目的 探究GP73是否通過上皮間質(zhì)轉(zhuǎn)化（EMT）影響肝癌細(xì)胞對奧沙利鉑耐藥。方法 采用實(shí)時熒光定量PCR（qRT-PCR）和Western blotting實(shí)驗(yàn)檢測GP73在肝癌患者癌旁和癌組織中的表達(dá)變化；Western blotting檢測GP73在SMMC7721、Bel-7404和HepG2細(xì)胞株中表達(dá)差異；采用濃度梯度遞增方法建立HepG2奧沙利鉑獲得性耐藥細(xì)胞株，通過MTT法檢測奧沙利鉑（2、4、8、16、32 μmol/L）對親本細(xì)胞和耐藥細(xì)胞活性的影響；采用Western blotting實(shí)驗(yàn)檢測HepG2親本細(xì)胞和耐藥細(xì)胞中GP73及上皮間質(zhì)轉(zhuǎn)化相關(guān)分子N-cadherin、Vimentin、E-cadherin的表達(dá)；利用siRNA/質(zhì)粒分別干擾或過表達(dá)GP73，采用MTT法檢測奧沙利鉑（2、4、8、16、32 μmol/L）對干擾GP73前后耐藥細(xì)胞活性的影響；通過Westernblotting實(shí)驗(yàn)檢測Vimentin、N-cadherin和E-cadherin的表達(dá)。結(jié)果 與癌旁組織比較，GP73 mRNA和蛋白水平在肝癌組織中均顯著上調(diào)（P<0.05）；與SMMC7721和Bel-7404GP73細(xì)胞比較，GP73在HepG2細(xì)胞中顯著高表達(dá)（P<0.05）。奧沙利鉑對親本和耐藥細(xì)胞的半數(shù)抑制濃度（IC₅₀）分別為（12.03±0.06）、（19.26±0.04）μmol/L；干擾GP73后奧沙利鉑對耐藥細(xì)胞的抑制率升高，8、16、32 μmol/L濃度組差異顯著（P<0.05）。與親本細(xì)胞比較，耐藥細(xì)胞中GP73、N-cadherin及Vimentin蛋白表達(dá)顯著上調(diào)（P<0.05），而E-cadherin蛋白表達(dá)顯著下調(diào)（P<0.05）。干擾GP73后，Vimentin、N-cadherin表達(dá)顯著下調(diào)，E-cadherin的表達(dá)顯著上調(diào)（P<0.05）；過表達(dá)GP73后，Vimentin、N-cadherin表達(dá)顯著上調(diào)，E-cadherin的表達(dá)顯著下調(diào)（P<0.05）。結(jié)論 GP73對人肝癌細(xì)胞奧沙利鉑耐藥具有促進(jìn)作用，其作用機(jī)制可能與調(diào)節(jié)EMT相關(guān)。

Objective To investigate whether GP73 affects oxaliplatin resistance of hepatocellular carcinoma through EMT. Methods qPCR and Western blot were used to detect expression of GP73 in adjacent normal tissues and HCC tissues. Western blotting was used to detect the expression difference of GP73 in SMMC7721, BEL-7404 and HepG2 cell lines. OXA resistant cell was established by long term and low dose OXA incubation. MTT assay was used to detect the effect of OXA (2, 4, 8, 16, 32 μmol/L) on the activity of non-resistant and resistant cells and evaluate the establishment of OXA acquired resistant cell lines. Western blotting was used to detect the expression of GP73, EMT markers N-cadherin, Vimentin and E-cadherin in non-resistant and OXA resistant cells. GP73 were knocked down and overexpressed by siRNA/plasmid transfection in OXA resistant HepG2 cell. MTT assay was used to detect the effects of oxaliplatin (2, 4, 8, 16, 32 μmol/L) on the activity of drug-resistant cells before and after GP73 interference. Western blotting was used to detect the expression of GP73, EMT markers N-cadherin、Vimentin and E-cadherin. Results Compared with adjacent tissues, the mRNA and protein levels of GP73 were significantly up-regulated in liver cancer tissues (P<0.05). Compared with SMMC7721 and Bel-7404GP73 cells, the expression of GP73 was significantly higher in HepG2 cells (P<0.05). The IC₅₀ values of oxaliplatin against parents and drug-resistant cells were (12.03±0.06), (19.26±0.04) μmol/L, respectively, respectively. After interfering with GP73, the inhibition rate of oxaliplatin on drug-resistant cells was increased, and there were significant differences in 8, 16 and 32 μmol/L concentration groups (P<0.05). Compared with parental cells, the protein expressions of GP73, N-cadherin and Vimentin in drug-resistant cells were significantly up-regulated (P<0.05), while the protein expression of E-cadherin was significantly down-regulated (P<0.05). After interference with GP73, the expression of Vimentin and N-cadherin was significantly down-regulated, while the expression of E-cadherin was significantly up-regulated (P<0.05). After overexpression of GP73, the expression of Vimentin and N-cadherin were significantly up-regulated, while the expression of E-cadherin was significantly down-regulated (P<0.05). Conclusion GP73 can promote oxaliplatin resistance in human hepatoma cells, and its mechanism may be related to regulating EMT.

R965

黑龍江省省屬高等學(xué)校基本科研業(yè)務(wù)費(fèi)（2019-KYYWF-0968）