Qilan preparation (芪藍顆粒) inhibits proliferation and induces apoptosis by down-regulating microRNA-21 in human Tca8113 tongue squamous cell carcinoma cells

DING Jiamin,XING Yifeng,CHEN Zuoliang,CHEN Wanlu,MA Zhongxiong,XIE Yunde,ZHOU Lin

DING Jiamin,CHEN Wanlu,MA Zhongxiong,XIE Yunde,Department of Oral Mucosal Diseases,Affiliated Stomatological Hospital of Fujian Medical University,Fuzhou 350000,China

XING Yifeng,School and Hospital of Stomatology,Fujian Medical University,Fuzhou 350000,China

CHEN Zuoliang,Department of Oral Mucosal Diseases,Xiamen Stomatological Hospital,School of Stomatology,Fujian Medical University,Xiamen 361003,China

ZHOU Lin,Department of Implantology,Affiliated Stomatological Hospital of Fujian Medical University,Fuzhou 350001,China

Abstract OBJECTIVE: The aim of this study was to examine the antitumor effects of Qilan preparation (芪藍顆粒) on oral squamous cell carcinoma (OSCC) in vitro and to investigate its underlying mechanisms of action.METHODS: Cell proliferation,cell cycle distribution and apoptosis were examined using cell counting kit-8 (CCK8)and flow cytometry (FCM).The expression of PTEN and PDCD4 were determined by western blot.Changes in miR-21 levels were quantified using TaqMan stem-loop real-time PCR.After miR-21 was transiently transfected into Tca8113 cells using Lipofectamine?3000,cell proliferation,apoptosis and miR-21 and PDCD4 expression levels were measured.RESULTS: Qilan preparation inhibited Tca8113 cell growth in a dose-and time-dependent manner by inducing apoptosis and cell cycle arrest in S-phase,decreasing miR-21 levels and increasing PTEN and PDCD4 expression.MiR-21 overexpression reversed the Qilan preparation-induced suppression of cell proliferation and induction of apoptosis while also blocking the increase in PDCD4.CONCLUSIONS: Our study revealed,for the first time,the ability of Qilan preparation to suppress TSCC cell growth and elucidated that Qilan preparation elicits its anti-cancer actions via either the miR-21/PDCD4 or PTEN pathway.

Keywords: microRNAs;squamous cell carcinoma of head and neck;apoptosis;cell cycle checkpoints;Qilan preparation

1.INTRODUCTION

MicroRNA-21 (miR-21),a genuine oncogene,5is overexpressed in a number of cancers including solid tumor like oral squamous cell carcinoma (OSCC).Recent studies have revealed that miR-21 is up-regulated in OSCC and considered to act as an apoptosis inhibitor by partly silencing tropomyosin 1 (TPM1).6MiR-21 also modulates cisplatin chemosensitivity and increases tumor cell invasion in OSCC by targeting programmed cell death 4 (PDCD4).7Additionally,miR-21 levels are reversely correlated with phosphatase and tensin homologue (PTEN),an miR-21 target gene,in OSCC.6,8PDCD4,a suppressor gene,can inhibit cancer cell growthviacell cycle arrest and the induction of apoptosis,which is under-expressed in OSCC tissues compared with normal oral tissues.

Based on these observations,we hypothesize that Qilan preparation might exert cytotoxic effects on OSCC cellsviaeither the miR-21/PTEN or miR-21/PDCD4 pathways.To test this hypothesis,we examined the association between miR-21/PTEN/PDCD4 expression and the effects of Qilan preparation on human Tca8113 TSCC cell proliferation,cell cycle and apoptosisin vitro.

2.METHODS

2.1.Plant materials

The four herbal medicines were screened individually.AR (Batch No.140304),Jiaogulan (Herba seu Radix Gynostemmatis Pentaphylli) (Batch No.140305) and CR(Batch No.140211) were purchased from the Xiamen YanLaiFu Pharmaceutical Co.,Ltd.,Fujian,China.Selenium-rich green tea (Batch No.140316) was purchased from Enshi ChunGui tea industry Co.,Ltd.,Hubei,China.They were authenticated carefully by one of the authors (Professor Huang Yiqi).The voucher specimens (AR No.Q1-20140526,Jiaogulan (Herba seu Radix Gynostemmatis Pentaphylli) No.Q2-20140526,CR No.Q3-20140526,selenium-rich green tea No.Q4-20140526) were deposited at the herbarium of the Xiamen Medicine Research Institute,Fujian,China.

2.2.Extraction and identification of Qilan preparation

In brief,the four herbs (740 g) were chopped into pieces and decocted with 8-fold volume of distilled water three times for 1 h each.After combination,the filtered and mixed suspensions from three decoctions were concentrated at 740 mg/mL under reduced pressure.Finally,the concentrated solution was diluted to a 100 mg/mL stock solution in basic medium and stored at 4 ℃after filtered through 0.45 and 0.22 μm filters.Fresh working drugs were prepared at the following concentrations in basic medium: 1.56,3.12,6.25,12.5 and 25 mg/mL.The same volume of basic medium was used as control or for the 0 mg/mL group.

The quality of Qilan preparation was measured by thin layer chromatography (TLC).The standards of astragaloside A,ginsenoside Rb1 and CR contrast material were used as positive references.

2.3.Cell culture and transfection

Human TSCC cell line Tca8113 was purchased from the ShangHai SBO Medical Biotechnology Co.LTD(Shanghai,China).Cells were cultured in RPMI1640(Hyclone,Logan,UT,USA).For functional analyses,Tca8113 cells were transfected with miR-21 mimics or the negative control RNA (NC RNA,which had the same number of basic groups but a different sequence compared with the miR-21 mimic) (GenePharma,Shanghai,China) using the Lipofectamine?3000 reagent(Invitrogen,Carlsbad,CA,USA) according to the manufacturer’s protocol.For each well in the 6-well plate,miR-21 mimic (60 nmol/L),NC (60 nmol/L) and 7.5 μL of the Lipofectamine?3000 transfection reagent was separately added to 125 μL of Opti-MEM?I medium(Gibco,Grand Island,NY,USA) and then mixed together to form the transfection complexes.The transfection complexes were added to the cells and incubated for 6 h before adding fresh medium.Twentyfour hours after transfection,the cells were exposed to Qilan preparation at a concentration of 6.25 or 0 mg/mL for an additional 24 h and then harvested for the following experiments.

2.4.Cell viability assay

Cells were seeded onto 96-well plates (Nunc,Copenhagen,Denmark) at a density of 1×104cells per well,incubated overnight,and then exposed to different concentrations of Qilan preparation in triplicate for 12,24 or 48 h,respectively.Cell viability was quantified using CCK-8 (Dojindo,Kumamoto,Japan).

2.5.Cell cycle analysis

Cells were seeded onto 6-well plates (Nunc,Copenhagen,Denmark) with 1 ×106cells per well and treated with various concentrations of Qilan preparation (1.56,3.125,6.25,12.5,or 25 mg/mL) for 12 h.After fixing the cell in cold 70% ethanol at 4 ℃ overnight,the cells were washed twice with cold PBS and then resuspended gently in propidium iodide (PI) staining solution (Beyotime,Shanghai,China),which consisted of 10 μL RNase A,25 μL PI staining solution and 0.5 mL buffer,and then incubated at 37℃ for 30 min in the dark.The samples were analyzed by flow cytometry (BD Accuri C6,San Diego,CA,USA) within 1 h of staining,and the percentage of cells in the G0/G1,S and G2/M phases were determined using FCS Express 4 plus (BD Accuri C6,San Diego,CA,USA).

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2.6.Cell apoptosis assay

Apoptosis analysis was performed according to the manufacturer’s instructions for the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences,San Diego,CA,USA).Then,the cells were harvested with 0.25%Trypsin without EDTA (Gibco,Grand Island,NY,USA),washed twice with cold PBS and resuspended in 100 μL of binding buffer containing 5 μL of FITC Annexin V and 5 μL of PI working solution at a concentration of 1×106cells/mL.Apoptosis was analyzed by flow cytometry (BD Accuri C6,San Diego,CA,USA) for at least 10 000 events within 1 h of staining.

2.7.RNA isolation,reverse transcription and TaqMan real-time PCR

Transfected or non-transfected Tca8113 cells were cultured in 6-well plates and exposed to different concentrations of Qilan preparation for 24 h.Total RNA was isolated using the TRIzol Reagent (Invitrogen,Carlsbad,CA,USA).A TaqMan stem-loop real-time PCR method was used to assess miR-21 expression using kits from Applied Biosystems (Foster City,CA,USA).

For each sample,we calculated the ΔCT value (targetreference).The mature miR-21 expression levels were quantified using an ABI Prism 7300 Sequence Detection System (Applied Biosystems,Foster City,CA,USA)with RNU44 as the normalization control 9,10.

2.8.Western blot

Transfected or non-transfected Tca8113 cells were cultured in 60-mm dishes and were exposed to different concentrations of Qilan preparation for 24 h.The cells were lysed in cell lysis buffer for 5 min at 4 ℃ and centrifuged at 12 000g(Sigma 1-15 PK,Berlin,German)for 30 min at 4 ℃.Protein concentration was determined with a BCA kit (Beyotime,Shanghai,China).After washing three times with TBST for 5 min each,the membranes were incubated with the secondary antibody conjugated to horseradish peroxidase (all diluted 1∶5000,Proteintech,Wuhan,China).After the membranes were washed three more times with TBST for 5 min each and then the proteins were detected using the enhanced chemiluminescence (ECL) Western Blot Detection Kit.Band intensities were measured with the AlphaView SA software (Alpha Innotech,San Leandro,CA,USA).Relative PDCD4 and PTEN protein expression was calculated relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

2.9.Statistical analysis

Data are presented as the mean ± standard deviation of three independent experiments.Statistical significance was determined by Student’s test or one-way analysis of variance followed by the least significant difference or Dunnett’s T3 multiple comparison test assuming homogeneity of variances or non-assumed homogeneity of variances using the SPSS 19.0 (IBM Corp.,Armonk,NY,USA) software.Descriptive and variance homogeneity tests were also performed.aP ＜0.05,bP ＜0.05,andcP ＜0.05 indicate statistical significance difference.

3.RESULTS

3.1.Effect of Qilan preparation on Tca8113 cell viability

The qualitative results of Qilan preparation by TLC was presented in the Supplementary file.Cell viability,determined by the CCK-8 assay,was significantly reduced at 1.56,3.12,6.25,12.5 and 25 mg/mL Qilan preparation (P ＜0.05) after treatment for 12,24 and 48 h,respectively (Table 1).

Table 1 Effect of Qilan preparation on Tca8113 cell viability in vitro

3.2.Qilan preparation induced apoptosis and S-phase cell cycle arrest in Tca8113 cells

The decrease in cell viability due to the Qilan preparation could be explained,in part,by the increased apoptosis determined by Annexin V-FITC and PI staining using flow cytometry.In the scatter plot from the double variable flow cytometry,the Q3 quadrant (FITC -/ PI -)shows living cells,the Q2 quadrant (FITC +/PI +) shows late apoptotic cells and the Q4 quadrant (FITC +/PI -)represents early apoptotic cells.In our study,the apoptosis rates include both the early and the late apoptotic cells.As assessed by flow cytometry and shown in Figure 1A and 1B,the number of apoptotic cells was significantly increased from 15.04% ± 1.67% to 69.74%± 4.63% when treated with concentrations of the Qilan preparation ranging from 3.12 to 25 mg/mL for 24 h compared with the control group (4.71% ± 1.65%,P ＜0.05).In addition,the reduction in the cell viability was associated with the S-phase cell cycle arrest.When cancer cells were treated with 3.12,6.25,12.5 or 25 mg/mLQilan preparation for 12 h,respectively,the number of cells in S-phase steadily increased from 38.64% ± 1.17%to 65.40% ± 1.96% compared with the untreated control cells (35.81% ± 1.16%,P ＜0.05) (Figure 3C,3D).These data indicate that the Qilan preparation was able to reduce cell viability by inducing apoptosis and blocking cell cycle progression.

3.3.Effect of Qilan preparation on the expression of miR-21 and its target proteins,PTEN and PDCD4,in Tca8113 cells

Using a TaqMan assay,we discovered a dose-dependent down-regulation of miR-21 after treatment with the Qilan preparation at concentrations ranging from 3.12 to 25 mg/mL compared with the control group (P ＜0.05,Figure 2A).

The inhibition of miR-21 expression was associated with an increase in PDCD4 and PTEN (Figure 2B,2C),two miR-21 targets.Peak stimulation of PDCD4 (2.48 ±0.12-fold) and PTEN (1.51 ± 0.06-fold) occurred at 6.25 and 12.5 mg/mL,respectively,with no further increases observed at the higher concentrations.There were still significantly increasing PDCD4 and PTEN levels at the higher concentrations compared with the control group(P ＜0.05,Figure 2B,2C).As tumor suppressor genes,PDCD4 and PTEN function as pro-apoptosis factors and are targeted by miR-21,at least partly explaining the apoptosis-inducing effect of the Qilan preparation.All of these data suggest a functional role of the Qilan preparation mediated by miR-21.

Figure 1 Effect of Qilan preparation on Tca8113 cell apoptosis and cell cycle

Figure 2 Effect of Qilan preparation on mir-21 expression and PDCD4 and PTEN in Tca8113 cells

3.4.Downregulation of miR-21 may contribute to Qilan preparation-induced cell growth inhibition and apoptosis induction

There were five groups including Control,NC RNA,Qilan preparation,NC RNA+Qilan preparation and miR-21+Qilan preparation group.Basic medium was used as a blank control (Control group).NC RNA group was used as a negative control.Qilan preparation group was used as a positive control.As shown in Figure 3A,we over-expressed miR-21 by approximately 14-fold compared to the NC RNA group and 29-fold compared to the NC RNA+Qilan preparation group with a miR-21 transfection mimic for 24 h before treatment with the Qilan preparation.Based on the results from the earlier experiments,we used 6.25 mg/mL as the effective concentration for the reverse test.A CCK-8 and apoptosis assay and Western blot were performed to determine the response of Tca8113 cells to miR-21 and the Qilan preparation.As shown in Figure 3B-D,overexpression of miR-21 partially reversed the antiproliferative and pro-apoptosis effects of the Qilan preparation.Compared with the NC RNA+Qilan preparation group (55.75% ± 3.63%),the cell viability of miR-21+Qilan preparation group was increased to 71.08%± 8.72%,but was still lower than the NC RNA group(97.83% ± 5.63%,P ＜0.05).In contrast,the percentage of cells undergoing apoptosis in the miR-21+Qilan preparation group decreased to 13.27% ± 0.44%compared with the NC RNA+Qilan preparation group(22.72% ± 1.52%),but was still higher than the NC group(P ＜0.05).Similar to these results,the expression level of the PDCD4 protein in the miR-21+Qilan preparation group was between that of the NC RNA group and the NC RNA+Qilan preparation group (P ＜0.05) (Figure 3E,3F).These data confirm the finding that miR-21 is a potential target of the Qilan preparation and that overexpression of miR-21 partly antagonized the anti-tumor actions of the Qilan preparation in human Tca8113 cellsin vitro.

Figure 3 Downregulation of miR-21 partially contributes to Qilan preparation-induced cell growth inhibition and apoptosis induction

4.DISCUSSION

According to modern medicine,cancer is well known as a disease of apoptosis deregulation and cell cycle dysfunction.Several reports have documented that TCM inhibits the growth and proliferation of various cancer cells through inducing apoptosis and/or cell cycle arrest.11Our present data reinforce this point and confirm the cytotoxicity of the Qilan preparation in human Tca8113 TSCC cells.We found that Qilan preparation treatment significantly inhibited Tca8113 cell viability in a dose-and time-dependent manner as determined by the CCK-8 assay.Moreover,Qilan preparation caused a dose-dependent induction of apoptosis and an accumulation of S phase cells,suggesting that the induction of apoptosis and the blockage of cells in Sphase are underlying mechanisms for Qilan preparationmediated growth inhibition.We hypothesize that Qilan preparation regulates growth arrest in S-phase by preventing proper DNA repair that causes the cell to undergo apoptosis.However,further study is necessary to determine the exact mechanism.

Medinaet al5demonstrated that miR-21 was a genuine oncomiR and that tumors could become addicted to oncomiRs,suggesting that the pharmacological inactivation of miRNAs such as miR-21 could be used to treat human cancers.This demonstration of the oncomiR addiction of miR-21 has far-reaching consequences for oncotherapy and suggests that miR-21 is an important therapeutic target for anti-tumor agents.Because overexpression of miR-21 affected tumor progression by down-regulating various target tumor suppressor genes in OSCC12,it is reasonable to hypothesize that Qilan preparation could regulate miR-21 expression in OSCC.In this study,we found that Qilan preparation treatment significantly decreased the abundance of miR-21 in Tca8113 cellsin vitro;similar results were observed in the cell viability and apoptosis assays.This corroborates with previous data that showed that miR-21 upregulation indicates poor prognosis and is an apoptosis inhibitor in TSCC.6To further confirm that miR-21 plays a role in the Qilan preparation mechanism in Tca8113 cells,we over-expressed miR-21 prior to Qilan preparation treatment and found that the cell viability and apoptosis were partially but significantly reversed.Because TCMs can have multiple targets and multifaceted functions,it is possible that there are other currently unknown target genes of the Qilan preparation responsible for the anticancer effects aside from just miR-21.However,our data suggest an anticancer function of the Qilan preparation mediated by miR-21.

It has been suggested that a single miRNA can simultaneously regulate a great number of target genes.13MiR-21 regulated genes include PDCD4,PTEN,TPM1 and CDK2AP1 in OSCC.PDCD4,a novel tumor suppressor gene,was originally identified to be upregulated in apoptotic cells14and has also been reported to be involved in apoptosis in cancer cell lines when those cells are exposed to anticancer agents.15PDCD4,which is under-expressed in OSCC,also appears to have an important role in the inhibition of cell cycle progression at S-phase.16PTEN functions as a protein phosphatase and mediates cell growth and apoptosisviathe phosphatidylinositol-3,4,5-trisphos-phate (PIP3)pathway.17,18Thus,we examined the contributions of the PDCD4 and PTEN proteins,and found that Qilan preparation treatment increased the expression of these two proteins in Tca8113 cells.Restoration of miR-21 levels significantly but incompletely blocked the increase in PDCD4 levels.One explanation for this observation is that one gene may be regulated by many miRs and that miR-21 may only be one of many factors that the Qilan preparation affects.Wanget al19demonstrated that ursolic acid (UA),a naturally occurring pentacyclic triterpene,exhibited potent proliferation inhibition and apoptosis induction in the human glioblastoma cell line U251 by suppressing the TGF-β1/miR-21/PDCD4 pathway.Berberine,a naturally occurring isoquinoline alkaloid that can be extracted from many medicinal herbs,has been reported to induce apoptosis in human multiple myeloma cells by decree-sing miR-21 levels.20Another report revealed that Matrine,one of the major alkaloids extracted from Kushen (Radix Sophorae Flavescentis),could inhibit breast cancer growthviathe miR-21/PTEN/Akt pathway in MCF-7 cells.21In accordance with previous studies,our data show that miR-21 is a molecular target involved in the regulation of apoptosis in cancer cell lines and rein-forces the notion that miRNAs,such as miR-21,can act as mediators of herbal medicines,by regulating target genes.Numerous studies have suggested that some traditional medicine extracts can induce tumor regression by arresting cell cycle at S phase.22Consistent with these previous papers,our cell cycle analysis data showed that the Qilan preparation caused a strong S-phase arrest in Tca8113 cells after 12 h treatment;however,the exact molecular mechanisms remain to be clarified.Previous results showed that reintroduction of PDCD4 could induce S-phase arrest of cell cycle in cancer cell lines.16,23

In our study,Qilan preparation induced S-phase arrest and increased the PDCD4 levels.However,overexpression of miR-21 partially reversed the up-regulation of PDCD4,but the portion of S-phase arrested cells induced by treatment with the Qilan preparation did not change (data not shown).The reason may be due to cell-type specificity of the PDCD4 actions.24For example,Jansenet al25reported that PDCD4 may enhance geldanamycin-induced G2-M arrest in UO-31 renal cancer cells.In a different ovarian cancer cell line,overexpression of PDCD4 induced cell cycle arrest as exemplified by an increased number of cells in the G2 or S phases.23However,in the colon carcinoma cell line RKO,PDCD4 did not alter cell cycle progression.26Taken together,it is possible that PDCD4 is not the direct molecular target of S-phase cell cycle arrest due to Qilan preparation treatment and further studies are needed to determine the exact mechanism of action.

In conclusion,we demonstrated in this study that Qilan preparation exhibits anti-proliferative and pro-apoptotic activities,as well as inducing S-phase cell cycle arrest in human Tca8113 TSCC cells.These results are produced,at least in part,by the preparation’s actions on the miR-21/PDCD4 or PTEN pathway (Figures 2,3).These findings help us to understand the effects and molecular mechanisms of the anti-cancer activity of Qilan preparation,which will aid in its application in the clinic.