect cell proliferation.Flow cytometry was used to detect the cell cycle,cell apoptosis and mitochondrial trans-membrane potential. The scuffing test and Transwell invasion assay were used to detect invasion and metastasis ability of cells. Moreover the expressions of gene and protein related with cell proliferation,invasion and metastasis were detected by Western Blot.Results The SSBP1 siRNA could obviously inhibit the expressions of SSBP1 mRNA and protein.As compared with negative control group and blank control group,after HepG2 cells were transfected by SSBP1 siRNA,the cell proliferation ability, cell proportion at phase G2and phase S, mitochondrial trans-membrane potential were obviously decreased,however, the cell proportion at phase G1and cell apoptosis rate were significantly increased (P<0.05). Meanwhile the expression levels of PCNA,Bcl-2 and MMP-9 proteins were obviously down-regulated,however, the expression levels of Bax proteins were significantly up-regulated (P<0.05).Conclusion The targeting silencing of SSBP1 gene can inhibit proliferation,invasion, metastasis of HepG2 cells,and can induce cell apoptosis via mitochondria pathway.

hepatocellular carcinoma; mitochondria single chain -DNA-binding protein;cell proliferation; invasion and metastasis

10.3969/j.issn.1002-7386.2017.17.004

063001 河北省唐山市人民醫(yī)院檢驗(yàn)科

R 735.7

A

1002-7386(2017)17-2578-04

2017-03-19)