NDRG2在热应激抗肝癌细胞侵袭中的作用及相关机制研究
摘要
原发性肝癌主要以肝细胞癌（Hepatocellular carcinoma，HCC）发生为主，为常见的恶性肿瘤之一，并且在世界各地发病率每年呈明显上升趋势，在我国其死亡率在消化系统恶性肿瘤中排第二位。由于肝癌常缺乏典型的临床表现，早期无明显症状，多数患者确诊时往往已处于晚期，出现肿瘤的远处转移。目前针对肝癌的主要治疗手段是对原发肿瘤的外科手术切除。肝癌一旦发生其他脏器的转移则明显影响预后，术后的生存率也随之降低。在肝癌导致死亡的病例中，肿瘤引发的侵袭、转移和术后复发是致死的主要原因。因此，寻找一种有效，安全的控制肝癌细胞侵袭、转移的治疗方法，对于降低肝癌的死亡率具有重要的意义。
肿瘤热疗是一种依靠物理能量使人体全身或局部加热，利用正常组织和肿瘤组织对温度耐受能力的差异，达到既能杀伤肿瘤细胞又不损伤正常细胞的治疗方法。研究发现，它不仅可以对肿瘤细胞产生直接或间接的杀伤致死效应，它还可干涉肿瘤转移的多个环节从而抑制肿瘤的侵袭和转移。尽管目前在热疗抗肿瘤方面的研究方面已取得了重大进展，但由于欠缺充分、具体的作用机制，所以影响了它在临床实践中的深入应用。
人N-Myc下游调节基因-2（N-Myc downstream regulated gene2，NDRG2）属于NDRG家族，是1999年由我校生化教研室首先利用削减杂交技术在人正常脑组织中克隆出的一个新基因（GenBank登录号： AF159092）。研究发现，它在肿瘤组织中呈现表达降低或不表达状态；因其具有抑制肿瘤细胞的增殖；促进肿瘤细胞的凋亡；抑制肿瘤细胞转移和侵袭等作用，由此可说明该基因为一抑癌基因。我们的研究还证实，NDRG2可参与到多种应激反应的过程中。那么，NDRG2是否在热应激抗肝癌细胞侵袭中发挥作用呢？我们能否从NDRG2入手去调控热疗抗肝癌细胞侵袭的信号通路呢？带着这样的疑问，我们进行了NDRG2在热疗引发的热应激抗肝癌细胞侵袭作用中的功能及其机制研究。
研究方法和结果：
1.研究对比了不同温度热作用对肝癌细胞迁移能力及体外侵袭能力的影响：将肝癌HepG-2和Huh-7细胞分别置于37C、39C、41C、43C、45C水浴中热作用30min后，通过细胞划痕实验检测细胞的迁移能力；侵袭小室（Transwell）实验检测细胞体外侵袭能力。结果显示：与37C (对照组)相比，41C、43C、45C组HepG-2和Huh-7细胞的迁移和侵袭能力均有不同程度的降低，其中45C组细胞迁移和侵袭能力下降尤其明显(P<0.05)。
2.研究不同温度热作用对肝癌细胞内NDRG2、MMP-2及MMP-9蛋白表达量的影响：将肝癌HepG-2和Huh-7细胞采用水浴加热的方式行37C、39C、41C、43C、45C热作用30min后，通过Western-blot实验检测NDRG2、MMP-2及MMP-9蛋白表达量。结果：与37C (对照组)相比，经45C热作用后肝癌HepG-2和Huh-7细胞的MMP-2及MMP-9分泌量明显下降，同时伴随细胞内NDRG2的表达量明显上升。
3.分析NDRG2在热应激抗肝癌细胞侵袭中的生物学功能：
（1）构建了NDRG2过表达和shRNA干涉的慢病毒载体，包装慢病毒并进行病毒滴度测定，感染肝癌HepG-2细胞后，利用载体携带的筛选标记进行稳定细胞株的筛选，最后采用Western-blot方法鉴定慢病毒稳转后NDRG2蛋白在细胞中的表达差异。（2）应用Transwell方法和Western-blot方法检测了对照组，单热处理组，NDRG2过表达组，及NDRG2过表达联合热处理组之间细胞侵袭力和细胞内NDRG2、MMP-2、MMP-9的表达情况。我们的研究结果发现：与对照组相比，给予NDRG2过表达的HepG-2细胞株45C、30min热处理后，细胞内NDRG2的表达明显增高，同时伴随细胞侵袭力、MMP-2和MMP-9的表达明显降低。（3）建立裸鼠荷瘤模型，并给予移植瘤局部45C的热作用，之后通过HE染色及免疫组化等实验技术观察肝癌细胞在裸鼠体内侵袭力及相应的NDRG2、MMP-2及MMP-9蛋白表达变化情况。结果显示，与对照组相比，45C热作用能有效抑制肿瘤对周围肌肉组织的侵袭作用，而干涉肿瘤细胞内NDRG2的表达则降低了热对肿瘤细胞侵袭的抑制作用。
4. NDRG2在热疗抗肝癌细胞侵袭中的分子机制研究：
（1）Western-blot方法检测了45C，30min热作用后12h内在空载体对照和NDRG2过表达的HepG-2细胞间HSP90，HSP70和HSP27蛋白的表达变化情况。结果显示，热作用后的12h内HSP27和HSP90在两组之间的各时间点均未见显著变化，而HSP70在热作用后6h表达量开始升高，但在两个组之间没有显著差异。（2）Western-blot方法检测了45C，30min热作用后12h内在空载体对照和NDRG2过表达的HepG-2细胞间MAPK通路中ERK1/2、JNK、p38MAPK总蛋白和磷酸化的表达变化情况。通过对实验结果的分析，我们发现对照组的HepG-2细胞在给予热处理后的2h之内，ERK1/2的磷酸化水平开始增加，在热处理后的4h又迅速的回降到基础水平，甚至到后面的时间磷酸化水平降得更低；可是NDRG2的过表达不仅降低了HepG-2细胞中ERK1/2的本底水平，还降低了热作用早期对ERK1/2的诱导；然而，在以上的整个过程中JNK和p38MAPK总蛋白和磷酸化的表达水平在对照和NDRG2过表达的HepG-2细胞间并没有发生显著性变化。（3）为了进一步验证上一实验结果，我们分别应用ERK1/2，p38MAPK和JNK三个激酶的抑制剂PD98059、SB203580和SP600125作用于NDRG2被敲除的HepG-2细胞，经过45C，30min热处理后，结果发现也只有加了PD98059（ERK1/2的抑制剂）组可以明显抑制肝癌细胞的侵袭和MMP-2、MMP-9蛋白的表达。
5.比对NDRG2联合热应激对肝癌细胞治疗的效果：
（1）应用Annexin V联合PI染色法检测37C，43C和45C热处理后的细胞凋亡情况，结果显示，空载体对照的HepG-2细胞的凋亡率会随着加热温度的增加而增加，于此同时伴随着坏死率的增加。然而，给予NDRG2过表达的HepG-2细胞43C的热处理，细胞的凋亡率可高达45C热作用于空载体对照组的水平，而细胞坏死率却没有随之增加。
（2）我们近一步应用Transwell法和Western-blot法检测了37C，43C和45C热处理后的细胞侵袭情况，空载体对照的HepG-2细胞的侵袭能力和MMP-2、MMP-9蛋白的表达会随着温度的增加而降低，其中在45C时这种变化最为显著。然而在给予NDRG2过表达的HepG-2细胞43C的热作用时，细胞的侵袭能力和MMP-2、 MMP-9蛋白的表达呈现显著降低的变化，并可达到空载体对照组45C的水平。
结论：
1.45C的热作用可明显抑制肝癌细胞的迁移和侵袭能力。
2. NDRG2参与了热应激抑制肝癌细胞侵袭的作用，并且热应激所诱导NDRG2的表达量变化与肝癌细胞的侵袭和转移能力呈现一种负相关性。
3.在热诱导NDRG2抑制肝癌细胞侵袭、转移能力的作用中，是通过抑制ERK1/2磷酸化的分子机制而实现的。
4.43C热联合NDRG2可对肝癌细胞产生较高的促凋亡和抗侵袭的作用效果。
Hepatocellular carcinoma (HCC) is one of the most frequent malignancies worldwide,accounting for85%to90%of primary liver cancers. However, HCC is difficult todiagnosis and has a rapid progress of the course due to lack of the typical clinicalmanifestations, classical symptoms in early stage, and mostly with serious liver cirrhosis.Surgery is the mainly treatment in early stage. The disease course is usually late whendiagnosed, so the patients have lost the optimal time of surgery. At the meanwhile, theHCC is easy to recurrence and distant metastasis even though the tumor has been removed.Invasion and metastasis are the most important biologic characteristics for malignantcarcinoma, whiel chiefly accounts for clinic death of cancer patients. Once it hasprogressed to the metastatic stage that is extremely difficult to treat and does not respondto non-operative treatment. Therefore, the inhibition of invasion and metastasis has been the key factor for the successful treatment of HCC.
Hyperthermia, a minimally invasive treatment with few side effects, has recentlybeen used for cancer therapy. A number of clinical and animal experiments have shownthat HT is a tolerable and clinically practical supplementary therapy for patients withadvanced malignant tumor, recurrent tumor and metastatic disease. HT exerts therapeuticeffects not only by delaying tumor growth but also by inhibiting metastasis. Althoughsome progress has been made in terms of assessing the biological effect of HT, themolecular mechanism that mediates the anti-metastatic effect of HT has not beenelucidated.
Human NDRG2gene was named as N-Myc downstream regulated gene2. It belongsto the NDRG family. NDRG2was first discovered and cloned from normal human braintissue by our laboratory in1999(Syld GenBank AF159092). It was indicated by someprevious researches that NDRG2appeared low expression in some tumors. The ectopicexpression of NDRG2suppresses the proliferation of tumor cells. In addition,accumulated evidence indicates that the absence of NDRG2expression in a variety ofcarcinomas contributes to increased tumor metastatic potential. All of these findingssuggest that NDRG2has tumor suppressor role. In addition, increasingly more effortshave aimed to determine the role of NDRG2under stress conditions.
At present, there was no report about relationship between HT and NDRG2oninvasion of HCC cells, so we decided to observe the changes of NDRG2in HCC cellsafter HT. So our research mainly sought to clarify the biological role of NDRG2ininvasion of HCC under HT. Understanding the molecular mechanism involved in HT mayhave valuable implications for developing optimized HT therapies for some NDRG2deficient cancers.
Methods and Results：
1. To assess the effects of anti-migration and anti-invasive of HT at different temperaturein HCC cells.
We examined anti-migration and anti-invasive effect of HT on HepG-2andHuh-7cells by subjecting them to water bath at different temperatures (37C,39C,41C,43C and45C) for30min. Cell wound healing assay and Matrigel invasionassay were performed to evaluate the effects of HT on migration and invasion of cells, respectively. The migration and invasion of cells were affected by a heat treatment.Compared with control group (37C), heat treated at45C significantly reduced thepercentage invaded cells in HepG-2and Huh-7, respectively.
2. To assess the expresses of MMP-2/9and NDRG2of HCC cells at differenttemperature of HT.To further confirm the anti-invasion effect of HT, we examined the expression ofNDRG2、MMP-2and MMP-9in HepG-2and Huh-7cells by subjecting them to waterbath at different temperatures (37C,39C,41C,43C and45C) for30min. Theexpression of MMP-2and MMP-9was significantly reduced by HT at45C.Meanwhile, Western-blot showed that expression of NDRG2was increased by heatshock at45C in both HepG-2and Huh-7cells.
3. To investigate the biological role of NDRG2in the invasion of HCC cells exposed toHT.
(1) We constructed the lentivirus vectors of NDRG2overexpression orNDRG2-shRNA to produce the lentiviral particles, and then infected the HepG-2cells. With long term selection of blasticidin or puromycin respectively, weestablished the stable cell lines with NDRG2overexpression or NDRG2knockdown.
(2) Matrigel invasion assay and Western-blot were performed to evaluate the effect ofHT on invasion of cells. In NDRG2overexpressing cells, HT at45C reduced theinvasive potential significantly, compared with HepG-2cells treated by HT alone.We next detected the expression of MMP-2and MMP-9. Western-blot revealed thatexpression of MMP-2and MMP-9was decreased in NDRG2overexpressing HepG-2cells and even lower after the cells were treated by HT.
(3) We further examined the anti-invasion potential of NDRG2in HCC-implantedmice. NDRG2dificient HepG-2cells were injected into nude mice. H&E staining ofhistological sections from mouse xenograft model revealed that HT suppressed theinvasion ability of HepG-2cells significantly. Malignant tumors slightly invaded intonearby tissues in HT treated mouse model. In contrast，suppression of NDRG2facilitate invasion of tumor nodules and reversed anti-invasive effect of HT inHepG-2cells with significant destruction of the muscle layer. The expression of bothMMP-2and MMP-9was also detected in tissues sections，and a representativeimmunohistochemical staining was presented. Consistent with H&E staining results， we found that down-regulation of NDRG2alleviated the repression of MMP-2andMMP-9expression by HT.
4. To elucidate the molecular mechanism of NDRG2mediated the anti-invasion effect ofHT.
(1) We examined the expression profiles of HSP27, HSP70and HSP90in HepG2cells of control and overexpression of NDRG2at different time points after cellswere subjected to HT at45C for30min. There was no change in expression levelsof HSP27and HSP90after HT between these two groups. HSP70expression wasinduced at6h and kept at high levels through12h after HT. However, the expressionpattern of HSP70was the same between these two groups.
(2) We investigated the effect of NDRG2on the downstream activation of the MAPKby detecting phosphorylated p38MAPK, ERK1/2and JNK which were induced byHT. The phosphorylation of ERK1/2increased up within2h, then declined to thebasal level rapidly4h after heat shock treatment, and even lower later. Moreover,overexpression of NDRG2abrogated the intrinsic and HT induced activation ofERK1/2pathway. HT was found to increase activation of p38MAPK in a timedependent manner, while the phosphorylation level of p38MAPK remainedunchanged in NDRG2overexpressing cells.
(3) To further confirm the role of NDRG2on ERK1/2activation, lentivirus-mediatedshRNA was employed to downregulate endogenous NDRG2expression in HepG-2cells. NDRG2deficient cells were treated with the ERK1/2inhibitor, PD98059,p38MAPK inhibitor, SB203580or JNK inhibitor, SP600125, respectively. Silence ofNDRG2expression significantly increased the invasion ability of HT treated cells.Meanwhile, PD98059treatment could significantly enhance the anti-invasive effectof HT and decrease MMP-2/MMP-9expression in NDRG2deficient HepG-2cells.These findings indicated that ERK1/2activation could be selectively inhibited byNDRG2expression in HepG-2cells.
5. To demonstrate the therapeutic effect of NDRG2combined with HT against HCC.
(1) We examined the type of cell death induced by HT at different temperature (37C,43C and45C) for30min, using double staining with Annexin V and PI. HepG-2cells treated at45C showed significantly higher rate of cell apoptosis, compared withthose treated at43C. HT at45C caused massive increase in number of necrotic cells in comparison with cells treated at43C. Intriguingly, we found that combination ofNDRG2expression and HT at43C resulted in increased level of apoptosis andreduced necrosis, compared with treatment of HT at45C.
(2) We examined anti-invasive effect of HT on HepG-2by subjecting them to waterbath at different temperatures (37C,43C and45C) for30min. Matrigel invasionassay were performed to evaluate the effects of HT on invasion of cells. The cellstreated at45C showed significantly lower percentage of invaded cells, comparedwith those treated at43C. The combined treatment of HT at43C and NDRG2expression decreased expression of MMP-2and MMP-9as well as invaded cellpercentage.
Conclusion：
1. HT at45C could inhibit migration and invasion of HCC cells.
2. NDRG2involves in HT inhibiting HCC cell invasion ability, moreover theexpression of NDRG2and cell invasion ability are negative correlation.
3. NDRG2mediates the anti-invasion effect of HT via inhibition of the ERK1/2signaling pathway.
4. NDRG2synergizes with HT to inhibit invasiveness of HCC cells and decreasespontaneous necrosis.
肿瘤热疗是一种依靠物理能量使人体全身或局部加热，利用正常组织和肿瘤组织对温度耐受能力的差异，达到既能杀伤肿瘤细胞又不损伤正常细胞的治疗方法。研究发现，它不仅可以对肿瘤细胞产生直接或间接的杀伤致死效应，它还可干涉肿瘤转移的多个环节从而抑制肿瘤的侵袭和转移。尽管目前在热疗抗肿瘤方面的研究方面已取得了重大进展，但由于欠缺充分、具体的作用机制，所以影响了它在临床实践中的深入应用。
人N-Myc下游调节基因-2（N-Myc downstream regulated gene2，NDRG2）属于NDRG家族，是1999年由我校生化教研室首先利用削减杂交技术在人正常脑组织中克隆出的一个新基因（GenBank登录号： AF159092）。研究发现，它在肿瘤组织中呈现表达降低或不表达状态；因其具有抑制肿瘤细胞的增殖；促进肿瘤细胞的凋亡；抑制肿瘤细胞转移和侵袭等作用，由此可说明该基因为一抑癌基因。我们的研究还证实，NDRG2可参与到多种应激反应的过程中。那么，NDRG2是否在热应激抗肝癌细胞侵袭中发挥作用呢？我们能否从NDRG2入手去调控热疗抗肝癌细胞侵袭的信号通路呢？带着这样的疑问，我们进行了NDRG2在热疗引发的热应激抗肝癌细胞侵袭作用中的功能及其机制研究。
研究方法和结果：
1.研究对比了不同温度热作用对肝癌细胞迁移能力及体外侵袭能力的影响：将肝癌HepG-2和Huh-7细胞分别置于37C、39C、41C、43C、45C水浴中热作用30min后，通过细胞划痕实验检测细胞的迁移能力；侵袭小室（Transwell）实验检测细胞体外侵袭能力。结果显示：与37C (对照组)相比，41C、43C、45C组HepG-2和Huh-7细胞的迁移和侵袭能力均有不同程度的降低，其中45C组细胞迁移和侵袭能力下降尤其明显(P<0.05)。
2.研究不同温度热作用对肝癌细胞内NDRG2、MMP-2及MMP-9蛋白表达量的影响：将肝癌HepG-2和Huh-7细胞采用水浴加热的方式行37C、39C、41C、43C、45C热作用30min后，通过Western-blot实验检测NDRG2、MMP-2及MMP-9蛋白表达量。结果：与37C (对照组)相比，经45C热作用后肝癌HepG-2和Huh-7细胞的MMP-2及MMP-9分泌量明显下降，同时伴随细胞内NDRG2的表达量明显上升。
3.分析NDRG2在热应激抗肝癌细胞侵袭中的生物学功能：
（1）构建了NDRG2过表达和shRNA干涉的慢病毒载体，包装慢病毒并进行病毒滴度测定，感染肝癌HepG-2细胞后，利用载体携带的筛选标记进行稳定细胞株的筛选，最后采用Western-blot方法鉴定慢病毒稳转后NDRG2蛋白在细胞中的表达差异。（2）应用Transwell方法和Western-blot方法检测了对照组，单热处理组，NDRG2过表达组，及NDRG2过表达联合热处理组之间细胞侵袭力和细胞内NDRG2、MMP-2、MMP-9的表达情况。我们的研究结果发现：与对照组相比，给予NDRG2过表达的HepG-2细胞株45C、30min热处理后，细胞内NDRG2的表达明显增高，同时伴随细胞侵袭力、MMP-2和MMP-9的表达明显降低。（3）建立裸鼠荷瘤模型，并给予移植瘤局部45C的热作用，之后通过HE染色及免疫组化等实验技术观察肝癌细胞在裸鼠体内侵袭力及相应的NDRG2、MMP-2及MMP-9蛋白表达变化情况。结果显示，与对照组相比，45C热作用能有效抑制肿瘤对周围肌肉组织的侵袭作用，而干涉肿瘤细胞内NDRG2的表达则降低了热对肿瘤细胞侵袭的抑制作用。
4. NDRG2在热疗抗肝癌细胞侵袭中的分子机制研究：
（1）Western-blot方法检测了45C，30min热作用后12h内在空载体对照和NDRG2过表达的HepG-2细胞间HSP90，HSP70和HSP27蛋白的表达变化情况。结果显示，热作用后的12h内HSP27和HSP90在两组之间的各时间点均未见显著变化，而HSP70在热作用后6h表达量开始升高，但在两个组之间没有显著差异。（2）Western-blot方法检测了45C，30min热作用后12h内在空载体对照和NDRG2过表达的HepG-2细胞间MAPK通路中ERK1/2、JNK、p38MAPK总蛋白和磷酸化的表达变化情况。通过对实验结果的分析，我们发现对照组的HepG-2细胞在给予热处理后的2h之内，ERK1/2的磷酸化水平开始增加，在热处理后的4h又迅速的回降到基础水平，甚至到后面的时间磷酸化水平降得更低；可是NDRG2的过表达不仅降低了HepG-2细胞中ERK1/2的本底水平，还降低了热作用早期对ERK1/2的诱导；然而，在以上的整个过程中JNK和p38MAPK总蛋白和磷酸化的表达水平在对照和NDRG2过表达的HepG-2细胞间并没有发生显著性变化。（3）为了进一步验证上一实验结果，我们分别应用ERK1/2，p38MAPK和JNK三个激酶的抑制剂PD98059、SB203580和SP600125作用于NDRG2被敲除的HepG-2细胞，经过45C，30min热处理后，结果发现也只有加了PD98059（ERK1/2的抑制剂）组可以明显抑制肝癌细胞的侵袭和MMP-2、MMP-9蛋白的表达。
5.比对NDRG2联合热应激对肝癌细胞治疗的效果：
（1）应用Annexin V联合PI染色法检测37C，43C和45C热处理后的细胞凋亡情况，结果显示，空载体对照的HepG-2细胞的凋亡率会随着加热温度的增加而增加，于此同时伴随着坏死率的增加。然而，给予NDRG2过表达的HepG-2细胞43C的热处理，细胞的凋亡率可高达45C热作用于空载体对照组的水平，而细胞坏死率却没有随之增加。
（2）我们近一步应用Transwell法和Western-blot法检测了37C，43C和45C热处理后的细胞侵袭情况，空载体对照的HepG-2细胞的侵袭能力和MMP-2、MMP-9蛋白的表达会随着温度的增加而降低，其中在45C时这种变化最为显著。然而在给予NDRG2过表达的HepG-2细胞43C的热作用时，细胞的侵袭能力和MMP-2、 MMP-9蛋白的表达呈现显著降低的变化，并可达到空载体对照组45C的水平。
结论：
1.45C的热作用可明显抑制肝癌细胞的迁移和侵袭能力。
2. NDRG2参与了热应激抑制肝癌细胞侵袭的作用，并且热应激所诱导NDRG2的表达量变化与肝癌细胞的侵袭和转移能力呈现一种负相关性。
3.在热诱导NDRG2抑制肝癌细胞侵袭、转移能力的作用中，是通过抑制ERK1/2磷酸化的分子机制而实现的。
4.43C热联合NDRG2可对肝癌细胞产生较高的促凋亡和抗侵袭的作用效果。
Hepatocellular carcinoma (HCC) is one of the most frequent malignancies worldwide,accounting for85%to90%of primary liver cancers. However, HCC is difficult todiagnosis and has a rapid progress of the course due to lack of the typical clinicalmanifestations, classical symptoms in early stage, and mostly with serious liver cirrhosis.Surgery is the mainly treatment in early stage. The disease course is usually late whendiagnosed, so the patients have lost the optimal time of surgery. At the meanwhile, theHCC is easy to recurrence and distant metastasis even though the tumor has been removed.Invasion and metastasis are the most important biologic characteristics for malignantcarcinoma, whiel chiefly accounts for clinic death of cancer patients. Once it hasprogressed to the metastatic stage that is extremely difficult to treat and does not respondto non-operative treatment. Therefore, the inhibition of invasion and metastasis has been the key factor for the successful treatment of HCC.
Hyperthermia, a minimally invasive treatment with few side effects, has recentlybeen used for cancer therapy. A number of clinical and animal experiments have shownthat HT is a tolerable and clinically practical supplementary therapy for patients withadvanced malignant tumor, recurrent tumor and metastatic disease. HT exerts therapeuticeffects not only by delaying tumor growth but also by inhibiting metastasis. Althoughsome progress has been made in terms of assessing the biological effect of HT, themolecular mechanism that mediates the anti-metastatic effect of HT has not beenelucidated.
Human NDRG2gene was named as N-Myc downstream regulated gene2. It belongsto the NDRG family. NDRG2was first discovered and cloned from normal human braintissue by our laboratory in1999(Syld GenBank AF159092). It was indicated by someprevious researches that NDRG2appeared low expression in some tumors. The ectopicexpression of NDRG2suppresses the proliferation of tumor cells. In addition,accumulated evidence indicates that the absence of NDRG2expression in a variety ofcarcinomas contributes to increased tumor metastatic potential. All of these findingssuggest that NDRG2has tumor suppressor role. In addition, increasingly more effortshave aimed to determine the role of NDRG2under stress conditions.
At present, there was no report about relationship between HT and NDRG2oninvasion of HCC cells, so we decided to observe the changes of NDRG2in HCC cellsafter HT. So our research mainly sought to clarify the biological role of NDRG2ininvasion of HCC under HT. Understanding the molecular mechanism involved in HT mayhave valuable implications for developing optimized HT therapies for some NDRG2deficient cancers.
Methods and Results：
1. To assess the effects of anti-migration and anti-invasive of HT at different temperaturein HCC cells.
We examined anti-migration and anti-invasive effect of HT on HepG-2andHuh-7cells by subjecting them to water bath at different temperatures (37C,39C,41C,43C and45C) for30min. Cell wound healing assay and Matrigel invasionassay were performed to evaluate the effects of HT on migration and invasion of cells, respectively. The migration and invasion of cells were affected by a heat treatment.Compared with control group (37C), heat treated at45C significantly reduced thepercentage invaded cells in HepG-2and Huh-7, respectively.
2. To assess the expresses of MMP-2/9and NDRG2of HCC cells at differenttemperature of HT.To further confirm the anti-invasion effect of HT, we examined the expression ofNDRG2、MMP-2and MMP-9in HepG-2and Huh-7cells by subjecting them to waterbath at different temperatures (37C,39C,41C,43C and45C) for30min. Theexpression of MMP-2and MMP-9was significantly reduced by HT at45C.Meanwhile, Western-blot showed that expression of NDRG2was increased by heatshock at45C in both HepG-2and Huh-7cells.
3. To investigate the biological role of NDRG2in the invasion of HCC cells exposed toHT.
(1) We constructed the lentivirus vectors of NDRG2overexpression orNDRG2-shRNA to produce the lentiviral particles, and then infected the HepG-2cells. With long term selection of blasticidin or puromycin respectively, weestablished the stable cell lines with NDRG2overexpression or NDRG2knockdown.
(2) Matrigel invasion assay and Western-blot were performed to evaluate the effect ofHT on invasion of cells. In NDRG2overexpressing cells, HT at45C reduced theinvasive potential significantly, compared with HepG-2cells treated by HT alone.We next detected the expression of MMP-2and MMP-9. Western-blot revealed thatexpression of MMP-2and MMP-9was decreased in NDRG2overexpressing HepG-2cells and even lower after the cells were treated by HT.
(3) We further examined the anti-invasion potential of NDRG2in HCC-implantedmice. NDRG2dificient HepG-2cells were injected into nude mice. H&E staining ofhistological sections from mouse xenograft model revealed that HT suppressed theinvasion ability of HepG-2cells significantly. Malignant tumors slightly invaded intonearby tissues in HT treated mouse model. In contrast，suppression of NDRG2facilitate invasion of tumor nodules and reversed anti-invasive effect of HT inHepG-2cells with significant destruction of the muscle layer. The expression of bothMMP-2and MMP-9was also detected in tissues sections，and a representativeimmunohistochemical staining was presented. Consistent with H&E staining results， we found that down-regulation of NDRG2alleviated the repression of MMP-2andMMP-9expression by HT.
4. To elucidate the molecular mechanism of NDRG2mediated the anti-invasion effect ofHT.
(1) We examined the expression profiles of HSP27, HSP70and HSP90in HepG2cells of control and overexpression of NDRG2at different time points after cellswere subjected to HT at45C for30min. There was no change in expression levelsof HSP27and HSP90after HT between these two groups. HSP70expression wasinduced at6h and kept at high levels through12h after HT. However, the expressionpattern of HSP70was the same between these two groups.
(2) We investigated the effect of NDRG2on the downstream activation of the MAPKby detecting phosphorylated p38MAPK, ERK1/2and JNK which were induced byHT. The phosphorylation of ERK1/2increased up within2h, then declined to thebasal level rapidly4h after heat shock treatment, and even lower later. Moreover,overexpression of NDRG2abrogated the intrinsic and HT induced activation ofERK1/2pathway. HT was found to increase activation of p38MAPK in a timedependent manner, while the phosphorylation level of p38MAPK remainedunchanged in NDRG2overexpressing cells.
(3) To further confirm the role of NDRG2on ERK1/2activation, lentivirus-mediatedshRNA was employed to downregulate endogenous NDRG2expression in HepG-2cells. NDRG2deficient cells were treated with the ERK1/2inhibitor, PD98059,p38MAPK inhibitor, SB203580or JNK inhibitor, SP600125, respectively. Silence ofNDRG2expression significantly increased the invasion ability of HT treated cells.Meanwhile, PD98059treatment could significantly enhance the anti-invasive effectof HT and decrease MMP-2/MMP-9expression in NDRG2deficient HepG-2cells.These findings indicated that ERK1/2activation could be selectively inhibited byNDRG2expression in HepG-2cells.
5. To demonstrate the therapeutic effect of NDRG2combined with HT against HCC.
(1) We examined the type of cell death induced by HT at different temperature (37C,43C and45C) for30min, using double staining with Annexin V and PI. HepG-2cells treated at45C showed significantly higher rate of cell apoptosis, compared withthose treated at43C. HT at45C caused massive increase in number of necrotic cells in comparison with cells treated at43C. Intriguingly, we found that combination ofNDRG2expression and HT at43C resulted in increased level of apoptosis andreduced necrosis, compared with treatment of HT at45C.
(2) We examined anti-invasive effect of HT on HepG-2by subjecting them to waterbath at different temperatures (37C,43C and45C) for30min. Matrigel invasionassay were performed to evaluate the effects of HT on invasion of cells. The cellstreated at45C showed significantly lower percentage of invaded cells, comparedwith those treated at43C. The combined treatment of HT at43C and NDRG2expression decreased expression of MMP-2and MMP-9as well as invaded cellpercentage.
Conclusion：
1. HT at45C could inhibit migration and invasion of HCC cells.
2. NDRG2involves in HT inhibiting HCC cell invasion ability, moreover theexpression of NDRG2and cell invasion ability are negative correlation.
3. NDRG2mediates the anti-invasion effect of HT via inhibition of the ERK1/2signaling pathway.
4. NDRG2synergizes with HT to inhibit invasiveness of HCC cells and decreasespontaneous necrosis.
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