阅读说明：本技术 Nudt21基因在制备治疗肺癌药物中的应用 (Application of NUDT21 gene in preparation of lung cancer treatment drug ) 是由 王立生 肖凤君 高川成 徐芹芹 吴祖泽 于 2020-03-23 设计创作，主要内容包括：本发明提供了NUDT21基因在制备治疗肺癌药物中的应用,涉及肺癌诊断和食疗技术领域,所述NUDT21基因的核苷酸序列如SEQ ID NO.1所示。通过对临床小细胞肺癌标本进行免疫组织化学染色分析和体内体外实验及TCGA数据集证明NUDT21与肺癌密切相关,沉默NUDT21明显促进了肿瘤的生长,高表达NUDT21降低了肿瘤的生长进程,同时NUDT21影响肺癌患者的生存期。因此可将NUDT21作为肺癌的一个基因靶点,进一步提高对于肺癌尤其是小细胞肺癌的诊断和治疗效果,从而促进对肺癌的精准医疗。(The invention provides an application of an NUDT21 gene in preparing a medicine for treating lung cancer, and relates to the technical field of lung cancer diagnosis and dietary therapy, wherein the nucleotide sequence of the NUDT21 gene is shown as SEQ ID No. 1. Immunohistochemical staining analysis, in vivo and in vitro experiments and TCGA data sets of clinical small cell lung cancer specimens prove that the NUDT21 is closely related to lung cancer, silent NUDT21 obviously promotes the growth of tumors, high-expression NUDT21 reduces the growth process of the tumors, and the NUDT21 influences the survival time of lung cancer patients. Therefore, the NUDT21 can be used as a gene target of lung cancer, the diagnosis and treatment effects of the lung cancer, especially small cell lung cancer, are further improved, and accurate treatment of the lung cancer is promoted.)

The application of the NUDT21 gene in preparing the medicine for treating lung cancer is characterized in that the nucleotide sequence of the NUDT21 gene is shown as SEQ ID No. 1.

2. The use of claim 1, wherein the NUDT21 affects the growth of lung cancer cells A549 including small cell lung cancer by modulating GLS1 alternative splicing in hypoxic microenvironments.

Technical Field

The invention belongs to the technical field of lung cancer diagnosis and treatment, and particularly relates to an application of an NUDT21 gene in preparation of a lung cancer treatment drug.

Background

At present, the diagnosis and treatment of lung cancer still face some difficult problems, especially Small Cell Lung Cancer (SCLC), which accounts for a smaller proportion of lung cancer diseases in the world than the common adenocarcinoma. However, over 40 ten thousand cases are reported each year, and the treatment options of a large number of people are rare and have poor curative effect, and the most common chemotherapy or radiotherapy has certain limitations.

Radiotherapy and chemotherapy are often used in the original diagnosis and treatment of lung cancer, and gene therapy is also used for treatment, but many adverse reactions and complications exist, and the lung cancer cannot be comprehensively diagnosed.

Disclosure of Invention

In view of the above, the invention aims to provide application of the NUDT21 gene in preparation of a drug for treating lung cancer, and the NUDT21 gene is used as a gene target of lung cancer, so that the treatment effect of the lung cancer can be further improved, and accurate medical treatment of the lung cancer is promoted.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of an NUDT21 gene in preparing a medicament for treating lung cancer, wherein the nucleotide sequence of the NUDT21 gene is shown as SEQ ID No. 1.

Preferably, the NUDT21 affects the growth of lung cancer cells a549, including small cell lung cancer, by modulating GLS1 alternative splicing in hypoxic microenvironments.

The invention provides an application of an NUDT21 gene in preparing a medicament for treating lung cancer, wherein the nucleotide sequence of the NUDT21 gene is shown as SEQ ID No. 1. The immunohistochemical staining analysis is carried out on clinical small cell lung cancer specimens, the expression of the NUDT21 in small cell lung cancer tissues is found to be reduced compared with that in marginal normal tissues, and the TCGA data set shows the correlation between the NUDT21 and the survival time of lung cancer patients; expression of NUDT21 is regulated in hypoxic microenvironments and appears to HIF-1 α mediate expression of NUDT 21; further experiments in hypoxic microenvironments showed that hypoxia altered the expression of NUDT21 resulting in an influence on carbohydrate metabolism (A, B in fig. 3) and glutamine metabolism (C in fig. 3), with consequent conversion of glutaminase isoforms (D, E in fig. 3). In vivo and in vitro experiments prove that the NUDT21 is closely related to lung cancer, and under the condition of no radiotherapy and chemotherapy, silencing NUDT21 obviously promotes the proliferation of A549, inhibits apoptosis and promotes the formation of clones. Further injecting silent NUDT21 and high expression NUDT 21A 549 into nude mice respectively, finding that silent NUDT21 obviously promotes the growth of tumors, and high expression NUDT21 reduces the growth process of the tumors. Therefore, the NUDT21 can be used as a gene target of lung cancer, the treatment effect on the lung cancer, especially on small cell lung cancer, is further improved, and accurate treatment on the lung cancer is promoted.

Drawings

FIG. 1 is a graph of immunohistochemical staining analysis of clinical small cell lung cancer specimens and correlation of lung cancer patient survival to NUDT21, wherein: a represents a representative image of NUDT21 staining in tissue; -is sample staining negative, + is weakly stained sample, + is moderately stained sample, + is strongly stained sample; b represents the H-score statistics of 15 pairs of samples; c represents Kaplan-Meier analysis, with expression of NUDT21 correlated with survival in patients with lung cancer (median survival increased from 46.881678009 months to 78.61 months; no change in NUDT 21: n ═ 2288, change in NUDT 21: n ═ 15); p <0.05 in comparison to control;

fig. 2 is a graph of the expression of NUDT21 in hypoxic microenvironments, wherein: a represents the expression of NUDT21 in a549 cells at different times under 1% oxygen conditions; b represents 1% O₂Culturing A549 cells, and detecting expression of HIFs and NUDT21 by western blot, wherein C represents the effect of 2h culture of A549 in DFO at different concentrations on expression of HIFs and NUDT21 Western blot, D represents the effect of silencing HIF-1 α on expression of NUDT21, E represents the effect of silencing HIF-2 α on expression of NUDT21, and the graph represents p<0.0001；

FIG. 3 is a graph of the effect of changes in expression of NUDT21 on glutamine metabolism in an oxygen microenvironment, wherein: a represents that A549 cells are cultured for 24h and 48h under the anoxic condition, and the expression of Gult1 and 3 is detected by Q-PCR; b represents that the Q-PCR method is used for detecting the expression of the Gult1 and Gult3 of A549 cells after NUDT21 is silenced; c represents that A549 cells are cultured for 24 hours and 48 hours under the anoxic condition, and the expression of GLS1 is detected by a Q-PCR method; d represents the expression of GLS1 of two splice variants of GAC and KGA in a549 cells under hypoxic conditions; e represents that the cultured A549 cells are cultured for 24 hours and 48 hours under the anoxic condition, and western blot detects the expression of NUDT21, GLS1, GAC and KGA subtypes; wherein p <0.05, p <0.01, p < 0.001;

fig. 4 is a graph of NUDT21 vs lung cancer, wherein: a represents silencing of A549 cells by NUDT21, and detection results by Q-PCR and western blot; b represents the effect of silencing NUDT21 on a549 cell proliferation; c represents the inhibition effect of NUDT21 on A549 cell apoptosis; d represents the clone formation of A549 cells after NUDT21 is silenced; e indicates that a549 cells were cultured under normal conditions and under hypoxic conditions for 15h, under which a549 cells had greater migratory capacity, p <0.05, p <0.01, p < 0.001.

FIG. 5 is a graph showing experimental results of a xenograft model in which: a represents Q-PCR detection of expression of NUDT21 in A549 cells; b indicates tumor growth was monitored 1 day, 7 days, 14 days, and 21 days after injection of a549 cells, respectively; c represents the change in body weight of the mice during the experiment; d represents the weight change of each group after tumor resection after the experiment is finished; e represents the comparison of tumor volumes of each group; f represents immunohistochemical detection of expression of NUDT21(70 ×); p <0.001 in the figure;

FIG. 6 is a diagram showing the structure of the expression protein of the NUDT21 gene, in which (left) is the structure of NUDT21(CPSF5) and (right) is the structure of CFim composed of it.

Detailed Description

The invention provides an application of an NUDT21 gene in preparing a medicament for treating lung cancer, wherein the nucleotide sequence of the NUDT21 gene is shown as SEQ ID No. 1. Preferably, the lung cancer of the present invention includes small cell lung cancer. The NUDT21 (also called CPSF5 or CFIm25) is used as a main regulator of shortening of 3' UTR, is a highly conserved CFIM component and is involved in the early stage of eukaryotic pre-mRNA aggregation, and the NUDT21 is mediated by hypoxia inducible factor HIF-1a (shown in figure 2), can regulate the generation and development of lung cancer by regulating alternative splicing and selective polyadenylation and is also involved in the survival and metabolism of lung cancer cells.

The NUDT21 gene is a mRNA precursor 3' end modifier, so that one member of the NUDT hydrolase protein superfamily has a NUDIX hydrolase structure domain, and the function of the NUDT hydrolase structure domain is similar to that of a real RNA binding protein. The expression protein NUDT21 (FIG. 6) of the NUDT21 gene of the present invention can bind to a specific RNA sequence, but because two of the four essential glutamic acid residues are lacking, the catalytic function and metal binding are ensured, and thus RNA cleavage is not possible.

In the invention, under the condition of low oxygen environment, the expression of the NUDT21 gene in the small cell lung cancer tissue is down-regulated. And most of small cell lung cancer tissue samples have negative H-SCORE SCOREs of the NUDT21 genes in the immunohistochemical detection, part of the small cell lung cancer tissue samples have weak positive H-SCORE SCOREs, a few of the small cell lung cancer tissue samples have moderate positive H-SCORE SCOREs, and the small cell lung cancer tissue samples have strong positive H-SCORE SCOREs in the immunohistochemical detection of the NUDT21 genes in punctured marginal normal tissues. Therefore, the H-SCORE of the NUDT21 gene was higher in the small cell lung cancer tissue-punctured borderline normal tissue than in the small cell lung cancer tissue, while the TCGA data set indicated that the survival of lung cancer patients was closely related to NUDT 21. Silencing the NUDT21 gene may be a potential target for promoting tumor growth and tumor metastasis.

The application of the NUDT21 gene provided by the present invention in the preparation of a drug for treating lung cancer will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.