Roles of Mir-144-ZFX pathway in growth regulation of non-small-cell lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide. Non-small cell lung carcinoma (NSCLC) accounts for most of the lung cancer cases and the prognosis of this disease remains poor despite decades of intensive investigation. Thus new insights into underlying mechanisms by which NSCLC develops are avidly needed as the basis for development of new lines of therapeutic strategies. The past decade has witnessed a growing interest on the regulatory roles of micro RNAs on various categories of malignancies. Related data has been well documented in carcinogenesis and pathophysiology of a variety of malignancies. Even so, there is a relative lack of data on roles of mir-144 in tumor biology and there has been no report showing the involvement of mir-144 in NSCLC development. METHODS/PRINCIPAL FINDING: From human NSCLC tumor tissue samples and cell culture samples, we found that the expression of mir-144 is associated with malignant phenotype of NSCLC. Further investigations showed that ectopic mir-144 expression dramatically inhibits NSCLC tumor cell growth and induces apoptosis as manifested by elevated apoptotic protein markers and flowcytometry change. Moreover, we also found that ZFX protein expression is also associated with malignant phenotype of NSCLC and knockdown of ZFX protein results in a similar effect as of ectopic mir-144 expression. Finally, we found that ZFX expression is highly adjustable upon presence of mir-144 and ectopic expression of ZFX dramatically dampens mir-144 action of tumor inhibition. CONCLUSIONS: Our results for the first time showed mir-144-ZFX pathway is involved in the development of NSCLC, which sheds a light for further investigations on underlying mechanisms toward better understanding and management of NSCLC

Ectopic ZFX expression rescues growth inhibitory effect of ectopic miR-144 expression in A549 cells.

<p>A. Western-blots of ZFX, cytochrome-c, caspase-3 and GAPDH in A549 cells with only mir-144 over-expression, or mir-144 over-expression combined with ZFX over-expression. The control group was transducted with two empty vector coding viruses. B. The growth curves of above cells. C, D and E. The representative pictures of Guava Nexin Assay results of above cells. F. Quantification of Guava Nexin Assay results, mean±SD, n = 3.</p

Levels of mir-144 and mir-451 are decreased NSCLC tumor tissues comparing to peri-tumor normal tissues.

<p>A and B. The relative expression levels of mir-144 and mir-451 in normal (n = 26) and tumor tissues (n = 26). Expression levels of mir-144 or mir-451 in tumor tissues were normalized to the expression of above microRNAs in the corresponding normal tissues from the same patient. C. The relative expression levels of mir-144 in low-grade (IA-IIB) (n = 14) and high-grade (IIIA-IV) lung adenocarcinoma (n = 12).</p

ZFX mRNA and protein expression in the human bronchial epithelial cell line and lung cancer cell lines.

<p>The effect of ectopic miR-144 expression on ZFX protein level in A549 cells. A. Relative mRNA expression levels of ZFX in A549, CRL-5875, HTB-183 and 16HBE14o- cells, mean±SD, n = 3. B. Western-blots of ZFX and GAPDH in above cells. C. Western-blots of ZFX and GAPDH in A549 cells with or without mir-144 over-expression. D. The scheme of the luciferase assay using pMIR-REPORT System to evaluate the direct inhibition of mir-144 on ZFX protein expression. E. The different effects of mir-144 on ZFX 3′-UTR and its mutant, mean±SD, n = 5.Mir-144 can efficiently inhibit the luciferase expression by binding to ZFX 3′-UTR, but has no effect on the luciferase expression when the binding sites were mutated on ZFX 3′-UTR.</p

Expression levels of mir-144 in A549, CRL-5875, HTB-183 and 16HBE14o- cells, mean±SD, n = 5.

<p>Expression levels of mir-144 in A549, CRL-5875, HTB-183 and 16HBE14o- cells, mean±SD, n = 5.</p

ZFX knockdown inhibits growth of A549 cells and promotes apoptosis of cells.

<p>A. Relative mRNA expression levels of ZFX in A549 with or without ZFX knockdown, mean±SD, n = 3. B. Western-blots of ZFX, cytochrome-c, caspase-3 and GAPDH in above cells. C. The growth curves of A549 cells with or without ZFX knockdown, mean±SD, n = 3. D and E. The representative pictures of Guava Nexin Assay results of A549 cells without and with ZFX knockdown, respectively. F: Quantification of Guava Nexin Assay results, mean±SD, n = 3.</p

Ectopic miR-144 expression inhibits growth of A549 cells and promotes apoptosis of the cells.

<p>A. Over-expression of mir-144 in A549 cells, mean±SD, n = 3. B. Growth curves of A549 cells with or without mir-144 over-expression, mean±SD, n = 3. C. Western-blots of A549 cells with or without mir-144 over-expression. D and E. The representative pictures of Guava Nexin Assay results of A549 cells without and with mir-144 over-expression, respectively. F: Quantification of Guava Nexin Assay results, mean±SD, n = 3.</p

ZFX expression levels are significantly higher in tumors when compared to their adjacent normal tissues.

<p>A. The relative expression levels of ZFX in normal (n = 26) and tumor tissues (n = 26). The relative levels of ZFX in tumors were obtained by normalizing against the expression of ZFX in the corresponding normal tissues from the same patient. B. The relative expression levels of ZFX in low-grade (IA-IIB) (n = 14) and high-grade (IIIA-IV) lung adenocarcinoma (n = 12).</p