Comprehensive Analysis of Potential Key Genes in Progression of Large Cell Neuroendocrine Lung Carcinoma

Background Pulmonary LCNEC (large-cell neuroendocrine carcinoma) is a kind of high-grade lung neuroendocrine tumors (NET). There’s an increase in the pathologically diagnosis of LCNECs during recent years. However, the underlying mechanisms of the progression of LCNEC still remains unclear. In this article, we utilized three GEO datasets to elucidate the signicant candidate genes and pathways in LCNEC: GSE1037, GSE11969, and GSE44447,including 17 LCNEC samples and 31 normal lung tissues, were analyzed using several bioinformatics methods to explore the key genes involved in the development of LCNEC. We identied ten hub genes which might be important in the pathogenesis of LCNEC. Then we performed western blot on specimens from four patients diagnosed with LCNEC in our hospital to validate these genes.


Abstract Background
Pulmonary LCNEC (large-cell neuroendocrine carcinoma) is a kind of high-grade lung neuroendocrine tumors (NET). There's an increase in the pathologically diagnosis of LCNECs during recent years.
However, the underlying mechanisms of the progression of LCNEC still remains unclear.

Methods
In this article, we utilized three GEO datasets to elucidate the signi cant candidate genes and pathways in LCNEC: GSE1037, GSE11969, and GSE44447,including 17 LCNEC samples and 31 normal lung tissues, were analyzed using several bioinformatics methods to explore the key genes involved in the development of LCNEC. We identi ed ten hub genes which might be important in the pathogenesis of LCNEC. Then we performed western blot on specimens from four patients diagnosed with LCNEC in our hospital to validate these genes.

Conclusion
The results of our study might provide new insights for the development of new biomarkers and therapeutic targets of LCNEC.

Background:
Large cell neuroendocrine carcinoma (LCNEC) accounts for nearly 3% of all lung cancers. It is a rare form of aggressive lung cancer with an extremely high proliferation rate [1]. This type of lung cancer occurs more frequently in male smokers who smokes heavily, whereas uncommon in females who do not smoke [2].LCNEC is often categorized in the NSCLC group. However, it is also a member of the lung neuroendocrine tumor (NET) group [3] because it arises from lung cells of neuroendocrine system. The WHO 2015 classi cation system is commonly used for nomenclature [4].
LCNEC is classi ed into three subsets according to molecular characteristics as well as clinical features [5].SCLC-like, NSCLC-like are major subsets and minor subset is carcinoid-like[6].The different classi cation of subsets lead to different management strategies [7].
Patients diagnosed of LCNEC have dismal prognosis [8].Compared with other types of lung cancers, LCNEC also has different metastatic patterns [9].Surgery is the primary treatment for early stage disease.
However, the cause and underlying molecular events of lung large cell neuroendocrine carcinoma are still far from clear. In our study, we applied integrated bioinformatics to identify key pathogenic genes involved in LCNEC and then validated those genes in LCNEC specimen tissues.

Methods: 1 Bioinformatics Analysis
In our research, we downloaded three microarray datasets from NCBI-GEO (NCBI-Gene Expression Omnibus database https://www.ncbi.nlm.nih.gov/geo) : GSE1037, GSE11969, GSE44447. The platform for GSE1037 is GPL962, which includes 19 normal lung samples and 8 LCNEC specimens. The platform for GSE11969 is GPL7015, which consists of 5 normal lung samples and 2 LCNEC samples. The platform for GSE44447 is GPL14550, which includes 7 normal lung samples and 6 LCNEC samples.
There are 17 LCNEC samples and 31 normal samples in total. We ltered differentially expressed genes using the limma software package. Samples with a corrected P-value of 0.05 and log fold change FC of 2 were considered DEGs.
Venn diagram was used to nd the common differentially expressed genes in the three datasets.

Specimen validations
This study was approved by Fujian Provincial Hospital Ethics Committee. The investigations conform to the principles in the declaration of Helsinki. Written consent of the patients were obtained.
Specimens of four patients diagnosed with LCNEC from Jan 2019 to Oct 2019 in our department was surgically resected. We only included specimens of histologically pure LCNEC patients. All cases met the WHO 2015 criteria for LCNEC. Western blot was performed on the four specimens. Procedures were carried out according to protocols. ImageJ was used to analyze band intensity. All analyses were performed using SPSS version 26.0 for Windows (SPSS Inc. Chicago. IL). p-values < 0.05 were considered statistically signi cant.

Discussion:
LCNEC is an aggressive tumor [29].It has unique biological and molecular features. Patients diagnosed with LCNEC have dismal prognosis. Since this tumor is extremely rare, it is very di cult to be clinically diagnosed. It is classi ed differently due to pathological features [30] [31]. LCNEC has similar features with small cell lung cancer, including aggressiveness, relations with smoking, extremely high rates of progression, certain gene expressions. Standard treatments of LCNEC and are still not well established.
Since current treatment results are not satisfactory. There's emerging need for better clari cation of the underlying features of LCNEC and discovery of new options for treatments.
In our study, we used bioinformatics method to reveal key genes and pathways involved in pathogenesis cycle, mitotic cell cycle checkpoint, HTLV-1 infection, adaptive immune system. we validated data using sample tissues dissected from LCNEC patients. Further we got three signi cant important genes: CCT4, CDC20 and KNTC1. They are all protein coding genes. CCT4 (Chaperonin Containing TCP1 Subunit 4) is related to pathways including cooperation of prefoldin and TriC/CCT in actin and tubulin folding ,and cargo tra cking to the periciliary membrane. Gene ontology annotations related to this gene include unfolded protein binding. CDC20 (Cell Division Cycle 20) is related to pathways like CDK-mediated phosphorylation and removal of Cdc6, as well as mitotic roles of polo like kinases. Gene ontology annotations related to this gene include enzyme binding and protein C-terminus binding. KNTC1's (Kinetochore Associated 1) related pathways are cell cycle, mitotic and mitotic prometaphase.
However, there are limitations to our study. Since LCNEC is an extremely rare disease, the collection of large data set is very di cult. For further studies, we would like to collaborate with other hospitals to gather more data on LCNEC.

Conclusion:
In conclusion, our study identi ed key genes in pulmonary large cell neuroendocrine carcinoma development, results showed that CCT4, CDC20 and KNTC1 are upregulated in lung large cell neuroendocrine carcinomas and they have the potential to become valuable therapeutic targets for LCNEC in the future.