Zhongliang Ma

MicroRNA-34a inhibits the proliferation and promotes the apoptosis of non-small cell lung cancer H1299 cell line by targeting TGFβR2.

MicroRNAs (MiRNAs) are small non-coding RNA molecules which act as important regulators of post-transcriptional gene expression by binding 3′-untranslated region (3′-UTR) of target messenger RNA (mRNA). In this study, we analyzed miRNA-34a (miR-34a) as a tumor suppressor in non-small cell lung cancer (NSCLC) H1299 cell line. The expression level of miR-34a in four different NSCLC cell lines, H1299, A549, SPCA-1, and HCC827, was significantly lower than that in the non-tumorigenic bronchial epithelium cell line BEAS-2B. In human NSCLC tissues, miR-34a expression level was also significantly decreased in pT2-4 compared with the pT1 group. Moreover, miR-34a mimic could inhibit the proliferation and triggered apoptosis in H1299 cells. Luciferase assays revealed that miR-34a inhibited TGFβR2 expression by targeting one binding site in the 3′-UTR of TGFβR2 mRNA. Quantitative real-time PCR (qRT-PCR) and Western blot assays verified that miR-34a reduced TGFβR2 expression at both mRNA and protein levels. Furthermore, downregulation of TGFβR2 by siRNA showed the same effects on the proliferation and apoptosis as miR-34a mimic in H1299 cells. Our results demonstrated that miR-34a could inhibit the proliferation and promote the apoptosis of H1299 cells partially through the downregulation of its target gene TGFβR2.

Direct repression of the oncogene CDK4 by the tumor suppressor miR-486-5p in non-small cell lung cancer

MicroRNAs are a class of non-coding single-stranded RNA, 20-23 nucleotide in length, which can be involved in the regulation of gene expression. Through binding with 3′-untranslated regions (3′-UTR), microRNAs can cause degradation of target mRNAs or inhibition of translation, and thus regulating the expression of genes at the post-transcriptional level. In this study, we found that miR-486-5p was significantly downregulated in non-small cell lung cancer (NSCLC) tissues and cell lines, suggesting that miR-486-5p might function as a tumor suppressor in lung cancer. Additionally, we showed that CDK4, an oncogene that plays an important role in cell cycle G1/S phase progression, was directly targeted by miR-486-5p. Furthermore, our data reveals that knockdown of CDK4 by siRNA can inhibit cell proliferation, promote apoptosis, and impede cell-cycle progression. In epigenetics, the upstream promoter of miR-486-5p was strongly regulated by methylation in NSCLC. Collectively, our results suggest that miR-486-5p could not only inhibit NSCLC by downregulating the expression of CDK4, but also be as a promising and potent therapy in the near future.

MiR-181a-5p inhibits cell proliferation and migration by targeting Kras in non-small cell lung cancer A549 cells.

MicroRNAs play important roles in carcinogenesis and tumor progress. Lung cancer is the leading cause of cancer mortality worldwide. In this study, the function of miR-181a-5p was investigated in non-small-cell lung cancer (NSCLC). Results showed that miR-181a-5p was significantly decreased in NSCLC tissues and cell lines. The proliferation and migration of A549 cells transfected with miR-181a-5p mimic was significantly inhibited. Luciferase activity assay results demonstrated that two binding sites of Kras could be directly targeted by miR-181a-5p. Furthermore, Kras was down-regulated by miR-181a-5p at both transcriptional and translational levels. SiRNA-mediated Kras down-regulation could mimic the effects of miR-181a-5p mimic in A549 cells. Our findings suggest that miR-181a-5p plays a potential role in tumor suppression by partially targeting Kras and has the potential therapeutic application in NSCLC patients.

MiR-146a-5p inhibits cell proliferation and cell cycle progression in NSCLC cell lines by targeting CCND1 and CCND2

Previous studies have indicated that miR-146a-5p acts as an oncogene in several types of cancer, yet a tumor suppressor gene in others. In non-small cell lung cancer (NSCLC), one report showed that it was downregulated and played the role of tumor suppressor. However, another study showed that miR-146a-5p was overexpressed in the serum of NSCLC patients compared to healthy controls. Therefore, it is obvious that further study of the function of miR-146a-5p in NSCLC is necessary to fully understand its importance. Herein, we have verified that miR- 146a- 5p acts as a tumor suppressor in NSCLC. Our data revealed that the expression level of miR-146a-5p was significantly decreased in several human NSCLC cell lines, and also less abundant in human NSCLC tissues, when compared with controls. Moreover, we observed that miR-146a-5p could suppress cell proliferation, both in vitro and in vivo. Our results also showed that miR-146a-5p directly targeted the 3′-UTR of CCND1 and CCND2 mRNAs as well as decreased their expression at both mRNA and protein levels, causing cell cycle arrest at the G0/G1 phase. Furthermore, siRNA-mediated downregulation of CCND1 or CCND2 yielded the same effects on proliferation and cell cycle arrest as miR-146a-5p upregulation did in the NSCLC cell lines. We confirmed that the expression of miR-146a-5p had negative relationship with CCND1 or CCND2. Besides, we also found that miR-146a-5p could inhibit tumor growth in xengroft mouse models, and CCND1 and CCND2 were downregulated in miR-146a-5p overexpressed xengroft tumor tissues. In summary, our results demonstrated that miR-146a-5p could suppress the proliferation and cell cycle progression in NSCLC cells by inhibiting the expression of CCND1 and CCND2.

tRF-Leu-CAG promotes cell proliferation and cell cycle in non-small cell lung cancer

tRNA‐derived RNA fragments (tRFs), non‐coding single‐stranded RNAs with 14–35 nt in length, were found to play important roles in gene regulation, even in carcinogenesis. In this study, we investigated the expression of tRF‐Leu‐CAG in human non‐small cell lung cancer (NSCLC) and its function in the cell proliferation and cell cycle of NSCLC. The expression level of tRF‐Leu‐CAG was detected in NSCLC tissues, cell lines, and sera. tRF‐Leu‐CAG RNA levels were higher in NSCLC tumor tissues than in normal tissues, and also upregulated in NSCLC cell lines. A significant relationship was observed between stage progression and tRF‐Leu‐CAG in NSCLC sera. We found that in H1299 cells, inhibition of tRF‐Leu‐CAG suppressed cell proliferation and impeded cell cycle. AURKA was also repressed with the knockdown of tRF‐Leu‐CAG. Thus, our study revealed that tRF‐Leu‐CAG may be involved in regulating AURKA and could be a new diagnostic marker and potential therapeutic target in NSCLC.

MiR-181a-5p promotes anoikis by suppressing autophagy during detachment induction in the mammary epithelial cell line MCF10A.

MicroRNAs (miRNAs) are small non-coding RNAs of ∼22 nucleotides (nt) in length that bind to the 3′UTRs of their target mRNAs, leading to translational inhibition or mRNA degradation (Bartel, 2009). miRNAs regulate the expression of hundreds of genes involved in various biological processes, such as apoptosis, migration, metastasis, and autophagy (Flynt and Lai, 2008; Frankel and Lund, 2012; Ventura and Jacks, 2009). Previous studies have indicated that miRNAs may act as oncogenes or antioncogenes in tumorigenesis of various cancers (Hammond, 2015). When considering the rate of failure in cancer treatment, metastasis has been pinpointed as a major cause (Nguyen and Massague, 2007). Whereas, anoikis, a type of programmed cell death resulted from the loss of cell-matrix interactions, can suppress tumor metastases. In contrast, resistance to anoikis will enhance metastases by enabling ECM-detached cancer cells to survive (Frisch and Francis, 1994). Recently, it has been reported that autophagy promoted cell survival during detachment-induced anoikis (Fung et al., 2008). In this study, we show that miRNA miR-181a-5p modulates anoikis in human mammary epithelial cell line (MCF10A); specifically, our data demonstrate that autophagy was significantly impaired in MCF10A cells stably-expressing miR-181a-5p, and that the effects of miR-181a-5p on anoikis were reversed by overexpression of autophagy-related gene, ATG5. In summary, we report a novel regulation of anoikis that involves the repression of autophagy by miR181a-5p. The expression of miR-181a-5p was upregulated during anoikis in mammary epithelial cell line MCF10A and breast cancer cell line MCF7. We examined the expression of dozens of miRNAs during anoikis in MCF10A cell line using quantitative real-time PCR and found that the expression levels of four miRNAs (miR-181a-5p, miR-143, miR-30b, and miR-338-5p) were upregulated. Notably, the expression level of miR-181a-5p showed the most significant increase. Furthermore, the expression of miR-181a-5p was detected in both MCF10A cell line and the breast cancer cell line MCF7 at 24 h and 48 h after detachment induction. In MCF10A cells, the expression of miR-181a-5p declined obviously at 24 h after detachment induction but increased at 48 h (Fig. 1A). However, in MCF7 cell line, the expression of miR181a-5p increased at both 24 h and 48 h after detachment induction (Fig. 1B). These data indicate that the expression of miR-181a-5p can be modulated in the detachment environment. Overexpression of miR-181a-5p attenuated anoikis resistance. To examine the role of miR-181a-5p during cell detachment, we generated MCF10A and MCF7 cell lines stably expressing miR-181a-5p using a lentivirus vector containing a precursor miR-181a-5p expression cassette. As shown in Fig. 1C, the expression of miR-181a-5p increased by 175 folds in MCF10A cells stably-expressing miR-181a5p, compared with the cells transfected with the empty vector (herein termed as control cells). PARP1 is one of the major cleavage targets of caspase-3 and it has been shown that cleavage of PARP1 promotes apoptosis (Soldani and Scovassi, 2002). Therefore, we examined the expression of cleaved PARP1 in MCF10A cells overexpressing miR-181a5p and found that 48 h after detachment induction, expression levels of cleaved PARP1 were increased significantly compared with that in control cells; however, we did not observe any significant change in MCF7 cells stably-expressing miR-181a-5p (Fig. 1D and 1E). Next, we performed FACS analysis to examine the rate of apoptosis in MCF10A cells overexpressing miR-181a-5p 48 h after detachment induction (Fig. 1F and 1G). This data revealed that the rate of apoptosis in miR-181a-5p overexpressing MCF10A cells increased significantly compared with that in MCF10A control cells (Fig. 1F and 1G). Nonetheless, there was no significant difference in the rate of apoptosis between the miR181a-5p overexpressing cells and the control cells in attachment culture. In addition, we performed colony formation assays and found that after 48 h in detachment culture followed by two weeks of attachment culture, the MCF10A cells overexpressing miR-181a-5p formed fewer colonies than the control cells (Fig. 1H and 1I). These results imply that miR-181a-5p could play an important role in suppression of anoikis resistance in MCF10A cells.

Epidermal growth factor receptor (EGFR): A rising star in the era of precision medicine of lung cancer

Lung cancer is a leading cause of cancer mortality worldwide. In tumors, the important role of noncoding RNA regulatory networks has been more and more reveal. EGFR has been identified as an oncogenic driver of NSCLC, especially activating mutations EGFR and its inhibition with specific TKIs can generate dramatic tumor responses. Studies have shown that EGFR plays significant roles in the progression of NSCLC. Subset analysis of the small proportion of patients with EGFR-mutant lung cancer showed a disease-free survival benefit, but was underpowered to detect a survival advantage. Herein, we highlight the progression of EGFR, noncoding RNA, and their roles in carcinogenesis. We also focus on anti-lung cancer drug development and EGFR-related drug resistance.

A simple and fast method for profiling microRNA expression from low-input total RNA by microarray

Analysis of mature microRNA (miRNA) expression is important to understand its physiological functions and pathological implications. Microarray is a powerful technology to profile global miRNA expression. In this study, we developed a rapid miRNA labeling method by which miRNA was directly labeled in total RNA for microarray detection. This method consists of RNA tailing by poly(A) polymerase, reverse transcription, and template‐switching by moloney murine leukemia virus (MMLV) reverse transcriptase. After these reactions, the small RNA cDNA was ready for labeling for microarray detection. The whole process of prearray sample preparation was dramatically shortened to 2 h. Furthermore, this method allows very limited starting total RNA (100 ng) for microarray analysis. Our data showed that the results from our method were highly consistent with that of real‐time polymerase chain reaction (PCR). 2012 IUBMB IUBMB Life, 64(7): 612–616, 2012

Tanshinones suppress non-small cell lung cancer through up-regulating miR-137.

Great progress has been made in the treatment of lung cancer, notably a diversification in therapeutic drugs including immunotherapies such as those based on PD-1. Among these, there are many anti-disease molecules extracted from Chinese traditional medicine, such as artemisinin and tanshinones. Tanshinones, including Tanshinone I (T1), Tanshinone IIA (T2A), and cryptotanshinone (CT), are the liposoluble constituents of Salia miltiorrhiza, a traditional herb, which has favorable medicinal value. Of particular interest is the cytotoxic effect that tanshinone has on tumor cells, which is thought to occur primarily through the induction of apoptosis and interdiction of cell cycle progression, angiogenesis, cell invasion, and metastasis. Previous studies have indicated that the apoptosis-inducing effect of tanshinones might effectively target and inhibit the growth of human lung cancer cells [1]. Thus, it is reasonable to speculate that tanshinones may play an important role in the treatment of non–small cell lung cancer (NSCLC). Studies have already shown that tanshinone molecules can inhibit the growth of lung cancer cells in vitro [2], and that the mechanism may be through the interruption of cell cycle progression and induction of cell apoptosis, resulting in the up-regulation of miR137 expression. MicroRNAs (miRNAs) are a class of single-stranded small noncoding RNAs, which range from 18–23 nucleotides in length, are encoded by an endogenous gene, and regulate gene expression at the post-transcriptional level. In the tumorigenesis process, miRNAs play a regulatory role and can govern the expressions of tumorassociated genes [3]. We proposed that tanshinones might inhibit NSCLC via regulating the expression of related miRNAs and tested this hypothesis by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Our results showed that tanshinones could up-regulate the expression of miR-137, as evidenced by higher expression of miR-137 in H1299 cells incubated with 4 μM T1, 4 μM T2A, or 5 μM CT (Hao-Xuan Bio-Tech Co., Ltd, Xi’an, China) for 48 h when compared with those in the control cells incubated with 5 μM dimethyl sulfoxide only (Supplementary Fig. S1A). Results from qRT-PCR assays also showed that the expression levels of the miRNAs tested, including let-7b/c, miR-25, miR-32, miR-34a, miR-92a/b, miR-363, and miR-367, were all significantly up-regulated by tanshinones (T1, T2A, and CT). We further found that miR-137 was significantly down-regulated in H1650 (P < 0.01), A549, 95-D, SPCA-1, H358, HCC-827, PC-9, and H23 cells (P < 0.001) when compared with that in the BEAS-2B control cells (Supplementary Fig. S1B). We also found that the effect of tanshinones on NSCLC was inhibited when cells were transfected with miR-137 inhibitor (RiboBio, Guangzhou, China) (Supplementary Fig. S1C,D). These results suggested an association between NSCLC carcinogenesis and the reduction in miR-137 expression. Based on the above findings, we chose the A549 and SPCA-1 cells to investigate the effect of miR-137 on NSCLC cell proliferation. A549 and SPCA-1 cells were transfected with an miR-137 mimic, and the transfection efficiency was evaluated by qRT-PCR, showing that the miR-137 levels did in fact increase in both cell lines (Fig. 1A). Our data revealed that transfection with miR-137 mimic resulted in an inhibition of cell proliferation in both A549 and SPCA-1 cells (Fig. 1B,C). When compared with cells transfected with negative control mimic (RIBOBIO), cells transfected with miR137 mimic yielded a significant decrease of NSCLC cell growth after 72 h. Additionally, the proportion of cells at G0/G1 phase increased by >10% compared with the control 48 h post-transfection with miR-137 mimic (Fig. 1D). These results demonstrated that miR-137 could inhibit the proliferation of NSCLC cells and cause G0/G1 cell cycle arrest. Next, we used TargetScan prediction programs (www.targetscan. org) to identify and propose ULK2 and IBTK as the potential targets for miR-137. ULK2 can act upstream of phosphatidylinositol

MicroRNA-107-5p suppresses non-small cell lung cancer by directly targeting oncogene epidermal growth factor receptor

MicroRNAs (miRNAs) are dysregulated in cancers, including human non-small cell lung cancer (NSCLC). The function of MicroRNA-107-5p (miR-107-5p) in NSCLC is not fully elucidated. Epidermal growth factor receptor (EGFR) is a cancer-driven gene in tumorigenesis. In this study, we found that miR-107-5p was significantly decreased in NSCLC tissues and NSCLC cell lines. Moreover, our results indicated that miR-107-5p could suppress cell proliferation, inhibit metastasis, impede cell cycle, and promote apoptosis via directly targeting EGFR. We also investigated roles of miR-107-5p in vivo. The results showed that it could inhibit tumor growth. Therefore, our study demonstrated that miR-107-5p not only suppressed the progression in NSCLC cells by inhibiting the expression of EGFR, but also could be a promising and a new potential therapeutic target for lung cancer.MicroRNAs (miRNAs) are dysregulated in cancers, including human non-small cell lung cancer (NSCLC). The function of MicroRNA-107-5p (miR-107-5p) in NSCLC is not fully elucidated. Epidermal growth factor receptor (EGFR) is a cancer-driven gene in tumorigenesis. In this study, we found that miR-107-5p was significantly decreased in NSCLC tissues and NSCLC cell lines. Moreover, our results indicated that miR-107-5p could suppress cell proliferation, inhibit metastasis, impede cell cycle, and promote apoptosis via directly targeting EGFR. We also investigated roles of miR-107-5p in vivo. The results showed that it could inhibit tumor growth. Therefore, our study demonstrated that miR-107-5p not only suppressed the progression in NSCLC cells by inhibiting the expression of EGFR, but also could be a promising and a new potential therapeutic target for lung cancer.