MiR-149-3p Suppressed Cell Growth And Invasion By Downregulating The Expression Of ATP8A1 In Non-small Cell Lung Cancer

IntroductionLung cancer is the leading cause of cancer-related deaths around the world and the majority of lung cancer cases are non-small cell lung cancer (NSCLC), which accounts for approximately 80% of all primary lung cancers. Despite the improvements in therapeutic modalities, the 5-year survival rate of NSCLC patients is still around 15%. Similar to other malignant tumors, the leading cause of poor prognosis is that it is rather difficult to eliminate total cancer cells, and due to the very high proliferative and metastatic capability of cancer cells, the remaining cancer would still cause relapse. Thus it is necessary to explore the molecular mechanisms and to facilitate the treatment of NSCLC. Recently, with the development in the field of cancer research, various novel therapeutic approaches including genetic therapy and molecular therapy have yielded improved therapeutic effects. For instance, derafenib and trametinib have been applied in clinical trials and manifested promising effects to improve overall survival of melanoma patients. However, given the heterogenous and diverse characteristics of malignant tumors and the rather complicated mechanisms, current new approaches remain unable to totally cure malignant tumors. Thus it is in urgent need to studies further and comprehensively on the underlying mechanisms of the origination and development of cancers.MicroRNAs (miRNAs) are a class of small non-coding RNA molecules,19-25 nucleotides in length that regulate gene expression post-transcriptionally by targeting mRNAs. MiRNAs bind to the 3’-untranslated region (3’UTR) of target mRNAs leading to translational repression or degradation of mRNA. Accumulating evidence shows that miRNAs play critical roles in the regulation of cancer initiation and progression. MiRNAs might serve as new therapeutic strategies for cancers, as they can act as tumor suppressors or as oncogenes dependent on their target mRNAs. For example, Zhao et al showed expression of the miR-497 was significantly decreased in renal cancer and associated with tumor stage, histological grade and lymph node metastases. Sun et al revealed that miR-646 was significantly decreased in osteosarcoma and inhibited cell proliferation, migration and invasion by targeting FGF2. Yang et al suggested that miR-221/222 were significantly upregulated in human glioma and promote glioma cell invasion and angiogenesis by targeting TIMP2 Compared with the normal tissues, the expression of miR-140 is down-regulated in cell carcinomas including lung cancer cells. The outcomes of recent studies have shown that miR-140s not only perform negative roles in tumor growth and metastasis but also suppress the migration and invasion of cancer cells by downregulating the expression of their special target genes.ATP8A1 has been described as an aminophospholipid translocase (APLT) or flippase which is responsible for the translocation of phosphatidyl-serine (PS) and phosphatidylethanolamine (PE) across lipid bilayers. The deficiency of ATP8A1 can result in the exposure of phosphatidyl-serine on the surface of the cells. The collapse of membrane lipid asymmetry with phosphatidyl-serine externalization is the hallmark of cells undergoing apoptosis. It has been identified that ATP8A1.plays critical roles in cell migration. However, the expression of miR-140-3p in NSCLC and its interaction with ATP8A1 remains unknown.The present study included 2 major parts and focused on the pathological role of miR-14-3p and ATP8A1 in NSCLC and their therapeutic potentials. In the 1st part, we used 4 search software such as TargetScan to predict the target gene of miR-140-3p as ATP8A1. Then we detected the expression of ATP8A1 and miR-140-3p in NSCLC by immunohistochemistry, northern blot, qRT-PCR and western blot. Next we used luciferase assay to confirm the interaction between ATP8A1 and miR-140-3p via miRNA::mRNA interaction. We subsequently analyzed the correlation between ATP8A1 expression and various clinical factors. In the 2nd part, we up-regulated the expression of miR-140-3p using miR-140-3p mimics in NSCLC cell lines SPC-A1 and H1299, and examined the change of proliferation, invasion, migration and apoptosis of NSCLC cells in vitro. Then we explored the impact of overexpressed ATP8A1 on the therapeutic effects of miR-140-3p mimics. We then established nude mice NSCLC xenograft models including SPC-A1 transfected with miR-140-3p mimics and normal control vectors, and studied the change of tumor growth.Part I Overexpression of ATP8A1 in NSCLC and Down-regulated miR-140-3p as Regulators-a Clinicopathological StudyMethods1. To uncover the molecular mechanism by which miR-140-3p performs a suppressive role in tumor development, four cited programs, miRanda, miRDB, miRWalk and Targetscan, were exploited to predicate the potential targets of the miR-140-3p in NSCLC cells. ATP8A1 was picked as most potential targets after we reviewed related papers.2. We first determined ATP8A1 expression in 75 NSCLC tissues and 20 adjacent normal ones using immunohistochemistry, and then checked the relative levels of ATP8A1 mRNA in 20 pairs of cancer tissues and adjacent normal ones using real-time RT-PCR and western blot.3. To further investigate the significance of ATP8A1 expression in NSCLC, we analyzed the association of ATP8A1 using immunohistochemistry with patients’ clinical parameters.4. The level of mature miR-140-3p in 16 pairs of tumor issues and their corresponding adjacent normal ones were determined using a bead-based miRNA expression profiling method. To further confirm the downregulated expression of miR-140-3p in lung carcinoma, we measured the relative expression levels of miR-140-3p normalized to U6 RNA in 20 pairs of cancer issues and adjacent normal ones using real-time RT-PCR analysis analysis.Results1. The upregulation of ATP8A1 expression in human NSCLC tissuesThe data analysis revealed that the protein expression was significantly overexpressed in NSCLC patients compared with normal lung tissues, the mRNA levels in 20 cancer tissues were obviously higher than those in their adjacent normal lung tissues. Furthermore, determination of ATP8A1 protein levels in 20 pairs of NSCLC tissues and their normal counterparts using western blot showed that the expression levels of ATP8A1 proteins were significantly up-regulated in NSCLC cells. Consist with the results above, the fluorescence of ATP8A1 proteins immunohistochemical stained in lung cancer tissues were distinctly stronger than that in adjacent normal lung tissues. All these data indicated that there should be a strong correlation between the enhanced expression of ATP8A1 and progression of the lung cancer.2. Correlation between ATP8A1 and clinical factors in NSCLC patientsThere was a significant correlation between ATP8A1 expression with tumor size, lymph node metastasis, differentiation classification and clinical stage. On the contrary, the ATP8A1 expression was not related to histology, gender and alcohol or cigarette history. Similar to these findings, age is not correlated to ATP8A1 expressoin in NSCLC patients.3. The downregulation of miR-140-3p expression in human NSCLC tissuesTWe found that miR-140 levels in most of tumor samples involving lung cancer tissue were observed to be lower than the levels in matched normal samples which is in line with the results reported previously. The relative expression level of miR-140-3p in normal lung tissue is 1.25 times higher than that in tumor tissue. To further confirm the downregulated expression of miR-140-3p in lung carcinoma, we measured the relative expression levels of miR-140-3p normalized to U6 RNA in 20 pairs of cancer issues and adjacent normal ones using real-time RT-PCR analysis. The results showed that expression levels of miR-140-3p in 19 out of 20 tumor issues were significantly lower than the levels of the microRNA in matched normal ones.4. ATP8A1 identified as a direct target of miR-140-3p in NSCLC cellThe wild-type 3’untranslated region of ATP8A1 which was supposed to be bound by miR-140-3p and its mutant without putative binding sites of miR-140-3p were fused directly downstream of the firefly luciferase gene (pLuc), respectively. Two constructed vectors, Luc-ATP8A1-3’UTR-WT and Luc-ATP8A1-3’UTR-MUT, were cotransfected with miR-140-3p mimics into SPC-A1 or H1299 cells, respectively. The luciferase activity of reporter protein encoded by the gene locating at Luc-ATP8A1-3’UTR-WT decreased by 0.54 fold in SPC-A1 cells and by 0.61 fold in H1299 cells respectively, compared with that of the reporter protein encoded by the gene lying in Luc-ATP8A1-3’UTR-MUT. However, there were no obvious differences in luciferase activities of reporter protein encoded by the gene situated at Luc-ATP8A1-3’UTR-WT in tumor cells when the vectors cotransfected with the control mimics. These data indicated that miR-140-3p inhibited the luciferase activity of reporter protein by directly binding to their putative binding sites which were mutated in the research. The results of further studies on the protein levels using western blot show that the relative level of ATP8A1 normalized to GAPDH decreased by 0.72 fold in SPC-A1 and by 0.59 fold in H1299 cells transfected with miR-140-3p mimics, compared with the level in NSCLC cells with the control mimics. To test if miR-140-3p could regulate ATP8A1 expression at mRNA level, the mRNA level of ATP8A1 was monitored in NSCLC cells transfected with miR-140-3p or the negative control mimics. Quantitative real-time PCR analysis revealed the mRNA level of ATP8A1 decreased by 0.67 fold in SPC-A1 and by 0.59 fold in H1299 cells, compared with the corresponding mRNA level in NSCLC cells with control mimics. Consequently, the increased level of miR-140-3p in NSCLC cell had a significant silencing impact on endogenous ATP8A1 expression both at the mRNA and protein level.Conclusion1. Our present study revealed that miR-140-3p down-regulated in NSCLC tissues compared to their normal lung tissue counterparts. In consistence with the results of miR-140-3p expression in other tumors, miR-140-3p is highly possible to play an inhibitor role in NSCLC.2. ATP8A1 up-regulated in NSCLC tissues compared to their normal lung tissue counterparts. Moreover, ATP8A1 correlated with critical clinical parameters including tumor size, cell differentiation, lymph node metastasis and clinical stage, which indicated potential role of ATP8A1 in the proliferation and migration of NSCLC tumor cells.3. By bioinformatics methods and luciferase assay, our results confirmed that ATP8A1 is the target gene of miR-140-3p. miR-140-3p is potentially an inhibitor in NSCLC which requires further validation of its function by in vitro and in vivo studies.Part Ⅱ MiR-140-3p suppressed cell growth and invasion by downregulating ATP8A1 in non-small cell lung cancer-an in vitro and in vivo studyMethods1. To study the effect of miR-140-3p on the cell growth, the mature miR-140 and their corresponding negative control mimics were transiently transfected into SPC-A1 or H1299 cells. And then colony formation assay and MTT assay was used to examine the change of proliferation of NSCLC tumor cells.2. To test whether miR-140-3p had an impact on the NSCLC cell migration and invasion, we carried out wound healing assays and transwell invasion assays. The mature miR-140 and their corresponding negative control mimics were transiently transfected into SPC-A1 or H1299 cells. And then cytometry was carried out to examine the apoptosis rate of the cells.3. To determine whether the increased level of ATP8A1 protein could reverse the tumor-suppressive effects of miR-140-3p, the vector overexpressing ATP8A1, pCDNA3.0::ATP8A1, was cotransfected with miR-140-3p mimics into SPC-A1 and H1299 cells, respectively. And then we detected the expression of ATP9A1 using western blot, and examined the proliferation by MTT assay.4. To study the impact of overexpression of miR-140-3p on NSCLC in vivo, we first transfected SPC-A1 with mature miR-140 and their corresponding negative control mimics. And the established nude mice xenograft model using 2 transfected tumor cells.Results1. Negative effect of miR-140-3p on cell viability and growth in vitro The levels of miR-140-3p in SPC-A1 transfected with miR-140-3p mimics increased by 8.2 fold while that in H1299 with the microRNA mimics by 7.8 fold, when compared with the miR-140-3p levels in tumor cells transfected with their negative control mimics. The cell viabilities of the lung cancer cells, SPC-A1 and H1299, were decreased by 0.6 and 0.7 fold after 48 hours post-transfection of miR-140-3p respectively, compared with that of the tumor cells transfected with negative control mimics according to the results of MTT assays. Similar to the consequences of MTT assays, the data obtained from colony-formation assays showed that the colony numbers of the two cell lines treated with miR-140-3p decreased to 36% of the numbers of the cells with negative control mimics for SPC-1A and 51% for H1299 cells approximately, indicating miR-140-3p performs a negative function in the long-term growth of NSCLC cells2. Negative effect of miR-140-3p on cell viability and growth in vivo At 25 days post-injection, the mean weight of the tumors with transfected miR-140-3p was about 38% of that of the tumors with negative control mimics. Thus, we suggested that the transfection of miR-140-3p into lung cancer cells can inhibit cell growth both in vitro and in vivo.3. The inhibition of cell migration and invasion but induction of cell apoptosis by miR-140-3pthe closure rate of SPC-A1 cells transfected with miR-140-3p mimics decreased by 0.37 fold while that of H1299 cells with the microRNA mimics by 0.34 fold, compared with the rate of tumor cells with control mimics. Similarly, the abilities in invasion of SPC-A1 reduced by 0.64 fold while that of H1299 by 0.71 fold after transfection with miR-140-3p mimics, when compared with the invasion abilities of the cancer cells transfected with negative control mimics. As described previously, apoptosis is a key process in cancer development and progression. It is interesting to know if miR-140-3p expression should affect the apoptosis of NSCLC cell. The flow cytometry analysis revealed that the apoptosis rate of SPC-A1 cells transfected with miR-140-3p increased by 0.44 fold while that of H1299 cell with the microRNA mimics by 0.51 fold, compared with the apoptosis rate in control groups. These data indicated that miR-140-3p functioned as a tumor suppressor that inhibited the growth, migratory and invasive phenotype of NSCLC cells but enhanced their programmed cell death.4. The impact of miR-140-3p on the NSCLC cell growth and invasion reversed by ATP8A1 overexpressionThe level of miR-140-3p in cell contransfected with the microRNA and pCDNA3.0::ATP8A1 decreased by 0.13 fold in SPC-A1 and by 0.15 fold in H1299 cell, compared with the miR-140-3p level in cells just transfected with miR-140 mimics, but increased by 3.8 fold in SPC-A1 and by 4.7 fold in H1299 cells, compared with the microRNA level in cells just with negative control mimics. The relative expression level of ATP8A1 protein in NSCLC cells cotranfected with miR-140-3p mimics and plasmids overexpressing ATP8A1 were close to those in NSCLC cells with control mimics but significantly higher than those in cells transfected only with miR-140-3p mimics. The colony number of SPC-A1 cells increased by 1.1 fold after ATP8A1 overexpressing while that of H1299 cells by 0.81 fold, compared with the colony number of the NSCLC cells only with miR-140-3p mimics. Likewise, the percentage of invading cells increased by 1.71 fold in SPC-A1 and by 1.91 fold in H1299 cells contransfected with miR-140-3p mimics and pCDNA3.0::ATP8A1, compared with that in NSCLC cells after tranfected only with miR-140-3p mimics. These data indicated that up-regulation of ATP8A1 expression could offset the negative impact of miR-140-3p on NSCLC cell growth and invasion partially.Conclusion1. MiR-140-3p was observed to perform its tumor suppressor function via its inhibition on cell growth, migration and invasion but its induction of cell apoptosis.2. The growth of NSCLC cells in nude mouse models were suppressed by overexpression of miR-140-3p. The increased level of intracellular ATP8A1 protein attenuated the inhibitor role of miR-140-3p in the growth and mobility of NSCLC cell.3. A regulation mechanism of miR-140-3p for the development and progression of NSCLC through downregulating the ATP8A1 expression was first discovered in the present study.