| BackgroundNon-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide because of its high incidence and mortality rate. Recurrence happens frequently after chemotherapeutic treatment. According to the cancer stem cell hypothesis, a small population of cells within tumors possesses stem-like properties, and is responsible for the initiation and progression of tumors, but also may be the source of tumor relapse. In recent years, substantial experimental evidence has been generated in support of the existence of lung cancer stem cells (LCSCs). Stem-like cells in lung tissue were first isolated from normal mouse lung using stem-cell surface makers, Sca-1+/CD34+, and were termed BASCs. Subsequently, CD133+ cells, which possess self-renewal capacity and can recapitulate lung tumors in immuno-deficient mice, were identified in human lung cancer samples. Refined investigation into the mechanisms required for NSCLC tumorigenesis and chemoresistance may lead to an improved survival rate for lung cancer patients.LCSCs have been enriched by three different methods, including sorting for CD133+ cell, selecting for side-population (SP) cells that efflux Hoechst dyes, or isolating cellular clusters which resemble self-replicating cells ("tumor spheres") from suspension cultures. However, these methods purify both LCSCs and some early precursor cells. LCSCs are believed to be resistant to chemotherapy, in part, due to over-expression of an ATP-binding cassette half-transporter, ABCG2, which pumps drugs out of the cells and provides a selective survival advantage. Thus, CSCs are rendered resistant to drug treatment, including chemotherapeutic drugs, and ABCG2 might serve as a marker for stem cells from various sources. Since LCSCs are relatively resistant to chemotherapy, we attempted to enrich LCSCs by consecutive passaging of a NSCLC cell line, A549, in mice treated with Paclitaxel. We also investigated the mechanisms regulate the self-renewal and tumorigenicity of LCSCs. Bmil is a member of the Polycomb group (PcG) genes, which are transcriptional repressors that play essential roles in the maintenance of appropriate gene expression during development. Previous studies have identified that Bmil was over-expressed in NSCLC and some other epithelial malignancies. In particular, Bmil over-expression is correlated with poor prognosis for lung cancer patients. It has recently been reported that Bmil is required for the proliferation of various types of differentiated cells and for the self-renewal of stem cells. Some studies have shown that Bmil was highly enriched in tumor-initiating stem cells. However, whether the requirement for Bmil in lung tumor development correlates with a requirement for Bmil to maintain the function of LCSCs has not yet been fully investigated.In our present study, we provide evidence that the expression of Bmil in NSCLC could be stably down-modulated by intratumoral injections with lentivirus-delivered Bmil-shRNA into xenografts in NOD/SCID mice. The remarkably decreased expression of Bmil is associated with the impaired key characteristics of LCSCs-self-renewal in vitro and initiating tumors in vivo. Down-regulation of Bmil paralleled increased P16INK4a, known as Bmil target. These observations lead us to conclude Bmil regulates self-renewal and tumorigencity of LCSCs by silencing some targets proteins including P16INK4a in NSCLC.ObjectiveTo observe the effect of down-modulation Bmil by intratumoral injecting with lentivirus-delivered Bmil shRNA into xenografts on the self-renewal in vitro and tumorigenicity in vivo of lung cancer stem cells (LCSCs).Methods1. Mouse xenograft models were made by injecting subcutaneously with A549 cells in NOD/SCID mice. To enrich LCSCs in A549-3rd cells by consecutive passaging of A549 in mice treated with Paclitaxel. The proportion of CD133+ cells been determined by fluorescence activated cell sorting (FACS) and the expression levels of Bmil and ABCG2 were detected by real-time quantitative RT-PCR and western blotting.2. Downregulation of Bmil in LCSCs by RNA interference. Bmil small hairpin RNA (shRNA) sequence and a nonspecific shRNA were designed and packaged into the recombinant lentivirus, named vsh-Bmil and vsh-NC respectively. Tumors were injected with vsh-Bmil, vsh-NC or PBS. The transfection efficency was confirmed by fluorescence microscope. And the expression of Bmil was detected by real-time PCR and western blotting.3. The spheres formation and xenograft formation ability of A549 cells, A549-3rd cells, A549-4th cells, A549-4th-Bmil cells and A549-4th-NC cells has been tested.Results1. There is a 12-fold increase (P=0.01) in the number of cells that are able to form spheres in A549-3rd cells compared with parental A549 cells, (15.6%±1.5%)and (1.2%±0.4%), respectively. Furthermore, spheres from A549-3rd cells could be passaged for at least 8 to 10 generations, while spheres from A549 cells could only be passaged 2 to 3 generations. Eight weeks after been injected, only one of the six mice injected with A549 cells had tumors, while five out of six mice injected with A549-3rd cells had tumors. Similarly, the proportion of CD 133+ cells was higher in A549-3rd compared to A549 cells after chemotherapy. And expression of Bmil and ABCG2 were both significantly increased (P=0.02) in A549-3rd cells compare to A549 cells.2. GFP expression was detected in the xenografted tumors 72 hours after lentivirus injection. The expression of Bmil in A549-4th-Bmil cells was significantly reduced (P=0.02) compared to A549-4th-NC cells and control A549-4th cells. Knockdown of Bmil expression resulted in a decrease in the levels of Bmil protein compared to the control cells, while the protein level of P16INK4a was significantly increased.3. After about 7 days of culture in serum-free medium, (0.9%±0.2%) of A549-4th-Bmil cells, (13.9%±1.1%) of A549-4th-NC cells, and (14.7%±1.3%) of A549-4th cells formed spheres. There was a 14-fold decrease (P=0.01) in the sphere-forming capacity in cells without Bmil. Although two out of six mice injected with A549-4th-Bmi 1 cells developed tumors, there were still significantly fewer tumors overall compared to mice injected with A549-4th-NC and A549-4th cells.Conclusion1. These results show that the self-renewal and xenograft formation potential of the A549-3rd cells has increased compared to A549 cells. Therefore, chemotherapeutic treatment selectively enhanced the number of LCSCs in the tumor by the third generation.2. The expression of Bmil in A549-4th-Bmil cells was significantly reduced by RNA interference, and knockdown of Bmil expression resulted in a decrease in the levels of Bmil protein compared to the control cells. While the increase level of P16INK4a was associated with the reduction of Bmil expression.3. Downregulation of Bmil in NSCLC reduces the ability of self-renewal in vitro and tumorigenicity in NOD/SCID mice of LCSCs by silencing some targets proteins including P16INK4a. |
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