慢病毒介导的RNA干扰抑制骨桥蛋白表达对肝癌细胞生物学行为的影响
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摘要
肝细胞肝癌(简称肝癌)是世界上最常见、恶性度最高的肿瘤之一，位居全球恶性肿瘤发病率的第6位、死因的第3位，在我国已成为恶性肿瘤的第2位死因。近几十年来尽管对肝癌的基础研究和诊断治疗都取的了显著进步，但肝癌的总体预后仍然很差，并没有显著的改善，5年生存率仅为5％左右。手术治疗仍是目前肝癌最有效的方法，但肝癌根治性切除后5年转移复发率高达60～70％，其远期疗效仍欠满意。术后转移复发已成为进一步提高肝癌疗效的主要障碍，临床上迫切需要寻找能预测肝癌转移复发的分子和新的干预治疗方法。
骨桥蛋白(Osteopontin，OPN)是一种分泌型的磷酸化糖蛋白，可与整合素和CD44结合，参与肿瘤细胞的信号转导等过程，许多研究显示其参与多种恶性肿瘤的发生和转移。我们前期与美国国立癌症研究所合作，应用cDNA arrays技术在全基因组范围内比较40例不伴与伴有肝内转移的肝癌之间、肝内转移灶和原发瘤之间基因表达谱的变化，经细胞及组织表达水平分析，证实OPN表达升高与肝癌转移密切相关，应用OPN抗体阻断其作用可抑制肝癌细胞的侵袭转移。后期研究我们还发现肝癌患者外周血OPN表达水平与术后肿瘤转移复发及病人的预后有关，提示OPN可作为肝癌复发转移的预测指标和治疗靶点，具有重要的临床应用价值。
为此，本课题采用目前国际上较先进的RNA干扰技术—慢病毒介导的microRNA(miRNA)干扰，抑制OPN表达并构建稳定的OPN表达沉默肝癌细胞株，通过体内、体外实验观察OPN沉默对肝癌细胞株生物学行为的影响，并探讨其相关机制。
第一部分慢病毒RNAi载体的构建
本部分的研究目的是构建慢病毒RNAi表达载体，并将其转染人高转移潜能肝癌细胞株HCCLM3(原位接种5周后肺转移率为100％，皮下接种5周后肺转移率达100％)，并筛选、获得稳定转染细胞株。
方法：首先体外化学合成OPN序列特异性pre-miRNA双链(OPNi-1、OPNi-2、OPNi-3、OPNi-4)和非特异性对照链OPNi-3M，并将双链前体克隆进线性质粒表达载体pcDNA ~(TM)6.2-GW／EmGFP-miR，而后将其转入感受态菌E．coli.One ShotTOP10，经克隆、筛选、测序以确保插入序列正确无误。进一步将质粒pcDNA~(TM)6.2-GW／EmGFP-miR, pDONR~(TM). 221和pLenti6／V5-DEST行快速BP／LR重组连接反应，形成新的质粒pLenti6-V5-GW／EmGFP-miR表达载体。通过转化大肠杆菌E．coli. One Shot Stb13~(TM)，挑取阳性克隆并大量扩增，筛选扩增pLenti6-V5-GW／EmGFP-miR以备包装病毒用。将质粒pLenti6-V5-GW／EmGFP-miR 3μg、包装质粒ViraPower ~(TM)．Packaging Mix(pLP1、plp2、pLP／VSVG)9μg、Lipofectamine ~(TM)2000 36μl混合，在10cm直径培养皿转染6×10~6个293FT细胞，37℃培养，72小时后收集含有病毒的细胞培养基上清，离心浓缩分装，-80℃储存。采用流式细胞仪检测EmGFP阳性细胞率的方法测定病毒滴度，公式为滴度(TU／ml)=[(GFP阳性细胞率×细胞总数)／体积]×病毒稀释倍数。采用不同的MOI值(单个细胞感染的病毒颗粒数)感染HCCLM3细胞，72小时后荧光显微镜下观察感染细胞GFP表达的阳性率和强度，选择GFP表达强的最小MOI值为最佳MOI值，最后，采用0.5μg／ml的Blasticidin(稻瘟菌素)反复筛选病毒感染细胞株，得到稳定转染细胞株。
结果：经测序证实插入的序列正确。构建的病毒滴度约为1×10~8 TU／ml。本实验的MOI=30。得到稳定转染细胞株4个：LM3-Lenti.OPNi-1、LM3-Lenti.OPNi-2、LM3-Lenti. OPNi-4、LM3-Lenti.OPNi-3M。HCCLM3细胞感染病毒Lenti.OPNi-3后，单克隆集落形成能力及增殖力显著下降，无法得到可以传代的病毒感染细胞株。故在后续有关Lenti. OPNi-3感染细胞株皆采用高滴度(MOI=50)病毒感染一定数量的HCCLM3细胞制备，以确保95％以上的细胞GFP表达阳性。应用Real-Time PCR检测5种病毒对OPN表达的抑制率分别为Lenti.OPNi-160％、Lenti. OPNi-272％、Lenti.OPNi-3 90％、Lenti.OPNi-451％、Lenti.OPNi-3M 14％，Western blot检测进一步证实上述结果。选取Lenti.OPNi-2和Lenti.OPNi-3作为后续研究的主要对象。发现病毒感染后细胞P-eIF2α表达水平无显著差异，提示Lenti.OPNi-3抑制肝癌细胞生长可能存在其它机制而非RNAi引起细胞的抗病毒反应。Lenti.OPNi-2对细胞增殖无显著影响。对照病毒Lenti.OPNi-3M对OPN表达无明显抑制作用。
结论：我们成功构建并筛选出具有较强的OPN表达抑制作用的慢病毒Lenti.OPNi-2和Lenti.OPNi-3。Lenti.OPNi-3转染可明显抑制HCCLM3细胞的克隆形成能力，且这种作用与RNAi导致的非特异性干扰素样诱导效应无关。Lenti.OPNi-2虽可抑制OPN表达，但对细胞克隆形成能力无显著影响。对照病毒Lenti.OPNi-3M对OPN表达和细胞增殖均无明显抑制作用。
第二部分抑制OPN表达对肝癌细胞生物学特性的影响
一、OPN表达沉默对高转移潜能肝癌细胞体外增殖和侵袭能力的影响
本部分的研究目的是观察OPN表达抑制后对肝癌细胞HCCLM3的增殖和侵袭能力的影响，并初步探讨其相关调控机制。
(1)OPN表达沉默对肝癌细胞增殖的影响
第一部分研究发现Lenti.OPNi-3和Lenti.OPNi-2的作用存在明显不同，为进一步验证和解释这一发现，我们用MTT实验检测Lenti.OPNi-3M、Lenti.OPNi-2、Lenti.OPNi-3三组病毒转染对HCCLM3细胞增殖力的影响。结果显示与空白对照组(Mock)相比，Lenti.OPNi-3组细胞生长明显受抑(p＜0.001)，而Lenti.OPNi-3M、Lenti.OPNi-2组细胞增殖力无显著改变(p=0.27，p=0.08)。
为进一步探讨其相关机制，我们采用Western blot检测细胞增殖相关的信号转导蛋白pMEK、pERK1／2的表达情况，结果显示Lenti.OPNi-3组细胞pERK1／2表达量明显下降(p＜0.001)，而Lenti.OPNi-2和Lenti.OPNi-3M组pERK1／2无明显改变(p=0.218，p=0.482)。各组GAPDH、NF-κB等的表达量无显著差异。提示pERK1／2表达的下调是特异性的，OPN可能参与HCCLM3细胞MEK／ERK1／2信号通路的调节。
为了进一步验证这一推论，我们单用重组人OPN蛋白(10μM)或联合MEK抑制剂U0126(20μM)作用于稳定转染Lenti.OPNi-3的HCCLM3细胞，约20分钟后检测细胞pMEK、pERK1／2表达情况，发现单用OPN蛋白组pMEK、pERK1／2表达明显高于联合组(p＜0.001)，提示OPN激活ERK1／2是通过pMEK信号途径。由于Lenti.OPNi-3组细胞pERK1／2下降明显、生长停滞，Lenti.OPNi-2对细胞增殖无明显影响，而两者对细胞OPN表达的抑制率不同(蛋白水平分别为95％和78％)，提示OPN可能参与HCCLM3细胞的增殖，并且要满足细胞增殖OPN的表达量不能低于基础表达量的5％(1-95％)。然而与Lenti.OPNi-3M比较，Lenti.OPNi-3对HepG2细胞(无转移潜能的肝癌细胞株)增殖影响无显著差异(p=0.216)，且Western blot显示pMEK和pERK1／2的表达也无差异(p=0.127)。类似的现象也见于病毒转染后的正常肝脏细胞CCL13。说明对于OPN低表达的HepG2、CCL13细胞，可能需要满足增殖所需的OPN量更少或根本就不需要，抑制OPN表达并不影响它们的正常生长。
上述结果表明，OPN参与HCCLM3细胞增殖相关的MAPK信号转导通路的调节，但是发挥这种增殖调节功能所需的基础OPN表达量很少，在HCCLM3细胞不能低于基础表达量的5％，在恶性度较低的HepG2细胞以及正常肝脏细胞甚至可能更少。这可能解释为什么一些研究结果显示抑制OPN表达对细胞的增殖没有影响，原因可能是即使剩余少量的OPN也足以满足增殖的需要。
(2)OPN表达沉默对肝癌细胞侵袭能力的影响
为进一步研究OPN与肝癌细胞侵袭转移的关系，我们应用Matrigel侵袭实验检测Lenti.OPNi-3M、Lenti.OPNi-2、Lenti.OPNi-3抑制OPN表达后，对HCCLM3侵袭力的影响。结果显示与空白对照组(28.8±2.6)相比，Lenti.OPNi-2(9.2±1.3)和Lenti.OPNi-3(7.8±1.5)组的穿过人工基底膜的细胞数明显减少(p＜0.001)，而Lenti.OPNi-2和Lenti.OPNi-3两组差别无显著意义(p=0.152)。为进一步探讨其相关机制，Western blot检测了三组细胞OPN表达沉默后，基质降解酶MMP-2、uPA和MMP-9的表达，结果显示与空白对照相比，Lenti.OPNi-2和Lenti.OPNi-3两组细胞MMP-2、uPA表达显著下降(p＜0.001)，而MMP-9表达无明显变化(p=0.067和p=0.099)。为进一步验证OPN与MMP-2的直接联系，我们分别用不同浓度的重组人OPN蛋白(10μM和20μM)刺激OPN表达沉默的HCCLM3细胞(Lenti.OPNi-3)，明胶酶谱法分析细胞培养基上清，发现MMP-2表达随着OPN浓度的升高而增加(p=0.003和p=0.002)。用10μM重组人OPN蛋白刺激HCCLM3细胞，并用免疫荧光抗体标记p65蛋白。分别在45min、70min和2h三个不同时间点共聚焦显微镜下观察的p65蛋白在细胞内的位置变化。发现随着OPN蛋白作用时间的延长，p65蛋白逐渐从胞浆内转移至胞核，而p65只有在活化的状态才能从胞浆转移至胞核参与调节信号转导。而Western blot对不同时间点胞浆、胞核内p65蛋白表达的检测也证实了上述免疫荧光的观测结果。
上述结果显示Lenti.OPNi-2、Lenti.OPNi-3抑制HCCLM3细胞的侵袭转移可能与降低MMP-2和uPA的表达有关。Lenti.OPNi-2不影响HCCLM3细胞增殖，却能显著抑制HCCLM3侵袭转移。而Lenti.OPNi-3对HCCLM3细胞增殖和转移都具有显著抑制作用。这提示我们肿瘤细胞增殖和转移两种不同生物学行为对OPN蛋白的基础表达量可能存在不同量的要求。对于HCCLM3细胞，如果OPN蛋白表达量低于22％(1-78％)，侵袭转移将受限，而低于5％(1-95％)细胞增殖也受到抑制。可见侵袭转移较增殖对OPN蛋白表达下调更敏感，其详细机制有待探讨。另外，还发现对于HCCLM3细胞，OPN可能通过NF-κB信号途径调节MMP-2和uPA的表达。
二、OPN表达沉默对高转移潜能肝癌细胞成瘤和肺转移的影响
本部分的研究目的是采用裸鼠人肝癌肺转移模型进一步验证不同程度的抑制OPN表达对肝癌细胞HCCLM3的侵袭、增殖能力的影响。
我们分别将转染Mock、Lenti.OPNi-2、Lenti.OPNi-3的三组HCCLM3细胞(5×10~6／只)裸鼠皮下接种，6周后观察裸鼠的成瘤和肺转移。结果显示Lenti.OPNi-3组裸鼠成瘤率(50％)明显低于Mock对照组和Lenti.OPNi-2组(100％)，且肿瘤体积较小(Lenti.OPNi-3组为341±502.6mm~3，而Lenti.OPNi-2组为3686.9±2206.7mm~3，Mock组为4423.6±1206.7mm~3)(p＜0.001)。肺组织切片检测发现Mock、Lenti.OPNi-2、Lenti.OPNi-3三组肺转移率分别为100％、66.7％和50％，单个肺转移灶的数目分别为14±2.4个、3.5±3.4个和1.2±1.3个，差异有显著性(p＜0.01)；且肺转移灶都以Ⅰ期为主，Mock组和Lenti.OPNi-2组还可见到第Ⅳ期转移灶。采用活体荧光成像分析系统观察发现Lenti.OPNi-3组没有成瘤的裸鼠在原接种部位仍然存在发荧光的肿瘤细胞，说明肿瘤细胞增殖非常缓慢，无法成瘤并非是由于RNA干扰可能导致的非特异性细胞毒性作用。
我们通过对裸鼠成瘤大小和肺转移率的检测，再次证实了体外实验的观察结果，进一步说明不同程度的沉默OPN表达对HCCLM3细胞的增殖和侵袭两种生物学行为的影响是不一样的，侵袭转移比增殖对OPN基础表达量的下调更为敏感。
Hepatocellular carcinoma (HCC) is one of the most common and aggressivemalignancies worldwide, ranking the fifth most important cancer in terms of numbersof cases and the third in terms of cancer mortality. And it has become the secondcancer killer in China since 1990s. Unfortunately, despite efforts to improve itsprognosis, the overall survival of patients with HCC is still dismal. Metastaticrecurrence after HCC resection is one of the major obstacles to prolonging survival.Therefore, there is a tremendous interest and urgency to search for molecules relatedto HCC metastasis that would provide new predictors for HCC metastasis as well asnew targets for intervention.
Osteopontin (OPN), a secreted phosphoglycoprotein, is a promoting factor fortumorigenicity and metastasis of cancer cells in several types of tumors through avariety of mechanisms. OPN binds with several integrins and CD44 variants in anRGD sequence-dependent and-independent manner. In our previous work, wecollaborated with the National Cancer Insititute (NCI) of USA to compare thedifference in gene expression profiles between HCCs without and with accompanyingintra-hepatic metastases using a cDNA microarray of. 9180 cDNA clones. We foundthat OPN was one of the leading genes that over-expressed in the HCCs withmetastasis. An OPN-neutralizing antibody efficiently blocked in vitro invasion and invivo pulmonary metastasis of HCC cells. Moreover, we also found that preoperativeplasma OPN level can be used as a predictive marker for tumor recurrence andprognosis of patients with HCC after operation. These suggest that OPN is associatedwith metastasis of HCC. However, the mechanism by which OPN mediatesprogression and metastasis of HCC remains unknown. So, in this study, we tried tofind out what happened with the metastatic HCC cell line (HCCLM3) if its highendogenous OPN level was knocked down using lentiviral-mediated RNAinterference, and explore the possible mechanisms.
PARTⅠConstruction and selection of Lentiviral-mediated RNAiexpression vectors
The pcDNA ~(TM)6.2-GW/EmGFP-miR expression vectors contain adouble-stranded oligonucleotide (ds oligo) encoding a pre-miRNA sequence forexpression in mammalian cells using a RNA PolymeraseⅡ(PolⅡ) promoter, thehuman cytomegalovirus (CMV) immediate early promoter. The pDONR~(TM)221 vectoris used as an intermediate to transfer the pre-miRNA expression cassette into thelentiviral expression plasmid (pLenti6/V5-DEST) using Gateway Technology. Weperformed the Rapid BP/LR recombination reaction between pDONR~(TM)221,pcDNA~(TM)6.2-GW/EmGFP-miR and pLenti6/V5-DEST to generate the pLenti6/V5-GW/EmGFP-miR expression vectors. The sequence for construction of four OPN(GenBank accession number NM_000582) pre-miRNA and one control pre-miRNAwere as follows:
Packaging vector (9μg) (pLP1, plp2, pLP/VSVG), 3μg pLenti6/V5-GW/EmGFP-miR expression plasmid with OPNi pre-miRNA or control pre-miRNAwere cotransfected into 293FT packaging cells with Lipofectamine 2000 (36μl). After48 h the virus-containing culture supernatants were centrifuged to pellet the cell debris and stored in 1 ml aliquots at -80℃. Before proceeding to transduce theHCCLM3 cell line and express the miRNA for RNAi analysis, we determined the titerof lentiviral stock using EmGFP tittering method, calculated the EmGFP lentivirustiters from the dilutions at which the percentage of EmGFP-positive cells fall withinthe range of 1-30％. This was to avoid analyzing dilution samples containing multipleintegrated lentiviral genomes, which may result in an underestimate of the viral titer.Titer is expressed as transducing units (TU)/ml. The following formula was used tocalculate the titer: [F×C/V]×D(F=frequency of GFP-positive cells (percentageobtained divided by 100) C=total number of cells in the well at the time oftransduction V=volume of inoculum in ml D=lentivirus dilution). The lentiviraltiter for this study was 1×10~8 TU/ml. To obtain optimal expression of miRNA andtherefore, the highest degree of target gene knockdown, the lentiviral constructionwas transfected into mammalian cell line using a suitable MOI. MOI was defined asthe number of virus particles per cell and generally correlates with the number ofintegration events and as a result, expression. Typically, miRNA expression levelsincreased as increasing MOI. The MOI was determined by detecting the EmGFPfluorescent protein, and the suitable MOI was 30 for HCCLM3 cell line. HCCLM3cells were transfected with lentiviral vectors in the presence of polybrene (6μg/ml).After day 3, the medium was replaced with fresh, complete medium containg0.5mg/ml Blasticidin to select for stably transfected cells. Four stably transfected cells(Lenti.OPNi-1, Lenti.OPNi-2, Lenti.OPNi-4, Lenti.OPNi-3M) were obtained.Interestingly, only Lenti.OPNi-3 could inhibit the proliferation of HCCLM3significantly, and no Blasticidin-resistant colonies were identified. Real-time PCRassays showed that Lenti.OPNi-1, Lenti.OPNi-2, Lenti.OPNi-3, Lenti.OPNi-4 andLenti.OPNi-3M could suppress the OPN expression by 60％, 72％, 90％, 52％and14％, respectively. In addition, no significant induction of P-elF2αwas found, whichindicated that the inhibitory effect of RNAi was not through the activation ofIFN-response genes.
PARTⅡInhibitory effect of silencing OPN expression on the in vitroproliferation and invasive abilities of human HCC cells
1. Effect of OPN silencing and HCC proliferation
Of these miRNA lentiviral vectors, we selected Lenti.OPNi-2 and Lenti.OPNi-3which mediated efficient OPN suppression to evaluate effects of OPN knockdown onin vitro proliferation of HCCLM3 cells. Cell proliferation was measured by cellularuptake of MTT (3,-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide).Each assay was performed three times. Lenti.OPNi-3, but not Lenti.OPNi-2, couldinhibit the cell proliferatin significantly (p＜0.001). Lenti.OPNi-3 induced a significantdecrease in MEK and ERK1/2 phosphorylation (p＜0.001), while in cells transfectedwith Lenti.OPNi-3M there were no alteration in the level of OPN and in the activationof downstream pathways (p=0.482). In addition, Lenti.OPNi-3 did not induceaspecific downregulation of endogenous genes such as GAPDH and NF-κB. Incontrast, the proliferation rate of HepG2 without OPN overexpression was lesssensitive to the inhibitory effect of the OPN knockdown (p=0.216), and no changewas observed in the phosphorylation of MEK and ERK1/2 (p=0.127). Theconstitutively low-level expression of the OPN in the HepG2 cells may explain thereason for the HepG2 resistance to the OPN RNAi. Similar result was observed inCCL13 cells. Thus, lentiviral mediated OPN knockdown does not appear to affectcells with low OPN expression. To further investigate whether OPN inducesphosphorylation of MEK and then phosphorylation of ERK1/2 in HCCLM3 cells, thecells transfected with Lenti.OPNi-3 were treated with 10μM exogenous recombinantOPN, pretreated with specific MEK inhibitor U0126 (20μM), or treated with OPNalone. Cell lysates were analyzed by Western blot at different time points. The dataindicated that MEK and ERK were readily activated in OPN-stimulated cells, peakingat 20 minutes, but not in the cells treated with U0126.
2. Effect of OPN silencing and tumor invasion
We evaluated whether the suppression of OPN expression would alter the in vitrometastatic phenotype of HCCLM3 cells. Cell invasion was measured using Matrigelcoated transwell-invasion chambers. Migrating numbers of HCC-LM3 cells wassignificantly inhibited by Lenti.OPNi-2 (9.2±1.3) and Lenti.OPNi-3 (7.8±1.5),compared with Mock (28.84±2.6) (p＜0.001). No significant difference in invasivenesswas observed between Mock and Lenti.OPNi-3M (p=0.221). Parallel experimentswere performed in triplicate and each filter was photographed at 200×magnification.Next, we evaluated the protein expression of several metastatic markers includingMMP-2, MMP-9 and uPA, which have been previously shown to be associated withOPN in models of breast cancer and melanoma. Interestingly, MMP-2 and uPA expression but not MMP-9 were decreased significantly in Lenti.OPNi-2 andLenti.OPNi-3(p＜0.001). Previous studies have shown that OPN stimulates activationof pro-MMP-2 through NF-κB in murine melanoma cells. To further determinewhether OPN have the same effect on translocation of p65 into the nucleus inHCCLM3 cell, the cells were treated with 10μM OPN in basal medium for 0-2h at37℃. Immunofluorescence analysis of NF-κB was performed at different time points.The data showed that OPN induces translocation of p65 into nucleus in atime-dependent manner. In the cells pretreated with OPN, the majority of p65 stainingresided in the cytoplasm upto 30 min. At 45 min and 70 min some nucleartranslocation of p65 were observed, peaking at 2h. Western blotting analysis gave afurther confirmation of translocation of p65 into the nucleus. In the OPN-untreatedcells, the p65 was localized mostly in the cytoplasm compared with the nucleus,whereas in the OPN-treated cells, it was translocated into the nucleus. In order tocheck whether OPN induced pro-MMP-2 production and activation in these cells, theconditioned medium was collected, and MMP-2 activity was analyzed by gelatinzymography. The levels of both the pro and active forms of MMP-2 in theOPN-treated cells were significantly higher compared with the levels of MMP-2 inOPN-untreated cells(p＜0.01).
Inhibitory effect of silencing OPN expression on the in vivotumor growth and lung metastasis of human HCC cells
The in vitro study supported that the hypothesis that OPN contributed totumorigenicity and metastatic capacity of HCCLM3 cells. To directly confirm this invivo, HCCLM3 cells (5×10~6) were transfected with or without the lentiviral vectors,and then subcutaneously inoculated into nude mice. Tumor growth was monitored. At2 week after the injection, the tumors originating from the cells transfected withLenti.OPNi-2 and PBS were measurable. At 6 weeks, the animals were killed forethical reasons given the tumor volume, and then the tumors were excised from mice.HCCLM3 cells mock-transfected (PBS) or transfected with Lenti.OPNi-2 developedlarge tumors 3500mm~3 within 6 weeks. However, growth of HCCLM3 cellstransfected with Lenti.OPNi-3 was markedly suppressed (p＜0.001), suggesting thatknockdown of OPN leads to significant inhibition of tumor growth in vivo.
In the sixth week, pulmonary metastatic lesions were detected in every mouse inPBS group, with mainly gradeⅠ-Ⅱand some gradeⅢ-Ⅳtumor clusters. The mice ofPBS group had an average of 14+2.4 tumor clusters per lung. However, Only 50% miceinfected with the Lenti.OPNi-3-treated cells developed lung metastases with mostly gradeⅠtumorclusters with a combined average of 1.2±1.3 tumor clusters per lung. The effect wasstatistically significant(p＜0.01). The same significant effect was also observed in themice infected with the Lenti.OPNi-2-treated cells(p＜0.01). We also monitored HCCLM3cells growth and metastasis with bioluminescence imaging every week. Interestingly,EmGFP fluorescent protein can be detected in the mice with no measurable tumormass, which suggests the proliferation of HCCLM3 cells transfected withLenti.OPNi-3 was inhibited significantly.
Conclusions
1. We constructed two lentiviral vectors (Lenti.OPNi-2 and Lenti.OPNi-3) whichcould significantly suppress the expression of OPN, and selected two stablytransfected HCCLM3 cell lines (LM3- Lenti.OPNi-2 and LM3- Lenti.OPNi-3M).
2. Osteopontin could promote HCCLM3 cell proliferation through activation ofMEK/MAPK signaling pathways, and little basal expression of OPN is requiredfor HCCLM3 proliferation. However, the cells with constitutively low-levelexpression of OPN (HepG2 and CCL13) were less sensitive to the inhibitoryeffect of the OPN knockdown.
3. Osteopontin stimulates HCCLM3 cell invasion through activation of NF-κB,MMP-2 and uPA. Both Lenti.OPNi-2 and Lenti.OPNi-3 can inhibit pulmonarymetastases of HCCLM3 cells significantly which suggest more basal expressionof OPN is required for HCCLM3 invasion than for HCCLM3 proliferation.
Potential application of this study
1. Long lasting RNAi-based gene knockdown can be achieved by lentiviral basedexpression systems driving the production of short hairpin RNA species (microRNA).Lentiviral-mediated gene silencing is a good way to assess one gene function.
2. Lenti.OPNi-3 significantly inhibits proliferation and invasion of HCCLM3 withoverexpression of OPN, but does not appear to affect CCL13 with low OPN expression.Maybe OPN will become a new therapeutic target for highly metastatic HCC, and Lentiviral vectors provide some insight into vector tools of the bilolgical targettherapy in liver cancer.
Novelties
1. To the best of our knowledge, this is the first report showing that OPN expressionis critical for tumorigenesis and proliferatin of human hepatocellular carcinomacells since knocking down the expression abolishes tumorigenicity.
2. In addition, our data for the first time show that different biological behaviors ofHCC have a different requirement for the basal expression level of OPN.
骨桥蛋白(Osteopontin，OPN)是一种分泌型的磷酸化糖蛋白，可与整合素和CD44结合，参与肿瘤细胞的信号转导等过程，许多研究显示其参与多种恶性肿瘤的发生和转移。我们前期与美国国立癌症研究所合作，应用cDNA arrays技术在全基因组范围内比较40例不伴与伴有肝内转移的肝癌之间、肝内转移灶和原发瘤之间基因表达谱的变化，经细胞及组织表达水平分析，证实OPN表达升高与肝癌转移密切相关，应用OPN抗体阻断其作用可抑制肝癌细胞的侵袭转移。后期研究我们还发现肝癌患者外周血OPN表达水平与术后肿瘤转移复发及病人的预后有关，提示OPN可作为肝癌复发转移的预测指标和治疗靶点，具有重要的临床应用价值。
为此，本课题采用目前国际上较先进的RNA干扰技术—慢病毒介导的microRNA(miRNA)干扰，抑制OPN表达并构建稳定的OPN表达沉默肝癌细胞株，通过体内、体外实验观察OPN沉默对肝癌细胞株生物学行为的影响，并探讨其相关机制。
第一部分慢病毒RNAi载体的构建
本部分的研究目的是构建慢病毒RNAi表达载体，并将其转染人高转移潜能肝癌细胞株HCCLM3(原位接种5周后肺转移率为100％，皮下接种5周后肺转移率达100％)，并筛选、获得稳定转染细胞株。
方法：首先体外化学合成OPN序列特异性pre-miRNA双链(OPNi-1、OPNi-2、OPNi-3、OPNi-4)和非特异性对照链OPNi-3M，并将双链前体克隆进线性质粒表达载体pcDNA ~(TM)6.2-GW／EmGFP-miR，而后将其转入感受态菌E．coli.One ShotTOP10，经克隆、筛选、测序以确保插入序列正确无误。进一步将质粒pcDNA~(TM)6.2-GW／EmGFP-miR, pDONR~(TM). 221和pLenti6／V5-DEST行快速BP／LR重组连接反应，形成新的质粒pLenti6-V5-GW／EmGFP-miR表达载体。通过转化大肠杆菌E．coli. One Shot Stb13~(TM)，挑取阳性克隆并大量扩增，筛选扩增pLenti6-V5-GW／EmGFP-miR以备包装病毒用。将质粒pLenti6-V5-GW／EmGFP-miR 3μg、包装质粒ViraPower ~(TM)．Packaging Mix(pLP1、plp2、pLP／VSVG)9μg、Lipofectamine ~(TM)2000 36μl混合，在10cm直径培养皿转染6×10~6个293FT细胞，37℃培养，72小时后收集含有病毒的细胞培养基上清，离心浓缩分装，-80℃储存。采用流式细胞仪检测EmGFP阳性细胞率的方法测定病毒滴度，公式为滴度(TU／ml)=[(GFP阳性细胞率×细胞总数)／体积]×病毒稀释倍数。采用不同的MOI值(单个细胞感染的病毒颗粒数)感染HCCLM3细胞，72小时后荧光显微镜下观察感染细胞GFP表达的阳性率和强度，选择GFP表达强的最小MOI值为最佳MOI值，最后，采用0.5μg／ml的Blasticidin(稻瘟菌素)反复筛选病毒感染细胞株，得到稳定转染细胞株。
结果：经测序证实插入的序列正确。构建的病毒滴度约为1×10~8 TU／ml。本实验的MOI=30。得到稳定转染细胞株4个：LM3-Lenti.OPNi-1、LM3-Lenti.OPNi-2、LM3-Lenti. OPNi-4、LM3-Lenti.OPNi-3M。HCCLM3细胞感染病毒Lenti.OPNi-3后，单克隆集落形成能力及增殖力显著下降，无法得到可以传代的病毒感染细胞株。故在后续有关Lenti. OPNi-3感染细胞株皆采用高滴度(MOI=50)病毒感染一定数量的HCCLM3细胞制备，以确保95％以上的细胞GFP表达阳性。应用Real-Time PCR检测5种病毒对OPN表达的抑制率分别为Lenti.OPNi-160％、Lenti. OPNi-272％、Lenti.OPNi-3 90％、Lenti.OPNi-451％、Lenti.OPNi-3M 14％，Western blot检测进一步证实上述结果。选取Lenti.OPNi-2和Lenti.OPNi-3作为后续研究的主要对象。发现病毒感染后细胞P-eIF2α表达水平无显著差异，提示Lenti.OPNi-3抑制肝癌细胞生长可能存在其它机制而非RNAi引起细胞的抗病毒反应。Lenti.OPNi-2对细胞增殖无显著影响。对照病毒Lenti.OPNi-3M对OPN表达无明显抑制作用。
结论：我们成功构建并筛选出具有较强的OPN表达抑制作用的慢病毒Lenti.OPNi-2和Lenti.OPNi-3。Lenti.OPNi-3转染可明显抑制HCCLM3细胞的克隆形成能力，且这种作用与RNAi导致的非特异性干扰素样诱导效应无关。Lenti.OPNi-2虽可抑制OPN表达，但对细胞克隆形成能力无显著影响。对照病毒Lenti.OPNi-3M对OPN表达和细胞增殖均无明显抑制作用。
第二部分抑制OPN表达对肝癌细胞生物学特性的影响
一、OPN表达沉默对高转移潜能肝癌细胞体外增殖和侵袭能力的影响
本部分的研究目的是观察OPN表达抑制后对肝癌细胞HCCLM3的增殖和侵袭能力的影响，并初步探讨其相关调控机制。
(1)OPN表达沉默对肝癌细胞增殖的影响
第一部分研究发现Lenti.OPNi-3和Lenti.OPNi-2的作用存在明显不同，为进一步验证和解释这一发现，我们用MTT实验检测Lenti.OPNi-3M、Lenti.OPNi-2、Lenti.OPNi-3三组病毒转染对HCCLM3细胞增殖力的影响。结果显示与空白对照组(Mock)相比，Lenti.OPNi-3组细胞生长明显受抑(p＜0.001)，而Lenti.OPNi-3M、Lenti.OPNi-2组细胞增殖力无显著改变(p=0.27，p=0.08)。
为进一步探讨其相关机制，我们采用Western blot检测细胞增殖相关的信号转导蛋白pMEK、pERK1／2的表达情况，结果显示Lenti.OPNi-3组细胞pERK1／2表达量明显下降(p＜0.001)，而Lenti.OPNi-2和Lenti.OPNi-3M组pERK1／2无明显改变(p=0.218，p=0.482)。各组GAPDH、NF-κB等的表达量无显著差异。提示pERK1／2表达的下调是特异性的，OPN可能参与HCCLM3细胞MEK／ERK1／2信号通路的调节。
为了进一步验证这一推论，我们单用重组人OPN蛋白(10μM)或联合MEK抑制剂U0126(20μM)作用于稳定转染Lenti.OPNi-3的HCCLM3细胞，约20分钟后检测细胞pMEK、pERK1／2表达情况，发现单用OPN蛋白组pMEK、pERK1／2表达明显高于联合组(p＜0.001)，提示OPN激活ERK1／2是通过pMEK信号途径。由于Lenti.OPNi-3组细胞pERK1／2下降明显、生长停滞，Lenti.OPNi-2对细胞增殖无明显影响，而两者对细胞OPN表达的抑制率不同(蛋白水平分别为95％和78％)，提示OPN可能参与HCCLM3细胞的增殖，并且要满足细胞增殖OPN的表达量不能低于基础表达量的5％(1-95％)。然而与Lenti.OPNi-3M比较，Lenti.OPNi-3对HepG2细胞(无转移潜能的肝癌细胞株)增殖影响无显著差异(p=0.216)，且Western blot显示pMEK和pERK1／2的表达也无差异(p=0.127)。类似的现象也见于病毒转染后的正常肝脏细胞CCL13。说明对于OPN低表达的HepG2、CCL13细胞，可能需要满足增殖所需的OPN量更少或根本就不需要，抑制OPN表达并不影响它们的正常生长。
上述结果表明，OPN参与HCCLM3细胞增殖相关的MAPK信号转导通路的调节，但是发挥这种增殖调节功能所需的基础OPN表达量很少，在HCCLM3细胞不能低于基础表达量的5％，在恶性度较低的HepG2细胞以及正常肝脏细胞甚至可能更少。这可能解释为什么一些研究结果显示抑制OPN表达对细胞的增殖没有影响，原因可能是即使剩余少量的OPN也足以满足增殖的需要。
(2)OPN表达沉默对肝癌细胞侵袭能力的影响
为进一步研究OPN与肝癌细胞侵袭转移的关系，我们应用Matrigel侵袭实验检测Lenti.OPNi-3M、Lenti.OPNi-2、Lenti.OPNi-3抑制OPN表达后，对HCCLM3侵袭力的影响。结果显示与空白对照组(28.8±2.6)相比，Lenti.OPNi-2(9.2±1.3)和Lenti.OPNi-3(7.8±1.5)组的穿过人工基底膜的细胞数明显减少(p＜0.001)，而Lenti.OPNi-2和Lenti.OPNi-3两组差别无显著意义(p=0.152)。为进一步探讨其相关机制，Western blot检测了三组细胞OPN表达沉默后，基质降解酶MMP-2、uPA和MMP-9的表达，结果显示与空白对照相比，Lenti.OPNi-2和Lenti.OPNi-3两组细胞MMP-2、uPA表达显著下降(p＜0.001)，而MMP-9表达无明显变化(p=0.067和p=0.099)。为进一步验证OPN与MMP-2的直接联系，我们分别用不同浓度的重组人OPN蛋白(10μM和20μM)刺激OPN表达沉默的HCCLM3细胞(Lenti.OPNi-3)，明胶酶谱法分析细胞培养基上清，发现MMP-2表达随着OPN浓度的升高而增加(p=0.003和p=0.002)。用10μM重组人OPN蛋白刺激HCCLM3细胞，并用免疫荧光抗体标记p65蛋白。分别在45min、70min和2h三个不同时间点共聚焦显微镜下观察的p65蛋白在细胞内的位置变化。发现随着OPN蛋白作用时间的延长，p65蛋白逐渐从胞浆内转移至胞核，而p65只有在活化的状态才能从胞浆转移至胞核参与调节信号转导。而Western blot对不同时间点胞浆、胞核内p65蛋白表达的检测也证实了上述免疫荧光的观测结果。
上述结果显示Lenti.OPNi-2、Lenti.OPNi-3抑制HCCLM3细胞的侵袭转移可能与降低MMP-2和uPA的表达有关。Lenti.OPNi-2不影响HCCLM3细胞增殖，却能显著抑制HCCLM3侵袭转移。而Lenti.OPNi-3对HCCLM3细胞增殖和转移都具有显著抑制作用。这提示我们肿瘤细胞增殖和转移两种不同生物学行为对OPN蛋白的基础表达量可能存在不同量的要求。对于HCCLM3细胞，如果OPN蛋白表达量低于22％(1-78％)，侵袭转移将受限，而低于5％(1-95％)细胞增殖也受到抑制。可见侵袭转移较增殖对OPN蛋白表达下调更敏感，其详细机制有待探讨。另外，还发现对于HCCLM3细胞，OPN可能通过NF-κB信号途径调节MMP-2和uPA的表达。
二、OPN表达沉默对高转移潜能肝癌细胞成瘤和肺转移的影响
本部分的研究目的是采用裸鼠人肝癌肺转移模型进一步验证不同程度的抑制OPN表达对肝癌细胞HCCLM3的侵袭、增殖能力的影响。
我们分别将转染Mock、Lenti.OPNi-2、Lenti.OPNi-3的三组HCCLM3细胞(5×10~6／只)裸鼠皮下接种，6周后观察裸鼠的成瘤和肺转移。结果显示Lenti.OPNi-3组裸鼠成瘤率(50％)明显低于Mock对照组和Lenti.OPNi-2组(100％)，且肿瘤体积较小(Lenti.OPNi-3组为341±502.6mm~3，而Lenti.OPNi-2组为3686.9±2206.7mm~3，Mock组为4423.6±1206.7mm~3)(p＜0.001)。肺组织切片检测发现Mock、Lenti.OPNi-2、Lenti.OPNi-3三组肺转移率分别为100％、66.7％和50％，单个肺转移灶的数目分别为14±2.4个、3.5±3.4个和1.2±1.3个，差异有显著性(p＜0.01)；且肺转移灶都以Ⅰ期为主，Mock组和Lenti.OPNi-2组还可见到第Ⅳ期转移灶。采用活体荧光成像分析系统观察发现Lenti.OPNi-3组没有成瘤的裸鼠在原接种部位仍然存在发荧光的肿瘤细胞，说明肿瘤细胞增殖非常缓慢，无法成瘤并非是由于RNA干扰可能导致的非特异性细胞毒性作用。
我们通过对裸鼠成瘤大小和肺转移率的检测，再次证实了体外实验的观察结果，进一步说明不同程度的沉默OPN表达对HCCLM3细胞的增殖和侵袭两种生物学行为的影响是不一样的，侵袭转移比增殖对OPN基础表达量的下调更为敏感。
Hepatocellular carcinoma (HCC) is one of the most common and aggressivemalignancies worldwide, ranking the fifth most important cancer in terms of numbersof cases and the third in terms of cancer mortality. And it has become the secondcancer killer in China since 1990s. Unfortunately, despite efforts to improve itsprognosis, the overall survival of patients with HCC is still dismal. Metastaticrecurrence after HCC resection is one of the major obstacles to prolonging survival.Therefore, there is a tremendous interest and urgency to search for molecules relatedto HCC metastasis that would provide new predictors for HCC metastasis as well asnew targets for intervention.
Osteopontin (OPN), a secreted phosphoglycoprotein, is a promoting factor fortumorigenicity and metastasis of cancer cells in several types of tumors through avariety of mechanisms. OPN binds with several integrins and CD44 variants in anRGD sequence-dependent and-independent manner. In our previous work, wecollaborated with the National Cancer Insititute (NCI) of USA to compare thedifference in gene expression profiles between HCCs without and with accompanyingintra-hepatic metastases using a cDNA microarray of. 9180 cDNA clones. We foundthat OPN was one of the leading genes that over-expressed in the HCCs withmetastasis. An OPN-neutralizing antibody efficiently blocked in vitro invasion and invivo pulmonary metastasis of HCC cells. Moreover, we also found that preoperativeplasma OPN level can be used as a predictive marker for tumor recurrence andprognosis of patients with HCC after operation. These suggest that OPN is associatedwith metastasis of HCC. However, the mechanism by which OPN mediatesprogression and metastasis of HCC remains unknown. So, in this study, we tried tofind out what happened with the metastatic HCC cell line (HCCLM3) if its highendogenous OPN level was knocked down using lentiviral-mediated RNAinterference, and explore the possible mechanisms.
PARTⅠConstruction and selection of Lentiviral-mediated RNAiexpression vectors
The pcDNA ~(TM)6.2-GW/EmGFP-miR expression vectors contain adouble-stranded oligonucleotide (ds oligo) encoding a pre-miRNA sequence forexpression in mammalian cells using a RNA PolymeraseⅡ(PolⅡ) promoter, thehuman cytomegalovirus (CMV) immediate early promoter. The pDONR~(TM)221 vectoris used as an intermediate to transfer the pre-miRNA expression cassette into thelentiviral expression plasmid (pLenti6/V5-DEST) using Gateway Technology. Weperformed the Rapid BP/LR recombination reaction between pDONR~(TM)221,pcDNA~(TM)6.2-GW/EmGFP-miR and pLenti6/V5-DEST to generate the pLenti6/V5-GW/EmGFP-miR expression vectors. The sequence for construction of four OPN(GenBank accession number NM_000582) pre-miRNA and one control pre-miRNAwere as follows:
Packaging vector (9μg) (pLP1, plp2, pLP/VSVG), 3μg pLenti6/V5-GW/EmGFP-miR expression plasmid with OPNi pre-miRNA or control pre-miRNAwere cotransfected into 293FT packaging cells with Lipofectamine 2000 (36μl). After48 h the virus-containing culture supernatants were centrifuged to pellet the cell debris and stored in 1 ml aliquots at -80℃. Before proceeding to transduce theHCCLM3 cell line and express the miRNA for RNAi analysis, we determined the titerof lentiviral stock using EmGFP tittering method, calculated the EmGFP lentivirustiters from the dilutions at which the percentage of EmGFP-positive cells fall withinthe range of 1-30％. This was to avoid analyzing dilution samples containing multipleintegrated lentiviral genomes, which may result in an underestimate of the viral titer.Titer is expressed as transducing units (TU)/ml. The following formula was used tocalculate the titer: [F×C/V]×D(F=frequency of GFP-positive cells (percentageobtained divided by 100) C=total number of cells in the well at the time oftransduction V=volume of inoculum in ml D=lentivirus dilution). The lentiviraltiter for this study was 1×10~8 TU/ml. To obtain optimal expression of miRNA andtherefore, the highest degree of target gene knockdown, the lentiviral constructionwas transfected into mammalian cell line using a suitable MOI. MOI was defined asthe number of virus particles per cell and generally correlates with the number ofintegration events and as a result, expression. Typically, miRNA expression levelsincreased as increasing MOI. The MOI was determined by detecting the EmGFPfluorescent protein, and the suitable MOI was 30 for HCCLM3 cell line. HCCLM3cells were transfected with lentiviral vectors in the presence of polybrene (6μg/ml).After day 3, the medium was replaced with fresh, complete medium containg0.5mg/ml Blasticidin to select for stably transfected cells. Four stably transfected cells(Lenti.OPNi-1, Lenti.OPNi-2, Lenti.OPNi-4, Lenti.OPNi-3M) were obtained.Interestingly, only Lenti.OPNi-3 could inhibit the proliferation of HCCLM3significantly, and no Blasticidin-resistant colonies were identified. Real-time PCRassays showed that Lenti.OPNi-1, Lenti.OPNi-2, Lenti.OPNi-3, Lenti.OPNi-4 andLenti.OPNi-3M could suppress the OPN expression by 60％, 72％, 90％, 52％and14％, respectively. In addition, no significant induction of P-elF2αwas found, whichindicated that the inhibitory effect of RNAi was not through the activation ofIFN-response genes.
PARTⅡInhibitory effect of silencing OPN expression on the in vitroproliferation and invasive abilities of human HCC cells
1. Effect of OPN silencing and HCC proliferation
Of these miRNA lentiviral vectors, we selected Lenti.OPNi-2 and Lenti.OPNi-3which mediated efficient OPN suppression to evaluate effects of OPN knockdown onin vitro proliferation of HCCLM3 cells. Cell proliferation was measured by cellularuptake of MTT (3,-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide).Each assay was performed three times. Lenti.OPNi-3, but not Lenti.OPNi-2, couldinhibit the cell proliferatin significantly (p＜0.001). Lenti.OPNi-3 induced a significantdecrease in MEK and ERK1/2 phosphorylation (p＜0.001), while in cells transfectedwith Lenti.OPNi-3M there were no alteration in the level of OPN and in the activationof downstream pathways (p=0.482). In addition, Lenti.OPNi-3 did not induceaspecific downregulation of endogenous genes such as GAPDH and NF-κB. Incontrast, the proliferation rate of HepG2 without OPN overexpression was lesssensitive to the inhibitory effect of the OPN knockdown (p=0.216), and no changewas observed in the phosphorylation of MEK and ERK1/2 (p=0.127). Theconstitutively low-level expression of the OPN in the HepG2 cells may explain thereason for the HepG2 resistance to the OPN RNAi. Similar result was observed inCCL13 cells. Thus, lentiviral mediated OPN knockdown does not appear to affectcells with low OPN expression. To further investigate whether OPN inducesphosphorylation of MEK and then phosphorylation of ERK1/2 in HCCLM3 cells, thecells transfected with Lenti.OPNi-3 were treated with 10μM exogenous recombinantOPN, pretreated with specific MEK inhibitor U0126 (20μM), or treated with OPNalone. Cell lysates were analyzed by Western blot at different time points. The dataindicated that MEK and ERK were readily activated in OPN-stimulated cells, peakingat 20 minutes, but not in the cells treated with U0126.
2. Effect of OPN silencing and tumor invasion
We evaluated whether the suppression of OPN expression would alter the in vitrometastatic phenotype of HCCLM3 cells. Cell invasion was measured using Matrigelcoated transwell-invasion chambers. Migrating numbers of HCC-LM3 cells wassignificantly inhibited by Lenti.OPNi-2 (9.2±1.3) and Lenti.OPNi-3 (7.8±1.5),compared with Mock (28.84±2.6) (p＜0.001). No significant difference in invasivenesswas observed between Mock and Lenti.OPNi-3M (p=0.221). Parallel experimentswere performed in triplicate and each filter was photographed at 200×magnification.Next, we evaluated the protein expression of several metastatic markers includingMMP-2, MMP-9 and uPA, which have been previously shown to be associated withOPN in models of breast cancer and melanoma. Interestingly, MMP-2 and uPA expression but not MMP-9 were decreased significantly in Lenti.OPNi-2 andLenti.OPNi-3(p＜0.001). Previous studies have shown that OPN stimulates activationof pro-MMP-2 through NF-κB in murine melanoma cells. To further determinewhether OPN have the same effect on translocation of p65 into the nucleus inHCCLM3 cell, the cells were treated with 10μM OPN in basal medium for 0-2h at37℃. Immunofluorescence analysis of NF-κB was performed at different time points.The data showed that OPN induces translocation of p65 into nucleus in atime-dependent manner. In the cells pretreated with OPN, the majority of p65 stainingresided in the cytoplasm upto 30 min. At 45 min and 70 min some nucleartranslocation of p65 were observed, peaking at 2h. Western blotting analysis gave afurther confirmation of translocation of p65 into the nucleus. In the OPN-untreatedcells, the p65 was localized mostly in the cytoplasm compared with the nucleus,whereas in the OPN-treated cells, it was translocated into the nucleus. In order tocheck whether OPN induced pro-MMP-2 production and activation in these cells, theconditioned medium was collected, and MMP-2 activity was analyzed by gelatinzymography. The levels of both the pro and active forms of MMP-2 in theOPN-treated cells were significantly higher compared with the levels of MMP-2 inOPN-untreated cells(p＜0.01).
Inhibitory effect of silencing OPN expression on the in vivotumor growth and lung metastasis of human HCC cells
The in vitro study supported that the hypothesis that OPN contributed totumorigenicity and metastatic capacity of HCCLM3 cells. To directly confirm this invivo, HCCLM3 cells (5×10~6) were transfected with or without the lentiviral vectors,and then subcutaneously inoculated into nude mice. Tumor growth was monitored. At2 week after the injection, the tumors originating from the cells transfected withLenti.OPNi-2 and PBS were measurable. At 6 weeks, the animals were killed forethical reasons given the tumor volume, and then the tumors were excised from mice.HCCLM3 cells mock-transfected (PBS) or transfected with Lenti.OPNi-2 developedlarge tumors 3500mm~3 within 6 weeks. However, growth of HCCLM3 cellstransfected with Lenti.OPNi-3 was markedly suppressed (p＜0.001), suggesting thatknockdown of OPN leads to significant inhibition of tumor growth in vivo.
In the sixth week, pulmonary metastatic lesions were detected in every mouse inPBS group, with mainly gradeⅠ-Ⅱand some gradeⅢ-Ⅳtumor clusters. The mice ofPBS group had an average of 14+2.4 tumor clusters per lung. However, Only 50% miceinfected with the Lenti.OPNi-3-treated cells developed lung metastases with mostly gradeⅠtumorclusters with a combined average of 1.2±1.3 tumor clusters per lung. The effect wasstatistically significant(p＜0.01). The same significant effect was also observed in themice infected with the Lenti.OPNi-2-treated cells(p＜0.01). We also monitored HCCLM3cells growth and metastasis with bioluminescence imaging every week. Interestingly,EmGFP fluorescent protein can be detected in the mice with no measurable tumormass, which suggests the proliferation of HCCLM3 cells transfected withLenti.OPNi-3 was inhibited significantly.
Conclusions
1. We constructed two lentiviral vectors (Lenti.OPNi-2 and Lenti.OPNi-3) whichcould significantly suppress the expression of OPN, and selected two stablytransfected HCCLM3 cell lines (LM3- Lenti.OPNi-2 and LM3- Lenti.OPNi-3M).
2. Osteopontin could promote HCCLM3 cell proliferation through activation ofMEK/MAPK signaling pathways, and little basal expression of OPN is requiredfor HCCLM3 proliferation. However, the cells with constitutively low-levelexpression of OPN (HepG2 and CCL13) were less sensitive to the inhibitoryeffect of the OPN knockdown.
3. Osteopontin stimulates HCCLM3 cell invasion through activation of NF-κB,MMP-2 and uPA. Both Lenti.OPNi-2 and Lenti.OPNi-3 can inhibit pulmonarymetastases of HCCLM3 cells significantly which suggest more basal expressionof OPN is required for HCCLM3 invasion than for HCCLM3 proliferation.
Potential application of this study
1. Long lasting RNAi-based gene knockdown can be achieved by lentiviral basedexpression systems driving the production of short hairpin RNA species (microRNA).Lentiviral-mediated gene silencing is a good way to assess one gene function.
2. Lenti.OPNi-3 significantly inhibits proliferation and invasion of HCCLM3 withoverexpression of OPN, but does not appear to affect CCL13 with low OPN expression.Maybe OPN will become a new therapeutic target for highly metastatic HCC, and Lentiviral vectors provide some insight into vector tools of the bilolgical targettherapy in liver cancer.
Novelties
1. To the best of our knowledge, this is the first report showing that OPN expressionis critical for tumorigenesis and proliferatin of human hepatocellular carcinomacells since knocking down the expression abolishes tumorigenicity.
2. In addition, our data for the first time show that different biological behaviors ofHCC have a different requirement for the basal expression level of OPN.
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