European Review for Medical and Pharmacological Sciences 2019; 23: 1093-1101 LncRNA BC200 regulates the cell proliferation and cisplatin resistance in non-small cell lung cancer via PI3K/AKT pathway B.-B. GAO, S.-X. WANG Cancer Treatment Center, Shandong Provincial Hospital Affiliated to Shandong University, Ji’nan, China. Abstract. – OBJECTIVE: Long non-coding mor stage and metastasis of NSCLC patients. RNA (lncRNA) exerts tissue specificity and reg- BC200 promotes the malignant progression of ulates the occurrence and progression of tu- NSCLC via regulating cisplatin-induced apopto- mors. Previous bioinformatics showed that sis of H1299/DDP cells. lncRNA BC200 is served as an oncogene. How- ever, the specific role of BC200 in lung cancer Key Words: (LC) is rarely reported. The aim of this study is BC200, NSCLC, Cisplatin resistance, Malignant pro- to elucidate the regulatory effects of BC200 on gression. tumor development and cisplatin resistance in non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: The expression Introduction level of BC200 in 76 pairs of NSCLC tissues and adjacent normal tissues was detected by quan- titative Real Time-Polymerase Chain Reaction Lung cancer (LC) is a common primary tumor (qRT-PCR). Correlation analyses were conduct- of the respiratory system, which is the leading ma- ed to investigate the relation between BC200 ex- lignancy throughout the world1,2. Non-small cell pression and prognosis of NSCLC patients. Sub- lung cancer (NSCLC) is the most common type sequently, BC200 expression in LC cell lines was of LC, accounting for about 80% of LC cases. detected. After construction of si-BC200 and si- According to the pathological types, NSCLC is NC, the cellular functions of LC cells were de- divided into squamous cell carcinoma, adenocar- tected through colony formation, flow cytometry 3,4 and transwell assay, respectively. Western blot cinoma and large cell carcinoma . About 75% of was performed to detect the protein expressions NSCLC patients are already in an advanced stage of key genes in the PI3K/AKT pathway in LC of diagnosis, leading to the low 5-year survival cells. Finally, cell counting kit-8 (CCK-8) assay rate3,4. Genetics factors, diet, unhealthy lifestyles was carried out to explore the effect of BC200 and precancerous lesions are all closely related to on cisplatin resistance of LC cells via calculat- ing IC50. NSCLC occurrence. It is reported that over 50% RESULTS: Higher expression of BC200 was of NSCLC patients experience micrometastasis found in NSCLC tissues than that of adjacent before radical surgery, which is the direct cau- normal tissues. BC200 expression was positive- se of postoperative metastasis and recurrence3-6. ly correlated with tumor stage, lymph node me- The comprehensive understanding of the molecu- tastasis and distant metastasis of NSCLC pa- lar mechanism of NSCLC contributes to develop tients, whereas not correlated to age and sex. effective therapeutic approaches and to improve Knockdown of BC200 inhibited proliferation, in- vasion and migration of LC cells. Western blot the clinical outcomes of affected people. Cur- results showed that protein expressions of PI3K, rently, surgical resection and chemotherapy are AKT and STAT3 were downregulated after BC200 the preferred options for treating NSCLC, which knockdown in LC cells. Additionally, the IC50 in can greatly prevent recurrence and metastasis of H1299/DDP cells transfected with si-BC200 was NSCLC. However, chemotherapy resistance is lower than in those transfected with si-NC. The the main cause of chemotherapy failure, remar- apoptotic rate in H1299 cells transfected with si- 5,6 BC200 was remarkably lower than those trans- kably limiting its clinical application . With the fected with si-NC. in-depth researches on NSCLC, it is believed that CONCLUSIONS: BC200 is highly expressed NSCLC is the result of genetic and environmen- in NSCLC, which is positively correlated with tu- tal factors. Alterations in certain oncogenes and Corresponding Author: Shengxi Wang, MD; e-mail: [email protected] 1093 B.-B. Gao, S.-X. Wang tumor-suppressor genes finally lead to disordered Patients and Methods cell proliferation, apoptosis and differentiation7,8. Non-coding RNAs have been well recognized in Sample Collection recent years. Among them, long non-coding RNA 76 pairs of NSCLC tissues and adjacent normal (lncRNA) is a kind of non-coding RNA with over tissues were surgically resected. Enrolled patients 200 bases in length9-11. LncRNAs may be differen- were all pathologically diagnosed as NSCLC and tially expressed in malignancies, which are closely received DDP (cisplatin) chemotherapy (Table I). related to the progression of tumors. Certain lncR- Based on the therapeutic outcomes of DDP che- NAs could be served as diagnostic and prognostic motherapy, patients were divided into DDP-sen- markers12,13. Functionally, lncRNA degrades or sitive group (n=31) and DDP-insensitive group inhibits the target mRNA, thereafter regulating (n=45). This study was approved by the Shandong target gene expressions14,15. Misexpression of ln- Provincial Hospital Affiliated to Shandong Uni- cRNA may result in multiple misexpressed pro- versity Ethics Committee and all patients signed teins14,15. Accumulating evidence has shown that the informed consent. lncRNAs are closely related to the occurrence and progression of malignancies, which are served as Cell Culture tumor hallmarks16. For example, lncRNA BC200 Human LC cell lines (SKMES1, A549, PC- is differentially expressed in tumors and is utilized 9, H358, H1299 and SPCA1) and human bron- as a biomarker17-19. Our previous studies20,21 have chial epithelial cell line (16HBE) were obtained already found that BC200 is highly expressed in from Cell Bank, Chinese Academy of Sciences. NSCLC as an oncogene, which promotes prolife- DDP-resistance H1299 cells (H1299/DDP) were ration and induces apoptosis of LC cells. However, obtained from Runcheng (Shanghai, China). Cel- the potential mechanism of BC200 in regulating ls were cultured in Dulbecco’s Modified Eagle NSCLC development has not been fully elucida- Medium (DMEM; Gibco, Rockville, MD, USA) ted. Through literature review22,23, we concluded containing 10% fetal bovine serum (FBS; Gibco, that BC200 is involved in regulating proliferation, Rockville, MD, USA) and maintained in a 5% apoptosis, differentiation and metastasis of tumor CO2 incubator at 37°C. cells. The specific role of BC200 in NSCLC still needs to be further investigated. Transfection In the present work, we first detected the H1299 and H1299/DDP cells in logarithmic expression level of BC200 in NSCLC tissues and growth phase were seeded in the 6-well plates at adjacent normal tissues. The regulatory effect of a density of 5×104 cells per well. Cells were tran- BC200 on cisplatin resistance of LC cells and sfected with si-BC200 or si-NC according to the NSCLC tissues were further explored. instructions of Lipofectamine 2000 (Invitrogen, Table I. Association of lncRNA BC200 expression with clinicopathologic characteristics of non-small cell lung cancer. lncRNA BC200 expression Parameters Number of cases Low High p-value Age (years) 0.488 <60 32 20 12 ≥60 44 24 20 Gender 0.096 Male 37 25 12 Female 39 19 20 T stage 0.017 T1-T2 43 30 13 T3-T4 33 14 19 Lymph node metastasis 0.019 No 45 31 14 Yes 31 13 18 Distance metastasis 0.010 No 60 40 20 Yes 16 4 12 1094 LncRNA BC200 and cisplatin resistance in non-small cell lung cancer Carlsbad, CA, USA). Culture medium was repla- Deoxyribose Nucleic Acid (cDNA). The sequen- ced 5-6 hours later. ce of specific BC200 RT primers was 5′-AGAC- CTGCCTGGGCAATATAGC-3′, glyceraldehy- Cell Proliferation Assay de 3-phosphate dehydrogenase (GAPDH) was Transfected cells were digested for the prepara- 5′-CATGAGAAGTATGACAACAGCCT-3′. tion of cell suspension (5×104/mL). 100 μL of the The sequences of specific BC200 PCR primers: suspension was added in each well of the 96-well forward, 5′-AGACCTGCCTGGGCAATATA- plates. 24 hours after cell adherence, 100 μL of GC-3′ and reverse, 5′-GTTGTTGCTTTGAGG- medium containing 0, 1, 2, 4, 8, 16, 32, and 64 μg/ GAAGTTACG-3′. The expression levels of BC200 mL DDP was added. Each concentration had 5 were normalized to GAPDH (forward, 5′-CAT- replicates. After cell culture for 48 h, 10 μL of cell GAGAAGTATGACAACAGCCT-3′ and reverse, counting kit-8 (CCK-8) solution (Dojindo, Kuma- 5′-AGTCCTTCCACGATACCAAAGT-3′). After moto, Japan) was added and the optical density the cDNA was amplified, qRT-PCR was perfor- was determined at the wavelength of 450 nm 1 med to detect the expressions of related genes. hour later. The IC50 was finally calculated. Western Blot Colony Formation Assay Cells were lysed for protein extraction. The 200 transfected cells were seeded in each well concentration of each protein sample was deter- of the 6-well plates for 2-week cell culture. The mined by a BCA (bicinchoninic acid) kit (Abcam, medium was replaced once a week. Cells were Cambridge, MA, USA). The protein sample was then washed with phosphate-buffered saline separated by gel electrophoresis and transferred (PBS), fixed with methanol for 20 min and stained to PVDF (polyvinylidene difluoride) membranes with 0.1% crystal violet for 20 min. Colonies were (Millipore, Billerica, MA, USA). After incuba- captured using a microscope. tion with the primary and secondary antibody (Cell Signaling Technology, Danvers, MA, USA), Flow Cytometry Analysis of Cell Apoptosis immunoreactive bands were exposed by enhan- H1299 and SPCA1 cells in logarithmic growth ced chemiluminescence method (ECL). phase were seeded in the 6-well plates. After transfection for 24 h, cells were centrifuged and Statistical Analysis resuspended in 500 μL of Binding Buffer. Sub- We used Statistical Product and Service Solu- sequently, cells were incubated with 5 μL of An- tions (SPSS) 22.0 software (IBM, Armonk, NY, nexin V-APC and 5 μL of 7-AAD in the dark for USA) for statistical analysis.