Morin inhibited lung cancer cells viability,growth,and migration by suppressing miR-135b and inducing its target CCNG2

Abstract

Lung cancer is one of the most severe threats with the highest mortality rate to humans in the world. Recently, morin has been reported to have anti-tumor properties observed in several types of cancers. However, its mechanism is still unclear. We assessed the influences of morin on cell viability, colony formation, and migration ability of A549 and employed microRNA array to identify the microRNAs affected by morin. We found that morin-treated A549 cells showed statistically decreased cell viability, colony formation, and migration rate when comparing with the dimethyl sulfoxide–treated cells. Microarray results showed that with the treatment of morin, the expression level of miR-135b significantly reduced compared the control group, suggesting that morin may exert its anti-cancer property by suppressing the expression of miR-135b. In addition, we found a potential binding site of miR-135b within 3′ untranslated region of CCNG2-encoding cyclin homolog cyclin-G2. We evidenced that miR-135b directly targets CCNG2, which could be a potential biomarker of lung cancer prognosis. Morin exerts its anti-tumor function via downregulating the expression of miR-135b that directly targets and represses CCNG2.

Introduction

Lung cancer is the most leading cause of cancer mortality worldwide and kills more people every year than breast, prostate, and colon cancers combined. ¹ Regardless of the intensive effort that has been spent on developing early detection methods and novel therapeutic targets, the overall 5-year survival rate remains low. ² Therefore, development of efficient therapeutic approaches for lung cancer patient is imperative. Platinum-based chemotherapy is currently the most effective treatment for lung cancer, especially non–small cell lung cancer (NSCLC). However, the intrinsic and developed resistance to chemotherapeutic drugs lead to poor prognosis. ³ Recently, researchers began to investigate the role of natural compounds against cancers.^4,5 Morin is a flavonoid that is originally isolated from the Moraceae family. It has broad physiological effects, such as cytotoxicity, antiperglycemic and anti-diabetic activity, anti-oxidant activity, anti-inflammatory and anti-allergic activities, chemopreventive activity, inhibition of proliferation and induction of apoptosis, modulation of signaling pathways, anti-hypertensive activity, and anti-bacterial activity. ⁶ Moreover, morin has been reported to have anti-cancer properties observed in several types of cancers.^7–9 However, the effects of morin in lung cancer require further investigations.

MicroRNAs (miRNAs) are small non-coding RNA molecules (about 22 nt long) that function as post-transcriptional regulators of gene expression. ¹⁰ Many miRNAs were found to have links with cancers and are referred to as “oncomirs” or tumor “suppressors” by regulating post-transcription of cancer-related genes. ¹¹ Dysfunction of miRNA expression was observed in many types of human malignant tumors.^12,13 MiR-135b has been reported to be upregulated in several malignant tumors and promotes cancer cell proliferation by targeting tumor suppressors. For example, in colorectal cancer (CRC), miR-135b has been found to promote tumor progression by targeting tumor suppressor pathways such as phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K) pathway ¹⁴ or the transforming growth factor beta (TGF-β) pathway. ¹⁵ In glioblastoma, it was demonstrated that miR-135b affected glioblastoma cell proliferation by regulating a tumor suppressor gene GK5. ¹⁶

There are eight species of cyclins (cyclins A through H) reported in mammals. Cooperating with cyclin-dependent protein kinase (CDK) inhibitors, cyclins influence eukaryotic cell cycle through regulating CDKs. The unconventional cyclin homolog cyclin-G2 is a protein encoded by the CCNG2 gene in humans. CCNG2 has been reported as a tumor suppressor in variety of cancers.^17,18 However, the mechanism of CCNG2 in lung cancer is still unclear. Here, we found that morin plays an important function to inhibit lung cancer progression by repressing miR-135b and then promotes the expression of tumor suppressor CCNG2.

Materials and methods

Cell culture and transfection

The lung cancer cell line A549 was purchased from the American Type Culture Collection (ATCC, Dallas, TX, USA). Cells were cultured in RPMI (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), penicillin (100 U/mL), and streptomycin (100 µg/mL) in a humidified atmosphere of 5% CO₂ at 37°C. Cell transfections were performed using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. This study was approved by the ethics committee of Affiliated Second Hospital, Mudanjiang Medical University.

Cell viability analysis

Cell Counting Kit-8 (CCK-8; Dojindo Molecular Laboratories, Kumamoto, Japan) was used to examine the cell proliferation. A volume of 2 × 10³ cells/well were plated on 96-well plates and cultured in 100 µL of RPMI medium for 24 h. Then, 10 µL of CCK-8 was added to each well and then the plate was incubated for 2 h at 37°C. Absorbance was measured at 450 nm using a microplate reader (Tecan, Switzerland).

Affymetrix miRNA 4.0 array

Total RNA was analyzed using an Affymetrix GeneChip miRNA Array v. 4.0 (Affymetrix, Santa Clara, CA, USA). A volume of 275 ng of total RNA was labeled with biotin using a 3DNA Array Detection FlashTag^™ Biotin HSR Kit (Genisphere, Hatfield, PA, USA) following the manufacturer’s instructions. The GeneChip^® miRNA 4.0 arrays contain 30,424 total mature miRNA probe sets and were washed and stained using the Affymetrix GeneChip Hybridization Wash and Stain Kit. The samples were scanned with the Affymetrix GeneChip Scanner 3000 7G (Affymetrix).

Cloning formation and transwell migration assay

Cloning formation assay was performed as described by Cifone et al. ¹⁹ Cultures were coded and scored in a blinded fashion by a second observer. For transwell migration assays, 2.5 × 10⁴ cells were plated in the top chamber with the non-coated membrane (24-well insert; pore size: 8 mm; BD Biosciences, Franklin Lakes, NJ). In this assay, cells were plated in serum-free medium with morin or dimethyl sulfoxide (DMSO), and the medium supplemented with serum was used as a chemoattractant in the lower chamber. After 24 h of incubation, cells on the lower surface of the membrane were stained with crystal violet.

Luciferase activity assay

Stable A549 cell lines that constitutively express human wild type (WT) and mutated (mut) CCNG2 reporters were generated as described below. Both the WT and mut 3′ untranslated region (3′-UTR) of CCNG2 were amplified from human complementary DNA (cDNA) by polymerase chain reaction (PCR). The purified PCR product was digested by restriction endonuclease SacI and HindIII. The digested fragments were inserted into pMIRREPORTER and validated by sequencing. A549 cells were co-transfected with WT or mut CCNG2 3′-UTR reporter plasmids and pRL-TK. pCIneo vector control or miR-135b plasmid was co-transfected with the Renilla luciferase plasmid (phRL-TK; Promega, Madison, WI, USA). After 36 h of transfection, Dual-Luciferase Reporter Assay System (Promega) was used to measure the firefly and Renilla luciferase activities, according to the manufacturer’s protocol.

Clinical specimen collection and messenger RNA extraction

Tumor specimens and patient information were collected with patients’ informed consents from Affiliated Second Hospital and Affiliated Hongqi Hospital, Mudanjiang Medical University, China. Tumor specimens were stored in liquid nitrogen for overnight and then smashed with mortar. Total RNAs were extracted from smashed specimens using TRIzol reagent (Invitrogen).

Quantitative reverse transcription PCR

Total RNA was isolated using TRIzol reagent (Invitrogen) according to the standard protocol. The CCNG2 messenger RNA (mRNA) expression levels were analyzed using TaqMan MicroRNA Assays (Applied Biosystems, Foster City, CA, USA). Specifically, 1 µg of total RNA was reverse transcribed via PrimeScript II first Strand cDNA Synthesis Kit (TaKaRa, Japan) according to the manufacturer’s instructions. The cDNA was amplified with a TaqMan 2× Universal Master Mix (Applied Biosystems, Waltham, MA, USA), while the miRNA-specific real-time PCR was performed using an ABI 7500 Real-Time PCR system. The miR-135b expression was normalized to that of RNU6B, and CCNG2 expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Western blot analysis

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA, USA) with vortex every 5 min for 30 min in total. The cell lysates were centrifuged at 12,000 r/min for 15 min at 4°C, and the supernatant was collected. Protein concentration was measured by the Bradford protein assay. Denatured proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The transferred membranes were blocked with 5% milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature and then incubated with CCNG2 antibody (Cell Signaling Technology) for overnight at 4°C. The membranes were washed for three times with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies in TBST for 1 h at room temperature. After washing three times with TBST, the immunoblotting signals were detected via the chemiluminescence method (ECL; Millipore).

The Kaplan–Meier plotter

NSCLC patients in the KM plotter database come from the Kaplan–Meier plotter (http://kmplot.com/analysis/). The database was established using gene expression data and survival information of NSCLC patients. The CCNG2 was entered into the database (http://kmplot.com/analysis/) to obtain Kaplan–Meier survival plots. mRNA expression of certain genes above or below the median separates the cases into high expression and low expression. Hazard ratio (and 95% confidence intervals) and log-rank p were calculated and displayed on the webpage.

Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism software (San Diego, CA) and described as mean ± standard deviation (SD). Significant differences were determined using the one-way ANOVA for three treatment groups, and Student’s t test was applied for comparing two groups. Statistical significance was considered as p < 0.05.

Results

Morin inhibits lung cancer cell viability, growth, and migration

To identify whether morin has inhibitory functions on lung cancer cells, we treated A549 cells with morin or DMSO. Using CCK-8 assay to detect cell viability, we found that A549 cells treated with morin showed statistically decreased cell viability compared to those treated with DMSO (Figure 1(a)). To investigate the impact of morin on cell growth ability, we then measured the cloning efficiency of A549 cells (treated with morin or DMSO) by counting clone number growing on soft agar. It was observed that cells treated with morin showed significantly lower cloning efficiency compared with the control group (treated with DMSO; Figure 1(b)). As morin has been reported to exhibit an inhibitory effect on epithelial–mesenchymal transition (EMT) process of breast cancer, we hypothesized that morin might inhibit the migration of lung cancer cells. To confirm our hypothesis, transwell migration assay was performed (Figure 1(c)). We found that morin-treated cells showed statistically decreased migration rate when compared to the DMSO-treated group. These results indicate that morin might inhibit lung cancer cell viability, growth, and migration.

Figure 1.

Morin inhibits cell viability, growth, and migration of lung cancer cells. A549 cells were treated with 50 µM of morin or DMSO. (a) CCK-8 assay was used to detected cell viability. (b) Cloning efficiency was measured by counting clone number growing on soft agar. (c) Migration assay was performed to compare the migration ability between A549 cells treated with and without morin. Data were presented as mean + SD from three independent experiments with triple replicates per experiment (**p < 0.01 represents significant difference compared to DMSO group).

Morin suppresses miR-135b in lung cancer cells

To investigate the mechanisms of morin regulating lung cancer cell viability, growth, and migration, we checked miRNAs expression levels of A549 by microarray after 24 h of treatment with morin. The microarray results showed that with the treatment of morin, the expression level of a previously reported “oncomirs”—miR-135b—reduced to only half of the control group. In addition, miR-135b was overexpressed in comparison to other miRNAs in the heat map (Figure 2(a)), which is consistent with the previous study that miR-135b is highly expressed in lung cancer. ²⁰ To validate the microarray results, we analyzed the miR-135b expression levels by quantitative reverse transcription PCR (qRT-PCR) at different time points after morin treatment (Figure 2(b)). The time course study further confirmed the microarray results that the expression levels of miR-135b became persistently reduced from 0–48 h after morin treatment. In addition, we confirmed the expression levels of other three miRNAs (miR-26, miR-137, and miR-144) by qRT-PCR, as they showed relatively decreased, elevated, and/or unchanged expression levels after treatment with morin. The qRT-PCR results were consistent with that of the microarray: Under the administration of morin, only miR-135b expression underwent highest decrease, while the other three miRNAs showed changes no more significant that of miR-135 (Supplementary Figure S1).

Figure 2.

Morin suppresses miR-135b in lung cancer cells. (a) A549 cells were treated with 50 µM of morin or DMSO, and after 24 h, the expression levels of miRNAs were checked by microarray. (b) A549 cells were treated with 50 µM of morin or DMSO for 0, 12, 24, and 48 h, and the miR-135b expression levels were analyzed by qRT-PCR. Data were presented as mean + SD from three independent experiments with triple replicates per experiment (**p < 0.01 represents significant difference compared to control group.

As miR-26 expression slightly decreased in both microarray and qRT-PCR results, we observed the impact of miR-26 on lung cancer cells by performing the cell viability and migration assays. We found that compared with the miR-NC-transfected control group, miR-26-transfected A549 showed relatively promoted cell viability (Supplementary Figure S2A) and migration (Supplementary Figure S2B). However, the influence of miR-26 on cell viability and migration is no more dramatic than that of miR-135b. These results illustrated that morin might suppress the expression of miR-135b in lung cancer cells.

MiR-135b directly targets CCNG2

Although miR-135b has been reported to target several genes, such as THBS2 ²¹ and YKT, ²² we hope to explore the connections between miR-135b and tumor-associated genes. We found that there is a potential binding sites of miR-135b within CCNG2 3′-UTR. We seeded the matching sites or mutant sites (red) between these two sequences (Figure 3(a)). The luciferase reporter assay was performed on A549 to detect the relative luciferase activities of WT and mutant CCNG2 reporters. Luciferase activity of CCNG2 was significantly repressed by miR-135b compared to miR-NC, whereas mutated miR-135b showed no effect on CCNG2 transcription compared with miR-NC (Figure 3(b)). We then transfected A549 cells with miR-135b or control miR-NC mimics. After 48 h, the expression levels of CCNG2 were analyzed by qRT-PCR and western blotting (Figure 3(c) and (d)). Overexpression of miR-135b led to significant increases of both mRNA and protein expression levels of CCNG2 compared to the control miR-NC mimics. Based on these results, we speculated that miR-135b might directly target CCNG2. To confirm our speculation, we determined the expression levels of miR-135b and CCNG2 in human lung cancer specimens (n = 41) via qRT-PCR (Figure 3(e)) and used Spearman’s correlation analysis to identify the correlation between the expression levels of miR-135b and CCNG2 in human lung cancer specimens. We found that the expression levels of CCNG2 were negatively correlated with that of miR-135b. These results demonstrate that CCNG2 is the direct target of miR-135b.

Figure 3.

MiR-135b directly targets CCNG2. (a) Putative seed-matching sites or mutant sites (red) between miR-135b and 3′-UTR of CCNG2. (b) Luciferase reporter assay was performed on A549 to detect the relative luciferase activities of WT and mut CCNG2 reporters. (c and d) A549 cells were transfected with miR-135b or control miR-NC mimics. After 48 h, the expression levels of CCNG2 were analyzed by qRT-PCR and western blotting. Data were presented as mean + SD from three independent experiments with triple replicates per experiment (** p < 0.01 represents statistical significance). (e) The expression levels of miR-135b and CCNG2 in human lung cancer specimens (n = 41) were determined by qRT-PCR analysis, and Spearman’s correlation analysis was used to determine the correlation between the expression levels of miR-135b and CCNG2 in human lung cancer specimens.

CCNG2 expression levels are induced by morin and acts as a positive biomarker of lung cancer prognosis

As we have found that in lung cancer cells, morin might suppress the expression of miR-135b, and CCNG2 was the direct target of miR-135b, we further investigated whether morin could influence the expression of CCNG2. We treated A549 with morin and analyzed the mRNA expression levels of CCNG2 at different time points (Figure 4(a)). We found that with the treatment of morin, CCNG2 mRNA expression levels consistently increased from 0–48 h. We then used Kaplan–Meier plotter to analyze the prognosis of lung cancer patients with high (red: n = 568) or low expression (black: n = 577) of CCNG2 (Figure 4(b)). It showed that patients with high CCNG2 expression had higher overall survival probability than those with low CCNG2 expression. This indicated that CCNG2 expression could be influenced by morin and acts as a positive biomarker of lung cancer prognosis.

Figure 4.

CCNG2 expression level is induced by morin and acts as a potential biomarker of lung cancer prognosis. (a) A549 cells were treated with 50 µM of morin for 0, 12, 24, and 48 h, and the CCNG2 expression levels were analyzed by qRT-PCR. Data were presented as mean + SD from three independent experiments with triple replicates per experiment (**p < 0.01 represents significant difference). (b) The Kaplan–Meier plotter was employed to analyze the prognosis of lung cancer patients with high (red: n = 568) or low expression (black: n = 577) of CCNG2.

As CCNG2 is involved in cell cycle regulation, we investigated whether other cell cycle member, such as the cell cycle kinase CDC2, can be affected by morin. We then treated A549 with 50 µM morin or DMSO and checked the expression level of CDC2 by qRT-PCR after 24 h. We also detected the protein levels of CDC2 by western blotting after 48 h of treatment. The results showed that the administration of morin reduced the expression of CDC2 at both the mRNA and protein levels (Supplementary Figure S3). These results support the involvement of morin in cell cycle regulation.

Overexpression of miR-135b reverses the suppression of morin in lung cancer

To further investigate whether the suppression of morin in lung cancer indeed functions via miR-135b, we transfected the morin-treated A549 cells with miR-135b mimics, or control. The western blotting results demonstrated that morin caused significant increase of CCNG2, and the addition of miR-135b reversed this phenomenon (Figure 5(a)). We used the CCK-8 assay to detect the cell viability at different conditions. The results showed that morin treatment alone led to significantly reduced cell viability compared to the control group (Figure 5(b)). However, overexpression of miR-135b with morin treatment together completely rescued the cell viability. We further studied the effect of morin and miR-135b on lung cancer cells using cloning formation assay. As shown in Figure 5(c), by comparing to the control group, the decreased cloning number caused by morin treatment could be reversed by the induction of miR-135b. In addition, the transwell assay demonstrated that when treating A549 with morin alone, the cells showed less migration rate compared to the control group, while transfecting the morin-treated cells with miR-135b, they showed the similar migration rate as the control group (Figure 5(d)). The results well support our hypothesis that overexpression of miR-135b could reverse the suppression of cell viability, growth, and migration by morin treatment, which indicates that the suppression ability of morin functions by regulating miR-135b and its target CCNG2.

Figure 5.

Overexpression of miR-135b reverses the suppression of morin in lung cancer. (a) A549 cells were transfected with miR-135b mimics, or control, and were treated with 50 µM morin or DMSO. After 48 h, the expression levels of CCNG2 were analyzed by western blotting. (b) CCK-8 assay was used to detected cell viability. (c) Cloning efficiency was measured by counting clone number growing on soft agar. (d) Migration assay was performed as described (**p < 0.01 indicates significant difference compared to miR-NC + DMSO group; ^##p < 0.01 indicates significant difference compared to miR-NC + morin group).

Discussion

Lung cancer is a multifactorial and complicated disease commanded by uncontrolled proliferation of malignant cancer cells, resulting from the unbalanced expression of oncogenes and tumor suppressor genes. Loss of function of tumor suppressors or overexpression of oncogenes leads to dysregulated cellular signaling cascades which result in the initiation and progression of cancer. ²³ Even though the chemotherapy displayed significant clinical activity against a variety of solid tumors, especially lung cancers, its clinical efficacy is hindered in many patients due to both intrinsic and acquired resistance to the drug.^24,25 To find an agent that is affordable, safe, and effective against cancer is the major goal of anti-cancer drug discovery. Thus considering the diverse bioactivity of the ingredients isolated from natural sources, the natural products targeting multidrug resistance of cancer cells have been studied in depth. ²⁶

The flavonoid morin, originally isolated from the Moraceae family, has been reported to display a variety of biological actions, such as anti-oxidant, anti-inflammatory, and anti-carcinogenic abilities. Morin impedes tumor progression in many cancer types. For example, Sivaramakrishnan et al. ²⁷ claimed that in hepatocellular carcinoma (HCC), morin has anti-cancer properties if the cancer results from diethyl maleate (DEM) induction. Chung et al. ⁹ found that combined with the telomerase inhibitor MST-312, morin could be a novel targeted therapy drug for improved cancer prognosis. Singh et al. ²⁸ demonstrated that morin plays a protective role against acrylamide (AA)-induced hepatotoxicity. They evaluated the levels of DNA-damage-related markers, the oxidative stress, as well as the hepatic inflammation. They found that even the AA induced severe hepatic injury, the combined administration of AA and morin lead to statistically significant improvement in all analyzed parameters. In addition, the group treated with highest concentration of morin showed the most obvious recovery.

However, in our study, we found that morin can largely impede the cell viability and proliferation of lung cancer cells. By using transwell migration assay, we showed that morin possessed the property to block lung cancer cell migration. The mechanism of morin to inhibit cancer progression has been under investigation. Gupta et al. ²⁹ found that morin was a potential compound to inhibit cancer cell growth through repressing the signal transducer and activator of transcription 3 (STAT3) pathway that is related to cell survival, proliferation, transformation, and cancer metastasis. Activated Src, Janus kinase 1 (JAK-1), and JAK-2 were inhibited by morin, all of which participate in STAT3 signaling pathway, while PLAS3, a protein inhibitor of STAT3, was upregulated by morin treatment. This suggests that morin suppresses tumor progression by repressing the STAT3 and downregulating STAT3-dependent gene expression.

MiRNAs are small non-coding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression. ¹⁰ Many of them were found to be “oncomirs” or tumor “suppressors” by regulating post-transcription of cancer-related genes. ¹¹ To investigate whether morin suppresses tumor cell growth via regulating miRNA functions, we used miRNA array to test the lung cancer cell line A549 and found that morin obviously suppressed the expression of miR-135b, which is a well-known oncogene. MiR-135b has been reported as an important downstream component in oncogenic pathways. ³⁰ Li et al. ¹⁵ revealed that more than half of HCC clinical specimens expressed high level of miR-135b, which was correlated with its genomic content. They also found that heat shock factor 1 (HSF1) level was positively related with the miR-135b expression in the HCC clinical specimens without miR-135b amplification. Moreover, miR-135b was reported to target tumor suppressors in diverse cancer types.^14,16 For example, Li et al. reported that the TGF-β receptor II (TGFBR2) was a target of miR-135b, through which miR-135b promoted CRC progression. ¹⁵ Yu et al. ¹⁶ revealed that miR-135b was related with the carcinogenesis and progression of glioblastoma and that the tumor suppressor GK5 was a target of miR-135b. As a tumor suppressor, GK5 was known to inhibit glial cell proliferation and be related to glioblastoma cell invasion. Thus, miR-135b and restoration of GK5 may represent a potential therapeutic strategy for patients with glioma.

CCNG2 has been revealed to regulate cell growth and proliferation as a tumor suppressor, and its reduced expression is correlated with diverse types of malignant tumors.^31–34 As CCNG2 was found to be a tumor suppressor in lung cancer, we hypothesized that CCNG2 might be a target of miR-135b in lung cancer. To confirm our hypothesis, we seeded the matching sites or mutant sites (red) between the miR-135b and 3′-UTR sequences of CCNG2 to investigate the relationship between miR-135b and CCNG2. Luciferase reporter assay showed that CCNG2 was the direct target of miR-135b. The quantitative PCR (qPCR) and western blotting further demonstrated that miR-135b suppressed the expression of CCNG2. Moreover, 41 cases of human lung cancer clinical specimens demonstrated the negative correlation between miR-135b and CCNG2 expression. Besides, we found that morin could cause enhanced expression levels of CCNG2. Notably, by using Kaplan–Meier plotter analysis, we found that lung cancer patients expressing high levels of CCNG2 had a longer overall survival rate compared to those with low CCNG2 expression level. This indicates that CCNG2 could be a potential positive biomarker of lung cancer prognosis.

In conclusion, our findings indicate that morin might be an efficient anti-cancer drug for lung cancer therapy. Morin might possess its anti-cancer property by suppressing the expression of an “oncomirs” miR-135b, which directly targets a tumor suppressor CCNG2, and CCNG2 could be a positive biomarker of lung cancer prognosis.