The Role And Mechanism Of HIPPI In Non-Small-Cell Lung Cancer

Background and purpose: Huntingtin-interacting protein 1 protein interactor(HIPPI)is a multifunctional protein that regulates the pathological apoptosis of neurons and the pathophysiological functions of many cells.HIPPI was originally found to interact with HIP-1 to form heterodimer,activate the caspase8-mediated apoptosis pathway in the nucleus,and play an important role in the neuronal death of Huntington’s disease.Later studies have shown that HIPPI,as an important transcription regulator in cells,can be combined with gene promoters to regulate the transcription of many genes.Previous studies have shown that HIPPI was involved in regulating the apoptosis of glioma cells and the proliferation of colorectal cancer cells.This study aims to analyze the expression changes of HIPPI in NSCLC,and explore the role of HIPPI in tumor cell biology through cell and animal experiments,and further explore its mechanism in NSCLC.Method: Quantitative RT-PCR and Western Blot were used to detect the m RNA and protein expression levels of HIPPI in 4 lung cancer cell lines and a normal bronchial epithelial cell line.The lung adenocarcinoma cell lines H157 and A549 were selected and transfected with HIPPI interference plasmids.CCK8,clone formation,flow cytometry,scratch experiment,Transwell invasion experiment and nude mouse xenograft model was performed to evaluate the effect of silenced HIPPI on tumor cellular biology behaviors,chemosensitivity and tumor growth in vivo.The gene microarray analysis was conducted to examine the gene expression profiles of HIPPI-knockdown and control NSCLC cells.GO and KEGG were used to analyze the functions and signal pathways of differentially expressed genes.Quantitative RT-PCR was used to verify the differentially expressed genes in cells and clinical cases.Besides,the fresh tumor tissues and adjacent non-tumor tissues was collected from 42 patients with NSCLC squamous carcinoma who underwent surgical operations in Tianjin Medical University General Hospital from January 2016 to September 2018.IHC was used to detect the expression level of HIPPI in cancerous and adjacent normal tissues.Results:1.Through cellular experiments,we found that HIPPI knockdown could inhibit the proliferation,colony formation,migration and invasion,induce cell apoptosis and cycle arrest,and improve cisplatin sensitivity in vitro.Silenced HIPPI also suppressed tumor growth in vivo.2.A total of 328 up-regulated genes and 513 down-regulated genes were screened in HIPPI knockdown NSCLC cells using gene microarray.GO analysis showed that the down-regulated differentially expressed genes(DEGs)are mainly distributed in the microtubule tissue center,chromosome region,centrosome,exert transcriptional regulatory activity,participate in biological processes such as DNA replication,cell cycle phase transition,mitotic cell cycle phase transition,double Strand break repair and DNA conformation changes.KEGG enrichment analysis revealed that these down-regulated DEGs were significantly enriched in DNA replication,homologous recombination(HR),insulin signaling pathway,p53 signaling pathway and cell cycle pathway.Further analysis shown that HIPPI could positively regulate the expression of MCM2,MCM6 and MCM8,which are key regulators of DNA replication.In addition,the levels of MCM2,MCM6 and MCM8 in NSCLC tissues were higher than that in adjacent normal tissues.3.IHC analysis showed that HIPPI was located in the cytoplasm and nucleus of lung cancer tissue,and the positive rate of HIPPI in NSCLC squamous carcinoma tissues was 40.5%(17/42),which was significantly higher than that of normal adjacent tissues(11.9%,5/42),and the difference was statistically significant(P<0.05).However,no statistical correlation was found between HIPPI expression and clinicopathological factors.Kaplan-meier results showed that the overall survival rate of patients with high HIPPI expression was lower than that of patients with low HIPPI expression(11.8% vs 55.6%,P=0.040).Conclusion: Targeting HIPPI inhibits the proliferation,migration,invasion and tumor growth in NSCLC,and it can also improve the sensitivity of tumor cells to cisplatin.The biological characteristics of HIPPI in NSCLC may be related to MCMs-mediated DNA replication pathway.Besides,the expression of HIPPI protein in NSCLC squamous carcinoma tissues is higher than that of normal paracancer tissues,and higher HIPPI levels predicts poor prognosis of patients.These findings suggest that HIPPI may be used as a potential diagnostic marker and therapeutic target,providing a new strategy for the diagnosis and treatment of NSCLC.