MicroRNA-183 Promotes Motility Of CD133~+/CD326~+ CSLCs Through PTPN4 Repression

BackgroundNon-small cell lung cancer(NSCLC) is one of the most common cancers worldwide and ranks as a leading cause of lung cancer mortality partly due to distant metastasis even in the early stages. An increasing number of studies have revealed that primary tumors contain a rare subpopulation of cells, termed cancer stem-like cells(CSLCs) or tumor initiating cells(TICs) that possess stronger invasiveness to form metastases in remote organs.MicroRNAs(miRNAs) are a class of small, endogenous, non-coding RNAs that negatively regulate gene expression by partial or entire complementary binding to 3′untranslatedregion(UTR) of mRNAs. Emerging evidence reveals that abnormal miRNA expression is relevant to the dysregulation of CSLCs in various cancers such as miR-181 and hepatic CSLCs, miR34 and pancreatic CSLCs, miR-340 and glioma CSLCs, miR-125 a and breast CSLCs, miR-17 and ovarian CSLCs, miR-146 a and colorectal CSLCs, and miR-320 and prostate CSLCs.Previous studies revealed that miR-183 can mediate the invasiveness and growth of NSCLC. However, the exact role of miR-183 in regulating the biological behavior of CSLCs in NSCLC remains unclear. Thus, a better recognition of abberant miR-183 in CSLCs might help to explain the potential mechanism of lung cancer bio-behavior.The thesis discusses these.Objectives1. Obtain the lung adenocarcinoma initiating cells from A549 cell line based on paclitaxel treatment combination with serum-free cultivation and to validate the spared cells can represent the CSLCs.2. Define the function of miR-183 in the metastatic capability of CD133+/CD326+ A549 CSLCs.3. Clarify the mechanisms of miR-183 on CD133+/CD326+ A549 CSLCs.Methods1. Detect the expression of miR-183 in CD133+/CD326+ A549 CSLCs.(1).Using combined paclitaxel with serum-free cultivation to obtain CSLCs. from A549.(2).Verifying the expression of miR-183 in CD133+/CD326+ A549 CSLCs by qRT-PCR..2. Studying the pro-invasive role of miR-183 in CD133+/CD326+ A549 CSLCs.(1). We established stable miR-183 overexpressed or knockdown CD133+/CD326+ CSLCs using a lentiviral system.(2). To confirm the relevant variation of miR-183 expression in CD133+/CD326+ CSLCs infected with miR-183 and anti-miR183 by qRT-PCR.(3).Studying the function of miR-183 in the motility of CD133+/CD326+ CSLCs by bioluminescence imaging and Transwell assay.3.Investigate the mechanisms of miR-183 on CD133+/CD326+ A549 CSLCs.(1). Using three publicly available databases including TargetScan, PicTar, and miRanda to searching for potential target genes of miR-183(2). We conducted a luciferase reporter assay to confirm whether PTPN4 was a direct target of miR-183. Verifying the expression of PTPN4 mRNA and protein levels in CD133+/CD326+ CSLCs of miR-183 knockdown by qRT-PCR and western blotting analyse. We performed a rescue Transwell assay by overexpressing PTPN4 in the presence or absence of miR-183 expression in CSLCs.(3). To further investigate whether the miR-183-induced modulation of PTPN4 is of clinical relevance.Results1. miR-183 expression was up-regulated in CD133+/CD326+ A549 CSLCsAccording to our previous study mentioned above, we successfully induced CD133+/CD326+ CSLCs from A549 cells and again affirmed that miR-183 was up-regulated in CD133+/CD326+ CSLCs compared to normal A549 cells. These data are in agreement with our previous report that demonstrated up-regulation of miR-183 by miRNA microarray and validation in the A549 cell line and primary samples.2. miR-183 promotes the motility of CD133+/CD326+ A549 CSLCs. in vivo and in vitroTo confirm the function of miR-183 in CD133+/CD326+ CSLCs, we established stable miR-183 overexpressed or knockdown CD133+/CD326+ CSLCs using a lentiviral system as described above. The relevant variation of miR-183 expression in cells infected with miR-183 and anti-miR183 was confirmed by qRT-PCR. To assess changes in cell migration, CD133+/CD326+ CSLCs that overexpressed miR-183 or the negative control were allowed to migrate through a Transwell membrane into complete media. Compared to the negative control, overexpression of miR-183 led to the promotion of the motility of CD133+/CD326+ CSLCs.Then, to evaluate cell invasion capability, miR-183 was overexpressed in CD133+/CD326+ CSLCs that were plated on membranes precoated with matrigel. miR-183 overexpression significantly promoted the invasion of CD133+/CD326+ CSLCs.In intravenous injection assays, bioluminescence imaging revealed that fluorescence signals in the miR-183-overexpressed group were significantly higher than the control group, which indicated that more metastasis is formed in the lungs after miR-183 overexpression. In order to rule out the possibility that the promotion of metastasis by miR-183 may in part be caused by increasing cell proliferation, we analyzed cell proliferation using the CCK8 assay. There were no differences in cell proliferation. To further investigate the pro-invasive role of miR-183, we examined the effects of inhibiting miR-183 on the phenotype of metastasis in CD133+/CD326+ CSLCs. As expected, inhibition of miR-183 markedly reduced the invasive capabilities of CD133+/CD326+ CSLCs. Collectively, our data suggest that miR-183 greatly contributes to the metastasis and invasion of CD133+/CD326+ CSLCs.3. PTPN4 mediates miR-183-induced migration and invasion in CD133+/CD326+ CSLCs.To explore the molecular mechanism of miR-183 on the motility of CD133+/CD326+ CSLCs, we searched for potential target genes of miR-183 using three publicly available databases including TargetScan, PicTar, and miRanda. The above algorithms indicated that PTPN4 was a theoretical target of miR-183, and it was then selected for further analysis. We conducted a luciferase reporter assay to confirm whether PTPN4 was a direct target of miR-183. PTPN4 wild-type(WT) or mutant(MUT) 3′-UTRs were cloned into pGL3 luciferase reporter vectors and co-transfected with miR- 183 or control mimics into HEK 293 T cells. The results revealed that miR-183 overexpression caused a clear decrease in. relative luciferase activity. Furthermore, qRT-PCR and western blotting analyses showed that miR-183 knockdown significantly promoted the PTPN4 mRNA and protein levels in CD133+/CD326+ CSLCs. Together, these results strongly support that miR-183 directly suppresses PTPN4 by means of mRNA degradation as well as translational repression.To further examine whether miR-183 promotes invasiveness of CD133+/CD326+ CSLCs through PTPN4, we performed a rescue experiment by overexpressing PTPN4 in the presence or absence of miR-183 expression in CSLCs. After co-transfection, the expression of PTPN4 was analyzed by western blot. Consistent with the expression of target proteins, miR-183 could enhance the invasiveness of CD133+/CD326+ CSLCs, and the decreased metastatic potential was also confirmed in PTPN4-overexpressing cells compared to control cells. Furthermore, concomitant overexpression of miR-183 and PTPN4 could partially abrogate miR-183-induced invasiveness of CD133+/CD326+ CSLCs. Thus, these findings demonstrate that PTPN4 is a functional target of miR-183.To further investigate whether the miR-183-induced modulation of PTPN4 is of clinical relevance, we first assessed the expression levels of PTPN4 in clinical lung adenocarcinoma tissues. PTPN4 levels were lower in lung adenocarcinoma tissues with metastasis compared to the non-metastasic lung adenocarcinoma tissues. We next correlated PTPN4 with miR-183 expression in the same lung adenocarcinoma specimens. A statistically significant inverse correlation was observed between PTPN4 mRNA levels and miR-183.ConclusionMiR-183 could suppress PTPN4 expression through binding to its 3′UTR and leading to degradation of PTPN4 mRNA as well as translational repression. Up-regulation of miR-183 in A549 CD133+/CD326+ CSLCs could play a proinvasive role by inverse repression of PTPN4 to promote lung adenocarcinoma CSLCs metastasis.