CN113981093B - Inpp4b基因表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用 - Google Patents

Inpp4b基因表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用 Download PDF

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CN113981093B
CN113981093B CN202111425206.5A CN202111425206A CN113981093B CN 113981093 B CN113981093 B CN 113981093B CN 202111425206 A CN202111425206 A CN 202111425206A CN 113981093 B CN113981093 B CN 113981093B
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李龙婕
郭艳杰
邹堃
邹杨
金肃年
王晓杰
王艺茜
李爽
于湛
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Abstract

本发明属于肿瘤学及肿瘤诊断领域，公开了INPP4B基因表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用。所述INPP4B为II型多磷酸肌醇4‑磷酸酶(inositolpolyphosphate4‑phosphatasetypeII,INPP4B)，其参与调控PI3K/AKT信号通路，是维持细胞中磷酸肌醇稳定的酶之一。本发明采用的分析方法发现在基因型不同的肺腺癌细胞中INPP4B表达量和放射敏感性存在差异，且INPP4B的表达水平和肺腺癌放射敏感性存在正相关关系，即INPP4B表达低的细胞，放疗敏感性差，INPP4B表达高的细胞，放疗敏感性高。在放疗抵抗A549细胞中，我们通过分子生物学手段提高INPP4B的表达，放疗抵抗细胞的放射敏感性随之升高。本发明提供了以INPP4B为靶点来预测肺腺癌放射敏感性，筛选放疗获益人群，为临床应用奠定基础。

Description

INPP4B基因表达量的检测剂在制备用于预测肺腺癌放疗敏感 性的试剂盒的应用
技术领域
本发明属于肿瘤学及肿瘤诊断领域，具体涉及INPP4B基因表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用。
背景技术
INPP4B最初在乳腺癌细胞中被认为是一种锚着独立性生长的抑制基因，作为乳腺癌的抑癌基因，参与PI3K信号通路的调节。在乳腺癌尤其是侵袭性较强的三阴性乳腺癌中经常可以检测到INPP4B的缺失，并且与肿瘤临床分级、肿瘤大小、激素受体缺失有关；INPP4B的缺失能够引起AKT的异常活化进而导致乳腺癌细胞的大量增殖。随后在前列腺癌、卵巢癌、恶性黑色素瘤、鼻咽癌、甲状腺癌等多种恶性肿瘤中也都观察到了相似的结果。近年来，随着研究的深入，INPP4B在一些肿瘤中显示出原癌基因的作用。即使在同一种肿瘤中，不同的研究者对INPP4B的表达和作用也得出了相反的结果。一项针对180例非小细胞肺癌组织的研究发现，12％的肺腺癌和25％的肺鳞癌中INPP4B拷贝数≤1，同时30％的肺腺癌和38％的肺鳞癌中存在INPP4B表达缺失。关于INPP4B与放化疗关系的研究很少，一项研究显示：INPP4B在放化疗抵抗的喉癌和肺癌细胞中表达升高，同时放化疗可以促进INPP4B表达。INPP4B在不同环境中显示出相反的作用反映其在肿瘤中极其复杂的功能。
放疗是恶性肿瘤重要治疗方法，70％的恶性肿瘤在治疗的不同阶段需要接受放疗。肺癌是全球发病率和死亡率最高的恶性肿瘤，放疗对肺癌治疗及其重要。对于较小的肿瘤，单纯放疗就可以达到根治；对于存在预后不良因素的术后患者，放疗可以减少复发；对于晚期患者放疗可以达到缓解症状，延长生存的目的。临床观察放疗只对部分患者治疗有效，但目前没有标志物预测放疗敏感性，无法做到个体化治疗。
发明内容
为了克服现有技术的不足，本发明提供INPP4B基因表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用，通过INPP4B基因表达量的检测剂制备试剂盒用于筛选肺腺癌患者中的放疗敏感人群，敏感人群进行放疗得到获益；对于不敏感人群不首选放疗以减轻患者身体损伤和经济负担。
本发明的上述目的是通过以下技术方案实现的：INPP4B基因表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用，其中所述试剂盒包括检测INPP4B基因表达量的检测剂。所述INPP4B为II型多磷酸肌醇4-磷酸酶(inositol polyphosphate 4-phosphatase type II,INPP4B)，结构如图1，其参与调控PI3K/AKT信号通路，是维持细胞中磷酸肌醇稳定的酶之一。
进一步的，所述检测剂于核酸或蛋白水平进行检测。
进一步的，所述检测剂用于执行以下任一种方法反转录聚合酶链反应、原位杂交法、Western Blot、ELISA、免疫组织化学。
进一步的，所述INPP4B基因表达量的检测剂检测对象选自非小细胞肺癌患者的血液、血清、血浆、组织或组织裂解液、细胞培养上清以及组织标本中的一种。
进一步的，所述肺腺癌放疗辐照仪器，具体包括用于肿瘤治疗的直线加速器和用于肿瘤放疗的放射源。
本发明与现有技术相比的有益效果是：可以治疗肿瘤的放疗疗法具有一定的副反应，而临床上只有部分患者对放疗敏感，而其余放疗抵抗患者在放疗后肿瘤没有得到治疗，反而出现了放疗副反应，本发明提供的INPP4B基因表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用可以通过检测INPP4B基因表达量预测肺腺癌放射敏感性，筛选放疗获益人群，使敏感人群得到放疗获益，不敏感人群减轻身体损伤及经济负担，真正实现对患者个体化针对性放疗。
附图说明
下面结合附图与具体实施方式对本发明作进一步说明
图1为本发明检测的INPP4B基因示意图；
图2为实施例1中检测不同细胞INPP4B mRNA表达情况柱状图；
图3为实施例1中检测不同细胞INPP4B蛋白表达情况示意图；
图4为实施例1中不同细胞照射后细胞克隆形成结果示意图；
图5为实施例1中Western blot检测A549-INPP4B+细胞系中目的蛋白的表达量变化示意图；
图6为实施例1步骤4中克隆形成在INPP4B高表达细胞系中的变化示意图(左侧为原始细胞，右侧为过表达细胞系)。
具体实施方式
下面通过具体实施例详述本发明，但不限制本发明的保护范围。如无特殊说明，本发明所采用的实验方法均为常规方法，所用实验器材、材料、试剂等均可从商业途径获得。
实施例1
证明INPP4B基因表达量与肺腺癌放疗敏感性的相关性
1.选择A549细胞、Calu-3细胞、H1650细胞、PC9细胞和H1975细胞进行研究。对A549细胞、Calu-3细胞、H1650细胞、PC9细胞和H1975细胞内INPP4B进行mRNA检测，检测结果如图2，结果显示INPP4B mRNA在不同背景的肺腺癌细胞系表达存在差异，其中H1650表达最多，其次为PC9、H1975、Calu-3、表达最少的是A549，仅为H1650的50％；为进一步明确蛋白质水平INPP4B的表达情况，对INPP4B mRNA表达分别为高(H1650)、中(H1975)、低(A549)的细胞进一步进行检测，检测结果如图3，结果显示在蛋白质水平，A549细胞表达INPP4B最少、H1975细胞表达INPP4B居中、H1650表达INPP4B最多。
2.不同基因背景肺腺癌细胞系放射敏感性检测：将需要照射的细胞放置到辐照仪器，细胞容器下放置1.5cm组织补偿，细胞容器上同样覆盖组织补偿，对SSD为100cm，将机架角转至180度，从下往上照射细胞面。辐照仪器使用Varian 23EX，采用6MV X线照射细胞，剂量率300cGy/min，空气条件下进行照射，照射面积为10cm*10cm，照射的总剂量为2Gy、4Gy、6Gy、8Gy、10Gy(如表1所示)。检测结果如图4，结果显示H1650细胞和H1975细胞对放射治疗较为敏感，在接受6Gy照射后已无细胞存活，而A549细胞对放射治疗不敏感，在6Gy剂量后仍有近一半细胞存活增殖。
表1照射剂量表
Figure BDA0003378050600000041
3.相关性分析INPP4B表达与肺腺癌放射敏感性之间的关系：不同基因背景肺腺癌细胞INPP4B表达结果显示：A549细胞INPP4B表达量最低，而查得在实验使用的细胞中该细胞放射敏感性最差；H1650细胞INPP4B表达量最高，而查得在实验使用的细胞中该细胞放射敏感性最高。经过统计学Pearson相关性分析，结果如表2，结果表明不同细胞放疗后的半数致死剂量和Western条带灰度值之间的相关性，P＝0.046(<0.05)，R2＝0.99，因此细胞放射敏感性和INPP4B表达二者之间存在正相关关系。
表2.半数致死剂量和蛋白表达相关性
Figure BDA0003378050600000051
*.在0.05级别(双尾)，相关性显著。
4.过表达INPP4B，证实INPP4B与放射敏感性之间存在相关性
1)构建INPP4B高表达细胞系：INPP4B基因CDS序列插入p cDNA3.1(+)载体，通过向A549细胞内转染INNP4B质粒，筛选INNP4B高表达细胞株，结果如图5，成功构建出A549-INNP4B+细胞株；
2)证实A549-INPP4B+细胞对放射敏感：细胞经2Gy X线照射，15天后细胞染色结果显示INNP4B过表达组细胞克隆形成减少(如图6)，证明INPP4B升高后，细胞对放射的敏感性增加，二者之间存在相关性。
由以上实验结果可确定INPP4B表达量与肺腺癌放射敏感性之间为正相关关系。
以上所述实施方式仅为本发明的优选实施例，而并非本发明可行实施的全部实施例。对于本领域一般技术人员而言，在不背离本发明原理和精神的前提下对其所作出的任何显而易见的改动，都应当被认为包含在本发明的权利要求保护范围之内。

Claims (6)

1.INPP4B表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用。
2.根据权利要求1所述的INPP4B表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用，其特征在于，所述检测剂于核酸水平进行检测。
3.根据权利要求2所述的INPP4B表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用，其特征在于，所述检测剂用于执行以下任一种方法：反转录聚合酶链反应、原位杂交法。
4.根据权利要求1所述的INPP4B表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用，其特征在于，所述检测剂于蛋白水平进行检测。
5.根据权利要求4所述的INPP4B表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用，其特征在于，所述检测剂用于执行以下任一种方法：WesternBlot、ELISA、免疫组织化学。
6.根据权利要求1所述的INPP4B表达量的检测剂在制备用于预测肺腺癌放疗敏感性的试剂盒的应用，其特征在于，所述肺腺癌放疗所使用辐照仪器，具体包括用于肿瘤治疗的直线加速器和用于肿瘤放疗的放射源。
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