MicroRNA expression and clinical outcome of small cell lung cancer

Abstract

The role of microRNAs in small-cell lung carcinoma (SCLC) is largely unknown. miR-34a is known as a p53 regulated tumor suppressor microRNA in many cancer types. However, its therapeutic implication has never been studied in SCLC, a cancer type with frequent dysfunction of p53. We investigated the expression of a panel of 7 microRNAs (miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a) in 31 SCLC tumors, 14 SCLC cell lines, and 26 NSCLC cell lines. We observed significantly lower miR-21, miR-29b, and miR-34a expression in SCLC cell lines than in NSCLC cell lines. The expression of the 7 microRNAs was unrelated to SCLC patients' clinical characteristics and was neither prognostic in term of overall survival or progression-free survival nor predictive of treatment response. Overexpression or downregulation of miR-34a did not influence SCLC cell viability. The expression of these 7 microRNAs also did not predict in vitro sensitivity to cisplatin or etoposide in SCLC cell lines. Overexpression or downregulation of miR-34a did not influence sensitivity to cisplatin or etoposide in SCLC cell lines. In contrast to downregulation of the miR-34a target genes cMET and Axl by overexpression of miR-34a in NSCLC cell lines, the intrinsic expression of cMET and Axl was low in SCLC cell lines and was not influenced by overexpression of miR-34a. Our results suggest that the expression of the 7 selected microRNAs are not prognostic in SCLC patients, and miR-34a is unrelated to the malignant behavior of SCLC cells and is unlikely to be a therapeutic target.

Figure 1. Ectopic overexpression or downregulation of miR34a in SCLC cell lines by microRNA precursor or inhibitor.

(A) miR-34a expression in NCI-N592 cells and NCI-H82 cells transfected with miR-34a precursor or inhibitor. The delta-delta Ct value was calibrated to the delta Ct value of miR34a in cells transfected with control miR precursor or inhibitor, respectively. (B) Cell cycle distribution of NCI-H82 cell transfected with control miR precursor, miR-34a precursor, control miR inhibitor, and miR-34a inhibitor. (C and D) Cell growth assay of NCI-H146 (C) and NCI-N592 (D) cells transfected with miR-34a inhibitor or control miR inhibitor. (E) Cell growth assay of NCI-H82 cells transfected with miR-34a precursor or control miR precursor. (F) Cell viability of NCI-N592 cells after repetitive transfection of control miR precursor or miR-34a precursor. Cells were counted and transfected again every three days. Error bars indicate standard deviation. p-values were calculated according to cell viability 5 days after transfection with the exception of microRNA precursor in NCI-N592 cell, which was calculated according to the number of cells counted 9 days after the first transfection.

Figure 2. Growth inhibition assay of NCI-H82 cells treated with (A and B) cisplatin and (C and D) etoposide.

Cells were transfected with the indicated oligonucleotides. Error bars indicate standard deviation of assays in triplicate.

Figure 3. Expression of miR-34a target genes in SCLC and NSCLC cancer cell lines.

(A) protein expression of miR-34a targets in cancer cell lines tranfected with control precursor (C) or miR-34a precursor (M). (B) mRNA expression of the AXL gene in cancer cells transfected with control precursor or miR-34a precursor. RNA was collected 24 hr after transfection. The expression of the gene in NCI-H82 and NCI-N592 cells were undetectable (UD).