BCL2, IRS1, AKT3, PTEN, and HIF1A expression levels in non-small cell lung cancer patients

Abstract

Background: Lung cancer is the leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are small noncoding molecules that play critical roles in cell proliferation, apoptosis, invasion, and metastasis, and they can target multiple genes at the mRNA level.

Materials and methods: Some online tools like TargetScan, miRDIP, miRmap, and miRanda were used to evaluate the validated target genes. Before choosing target genes, we took advantage of some bioinformatics tools including STRING, GeneMANIA, and TRED to predict the target genes. Finally, the expression levels of the target genes were measured in non-small cell lung cancer (NSCLC) tumor and their adjacent normal tissues via SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).

Results: According to bioinformatics tools, BCL2 and AKT3 were selected as target genes for miR-15/16, and BCL2 was shown to demonstrate a robust negative correlation with miR-15a in our previous analysis of NSCLC tumor samples. Furthermore, we found a significant correlation between BCL2 expression level and stage Ⅲ (p = 0.04). PTEN was assumed as a validated target gene of miR-21 that presented a significant decrease in tumor tissues compared to adjacent normal tissues. IRS1 was assigned as a target gene of miR-126/miR-128, and finally, HIF1A was selected as the target gene of miR-210. There was a significant negative association between IRS1 expression level and miR-126/miR-128, but a positive correlation was demonstrated between miR-210 and HIF1A at mRNA level.

Conclusion: Restoration of miR-15/16, miR-126, and miR-128 in NSCLC might be therapeutic candidates to control cell proliferation and apoptosis.

Keywords

AKT / BCL2 / HIF1A / IRS1 / miRNAs / non-small cell lung cancer / PTEN

References

[1] Ferlay J , Soerjomataram I , Dikshit R , et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015; 136 (5): E359- E386.

[2] Peters S , Adjei AA , Gridelli C , Reck M , Kerr K , Felip E , et al. Metastatic non-small-cell lung cancer (NSCLC): ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2012; 23 (suppl 7): vii56- vii64.

[3] Atlanta . American Cancer Society: Cancer Facts & Figures 2012. American Cancer Society; 2012;

[4] West H , Harpole D , Travis W . Histologic considerations for individualized systemic therapy approaches for the management of non-small cell lung cancer. Chest. 2009; 136 (4): 1112- 1118.

[5] Hammerman PS , Sos ML , Ramos AH , et al. Mutations in the DDR2 kinase gene identify a novel therapeutic target in squamous cell lung cancer. Cancer Discov. 2011; 1 (1): 78- 89.

[6] Shiraishi K , Kunitoh H , Daigo Y , et al. A genome-wide association study identifies two new susceptibility loci for lung adenocarcinoma in the Japanese population. Nature Genet. 2012; 44 (8): 900- 903.

[7] Xu C , Xie D , Yu SC , et al. β-catenin/POU5F1/SOX2 transcription factor complex mediates IGF-I receptor signaling and predicts poor prognosis in lung adenocarcinoma. Cancer Res. 2013; 73 (10): 3181- 3189.

[8] Filipits M . New developments in the treatment of squamous cell lung cancer. Curr Opin Oncol. 2014; 26 (2): 152- 158.

[9] Yang LL , Zhang XC , Chuai SK , et al. Integrative analyses of lung squamous cell carcinoma in ten Chinese patients with transcriptome sequencing. J Genet Genomics. 2015; 42 (10): 579- 587.

[10] Kenfield SA , Wei EK , Stampfer MJ , Rosner BA , Colditz GA . Comparison of aspects of smoking among the four histological types of lung cancer. Tob Control. 2008; 17 (3): 198- 204.

[11] Farazi TA , Spitzer JI , Morozov P , Tuschl T . miRNAs in human cancer. J Pathol. 2011; 223 (2): 102- 115.

[12] Tafsiri E , Darbouy M , Shadmehr MB , Zagryazhskaya A , Alizadeh J , Karimipoor M . Expression of miRNAs in non-small-cell lung carcinomas and their association with clinico-pathological features. Tumor Biol. 2015; 36 (3): 1603- 1612.

[13] Poortahmasebi V , Poorebrahim M , Najafi S , et al. How hepatitis C virus leads to hepatocellular carcinoma: a network-based study. Hepat Mon. 2016; 16 (2): e36005.

[14] Sun PL , Sasano H , Gao H . Bcl-2 family in non-small cell lung cancer: its prognostic and therapeutic implications. Pathol Int. 2017; 67 (3): 121- 130.

[15] Sun H , Lesche R , Li DM , et al. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc Nat Acad Sci. 1999; 96 (11): 6199- 6204.

[16] Al-Saad S , Donnem T , Al-Shibli K , Persson M , Bremnes RM , Busund LT . Diverse prognostic roles of Akt isoforms, PTEN and PI3K in tumor epithelial cells and stromal compartment in non-small cell lung cancer. Anticancer Res. 2009; 29 (10): 4175- 4183.

[17] Han C , Cho J , Moon J , et al. Clinical significance of insulin receptor substrate-I down-regulation in non-small cell lung cancer. Oncol Rep. 2006; 16 (6): 1205- 1210.

[18] Minakata K , Takahashi F , Nara T , et al. Hypoxia induces gefitinib resistance in non-small-cell lung cancer with both mutant and wild-type epidermal growth factor receptors. Cancer Sci. 2012; 103 (11): 1946- 1954.

[19] Giatromanolaki A , Koukourakis MI , Sivridis E , et al. Relation of hypoxia inducible factor 1α and 2α in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival. Br J Cancer. 2001; 85 (6): 881- 890.

[20] Karpathiou G , Sivridis E , Koukourakis M , et al. Autophagy and Bcl-2/BNIP3 death regulatory pathway in non-small cell lung carcinomas. APMIS. 2013; 121 (7): 592- 604.

[21] McCarroll JA , Gan PP , Erlich RB , et al. TUBB3/βIII-tubulin acts through the PTEN/AKT signaling axis to promote tumorigenesis and anoikis resistance in non-small cell lung cancer. Cancer Res. 2015; 75 (2): 415- 425.

[22] Scrima M , De Marco C , Fabiani F , et al. Signaling networks associated with AKT activation in non-small cell lung cancer (NSCLC): new insights on the role of phosphatydil-inositol-3 kinase. PLoS One. 2012; 7 (2): e30427.

[23] Hu Y , He K , Wang D , et al. TMEPAI regulates EMT in lung cancer cells by modulating the ROS and IRS-1 signaling pathways. Carcinogenesis. 2013; 34 (8): 1764- 1772.

[24] Snel B , Lehmann G , Bork P , Huynen MA , et al. STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene. Nucleic Acids Res. 2000; 28 (18): 3442- 3444.

[25] Jiang C , Xuan Z , Zhao F , Zhang MQ . TRED: a transcriptional regulatory element database, new entries and other development. Nucleic Acids Res. 2007; 35 (suppl 1): D137- D140.

[26] Kohl M , Wiese S , Warscheid B . Cytoscape: software for visualization and analysis of biological networks. in Data Mining in Proteomics. Springer; 2011: 291- 303.

[27] Dennis G . DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 2003; 4 (5): P3.

[28] Kiraz Y , Adan A , Kartal Yandim M , Baran Y . Major apoptotic mechanisms and genes involved in apoptosis. Tumour Biol. 2016; 37 (7): 8471- 8486.

[29] Kaira K , Yamamoto N . Prognostic and predictive factors in resected non-small-cell lung cancer. Expert Opin Med Diagn. 2010; 4 (5): 373- 381.

[30] Ibrahim N , Nazimi AJ , Ajura AJ , Nordin R , Latiff ZA , Ramli R . The clinical features and expression of bcl-2, cyclin D1, p53, and proliferating cell nuclear antigen in syndromic and non-syndromic keratocystic odontogenic tumor. J Craniofac Surg. 2016; 27 (5): 1361- 1366.

[31] Deng X , Gao F , Flagg T , Anderson J , May WS . Bcl2's flexible loop domain regulates p53 binding and survival. Mol Cell Biol. 2006; 26 (12): 4421- 4434.

[32] Laudanski J , Niklinska W , Burzykowski T , Chyczewski L , Niklinski J . Prognostic significance of p53 and bcl-2 abnormalities in operable nonsmall cell lung cancer. Eur Respir J. 2001; 17 (4): 660- 666.

[33] Coutinho-Camillo CM , Lourenço SV , de Araújo Lima L , Kowalski LP , Soares FA . Expression of apoptosis-regulating miRNAs and target mRNAs in oral squamous cell carcinoma. Cancer Genet. 2015; 208 (7-8): 382- 389.

[34] Han J , Chen Q . MiR-16 modulate temozolomide resistance by regulating BCL-2 in human glioma cells. Int J Clin Exp Pathol. 2015; 8 (10): 12698- 12707.

[35] Xu L , Wu Z , Chen Y , Zhu Q , Hamidi S , Navab R . MicroRNA-21 (miR-21) regulates cellular proliferation, invasion, migration, and apoptosis by targeting PTEN, RECK and Bcl-2 in lung squamous carcinoma, Gejiu City, China. PLoS One. 2014; 9 (8): e103698.

[36] Sun Y , Bai Y , Zhang F , Wang Y , Guo Y , Guo L . miR-126 inhibits non-small cell lung cancer cells proliferation by targeting EGFL7. Biochem Biophys Res Commun. 2010; 391 (3): 1483- 1489.

[37] Hu J , Cheng Y , Li Y , et al. microRNA-128 plays a critical role in human non-small cell lung cancer tumourigenesis, angiogenesis and lymphangiogenesis by directly targeting vascular endothelial growth factor-C. Eur J Cancer. 2014; 50 (13): 2336- 2350.

[38] Liang L , Zhou T , Jiang J , Pierce JH , Gustafson TA , Frank SJ . Insulin receptor substrate-1 enhances growth hormone-induced proliferation. Endocrinology. 1999; 140 (5): 1972- 1983.

[39] Niswender KD , Morrison CD , Clegg DJ , et al. Insulin activation of phosphatidylinositol 3-kinase in the hypothalamic arcuate nucleus. Diabetes. 2003; 52 (2): 227- 231.

[40] Liang L , Jiang J , Frank SJ . Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation. Endocrinology. 2000; 141 (9): 3328- 3336.

[41] Geng Y , Ju Y , Ren F , et al. Insulin receptor substrate 1/2 (IRS1/2) regulates Wnt/β-catenin signaling through blocking autophagic degradation of dishevelled2. J Biol Chem. 2014; 289 (16): 11230- 11241.

[42] Dearth RK , Cui X , Kim HJ , et al. Mammary tumorigenesis and metastasis caused by overexpression of insulin receptor substrate 1 (IRS-1) or IRS-2. Mol Cell Biol. 2006; 26 (24): 9302- 9314.

[43] Mardilovich K , Pankratz SL , Shaw LM . Expression and function of the insulin receptor substrate proteins in cancer. Cell Commun Signaling. 2009; 7: 14.

[44] Zhang J , Du Y , Lin Y , et al. The cell growth suppressor, mir-126, targets IRS-1. Biochem Biophys Res Commun. 2008; 377 (1): 136- 140.

[45] Zhao X , Zhu D , Lu C , Yan D , Li L , Chen Z . MicroRNA-126 inhibits the migration and invasion of endometrial cancer cells by targeting insulin receptor substrate 1. Oncol Lett. 2016; 11 (2): 1207- 1212.

[46] Wu L , Shi B , Huang K , Fan G . MicroRNA-128 suppresses cell growth and metastasis in colorectal carcinoma by targeting IRS1. Oncol Rep. 2015; 34 (5): 2797- 2805.

[47] Meng F , Henson R , Wehbe-Janek H , Ghoshal K , Jacob ST , Patel T . MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology. 2007; 133 (2): 647- 658.

[48] Wu X , Senechal K , Neshat MS , Whang YE , Sawyers CL . The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway. Proc Nat Acad Sci. 1998; 95 (26): 15587- 15591.

[49] Musilova K , Mraz M . MicroRNAs in B-cell lymphomas: how a complex biology gets more complex. Leukemia. 2015; 29 (5): 1004- 1017.

[50] Soria JC , Lee HY , Lee JI , et al. Lack of PTEN expression in non-small cell lung cancer could be related to promoter methylation. Clin Cancer Res. 2002; 8 (5): 1178- 1184.

[51] Aqeilan RI , Calin GA , Croce CM . miR-15a and miR-16-1 in cancer: discovery, function and future perspectives. Cell Death Differ. 2010; 17 (2): 215- 220.

[52] Xue G , Yan HL , Zhang Y , et al. c-Myc-mediated repression of miR-15-16 in hypoxia is induced by increased HIF-2α and promotes tumor angiogenesis and metastasis by upregulating FGF2. Oncogene. 2015; 34 (11): 1393- 1406.

[53] Semenza GL . HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol. 2000; 88 (4): 1474- 1480.

[54] Semenza GL . Targeting HIF-1 for cancer therapy. Nat Rev Cancer. 2003; 3 (10): 721- 732.

[55] Lau SK , Boutros PC , Pintilie M , et al. Three-gene prognostic classifier for early-stage non-small-cell lung cancer. J Clin Oncol. 2007; 25 (35): 5562- 5569.

[56] Dang K , Myers K . The role of hypoxia-induced miR-210 in cancer progression. Int J Mol Sci. 2015; 16 (3): 6353- 6372.

[57] Wang H , Flach H , Onizawa M , Wei L , McManus MT , Weiss A . Negative regulation of Hif1a expression and TH17 differentiation by the hypoxia-regulated microRNA miR-210. Nat Immunol. 2014; 15 (4): 393- 401.

RIGHTS & PERMISSIONS

2025 The Author(s). Malignancy Spectrum published by John Wiley & Sons Australia, Ltd on behalf of Higher Education Press.