張?zhí)N蘊, 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沉默對肝癌細胞增殖的影響及分子機制研究
張?zhí)N蘊, 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)對肝癌細胞增殖的影響并探討其分子機制。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,以及永生化肝細胞和肝癌細胞系中的mRNA和蛋白表達水平;在肝癌細胞中轉(zhuǎn)染靶向沉默TRIM44的shRNA,Wes-tern blot實驗檢測對TRIM44的沉默效果;MTS實驗分析TRIM44沉默對肝癌細胞活力的影響,EdU標記實驗檢測TRIM44沉默對肝癌細胞DNA合成能力的影響;軟瓊脂集落形成實驗檢測TRIM44沉默對肝癌細胞錨定非依賴生長能力的影響;Western blot法檢測TRIM44沉默對哺乳動物雷帕霉素靶蛋白(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沉默的肝癌細胞,并通過MTS實驗分析肝癌細胞的活力。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胞系中高于永生化肝細胞;TRIM44沉默可抑制肝癌細胞的活力、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沉默對肝癌細胞活力的抑制作用。結(jié)論:TRIM44沉默可能通過下調(diào)mTOR活性抑制肝癌細胞增殖。
三結(jié)構(gòu)域蛋白44; 肝細胞癌; 哺乳動物雷帕霉素靶蛋白
三結(jié)構(gòu)域蛋白(tripartite motif-containing protein,TRIM)家族是細胞生物學功能的關(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)域為特征[7]。研究表明TRIM家族與細胞增殖、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[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)文獻報道TRIM44有助于多種病理學狀態(tài),如癌癥、發(fā)育障礙、神經(jīng)退行性變和病毒感染[12]。也有研究證實了TRIM44在非小細胞肺癌、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)展中的作用研究報道。本研究將檢測TRIM44在肝癌細胞、永生化肝細胞及正常肝組織中的表達差異,并在肝癌細胞中用shRNA沉默TRIM44的表達,進而觀察肝癌細胞增殖的變化及探討其分子機制,以分析TRIM44對HCC進展的影響,為鑒定HCC藥物治療靶點提供新的依據(jù)。
1 材料
2 方法
2.1 細胞培養(yǎng) SK-Hep-1、PLC/PRF/5、Huh-7和MIHA細胞均培養(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細胞或PLC/PRF/5細胞于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 收集適量細胞,用1 mL TRIczol裂解細胞,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。每組實驗重復3次。以2-ΔΔCt計算目的基因的相對表達水平。
表1為主成分的特征值和方差貢獻率。累積方差貢獻率是確定需要采用的主成分個數(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數(shù)。PC1、PC2的方差貢獻率分別為70.259%、23.212%,前2個主成分解釋了總方差的近93.47%,故可以選擇前2個主成分來取代45個原始變量。
2.4 Western blot實驗 收集細胞,加入相應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)染細胞72 h后提取細胞總蛋白,測定濃度后,用Western blot法檢測樣本中的TRIM44蛋白及內(nèi)參照GAPDH表達水平,并比較shControl和shTRIM44轉(zhuǎn)染后,TRIM44蛋白表達差異。
2.6 MTS實驗 細胞轉(zhuǎn)染質(zhì)粒24 h后,消化細胞計數(shù),取8 000個細胞接種于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。每個樣品重復3次。
2.7 EdU標記實驗 細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后運用Click-iT EdU Imaging Kits進行EdU標記實驗,實驗操作嚴格按試劑盒說明書進行。
2.8 軟瓊脂集落形成實驗 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。細胞轉(zhuǎn)染質(zhì)粒24 h后,消化細胞并計數(shù),取1 000個細胞加入含有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)計學處理
采用SPSS 19.0軟件進行統(tǒng)計學分析,計量資料用均數(shù)±標準差(mean±SD)表示。對兩樣本均數(shù)間比較采用配對t檢驗。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。以P<0.05為差異有統(tǒng)計學意義。
1 TRIM44在肝癌組織和癌旁組織中的表達差異
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胞中高表達,提示TRIM44可能有利于肝癌細胞生長。
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在不同細胞系中的表達差異
我們通過RT-qPCR分析了TRIM44 的mRNA水平在不同細胞中的表達差異,結(jié)果顯示TRIM44的mRNA水平在肝癌細胞系中顯著高于永生化肝細胞,見圖3。另外,我們也用Western blot法檢測了TRIM44蛋白在不同細胞中的表達差異,結(jié)果顯示TRIM44 蛋白水平在肝癌細胞系中也顯著高于永生化肝細胞,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 檢測永生化肝細胞和肝癌細胞中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法檢測永生化肝細胞和肝癌細胞中TRIM44的蛋白水平
3 TRIM44沉默對肝癌細胞活力的影響
我們首先選取了TRIM44表達較高的2種肝癌細胞SK-Hep-1和PLC/PRF/5,并在細胞中轉(zhuǎn)染了靶向沉默TRIM44表達的shRNA質(zhì)粒shTRIM44及對照質(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種細胞中的表達,見圖5。在TRIM44有效沉默的基礎(chǔ)上,我們進行了MTS實驗,結(jié)果發(fā)現(xiàn)沉默TRIM44表達明顯抑制了SK-Hep-1和PLC/PRF/5細胞的活力,且隨著天數(shù)的增加,抑制趨勢更加明顯;在第5 天時,TRIM44沉默對SK-Hep-1和PLC/PRF/5細胞活力的抑制率分別達到50.8%和47.5%,差異具有統(tǒng)計學顯著性(P<0.01),見圖6。這說明TRIM44沉默可顯著抑制肝癌細胞的活力。
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細胞中shTRIM44的沉默效率
4 TRIM44對肝癌細胞DNA合成能力的影響
我們在SK-Hep-1細胞中沉默了TRIM44的表達并進行了EdU標記實驗。結(jié)果顯示TRIM44沉默組的EdU陽性細胞數(shù)比對照組減少約46.4%,說明TRIM44沉默可顯著抑制肝癌細胞的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SK-Hep-1 和 PLC/PRF/5細胞活力的影響
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標記實驗檢測TRIM44沉默對SK-Hep-1細胞DNA合成能力的影響
5 TRIM44對肝癌細胞錨定非依賴生長能力的影響
我們在SK-Hep-1細胞中沉默了TRIM44的表達并進行了軟瓊脂集落形成實驗,結(jié)果顯示TRIM44沉默的SK-Hep-1細胞形成集落的數(shù)目比對照細胞減少55%,說明TRIM44沉默可顯著抑制肝癌細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 軟瓊脂集落形成實驗檢測TRIM44沉默對SK-Hep-1細胞錨定非依賴生長能力的影響
6 mTOR在TRIM44沉默抑制肝癌細胞增殖中的作用
我們通過Western blot法檢測了TRIM44沉默的SK-Hep-1細胞中的mTOR及mTOR活性形式p-mTOR的蛋白水平,結(jié)果顯示TRIM44對mTOR表達未見顯著影響,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。為進一步分析mTOR在TRIM44沉默抑制肝癌細胞增殖中的作用,我們在沉默TRIM44的同時用2 μmol/L的mTOR激活劑MHY1485處理細胞,Western blot法檢測發(fā)現(xiàn)p-mTOR的蛋白水平得到明顯恢復,見圖10。而與此同時,MTS實驗分析結(jié)果顯示MHY1485也明顯拮抗了TRIM44沉默對肝癌細胞活力的抑制作用,見圖11。這些發(fā)現(xiàn)揭示TRIM44可能通過下調(diào)mTOR信號通路進而抑制肝癌細胞增殖。
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mTOR和p-mTOR蛋白水平的影響
近年來,許多文獻報道了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與淋巴結(jié)轉(zhuǎn)移和復發(fā)有關(guān),也可作為患者不良預后的預測因子[14],并且TRIM44的表達可增加巴雷特食管病發(fā)展為高度異型增生,最后進展為食管癌[13, 15]。在對非小細胞肺癌的研究中也發(fā)現(xiàn)TRIM44的高表達與腫瘤的低分化、TMM分期、淋巴結(jié)轉(zhuǎn)移、惡性腺瘤亞型和不良預后有關(guān),TRIM44過表達能通過mTOR信號通路促進上皮間質(zhì)轉(zhuǎn)化過程,進而促進腫瘤的侵襲和轉(zhuǎn)移,并且加快了G1/S期轉(zhuǎn)變,促進了腫瘤細胞增殖[12]。這些研究證實TRIM44是腫瘤形成和進展的一個重要因素。本研究主要關(guān)注于TRIM44對肝癌細胞增殖的影響,我們發(fā)現(xiàn)TRIM44在肝癌細胞及肝癌組織中是高表達的,TRIM44沉默能抑制肝癌細胞增殖。有文獻報道在腫瘤進展中TRIM44與mTOR信號通路密切相關(guān)[12, 15],并且mTOR活化有利于肝癌細胞增殖[16]。我們的進一步研究也發(fā)現(xiàn)TRIM44沉默可降低mTOR的活性,并且mTOR激活劑可拮抗TRIM44沉默對肝癌細胞增殖的抑制作用,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激動劑MHY1485對 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實驗分析MHY1485對TRIM44沉默的SK-Hep-1細胞活力的影響
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信號通路是細胞多種功能的關(guān)鍵調(diào)節(jié)因子,如細胞生長、增殖和代謝[18-19]。mTOR下游的靶點有4E-BP1、S6K、SREBP、ATG13、Akt、SGK等,通過這些靶點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信號通路與HCC的發(fā)生發(fā)展的關(guān)系十分密切,研究證實mTOR信號通路活化與HCC的低分化、惡性增殖、預后不良、早期復發(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沉默對肝癌細胞增殖的抑制作用。這結(jié)果提示TRIM44位于mTOR信號通路的上游。據(jù)研究顯示TRIM家族中多個成員具有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信號通路從而抑制肝癌細胞增殖的分子機制需要進一步的驗證。
[1] DeSantis CE, Lin CC, Mariotto AB, et al. Cancer treatment and survivorship statistics[J]. CA Cancer J Clin, 2014, 64(4):252-271.
[2] Siegel R, Ma J, Zou Z, et al. Cancer statistics[J]. CA Cancer J Clin, 2014, 64(1):9-29.
[3] Forner A, Gilabert M, Bruix J, et al. Reply: heterogeneity of intermediate-stage HCC necessitates personalized management including surgery[J]. Nat Rev Clin Oncol, 2015, 12(1):10.
[4] Bosch FX, Ribes J, Díaz M, et al. Primary liver cancer: worldwide incidence and trends[J]. Gastroenterology, 2004, 127(5 Suppl 1):S5-S16.
[5] Kanwal F, El-Serag HB, Ross D. Surveillance for hepatocellular carcinoma: can we focus on the mission?[J]. Clin Gastroenterol Hepatol, 2015, 13(4):805-807.
[6] Psyrri A, Arkadopoulos N, Vassilakopoulou M, et al. Pathways and targets in hepatocellular carcinoma[J]. Expert Rev Anticancer Ther, 2012, 12(10):1347-1357.
[7] Ikeda K, Inoue S. TRIM proteins as RING finger E3 ubiquitin ligases[J]. Adv Exp Med Biol, 2012, 770:27-37.
[8] Zhang J, Fang L, Zhu X, et al. Ro52/SSA sensitizes cells to death receptor induced apoptosis by down-regulating c-FLIP(L)[J]. Cell Biol Int, 2012, 36(5):463-468.
[9] Schwamborn JC, Berezikov E, Knoblich JA. The TRIM-NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural progenitors[J].Cell, 2009, 136 (5):913-925.
[10]Uchil PD, Pawliczek T, Reynolds TD, et al. TRIM15 is a focal adhesion protein that regulates focal adhesion disassembly[J]. J Cell Sci, 2014, 127(pt 18):3928-3942.
[11]Wang Y, He D, Yang L, et al. TRIM26 functions as a novel tumor suppressor of hepatocellular carcinoma and its downregulation contributes to worse prognosis[J]. Biochem Biophys Res Commun, 2015, 463(3):458-465.
[12]Xing Y, Meng Q, Chen X, et al. TRIM44 promotes proliferation and metastasis in non-small cell lung cancer via mTOR signaling pathway[J]. Oncotarget, 2016,7(21):30479-30491.
[13]Ong CA, Shapiro J, Nason KS, et al. Three-gene immunohistochemical panel adds to clinical staging algorithms to predict prognosis for patients with esophageal adenocarcinoma[J]. J Clin Oncol, 2013, 31(12):1576-1582.
[14]Kashimoto K, Komatsu S, Ichikawa D, et al. Overexpression of TRIM44 contributes to malignant outcome in gastric carcinoma[J]. Cancer Sci, 2012, 103(11):2021-2026.
[15]Ong CA, Shannon NB, Ross-Innes CS, et al. Amplification of TRIM44: pairing a prognostic target with potential therapeutic strategy[J]. J Natl Cancer Inst, 2014, 106(5):dju050.
[16]Choi KJ, Baik IH, Ye SK, et al. Molecular targeted therapy for hepatocellular carcinoma: present status and future directions[J]. Biol Pharm Bull, 2015, 38(7): 986-991.
[17]Cargnello M, Tcherkezian J, Roux PP. The expanding role of mTOR in cancer cell growth and proliferation[J]. Mutagenesis, 2015, 30(2):169-176.
[18]Xu K, Liu P, Wei W. mTOR signaling in tumorigenesis[J]. Biochim Biophys Acta, 2014, 1846(2):638-654.
[19]費洪榮, 趙 瑩, 王桂玲, 等. PI3K/mTOR雙重抑制劑PF-04691502 誘導人胃癌SGC-7901 細胞凋亡[J]. 中國病理生理雜志, 2013, 29( 11):1962-1965.
[20]Matter MS, Decaens T, Andersen JB, et al. Targeting the mTOR pathway in hepatocellular carcinoma: current state and future trends[J]. J Hepatol, 2014, 60(4):855-865.
[21]Urano T, Usui T, Takeda S, et al. TRIM44 interacts with and stabilizes terf, a TRIM ubiquitin E3 ligase[J]. Biochem Biophys Res Commun, 2009, 383(2):263-268.
[22]Pineda CT, Potts PR. Oncogenic MAGEA-TRIM28 ubi-quitin ligase downregulates autophagy by ubiquitinating and degrading AMPK in cancer[J]. Autophagy, 2015, 11(5):844-846.
[23]Dong LX, Sun LL, Zhang X, et al. Negative regulation of mTOR activity by LKB1-AMPK signaling in non-small cell lung cancer cells[J]. Acta Pharmacol Sin, 2013, 34(2):314-318.
(責任編輯: 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
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△通訊作者 Tel: 023-65358274; E-mail: cqmdwyz@yahoo.com.cn