乙肝病毒x蛋白调控Mcl-1选择性剪接的机制及其对肝癌发生发展的影响

Hepatitis B virus x protein (HBx) has been proved to play a critical role in hepatocarcinogenesis caused by HBV， but the molecular mechanism is currently unclear. In our previous study, hepatoma cells with HBV infection showed more resistant to apoptosis. The potential mechanism was that HBx might disturb the balance between pro-apoptotic Mcl-1S (short form)and anti-apoptotic Mcl-1L (long form), which are produced by precusor Mcl-1 mRNA alternative splicing. Moreover, our lncRNA arrays revealed that HBx was able to up-regulate the expression of metastasis-associated in lung adenocarcinoma transcript 1 (MALAT1). Furthermore, it has been reported that MALAT1 could modulate RNA alternative splicing by interaction with SR splicing factors. Therefore we hypothesized that HBx may exert anti-apoptotic effect by inducing MALAT1 expression and then modulating precusor Mcl-1 mRNA alternative splicing. In the present study, we will determine whether HBx could enhance MALAT1 expression in HBx+/- cell lines and hepatocellular carcinoma tissues, and study the effects of MALAT1 on Mcl-1 alternative splicing and anti-apoptotic property of hepatoma cells by loss/gain of function strategy, and explore the underlying mechanisms. Our study may help to elucidate the functional mechanisms of HBx in hepatocarcinogenesis and provide novel therapeutic targets for HBV-related hepatocellular carcinoma.

我们前期研究发现HBV感染的肝癌细胞具有更强的抗凋亡特性，其机制是HBx影响了Mcl-1选择性剪接产生的促凋亡蛋白Mcl-1S和抗凋亡蛋白Mcl-1L之间的平衡；lncRNA array显示HBx可上调MALAT1表达，且有研究表明MALAT1可通过与SR剪接因子相互作用而改变RNA剪接模式。据此我们提出假设：HBx可通过调节MALAT1表达进而调控Mcl-1的选择性剪接来发挥其抗凋亡作用。本研究将进一步证明HBx可上调MALAT1，通过loss/gain of function策略验证MALAT1调控Mcl-1选择性剪接及其对肝癌细胞抗凋亡特性和对索拉非尼治疗应答的影响，并进一步研究MALAT1与剪接因子之间的相互作用，以此揭示HBx调控Mcl-1选择性剪接的机制及其对肝癌肿瘤学特性的影响。本研究有助于阐明HBx在肝癌发生发展中的作用，并为寻找新的肝癌治疗靶点提供理论依据。

肝细胞癌(Hepatocellular carcinoma, HCC) 是临床最常见的恶性肿瘤之一，其发病率近年来呈持续上升趋势，死亡率在恶性肿瘤中仅次于胃癌、肺癌而居第三位。乙肝病毒(Hepatitis B virus, HBV)感染是HCC发生的首位危险因素，HBV感染的HCC肿瘤生物学行为在临床进程中较非HBV感染者更为激进。HBx(Hepatitis B virus x)蛋白是乙肝病毒致癌的重要因素，但其调控肝癌细胞凋亡的作用仍存在争议，我们前期研究发现：HBV感染的肝癌细胞表现出更强的抗凋亡特性，其机制可能是HBx影响了Mcl-1 mRNA 选择性剪接后的两种不同蛋白Mcl-1S和Mcl-1L(Mcl-1S/Mcl-1L)之间的平衡。.长链非编码RNA(Long non-coding RNA, lncRNA)对基因表达起着重要的调控作用，广泛参与机体的生理和病理过程，多项研究表明lncRNA可在转录后水平指导mRNA前体的选择性剪接。我们前期通过lncRNA array 发现HBx可诱导肝癌HepG2细胞系包括AGAP1-IT1在内的部分lncRNA的差异性表达，且有研究表明AGAP1-IT1可通过与SR剪接因子相互作用进而改变mRNA前体的剪接模式，这提示AGAP1-IT1可能在HBx致肝癌的过程中发挥着重要作用。.通过实验我们在HBx阳性和阴性(HBx+/-)肝癌细胞系及临床标本中发现HBx上调AGAP1-IT1的表达，且通过loss/gain of function 策略验证AGAP1-IT1通过与影响SF2/ASF表达和磷酸化水平，募集SF2/ASF结合到Mcl-1 mRNA exon 2 的剪接增强子，以此来揭示HBx调控Mcl-1选择性剪接而影响乙肝相关肝癌肿瘤学特性的机制。首次提出在AGAP1-IT1抑制剂和索拉菲尼肝癌耐药株中的协同杀伤作用。.综上所述，本研究以Mcl-1 mRNA前体选择性剪接为切入点，证明HBx可以通过调节 AGAP1-IT1的表达进而调控mRNA选择性剪接而发挥其致癌作用的假设，揭示出一条由HBx→ AGAP1-IT1→mRNA 选择性剪接→乙肝相关肝癌肿瘤生物学特性的调控途径，为今后相关领域的进一步研究奠定基础，并为寻找新的乙肝相关肝癌靶向治疗措施提供理论依据。