摘要
研究目的：研究BRAF基因及其信号通路RAS-BRAF-MEK-ERK与甲状腺乳头状癌发生的相关性及其部分作用机制。研究方法：收集73例散发的甲状腺乳头状癌患者及16例同期甲状腺瘤患者(对照组)的临床资料及标本。采用免疫组织化学和免疫印迹方法检测两组标本组织中的RAS,BRAF, MEK和ERK蛋白表达状况,探讨各蛋白表达和疾病的相关性；研究RAS-BRAF-MEK-ERK信号通路在甲状腺乳头状癌发生中可能的作用。
通过PCR和测序技术,对73例散发的甲状腺乳头状癌患者BRAF基因进行了突变筛查,探讨BRAF突变与甲状腺乳头状癌发病的相关性。
同时根据BRAF的mRNA序列选择2个靶序列并根据它们设计合成2对SiRNA干扰序列,脂质体转染干扰序列进入甲状腺癌细胞株SW579,运用RT-PCR和免疫荧光技术检测干扰的效果。对干扰成功的细胞系,进行细胞增殖,细胞周期(?)(?)RAS-BRAF-MEK-ERK信号通路相关蛋白的检测。
研究结果：RAS, BRAF,磷酸化MEK1/2和磷酸化ERK1/2表达水平在甲状腺乳头状癌组都明显高于甲状腺瘤组(p<0.05)；磷酸化ERK1/2蛋白在甲状腺乳头状癌组织中呈现了明显的细胞核分布,相比甲状腺瘤组织核内磷酸化ERK蛋白增加(p<0.05)；BRAF蛋白的表达与甲状腺乳头状癌的肿块大小、淋巴结转移以及临床分期相关(p<0.05)。
我们在73例甲状腺乳头状癌组织筛查到了42例存在V600E突变,突变率为57.5%。而在良性甲状腺瘤中未发现突变。而且,BRAF突变与甲状腺乳头状癌的淋巴转移以及临床分期相关(p<0001)。
2种siRNA干扰核苷酸转染甲状腺癌SW579细胞系后,显著地抑制了BRAFmRNA水平及蛋白水平的表达,分析干扰了BRAF基因的SW579细胞后,发现SW579的生长增殖受到抑制,细胞周期发生改变,G1/S期增加,同时,RAS-BRAF-MEK-ERK信号通路的激活受到抑制。
结论：
1. RAS,BRAF,磷酸化MEK1/2及磷酸化ERK1/2在甲状腺乳头状癌中高表达,可能与甲状腺乳头状癌发生及其淋巴结转移、临床分期相关。
2. BRAF可能通过激活MEK-ERK信号通路而发挥其生物作用。
3.甲状腺乳头状癌BRAF基因突变率为57.5%。BRAF基因突变与甲状腺乳头状癌发生及其淋巴结转移、临床分期相关。
4.干扰BRAF可能通过失活RAS-BRAF-MEK-ERK信号通路而抑制甲状腺癌细胞的生长和增殖。
Aims:To invstigate the association and mechanism between activity of BRAF and RAS-BARF-MAPK signal pathway in papillary thyroid cancer.
Methods:73cases of papillary thyroid cancer and16cases of thyroid gland benign with detailed clinical data were collected. Then we detected the expression of RAS, BRAF, MEK and ERK protein in all tumor specimens and thyroid benign tissues using immunohistochemistry and Western blot. The relation between the variation of the protein expression and diseases was established. We explored the effect of RAS-BRAF-MEK-ERK signaling pathway in papillary thyroid cancer.
To explore the correlation bewteen BARF mutation and papillary thyroid cancer, BRAF gene mutations in papillary thyroid cancer and were detected by PCR and sequencing technology. At the same time, we selected2targeting sequences and designed2pairs SiRNA interference sequences, then transfected them into SW579cell line with liposome and detected interference effect using RT-PCR and immunofluorescence. The interfered successfully cell line was detected for cell proliferation, cell cycle and the RAS-BRAF-MEK-ERK signaling pathway.
Results:
The expression of RAS, BRAF, MEK and ERK protein in thyroid cancer tissues is higher than the benign thyroid tissue, p<0.05,. Phosphorylated ERK protein in thyroid cancer presents a significant nuclear distribution compared to benign thyroid tissues p<0.05.
We screened42cases BARF V600D mutations in73cases of thyroid cancer. The mutation rate is57.5%. However, no mutation was found in benign thyroid.
To investigate effect of BRAF in tumor development, we use2siRNA interference oligonucleotides to transfect into SW579cell line. Suggesting BRAF mRNA level and protein expression are significantly inhibited. Through analyzing the SW579cell line after successfully interfere, the results show that the growth of SW579proliferation is inhibited, cell cycle changes, G1/S phase increase, at the same time, RAS-BRAF-MEK-ERK signaling pathway is inhibited.
Conclusions:
1. The expression of RAS, BRAF and phosphorylated MEK, in papillary thyroid cancer is higher than benign tissues.
2. The expression of phosphorylated ERK in nuclear was increased and the RAS-BRAF-MEK-ERK signaling pathway may be activated in papillary thyroid cancer
3. The expression and mutation of BRAF was associated with lymph node metastasis and clinical stage of papillary thyroid cancer.
4. Thyroid cancer cell growth and proliferation are inhibited through BRAF interference to inactivated RAS-BRAF-MEK-ERK signal pathway.
引文
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