張?zhí)N蘊(yùn)， 田 藝， 周 偉， 孫 卯， 吳永忠

(重慶市腫瘤研究所，重慶 400030)

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TRIM44沉默對(duì)肝癌細(xì)胞增殖的影響及分子機(jī)制研究

張?zhí)N蘊(yùn)， 田 藝， 周 偉， 孫 卯， 吳永忠△

(重慶市腫瘤研究所，重慶 400030)

目的： 研究三結(jié)構(gòu)域蛋白44(tripartite motif-containing protein 44, TRIM44)對(duì)肝癌細(xì)胞增殖的影響并探討其分子機(jī)制。方法:RT-qPCR和Western blot法分別檢測TRIM44在正常肝組織、肝癌組織和癌旁組織，以及永生化肝細(xì)胞和肝癌細(xì)胞系中的mRNA和蛋白表達(dá)水平；在肝癌細(xì)胞中轉(zhuǎn)染靶向沉默TRIM44的shRNA，Wes-tern blot實(shí)驗(yàn)檢測對(duì)TRIM44的沉默效果；MTS實(shí)驗(yàn)分析TRIM44沉默對(duì)肝癌細(xì)胞活力的影響，EdU標(biāo)記實(shí)驗(yàn)檢測TRIM44沉默對(duì)肝癌細(xì)胞DNA合成能力的影響；軟瓊脂集落形成實(shí)驗(yàn)檢測TRIM44沉默對(duì)肝癌細(xì)胞錨定非依賴生長能力的影響；Western blot法檢測TRIM44沉默對(duì)哺乳動(dòng)物雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)總蛋白及磷酸化水平的影響；用mTOR激活劑MHY1485處理TRIM44沉默的肝癌細(xì)胞，并通過MTS實(shí)驗(yàn)分析肝癌細(xì)胞的活力。結(jié)果:TRIM44的mRNA和蛋白水平在肝癌組織中高于癌旁組織和正常肝組織，TRIM44的mRNA和蛋白水平在肝癌細(xì)胞系中高于永生化肝細(xì)胞；TRIM44沉默可抑制肝癌細(xì)胞的活力、DNA合成能力及錨定非依賴生長能力；TRIM44沉默可降低mTOR磷酸化水平，MHY1485拮抗TRIM44沉默對(duì)肝癌細(xì)胞活力的抑制作用。結(jié)論:TRIM44沉默可能通過下調(diào)mTOR活性抑制肝癌細(xì)胞增殖。

三結(jié)構(gòu)域蛋白44； 肝細(xì)胞癌； 哺乳動(dòng)物雷帕霉素靶蛋白

三結(jié)構(gòu)域蛋白(tripartite motif-containing protein，TRIM)家族是細(xì)胞生物學(xué)功能的關(guān)鍵調(diào)節(jié)因子，TRIM家族成員含有多域的E3泛素連接酶，并以N端存在三結(jié)構(gòu)域?yàn)樘卣鱗7]。研究表明TRIM家族與細(xì)胞增殖、分化、凋亡、遷移和初始免疫等有關(guān)[7-10]。目前，TRIM蛋白家族成員TRIM16、TRIM3、TRIM28、TRIM40和TRIM26在惡性腫瘤中的作用也逐漸被證實(shí)[11]。而TRIM44的功能研究較少，已知TRIM44含有B box、coiled coil和zinc finger 結(jié)構(gòu)域。由于zinc finger結(jié)構(gòu)域在泛素特異性蛋白酶(ubiquitin-specific protease，USP)家族也有發(fā)現(xiàn)，因此TRIM44可能發(fā)揮“USP樣TRIM”功能抑制泛素化過程。據(jù)文獻(xiàn)報(bào)道TRIM44有助于多種病理學(xué)狀態(tài)，如癌癥、發(fā)育障礙、神經(jīng)退行性變和病毒感染[12]。也有研究證實(shí)了TRIM44在非小細(xì)胞肺癌、食管腺癌和胃癌發(fā)展中的作用[12-14]。然而，目前尚無TRIM44在HCC發(fā)生發(fā)展中的作用研究報(bào)道。本研究將檢測TRIM44在肝癌細(xì)胞、永生化肝細(xì)胞及正常肝組織中的表達(dá)差異，并在肝癌細(xì)胞中用shRNA沉默TRIM44的表達(dá)，進(jìn)而觀察肝癌細(xì)胞增殖的變化及探討其分子機(jī)制，以分析TRIM44對(duì)HCC進(jìn)展的影響，為鑒定HCC藥物治療靶點(diǎn)提供新的依據(jù)。

材 料 和 方 法

1 材料

2 方法

2.1 細(xì)胞培養(yǎng) SK-Hep-1、PLC/PRF/5、Huh-7和MIHA細(xì)胞均培養(yǎng)于含10% 胎牛血清、1% 青-鏈酶素的DMEM培養(yǎng)基中；HepG2細(xì)胞培養(yǎng)于含10%胎牛血清、1% 青-鏈酶素的MEM培養(yǎng)基中。所有細(xì)胞均在5% CO2、37 ℃培養(yǎng)箱中常規(guī)培養(yǎng)。

2.2 質(zhì)粒轉(zhuǎn)染 接種8×104SK-Hep-1細(xì)胞或PLC/PRF/5細(xì)胞于6孔板中，24 h后更換為無抗生素的DMEM培養(yǎng)基。將2 μg質(zhì)粒與6 μL質(zhì)粒轉(zhuǎn)染試劑混勻后，靜置15 min，均勻滴加于6孔板中。24 h后更換為常規(guī)培養(yǎng)基。

2.3 RT-qPCR 收集適量細(xì)胞，用1 mL TRIczol裂解細(xì)胞，提取RNA。取米粒大小正常肝組織、肝癌組織或癌旁組織于流式管中，加入1 mL TRIzol，用組織勻漿機(jī)破碎組織，并提取組織RNA。取1 μg RNA逆轉(zhuǎn)錄成cDNA，隨后使用FastStart Universal SYBR Green Master Mix進(jìn)行PCR，以β-actin為內(nèi)參照。TRIM44的上游引物5’-GCGAGGCCGAAGAAGACAAC-3’,下游引物為5’-TCCGGACACTTCCTCTTGGC；β-actin的上游引物為5’-CTCTTCCAGCCTTCCTTCCT,下游引物為5’-AGCACTGTGTTGGCGTACAG-3’。PCR反應(yīng)條件為 95 ℃ 2 min； 94 ℃ 20 s，60 ℃20 s，72 ℃ 20 s，75 ℃ 1 s，循環(huán)34次。每組實(shí)驗(yàn)重復(fù)3次。以2-ΔΔCt計(jì)算目的基因的相對(duì)表達(dá)水平。

表1為主成分的特征值和方差貢獻(xiàn)率。累積方差貢獻(xiàn)率是確定需要采用的主成分個(gè)數(shù)的主要依據(jù),其次才是特征值。表中第一主成分(PC1)的特征值λ1=0.047,第二主成分(PC2)的特征值λ2=0.016,所有主成分的特征值均小于1,均不能用以確定主成分的個(gè)數(shù)。PC1、PC2的方差貢獻(xiàn)率分別為70.259%、23.212%,前2個(gè)主成分解釋了總方差的近93.47%,故可以選擇前2個(gè)主成分來取代45個(gè)原始變量。

2.4 Western blot實(shí)驗(yàn) 收集細(xì)胞，加入相應(yīng)體積含蛋白酶抑制劑的RIPA裂解緩沖液，置于冰中搖床上裂解15 min，16 000×g離心5 min，吸取上清，用BCA法測定蛋白濃度。取米粒大小正常肝組織、肝癌組織或癌旁組織于流式管中，加入1 mL 含蛋白酶抑制劑的RIPA裂解緩沖液，用組織勻漿機(jī)破碎組織，破碎后將流式管置于冰中15 min，轉(zhuǎn)移樣本至EP管中，16 000×g離心5 min，吸取上清，用BCA法測定蛋白濃度。取30 μg蛋白上樣，SDS-PAGE分離蛋白后，轉(zhuǎn)移蛋白至NC膜，5%脫脂牛奶封閉1 h，稀釋 I 抗(TRIM抗體、mTOR抗體和p-mTOR抗體均1∶1 000稀釋,GAPDH抗體以1∶5 000稀釋)，4 ℃搖床孵育過夜，II 抗室溫下?lián)u床孵育2 h，ECL顯影。以GAPDH為內(nèi)參照。

2.5TRIM44沉默效果檢測 shControl和shTRIM44轉(zhuǎn)染細(xì)胞72 h后提取細(xì)胞總蛋白，測定濃度后，用Western blot法檢測樣本中的TRIM44蛋白及內(nèi)參照GAPDH表達(dá)水平，并比較shControl和shTRIM44轉(zhuǎn)染后，TRIM44蛋白表達(dá)差異。

2.6 MTS實(shí)驗(yàn) 細(xì)胞轉(zhuǎn)染質(zhì)粒24 h后，消化細(xì)胞計(jì)數(shù)，取8 000個(gè)細(xì)胞接種于96孔板。分別在轉(zhuǎn)染后 2 d、3 d、4 d、5 d加入20 μL MTS試劑，5% CO2、37 ℃培養(yǎng)箱中靜置2 h，于490 nm處讀取樣品吸光度。每個(gè)樣品重復(fù)3次。

2.7 EdU標(biāo)記實(shí)驗(yàn) 細(xì)胞接種于含無菌蓋玻片的6孔板中，24 h后轉(zhuǎn)染質(zhì)粒，3 d后運(yùn)用Click-iT EdU Imaging Kits進(jìn)行EdU標(biāo)記實(shí)驗(yàn)，實(shí)驗(yàn)操作嚴(yán)格按試劑盒說明書進(jìn)行。

2.8 軟瓊脂集落形成實(shí)驗(yàn) 將1.2%的瓊脂糖凝膠與含有20%血清的2×DMEM培養(yǎng)基等體積充分混勻，加入6孔板中，作為底層瓊脂糖凝膠，室溫凝固。細(xì)胞轉(zhuǎn)染質(zhì)粒24 h后，消化細(xì)胞并計(jì)數(shù)，取1 000個(gè)細(xì)胞加入含有20%血清的2×DMEM培養(yǎng)基中，混勻后再與0.7%的瓊脂糖凝膠等體積充分混勻作為上層膠加入鋪有底層膠的6孔板中，置于5% CO2、37 ℃培養(yǎng)箱中培養(yǎng)。每3 d向6孔板中加入若干滴培養(yǎng)基，3 周后觀察集落形成，0.05%結(jié)晶紫染色。

3 統(tǒng)計(jì)學(xué)處理

采用SPSS 19.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析，計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。對(duì)兩樣本均數(shù)間比較采用配對(duì)t檢驗(yàn)。多樣本均數(shù)間比較采用單因素方差分析，各組均數(shù)的兩兩比較采用SNK-q檢驗(yàn)。以P<0．05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 TRIM44在肝癌組織和癌旁組織中的表達(dá)差異

RT-qPCR分析結(jié)果顯示TRIM44的mRNA水平在肝癌組織中顯著高于癌旁組織和正常肝組織，見圖1；Western blot法檢測結(jié)果也顯示TRIM44的蛋白水平在肝癌組織中也顯著高于癌旁組織和正常肝組織，見圖2。以上結(jié)果說明TRIM44在肝癌組織和肝癌細(xì)胞中高表達(dá)，提示TRIM44可能有利于肝癌細(xì)胞生長。

Figure 1. The mRNA level of TRIM44 in the normal liver tissues (2 cases), HCC (4 cases) and adjacent nontumor liver tissues (4 cases) determined by RT-qPCR. N: adjacent nontumor liver tissue； T: HCC tissue. Mean±SD.**P<0．01vsN.

圖1 RT-qPCR 檢測正常肝組織、肝癌組織和癌旁組織中TRIM44的mRNA水平

Figure 2. The protein level of TRIM44 in the normal liver tissues (2 cases), HCC (4 cases) and adjacent nontumor liver tissues (4 cases) determined by Western blot. N: adjacent nontumor liver tissue； T: HCC tissue. Mean±SD.**P<0.01vsN.

圖2 Western blot法檢測正常肝組織、肝癌組織和癌旁組織中TRIM44的蛋白水平

2 TRIM44在不同細(xì)胞系中的表達(dá)差異

我們通過RT-qPCR分析了TRIM44 的mRNA水平在不同細(xì)胞中的表達(dá)差異，結(jié)果顯示TRIM44的mRNA水平在肝癌細(xì)胞系中顯著高于永生化肝細(xì)胞，見圖3。另外，我們也用Western blot法檢測了TRIM44蛋白在不同細(xì)胞中的表達(dá)差異，結(jié)果顯示TRIM44 蛋白水平在肝癌細(xì)胞系中也顯著高于永生化肝細(xì)胞，見圖4。

Figure 3.The mRNA level of TRIM44 in immortalized hepatocytes and hepatoma cell lines determined by RT-qPCR. Mean±SD.n=3.*P<0．05vsMIHA.

圖3 RT-qPCR 檢測永生化肝細(xì)胞和肝癌細(xì)胞中TRIM44的mRNA水平

Figure 4. The protein level of TRIM44 in immortalized hepatocytes and hepatoma cell lines determined by Western blot. Mean±SD.n=3.*P<0．05vsMIHA.

圖4 Western blot法檢測永生化肝細(xì)胞和肝癌細(xì)胞中TRIM44的蛋白水平

3 TRIM44沉默對(duì)肝癌細(xì)胞活力的影響

我們首先選取了TRIM44表達(dá)較高的2種肝癌細(xì)胞SK-Hep-1和PLC/PRF/5，并在細(xì)胞中轉(zhuǎn)染了靶向沉默TRIM44表達(dá)的shRNA質(zhì)粒shTRIM44及對(duì)照質(zhì)粒shControl，用Western blot法檢測沉默效率，結(jié)果顯示shTRIM44明顯沉默了TRIM44在這2種細(xì)胞中的表達(dá)，見圖5。在TRIM44有效沉默的基礎(chǔ)上，我們進(jìn)行了MTS實(shí)驗(yàn)，結(jié)果發(fā)現(xiàn)沉默TRIM44表達(dá)明顯抑制了SK-Hep-1和PLC/PRF/5細(xì)胞的活力，且隨著天數(shù)的增加，抑制趨勢更加明顯；在第5 天時(shí)，TRIM44沉默對(duì)SK-Hep-1和PLC/PRF/5細(xì)胞活力的抑制率分別達(dá)到50.8%和47.5%，差異具有統(tǒng)計(jì)學(xué)顯著性(P<0．01)，見圖6。這說明TRIM44沉默可顯著抑制肝癌細(xì)胞的活力。

Figure 5.The silencing efficiency of shTRIM44 in SK-Hep-1 and PLC/PRF/5 cells analyzed by Western blot. Mean±SD.n=3.*P<0．05vsshControl.

圖5 Western blot法檢測SK-Hep-1 和 PLC/PRF/5細(xì)胞中shTRIM44的沉默效率

4 TRIM44對(duì)肝癌細(xì)胞DNA合成能力的影響

我們在SK-Hep-1細(xì)胞中沉默了TRIM44的表達(dá)并進(jìn)行了EdU標(biāo)記實(shí)驗(yàn)。結(jié)果顯示TRIM44沉默組的EdU陽性細(xì)胞數(shù)比對(duì)照組減少約46.4%，說明TRIM44沉默可顯著抑制肝癌細(xì)胞的DNA合成能力，見圖7。

Figure 6. The effects ofTRIM44 knockdown on the viability of SK-Hep-1 (A) and PLC/PRF/5 (B) cells measured by MTS assay. Mean±SD.n=3.**P<0．01vsshControl.

圖6 MTS檢測TRIM44沉默對(duì)SK-Hep-1 和 PLC/PRF/5細(xì)胞活力的影響

Figure 7.The effect ofTRIM44 knockdown on the DNA synthesis in SK-Hep-1 cells detected by EdU incorporation assay. Mean±SD.n=3.*P<0．05vsshControl.

圖7 EdU標(biāo)記實(shí)驗(yàn)檢測TRIM44沉默對(duì)SK-Hep-1細(xì)胞DNA合成能力的影響

5 TRIM44對(duì)肝癌細(xì)胞錨定非依賴生長能力的影響

我們在SK-Hep-1細(xì)胞中沉默了TRIM44的表達(dá)并進(jìn)行了軟瓊脂集落形成實(shí)驗(yàn),結(jié)果顯示TRIM44沉默的SK-Hep-1細(xì)胞形成集落的數(shù)目比對(duì)照細(xì)胞減少55%，說明TRIM44沉默可顯著抑制肝癌細(xì)胞的錨定非依賴生長能力，見圖8。以上結(jié)果揭示TRIM44沉默明顯抑制了肝癌細(xì)胞增殖。

Figure 8.The effect ofTRIM44 knockdown on the ability of anchorage-independent growth in SK-Hep-1 cells observed by the method of colony formation on the soft agar. Mean±SD.n=3.*P<0．05vsshControl.

圖8 軟瓊脂集落形成實(shí)驗(yàn)檢測TRIM44沉默對(duì)SK-Hep-1細(xì)胞錨定非依賴生長能力的影響

6 mTOR在TRIM44沉默抑制肝癌細(xì)胞增殖中的作用

我們通過Western blot法檢測了TRIM44沉默的SK-Hep-1細(xì)胞中的mTOR及mTOR活性形式p-mTOR的蛋白水平，結(jié)果顯示TRIM44對(duì)mTOR表達(dá)未見顯著影響，而能明顯抑制p-mTOR的蛋白水平，提示TRIM44沉默降低了mTOR的活性，見圖9。為進(jìn)一步分析mTOR在TRIM44沉默抑制肝癌細(xì)胞增殖中的作用，我們在沉默TRIM44的同時(shí)用2 μmol/L的mTOR激活劑MHY1485處理細(xì)胞，Western blot法檢測發(fā)現(xiàn)p-mTOR的蛋白水平得到明顯恢復(fù)，見圖10。而與此同時(shí)，MTS實(shí)驗(yàn)分析結(jié)果顯示MHY1485也明顯拮抗了TRIM44沉默對(duì)肝癌細(xì)胞活力的抑制作用，見圖11。這些發(fā)現(xiàn)揭示TRIM44可能通過下調(diào)mTOR信號(hào)通路進(jìn)而抑制肝癌細(xì)胞增殖。

Figure 9.The effect ofTRIM44 knockdown on the protein levels of mTOR and p-mTOR measured by Western blot. Mean±SD.n=3.*P<0．05vsshControl.

圖9 Western blot法檢測TRIM44沉默對(duì)mTOR和p-mTOR蛋白水平的影響

討 論

近年來，許多文獻(xiàn)報(bào)道了TRIM44與腫瘤的關(guān)系。研究發(fā)現(xiàn)TRIM44在胃癌組織中的高表達(dá)與淋巴結(jié)轉(zhuǎn)移和復(fù)發(fā)有關(guān)，也可作為患者不良預(yù)后的預(yù)測因子[14]，并且TRIM44的表達(dá)可增加巴雷特食管病發(fā)展為高度異型增生，最后進(jìn)展為食管癌[13, 15]。在對(duì)非小細(xì)胞肺癌的研究中也發(fā)現(xiàn)TRIM44的高表達(dá)與腫瘤的低分化、TMM分期、淋巴結(jié)轉(zhuǎn)移、惡性腺瘤亞型和不良預(yù)后有關(guān)，TRIM44過表達(dá)能通過mTOR信號(hào)通路促進(jìn)上皮間質(zhì)轉(zhuǎn)化過程，進(jìn)而促進(jìn)腫瘤的侵襲和轉(zhuǎn)移，并且加快了G1/S期轉(zhuǎn)變，促進(jìn)了腫瘤細(xì)胞增殖[12]。這些研究證實(shí)TRIM44是腫瘤形成和進(jìn)展的一個(gè)重要因素。本研究主要關(guān)注于TRIM44對(duì)肝癌細(xì)胞增殖的影響，我們發(fā)現(xiàn)TRIM44在肝癌細(xì)胞及肝癌組織中是高表達(dá)的，TRIM44沉默能抑制肝癌細(xì)胞增殖。有文獻(xiàn)報(bào)道在腫瘤進(jìn)展中TRIM44與mTOR信號(hào)通路密切相關(guān)[12, 15]，并且mTOR活化有利于肝癌細(xì)胞增殖[16]。我們的進(jìn)一步研究也發(fā)現(xiàn)TRIM44沉默可降低mTOR的活性，并且mTOR激活劑可拮抗TRIM44沉默對(duì)肝癌細(xì)胞增殖的抑制作用，這些發(fā)現(xiàn)揭示TRIM44沉默可能通過下調(diào)mTOR的活性抑制肝癌細(xì)胞生長。

Figure 10.The effect ofTRIM44 knockdown with or without mTOR agonist MHY1485 on the protein levels of p-mTOR determined by Western blot. Mean±SD.n=3.*P<0．05vsshControl;#P<0．05vsshTRIM44.

圖10 Western blot法檢測mTOR激動(dòng)劑MHY1485對(duì) p-mTOR蛋白水平的影響

Figure 11.The effect of mTOR agonist MHY1485 on the viability ofTRIM44-silenceing SK-Hep-1 cells analyzed by MTS assay. Mean±SD.n=3.**P<0.01vsshTRIM44.

圖11 MTS實(shí)驗(yàn)分析MHY1485對(duì)TRIM44沉默的SK-Hep-1細(xì)胞活力的影響

mTOR是一種高度保守的蛋白激酶，屬于磷脂酰肌醇3-激酶相關(guān)激酶[phosphoinositide 3-kinase (PI3K)-related kinase，PIKK]家族[17]。mTOR信號(hào)通路是細(xì)胞多種功能的關(guān)鍵調(diào)節(jié)因子，如細(xì)胞生長、增殖和代謝[18-19]。mTOR下游的靶點(diǎn)有4E-BP1、S6K、SREBP、ATG13、Akt、SGK等，通過這些靶點(diǎn)mTOR可調(diào)節(jié)蛋白和脂類合成、溶酶體生物合成、自噬、能量代謝、細(xì)胞生長、凋亡和血管生成等生理過程。其上游的調(diào)節(jié)因子有TSC1/2、Rheb、RAG、AMPK等[18]。目前，已發(fā)現(xiàn)mTOR信號(hào)通路與HCC的發(fā)生發(fā)展的關(guān)系十分密切，研究證實(shí)mTOR信號(hào)通路活化與HCC的低分化、惡性增殖、預(yù)后不良、早期復(fù)發(fā)高度相關(guān)[20]。并且我們的研究也發(fā)現(xiàn)TRIM44沉默下調(diào)了mTOR的磷酸化水平，而mTOR激活劑明顯減弱了TRIM44沉默對(duì)肝癌細(xì)胞增殖的抑制作用。這結(jié)果提示TRIM44位于mTOR信號(hào)通路的上游。據(jù)研究顯示TRIM家族中多個(gè)成員具有E3泛素連接酶活性，并且能泛素化和降解AMPK[21-22]。AMPK位于mTOR上游，是其活性抑制因子[23]。而TRIM44具有穩(wěn)定TRIM家族成員的功能[21]，因此TRIM44可能通過維持TRIM家族成員的E3泛素連接酶活性促進(jìn)AMPK的泛素化降解，最終上調(diào)mTOR的活性，反之，TRIM44沉默則抑制mTOR的活性。這一可能的TRIM44沉默下調(diào)mTOR信號(hào)通路從而抑制肝癌細(xì)胞增殖的分子機(jī)制需要進(jìn)一步的驗(yàn)證。

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(責(zé)任編輯： 陳妙玲， 羅 森)

Effect of TRIM44 silencing on proliferation of hepatocellular carcinoma cells and its molecular mechanism

ZHANG Yun-yun, TIAN Yi, ZHOU Wei, SUN Mao, WU Yong-zhong

(ChongqingCancerInstitute,Chongqing400030China.E-mail:cqmdwyz@yahoo.com.cn)

AIM: To investigate the effect of tripartite motif-containing protein 44 (TRIM44) on the proliferation of hepatocellular carcinoma (HCC) cells and to study the molecular mechanism. METHODS: The expression of TRIM44 at mRNA and protein levels in normal liver tissues, HCC tissues, adjacent nontumor liver tissues, immortalized hepatocytes and hepatoma cell lines was determined by RT-qPCR and Western blot, respectively. The silencing ofTRIM44 was conducted by transfection of vector expressing shRNA targetingTRIM44 (shTRIM44) in the HCC cells, and the protein level of TRIM44 was measured by Western blot. The viability of the HCC cells was analyzed by MTS assay. The DNA synthesis of HCC cells was detected by Click-iT EdU Imaging Kit. The ability of anchorage-independent growth was determined by the method of colony formation on the soft agar. The effects of TRIM44 on the total protein and phosphorylation of mammalian target of rapamycin (mTOR) levels were measured by Western blot. The HCC cells were transfected with shTRIM44 and treated with mTOR agonist MHY1485, and the cell viability was analyzed by MTS assay. RESULTS: The mRNA and protein levels of TRIM44 in the HCC tissues were significantly higher than those in the adjacent nontumor liver tissues and normal liver tissues. In addition, the mRNA and protein levels of TRIM44 in the hepatoma cell lines were significantly higher than those in the immortalized hepatocytes.TRIM44 silencing significantly inhibited the viability of HCC cells and reduced the abilities of DNA synthesis and anchorage-independent growth of the HCC cells.TRIM44 silencing decreased the phosphorylation level of mTOR protein. MHY1485 significantly antagonized the inhibitory effect ofTRIM44 silence to the viability of HCC cells. CONCLUSION:TRIM44 silencing inhibits the proliferation of HCC cells possibly through down-regulating the activity of mTOR.

Tripartite motif-containing protein 44; Hepatocellular carcinoma; Mammalian target of rapamycin

1000- 4718(2016)11- 1972- 07

2016- 05- 30

2016- 09- 23

R735.7; R730.23

A

10.3969/j.issn.1000- 4718.2016.11.009

雜志網(wǎng)址： http://www.cjpp.net

△通訊作者 Tel: 023-65358274; E-mail: cqmdwyz@yahoo.com.cn