高迁移率族蛋白B1在非小细胞肺癌中功能的相关研究
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摘要
应用Western blot和荧光定量PCR技术检测HMGB1在几种常见的非小细胞肺癌细胞株以及非小细胞肺癌临床组织标本中的蛋白及转录水平的表达情况,在此基础上,应用免疫组化技术检测HMGB1蛋白在172例具有完整临床资料和随访信息的非小细胞肺癌组织中的表达情况,回顾性分析HMGB1蛋白在非小细胞肺癌中的临床病理特征。在初步确定HMGB1与非小细胞肺癌的关系后,进一步采用RNAi技术,构建HMGB1的慢病毒干扰载体,筛选获取稳定下调HMGB1表达的非小细胞肺癌A549/HMGB1-的细胞株,并在此基础上开展一系列细胞水平的实验,分析HMGB1表达下调后,细胞的生物学特性的改变情况。结果发现HMGB1基因是肺癌的增殖相关基因,而且HMGB1基因与肺癌细胞的侵袭能力密切相关,HMGB1的下调表达可以抑制非小细胞肺癌细胞的侵袭转移能力,研究结果证实HMGB1是非小细胞肺癌的转移促进基因。
BACKGROUND & OBJECTIVE
Lung cancer is the most common lung malignant tumor in the world at present. In recent years, along with the many environmental factor influence, the morbidity rate and mortality rate of lung cancer were rapid rise in the world. About 80% of lung cancers are non-small cell lung cancers (NSCLC). The main cause of death in NSCLC patients is the invasion and metastasis of tumors. It is critical to elucidate molecular mechanisms of metastasis and find the key factors controlling tumor metastasis, it will also be helpful to develop the effective preventive and therapeutic strategies. So it becomes the present big issue to investigate the molecular mechanism of NSCLC invasion and metastasis and to look for novel related molecular marker and target.
High-mobility group box 1 (HMGB1), an important member of the high-mobility group protein superfamily, has been implicated in a variety of biologically important processes, including transcription, DNA repair, V(D)J recombination, differentiation, development, and extracellular signaling. recent studies indicated that the overexpression of HMGB1 inhibit apoptosis of cancer cells and related to KIT mutation and genes related to tumor growth and invasion. Increased expression of HMGB1 with its receptor, receptor for advanced glycation end products (RAGE), was associated with the proliferation and metastasis of many tumor types, including breast cancer , hepatocellular carcinoma, melanoma, Prostate cancer, gastric cancer, and colorectal cancer. However, there is no report about the possible role of increased expression of HMGB1 in progression of NSCLC
METHODS
1. Expression analysis of HMGB1 in NSCLC by real-time PCR and Western blot
To determine correlation of HMGB1 with NSCLC, real-time PCR and Western blot analysis were performed to evaluate the expression levels of HMGB1 transcripts and protein in five lung cancer cell lines with different metastatic potential, WI-38 cell lines (human embryonic lung cell lines) and NSCLC tissue samples.
2. Expression of HMGB1 in colorectal carcinomas and its clinical significance
Protein expression of HMGB1 in normal lung tissue and NSCLC and its possible clinical significance in carcinogenesis, progression, invasion and metastasis were studied by immunohistochemistry.
3. Lentivirus-mediated silencing of HMGB1 gene and the influence on human NSCLC cells
Two short hairpin RNAs were designed to silence HMGB1 expression, and recombinant lentivirus vectors under the control of the U6 promoter with four short hairpin RNAs were constructed. 293FT cells were co-transfected with recombinant lentivirus vectors and adjunctive plasmid. Virus supernatants were harvested and stored in -80℃before used in the next experiments, virus titer was determined as routine.
A549, a kind of human NSCLC cells, were infected with virus supernatants obtaining HMGB1 specific RNAi lentiviral vectors, A549 infected with virus supernatants obtaining mocked RNAi lentiviral vectors were regarded as controls. Cells HMGB1 stably silenced were screened by blasticidin. Quantitative RT-PCR and Western blot analysis were used to examine the effectiveness of RNA interference. Effects of HMGB1 silence on cell proliferation was assessed by MTT assay, plate colony formation assay and flow cytometry in vitro. Moreover, motility and migration of tumor cells were determined by scratch-wound healing assay and in vitro Boyden chamber assay
RESULTS
1. Expression of HMGB1 in the samples of cell lines and tissues from NSCLC by real time RT-PCR and western blot
The results showed that the expression levels of HMGB1 mRNA in the 5 cell lines of NSCLC were higher than that of in WI-38 cell line originated from lung of human embryo. Compared with paired normal lung tissues, a significant increasing in the expression levels of HMGB1 mRNA was noted in 20 samples of primary NSCLC tissue. The over-expression of HMGB1 gene in NSCLC tissues was not correlated with the pathological types of lung cancer. We then examined the protein expression of HMGB1 protein in cell lines and tissues of NSCLC and normal controls. The expression level of HMGB1 protein was consistent with that of mRNA on the whole.
2. Expression of HMGB1 protein in NSCLC tissues by immunoh- istochemistry and its clinical significance
The positive expression of HMGB1 was found in 59.88% (103/172) of NSCLC tissues. The rates of expression of HMGB1 in lung squamous carcinoma, lung adenocarcinoma and adeno-squamous carcinoma were 50.77% (33/65), 58.54% (48/82) and 42.10% (8/19), respectively. The rate of the positive expression of HMGB1 protein in 115 samples of normal lung tissue was 32.17% (37/115). Compared with normal tissues, a significant increasing in expression of HMGB1 protein was observed in primary NSCLC tissues (P<0.05). No significant associations were found between HMGB1 expression and age, gender, tumor size and tumor type of pathology (P>0.05). However, HMGB1 expression was positively correlated with lymph node metastasis and TNM stage (P<0.05).
3. Effects of HMGB1 silencing on biological behaviors of human cells of NSCLC
1) Establishment of A549 cells stably HMGB1 silenced by RNA interference
We constructed plasimids that expressed short hairpin RNAs that were targeted against HMGB1 by lentiviral vector. Two different sequences were originally selected for targeting HMGB1 gene. We infected A549 cells with lentival vectors and examined HMGB1 mRNA and protein expression, and found HMGB1 siRNA S1 was the most effective at blocking HMGB1 expression (78.3%). The siRNA S1 was selected to be investigated in our next experiments.
2) Effects of HMGB1 silencing on the biological behaviors of human A549 cells
A significantly time-dependent inhibitory proliferation was found in A549/HMGB1- cells as compared with A549/mock and A549 cells by in vitro MTT assay(P<0.05). In addition, A549/HMGB1- cells had a significant impaired ability to form colonies in plates as compared with A549/mock and A549 cells(P<0.05). Interestingly, A549/HMGB1- cells showed a significantly decreased amount in S period by flow cytometry. The results indicated that silence of HMGB1 partially blocked proliferation of A549 cells.
To evaluate the role of HMGB1 silencing on movement and migration of A549 cells, we performed in vivo wound healing experiment. The results showed that each group had singifcant difference in different time (P<0.05). A549/HMGB1- cell had significantly reduced movement and migration compared with A549/mock and A549. There was also significant difference in different time among three groups (P<0.05).
A549/HMGB1- cell had significantly reduced invasiveness as compared with A549/mock and A549 cells as determined by in vitro boyden cave assay (P<0.05), suggesting that HMGB1 silencing inhibited the invasive abilities of A549 cells.
CONCLUSION
1. HMGB1 expression might be an independent prognostic indicator for the survival of patients with NSCLC
2. HMGB1 can promote proliferation and migration of NSCLC cells.
HMGB1 gene significantly correlates to proliferation, invasion and metastasis of NSCLC.
BACKGROUND & OBJECTIVE
Lung cancer is the most common lung malignant tumor in the world at present. In recent years, along with the many environmental factor influence, the morbidity rate and mortality rate of lung cancer were rapid rise in the world. About 80% of lung cancers are non-small cell lung cancers (NSCLC). The main cause of death in NSCLC patients is the invasion and metastasis of tumors. It is critical to elucidate molecular mechanisms of metastasis and find the key factors controlling tumor metastasis, it will also be helpful to develop the effective preventive and therapeutic strategies. So it becomes the present big issue to investigate the molecular mechanism of NSCLC invasion and metastasis and to look for novel related molecular marker and target.
High-mobility group box 1 (HMGB1), an important member of the high-mobility group protein superfamily, has been implicated in a variety of biologically important processes, including transcription, DNA repair, V(D)J recombination, differentiation, development, and extracellular signaling. recent studies indicated that the overexpression of HMGB1 inhibit apoptosis of cancer cells and related to KIT mutation and genes related to tumor growth and invasion. Increased expression of HMGB1 with its receptor, receptor for advanced glycation end products (RAGE), was associated with the proliferation and metastasis of many tumor types, including breast cancer , hepatocellular carcinoma, melanoma, Prostate cancer, gastric cancer, and colorectal cancer. However, there is no report about the possible role of increased expression of HMGB1 in progression of NSCLC
METHODS
1. Expression analysis of HMGB1 in NSCLC by real-time PCR and Western blot
To determine correlation of HMGB1 with NSCLC, real-time PCR and Western blot analysis were performed to evaluate the expression levels of HMGB1 transcripts and protein in five lung cancer cell lines with different metastatic potential, WI-38 cell lines (human embryonic lung cell lines) and NSCLC tissue samples.
2. Expression of HMGB1 in colorectal carcinomas and its clinical significance
Protein expression of HMGB1 in normal lung tissue and NSCLC and its possible clinical significance in carcinogenesis, progression, invasion and metastasis were studied by immunohistochemistry.
3. Lentivirus-mediated silencing of HMGB1 gene and the influence on human NSCLC cells
Two short hairpin RNAs were designed to silence HMGB1 expression, and recombinant lentivirus vectors under the control of the U6 promoter with four short hairpin RNAs were constructed. 293FT cells were co-transfected with recombinant lentivirus vectors and adjunctive plasmid. Virus supernatants were harvested and stored in -80℃before used in the next experiments, virus titer was determined as routine.
A549, a kind of human NSCLC cells, were infected with virus supernatants obtaining HMGB1 specific RNAi lentiviral vectors, A549 infected with virus supernatants obtaining mocked RNAi lentiviral vectors were regarded as controls. Cells HMGB1 stably silenced were screened by blasticidin. Quantitative RT-PCR and Western blot analysis were used to examine the effectiveness of RNA interference. Effects of HMGB1 silence on cell proliferation was assessed by MTT assay, plate colony formation assay and flow cytometry in vitro. Moreover, motility and migration of tumor cells were determined by scratch-wound healing assay and in vitro Boyden chamber assay
RESULTS
1. Expression of HMGB1 in the samples of cell lines and tissues from NSCLC by real time RT-PCR and western blot
The results showed that the expression levels of HMGB1 mRNA in the 5 cell lines of NSCLC were higher than that of in WI-38 cell line originated from lung of human embryo. Compared with paired normal lung tissues, a significant increasing in the expression levels of HMGB1 mRNA was noted in 20 samples of primary NSCLC tissue. The over-expression of HMGB1 gene in NSCLC tissues was not correlated with the pathological types of lung cancer. We then examined the protein expression of HMGB1 protein in cell lines and tissues of NSCLC and normal controls. The expression level of HMGB1 protein was consistent with that of mRNA on the whole.
2. Expression of HMGB1 protein in NSCLC tissues by immunoh- istochemistry and its clinical significance
The positive expression of HMGB1 was found in 59.88% (103/172) of NSCLC tissues. The rates of expression of HMGB1 in lung squamous carcinoma, lung adenocarcinoma and adeno-squamous carcinoma were 50.77% (33/65), 58.54% (48/82) and 42.10% (8/19), respectively. The rate of the positive expression of HMGB1 protein in 115 samples of normal lung tissue was 32.17% (37/115). Compared with normal tissues, a significant increasing in expression of HMGB1 protein was observed in primary NSCLC tissues (P<0.05). No significant associations were found between HMGB1 expression and age, gender, tumor size and tumor type of pathology (P>0.05). However, HMGB1 expression was positively correlated with lymph node metastasis and TNM stage (P<0.05).
3. Effects of HMGB1 silencing on biological behaviors of human cells of NSCLC
1) Establishment of A549 cells stably HMGB1 silenced by RNA interference
We constructed plasimids that expressed short hairpin RNAs that were targeted against HMGB1 by lentiviral vector. Two different sequences were originally selected for targeting HMGB1 gene. We infected A549 cells with lentival vectors and examined HMGB1 mRNA and protein expression, and found HMGB1 siRNA S1 was the most effective at blocking HMGB1 expression (78.3%). The siRNA S1 was selected to be investigated in our next experiments.
2) Effects of HMGB1 silencing on the biological behaviors of human A549 cells
A significantly time-dependent inhibitory proliferation was found in A549/HMGB1- cells as compared with A549/mock and A549 cells by in vitro MTT assay(P<0.05). In addition, A549/HMGB1- cells had a significant impaired ability to form colonies in plates as compared with A549/mock and A549 cells(P<0.05). Interestingly, A549/HMGB1- cells showed a significantly decreased amount in S period by flow cytometry. The results indicated that silence of HMGB1 partially blocked proliferation of A549 cells.
To evaluate the role of HMGB1 silencing on movement and migration of A549 cells, we performed in vivo wound healing experiment. The results showed that each group had singifcant difference in different time (P<0.05). A549/HMGB1- cell had significantly reduced movement and migration compared with A549/mock and A549. There was also significant difference in different time among three groups (P<0.05).
A549/HMGB1- cell had significantly reduced invasiveness as compared with A549/mock and A549 cells as determined by in vitro boyden cave assay (P<0.05), suggesting that HMGB1 silencing inhibited the invasive abilities of A549 cells.
CONCLUSION
1. HMGB1 expression might be an independent prognostic indicator for the survival of patients with NSCLC
2. HMGB1 can promote proliferation and migration of NSCLC cells.
HMGB1 gene significantly correlates to proliferation, invasion and metastasis of NSCLC.
引文
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[2] Bianchi ME. Beltrame M: Upwardly mobile proteins. Workshop: the role of HMG proteins in chromatin structure, gene expression and neoplasia. EMBO Rep 2000, 1:109-114.
[3] Muller S, Scaffidi P, Degryse B, Bonaldi T, Ronfani L, Agresti A, Beltrame M. Bianchi ME: New EMBO members' review: the double life of HMGB1 chromatin protein: architectural factor and extracellular signal. Embo J 2001, 20:4337-4340.
[4] Kokkola R, Andersson A, Mullins G, Ostberg T, Treutiger CJ, Arnold B, Nawroth P, Andersson U, Harris RA, Harris HE: RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages. Scand J Immunol 2005, 61:1-9.
[5] Merenmies J, Pihlaskari R, Laitinen J, Wartiovaara J, Rauvala H: 30-kDa heparin-binding protein of brain (amphoterin) involved in neurite outgrowth. Amino acid sequence and localization in the filopodia of the advancing plasma membrane. J Biol Chem 1991, 266:16722-16729.
[6] Brezniceanu ML, Volp K, Bosser S, Solbach C, Lichter P, Joos S, Zornig M: HMGB1 inhibits cell death in yeast and mammalian cells and is abundantly expressed in human breast carcinoma. Faseb J 2003, 17:1295-1297.
[7] Chuma M, Saeki N, Yamamoto Y, Ohta T, Asaka M, Hirohashi S, Sakamoto M: Expression profiling in hepatocellular carcinoma with intrahepatic metastasis: identification of high-mobility group I (Y) protein as a molecular marker of hepatocellular carcinoma metastasis. Keio J Med 2004, 53:90-97.
[8] Volp K, Brezniceanu ML, Bosser S, Brabletz T, Kirchner T, Gottel D, Joos S, Zornig M: Increased expression of high mobility group box 1 (HMGB1) is associated with an elevated level of the antiapoptotic c-IAP2 protein in human colon carcinomas. Gut 2006, 55:234-242.
[9] Wu D, Ding Y, Wang S, Zhang Q, Liu L: Increased expression of high mobility group box 1 (HMGB1) is associated with progression and poor prognosis in human nasopharyngeal carcinoma. J Pathol 2008, 216:167- 175.
[10] Liu PL, Tsai JR, Hwang JJ, Chou SH, Cheng YJ, Lin FY, Chen YL, Hung CY, Chen WC, Chen YH, Chong IW: HMGB1-Mediated MMP-9 Expression in Non-Small Cell Lung Cancer Contributes to Tumor Cell Invasiveness. Am J Respir Cell Mol Biol 2009.
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