（复旦大学遗传工程国家重点实验室-生命科学学院遗传学研究所 上海 200438）

（复旦大学遗传工程国家重点实验室-生命科学学院遗传学研究所 上海 200438）

目的研究miR-132在非小细胞肺癌（non-small cell lung cancer，NSCLC）细胞系H1299中的作用及相应的靶基因。方法real-time PCR检测7种细胞系中miR-132的表达量。用CCK-8检测稳定株细胞H1299-p EGP-miR-132与H1299-p EGP-miR-null的生长速率的变化。使用稳定株细胞H1299检测miR-132对于细胞克隆形成、细胞迁移和细胞侵袭的影响。用Transwell结合matrigel的方法检测H1299-p EGP-miR-132细胞与H1299-p EGP-miR-null细胞在72 h时细胞迁移和侵袭的变化，同时在H1299-p EGP-miR-132细胞中使用anti-miR-132进行了相关的实验验证。运用生物信息学方法预测miR-132的靶基因，并运用双荧光实验、real-time PCR和Western blot验证。在H1299-p EGP-miR-132细胞中共同转染anti-miR-132和PTCH1 siRNA检测其对细胞的增殖、迁移和侵袭的影响。结果miR-132在肺癌细胞系中呈现高表达，在NSCLC细胞H1299中miR-132通过靶向调节PTCH 1基因的表达发挥作用。H1299-pEGP-miR-132细胞的增殖（P＜0.01）、迁移（P＜0.05）、侵袭（P＜0.05）能力明显高于对照组。在H1299-pEGP-miR-132细胞中转入PTCH 1 siRNA后细胞增殖能力升高（P＜0.05），迁移和侵袭能力降低（P＜0.001）。H1299-p EGP-miR-132细胞15天形成的克隆数目是对照组细胞的1.41倍（P＜0.05）。Western blot法检测显示miR-132使H1299中PTCH1蛋白表达量显著降低（0.68倍），anti-miR-132使PTCH1表达量显著升高（1.97倍）。结论miR-132可能参与NSCLC的细胞增殖、迁移和侵袭。它的致癌性部分归因于对Hh信号通路关键的负调控蛋白PTCH1的调节。

miR-132； hedgehog信号通路； 非小细胞肺癌； PTCH 1； 细胞迁移和侵袭

*This work was supported by Shanghai Municipal Natural Science Foundation（l4ZRl403700）.

microRNAs（miRNAs）are a family of evolutionarily conserved small non-coding RNAs that constitute a prevalent gene regulation.They negatively regulate their target genes in a sequence-specific manner［1］.Recent advanced studies have shown that miRNAs regulate multitudinous biological process and repress the expression of functional cancer-related genes［2］.miRNAs associated with tumorigenesis display either tumor-promoting properties or tumor-suppressing characteristics.For instance，miR-212 in lung cancer［3］and miR-372/373 in testicular germ cell tumor［4］act as oncogenes.But on the contrary，miR-34a in neuroblastoma cells［5］and miR-122 in hepatocellular carcinoma［6］play the part of tumor suppressors.Lung cancer is the major cause of the cancer death all over the world，dysregulated expression of specific miRNAs could contribute to lung tumorigenesis［7］.

miR-132 is located on 17p13.3，which has been identified as an important regulator of many neurotrophic actions，including neurite outgrowth［8］，dopamine neurons differentiation［9］，recognition memory and synaptic plasticity［10］，and adult brain function［11］.In recent years，many advanced studies have showed that dysregulation of miR-132 was correlated with human disease，such as schizophrenia［11］，Alzheimer′s［12］，HIV［13］，and the most widely distributed human lung cancer［14］.However，the mechanistic role of miR-132 especially in non-small cell lung cancer（NSCLC）remains largely unknown.

Hedgehog（Hh）signaling pathway，which is composed of a series of repressive interactions［15］，was initially identified in Drosophila as a crucial mediator of several important processes during embryogenesis［16］.But its inappropriate reactivation in adults has been related to several totally different human cancers［17］.Signaling from this pathway is triggered via the Hh extracellular proteins binding to the Patched receptor（Ptch），thus inducing the suppression of inhibitory function of Ptch on Smoothened（Smo），and subsequently it activates the transcriptional activity of Gli factors［18］.In the absence of Hh ligands，the activity of Smo is restrained by Ptch，thus repressing the downstream cascade signaling［19］.Ptch1，a membrane receptor for Hh ligand，is more widely expressed and well characterized［20］.

Here，we report the identification of miR-132 in NSCLC cell.We further proved miR-132 negatively modulates PTCH 1 expression in NSCLC cell directly，indicating that miR-132 may have an important role in Hedgehog pathway.Our investigations will be helpful to illuminate the functions of miRNA and their impact on cell proliferation，migration and invasion.

Materials and Methods

Cell cultureHuman NSCLC cell line H1299（American Type Culture Collection，Manassas，VA，USA）and bulk-selected H1299 cells were grown in RPMI 1640 medium，supplemented with 10%FBS，2.383 mg/m L HEPES and 0.11 mg/mL sodium pyruvate at 37℃in a humidified atmosphere of 5% CO2.All cell culture media and reagents［the RPMI 1640 medium，fetal bovine serum（FBS），HEPES，Non-Essential Amino Acids and Sodium Pyruvate］were obtained from Invitrogen（Carlsbad，CA，USA）.

Expressing constructsHsa-miR-132 precursor genomic fragments were amplified by PCR.The PCR primers for hsa-miR-132 were：5′-CGCGGATCCTCGAGCCTCGGTGGACTC-3′and 5′-CTAGCTAGCCGCGGTGCTGATCAACG-3′.The vector construction process was performed as previously described［3］.The construct was designated as“p EGP-miR-132”.

Transient transfection and bulk-selected Hl299 cellsAll transfection were carried out using LipofectamineTM2000（Invitrogen）according to the manufacturer′s procedure.After 48 hours，H1299 cells transfected with p EGP-miR-132 were selected with 5μg/m L puromycin（Sigma，St Louis，MO，USA）for two weeks.The bulkselected H1299 cells stably expressing miR-132 were maintained in RPMI 1640 with 2.5μg/m L puromycin.Bulk cultures were used to avoid clone specific effects.

Reverse transcription and Quantitative RT-PCRTrizol（Invitrogen）was used to isolate total RNA，c DNA synthesis was performed using Primer Script RT reagent Kit（Ta KaRa，Tokyo，Japan）according to the manual.The specific reverse transcription（RT）primer of miR-132 was 5′-GTCG-TATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCGACCA-3′.Real-time quantitative PCR（RT-qPCR）was run on a Light-Cycler Roche 480（Roche Applied Science，Shanghai，China）with reactions consisted of a hot start（10 min at 95℃），40 cycles of 15 sec at 95℃and 60 sec at 60℃.Transcript levels for each target gene were normalized to 18srRNA levels by using comparative threshold cycle（Ct）method，in which fold difference= 2-（target geneΔCt-reference geneΔCt）；or by comparative CT methods，in which fold difference=2-ΔCt.Each sample was run in triplicate to ensure accuracy.Relative expression levels of miR-132，PTCH 1 and 18srRNA were detected.The primers of miR-132 were 5′-CGGCGGTAACAGTCTACAGCCA-3′and 5′-GTGCAGGGTCCGAGGT-3′，PTCH 1 were 5′-CGCTTTCTACATCTACCTGA-3′and 5′-AAGGGAACTGGGCATACTC-3′，18srRNA were 5′-TTGGTGGAGCGATTTGTCTG-3′and 5′-ATCTCGGGTGGCTGAACG-3′.

RNA interferenceMature miR-132 mimics（miR-132m）and miR-132 inhibitor（anti-miR-132）were synthesized by GenePharma（Shanghai，China）.The sequences of miR-132 mimics were 5′-UAACAGUCUACAGCCAUGGUCG-3′and 5′-ACCAUGGCUGUAGACUGUUAUU-3′.The sequence of miR-132 inhibitor was 5′-CGACCAUGGCUGUAGACUGUUA-3′.

Cell proliferation assayCell Counting Kit-8（Dojindo Laboratories，Kumamoto，Japan）was used to perform cell proliferation assays according to the protocol of the manufacturers on day 1，2，3 and 4 post-plating.Briefly，5000 cells were plated in 96-well plate per well and 10μL CCK-8 was added to each well when examined.Absorbance was measured at 450 nm and 630 nm（used as reference）with a Microplate Reader ELx808（Bio-Tek Instruments，Winooski，VT，USA）.Each experiment was performed in sextuplicate.

Cell migration and invasion assayThese were performed as previously described［3］.Transwell plates（8-μm poresize，6.5-mm diameter，Corning）with filters pre-coated with MATRIGEL Basement Membrane Matrix（coating concentration of 1 mg/ m L；BD Biosciences，USA）according to the manual.Briefly，cells（5×104）in 0.1 mL media containing 1%FBS were seeded into the upper chamber，with 0.6 mL of medium（10%FBS）under the upper chamber.Then put the plates into a humidified incubator（37℃and 5%CO2）for 72 hours.After removal of the Chambers，the non-invading cells were wiped off with cotton swab.After the cells under the chamber were fixed with methanol，stained with DAPI（Sigma）and washed with phosphatebuffered saline（PBS），all of the cells on the bottom of the chamber were counted under the microscope.The procedure of migration assay was the same as above，apart from that the insert Transwell chambers without Matrigel and medium containing 10%FBS was used for the cell suspensions.

Colony formation assayColony formation assays in soft agar were performed as reported before［3］.Base layer for each well was made by medium with 0.7%agar and 20%fetal bovine serum.Totally 5000 viable cells were harvested and suspended in medium containing 0.35%agar（Cat.A9045，Sigma）.Then cells were plated on the base layer，and laid with medium supplemented with 0.7%agar.Plate was maintained at 37℃in 5% CO2，incubated for 15 days，and then photographed using a light microscope.The numbers of colonies with diameters≥0.3 mm were counted.

Dual luciferase assayH1299 cells were cotransfected in 24-well plates with 50 pmol miR-132m，1μg firefly luciferase reporter containing the wild-or mutant-type putative target site in 3′-UTR of the genes and 0.15μg Renilla luciferase control vector p RL-TK（Promega，Madison，WI，USA）.Assays were assessed 48 h later using a Spectra Max M5 plate reader（Molecular Devices，Sunnyvale，CA）.Co-transfections of p EGP-miR-132，luciferase report construct and p RL-TK were performed with the same procedure.

ImmunoblottingWhole cells lysed in RIPA buffer（CW，Shanghai）supplemented with protease inhibitors（100μg/mL PMSF，Sigma-Aldrich；Complete Protease Inhibitor Cocktail，Roche）.Protein extracted from lysates was submitted to SDS-PAGE（10%）and transferred to polyvinylidene difluoride plus membrane（Micron Separation，Inc.）.Membranes were incubated in 5%non-fat milk prepared with TBS-containing 0.1%Tween-20（TBST）at room temperature（RT）for 1.5 h，and the membranes were immunoblotted with anti-PTCH1 rabbit antibody（Sigma）or with anti-tubulin mouse antibody（Sigma）overnight at 4℃，washed two times using TBS containing 0.1%Tween-20 at RT，and then incubated in secondary antibodies against rabbit or mouse IgG（1∶5 000，KPL，Gaithersburg，USA）for 1.5 h at RT.The membranes were washed three times and developed using ECL chemiluminescence（Thermo Scientific，Bremen，Germany）following the instructions of the manufacturer.

Statistical AnalysisEach value was derived from three independent experiments and data were presented as±s.Samples were analyzed by twotailed unpaired Student′s t test，and P values＜0.05 were considered as being statistically significant.We used（1），（2）and（3）to represent P values＜0.05，＜0.01 and＜0.001 respectively.All histograms were drawn using Graph Pad Prism 5.

Results

Expression of miR-l32 in cell linesWe first detected the expression of miR-132 in H1299 and six other cell lines using RT-qPCR.As showed in figure 1A，comparing with normal lung cells Beas-2B，relative expression level of miR-132 in H1299 almost tripled.

The expression construct and the stable lineWe next established H1299 transfectants stably expressing miR-132（H1299-p EGP-miR-132）to investigate the impact of miR-132 on cellular functions.The control clone stably expressing GFP was designated as“H1299-p EGP-miR-null”.The expression level of miR-132 in bulk-selected stable cells was detected by real-time PCR（Fig 1B）.Compared with the expression of miR-132 in H1299-p EGP-miR-null，that in H1299-p EGP-miR-132 was enhanced about 18 folds.

Overexpression of miR-l32 promoted cell proliferationTo explore the effect of miR-132 on cell viability，we performed cell proliferation analysis by Cell Counting Kit-8.Compared with H1299-p EGP-miR-null cells，H1299-p EGP-miR-132 showed a significant increase in cell growth rate（Fig 1C）.

To further confirm the results，we used synthetic miRNA inhibitor（anti-miR-132）to knock down the expression of miR-132，and then performed cell proliferation assay.As shown in figure 1D，in H1299-pEGP-miR-132 cells，anti-miR-132 partially decreased cell proliferation compared with negative control（antimiR-NC）on day 3 and day 4.

Fig l Hl299 cells stably expressing miR-l32 affected cell proliferation and anchorage-independent cell growthA，B：The expression level of miR-132.Real-time PCR showed that miR-132 increased in H1299 and in H1299-p EGP-miR-132 cells compared with H1299-p EGP-miR-null cells.C：Cell proliferation assays of H1299-p EGP-miR-132 and H1299-p EGP-miR-null cells.Stably overexpressing miR-132 promoted cell proliferation.D：The effect of miR-132 on cell proliferation was partially reversed by anti-miR-132.Each performed in sextuplicate（C，D）.E：H1299 cells stably expressing miR-132 promoted anchorage-independent cell growth.Totally 5000 cells were plated in soft-agar in each well of 6-well plate.After incubation for 15 days，colonies were examined under a microscope and colonies with diameter more than 3 mm were counted.Each assay performed in triplicate.

Overexpression of miR-l32 accelerated anchorageindependent growthWe further assessed the effect of miR-132 on anchorage-independent growth，a hallmark of tumor progression.After 15 days′growth，compared with H1299-pEGP-miR-null cells，H1299-pEGP-miR-132 cells showed significantly increased number of colonies and larger clones in soft agar（Fig 1E）.The result was confirmed in three independent experiments，suggesting that miR-132 overexpression promoted anchorage-independent growth.Overexpression of miR-132 improved the capacity of cell migration and invasion.

We investigated the role of miR-132 on cell migration and invasion，a critical determinant of malignant progression and metastases.Fig 2A was the representative picture of cell migration results（cell invasion picture not show）.A significant increase in cell migration and invasion was found in H1299-p EGP-miR-132 compared with H1299-p EGP-miR-null（Fig 2B，C）.When H1299-p EGP-miR-132 cells were transfected with anti-miR-132，the number of cell migration and invasion decreased（Fig 2D，E）.The cell migration and invasion results were in consistent with the results of anchorage-independent growth in H1299-p EGP-miR-132 cells.

Fig 2 miR-l32 promoted Hl299 cell migration and invasion and could partially be rescued by anti-miR-l32A：The representative picture of cell migration（cell invasion picture not show）.B，C：miR-132 improved the capacity of cell migration and invasion.D，E：The effect of miR-132 on cell migration and invasion was partially reversed by anti-miR-132.B-E：5×104 H1299-pEGP-miR-132 or H1299-pEGP-miR-null cells grown in 0.1 mL media（1%FBS）were seeded into upper chambers of Transwell uncoated（B，D；migration assay）or Matrigel matrix-coated（C，E；invasion assay）.72 hours later，the cells migrated through the bottom of the Transwell were stained and counted under a reverse microscope.Each assay performed in triplicate.

The results of bulk-selected cells supported a functional role for miR-132 in mediating cell proliferation，anchorage-independent growth and cell migration and invasion，suggesting that miR-132 may have oncogenic properties.

PTCH l is a target of miR-l32We first carried out computational identification of miRNA target genes with complementary sites of miR-132 in their 3′-UTR using computer algorithm，such as miRanda（http：//www.microrna.org/），miRWalk（http：//mirwalk.uni-hd.de/），and Targetscan（http：//www.targetscan.org/）.Of all the genes，PTCH 1 aroused our interest.As shown in figure 3A，miR-132 mimics decreased the reporter activity about 30%in the PTCH 1-3′UTR wild-type（wt-PTCH 1）co-transfections compared with that of the PTCH 1-3′UTR-mutant（mt-PTCH 1）cotransfections.Similar result was found when miR-132 expression construct p EGP-miR-132 was applied for dual luciferase experiment（Fig 3A）.The real time PCR and Western blot showed that overexpressing miR-132 decreased the m RNA and protein level of PTCH 1.When anti-miR-132 was transfected into H1299-p EGP-miR-132 cells，the decreased expression of PTCH1 could be partially reversed（Fig 3B）.

We further performed cell proliferation assay，cell migration and invasion assay in H1299-p EGP-miR-132 cells co-transfected with PTCH 1 siRNA and anti-miR-132（Fig 3C，E，F）.As shown in Figure 3，PTCH 1 siRNA and anti-miR-132 cotransfection promoted cell proliferation（Fig 3C），but inhibited cell migration and invasion（Fig 3D，E，F）.Similar results were obtained in H1299-p EGP-miR-132 cells transfected PTCH 1 siRNA alone（Fig 3D，E，F）.The result suggested that PTCH 1 played a part in the H1299-p EGP-miR-132 cell proliferation other than migration and invasion.

Discussion

miRNAs are a family of evolutionary conserved small non-coding RNAs processed from much longer primary transcripts that constitute a prevalent gene regulation［21-22］.By regulating the target genes，miRNAs function as critical mediator of numerous cellular processes，including cell growth，cell differentiation，proliferation，and apoptosis［23-24］.Thus abnormal expression of specific miRNAs in kinds of tissues could contribute to tumorigenesis［3-4］.

In recent years，much work has shed light on the significance of miR-132 in tumorigenesis.Zhang，et al［25］found that miR-132 is significantly decreased in breast cancer and functions as a tumor suppressor by targeting HN1.Luo，et al［26］found that miR-132/212 cluster inhibits the growth of lung cancer xenografts in nude mice.Furthermore，Jiang et al.［27］showed that forced expression of the miR-212/132 cluster suppresses A549 and H1299 cells proliferation.From our observations，overexpression of miR-132 in NSCLC cell H1299 improved the capacity of cell proliferation，anchorage-independent growth，cell migration and invasion.MiRNAs are known to function by regulating cell-specific target genes［28］，and it can be down-regulated and up-regulated depending on the type，location and stage of the cancer.For example，miR-96 is down-regulated in pancreatic cancer tissues and cell lines［29］，but it is up-regulated in hepatocellular carcinoma［30］.The effects of miR-132/212 cluster and miR-132 in H1299 cells were inconsistent.Similar outcome has been reported on miR-183/-96/-182 cluster and miR-183 in breast cancer cell T47D［31-32］.These conflicting results suggested that these miRNAs of the same cluster are transcribed or processed in different ways and function separately and differently［31］.

PTCH 1 is a membrane receptor for Hh ligand in Hh signaling pathway.We here documented that PTCH 1 is a direct target gene of miR-132.The ability of miR-132 to regulate PTCH1 protein level seems direct as it binds to the 3′UTR region of PTCH 1 mRNA，with the complementarity to the miR-132 seed region.Enforced miR-132 expression reduces cellular PCTH1 expression whereas anti-miR-132 partially rescues the PTCH1 protein expression.Generally，PTCH1 functions catalytically to protect Smo from localizing on the cell surface，thus suppressing the signal transducing by Smo in Hh signaling pathway［33］.Inappropriate reactivation of Hh signaling pathway in adults has been related to several totally different human cancers［17，34］. Aberrant activation of the Hh pathway in cancers can be caused by overexpressing Hh ligand binding to PTCH1，which is equal to genetic loss of PTCH1［35］.Previous study has shown that Hh ligands drive specific-type cell proliferation［34］，which is consistent with our observation.However，to ask why the effects of miR-132 overexpressing and PTCH1 silencing on behavior of cells were not an exact match，a reasonable explanation is that miR-132 could regulate other genes which participate in cell migration and invasion，opposing to the function of PTCH1.

Fig 3 PTCH l was targeted by miR-l32 and partially mediated the role of miR-l32A：miR-132 overexpression decreased the luciferase activity of PTCH 1-3′UTR wide-type（wt-3′UTR），compared with that of PTCH 1-3′UTR-mutant（mt-3′UTR）.B：Real-time PCR and Western blot showed the endogenous expression of PTCH1 in the following samples：H1299-pEGP-miR-132 and H1299-pEGP-miR-null，and H1299-pEGP-miR-132 cells transfected with anti-miR-132 or anti-miR-NC.It revealed that overexpression of miR-132 repressed the endogenous expression of PTCH1 compared with that of the control，and anti-miR-132 could partially rescue the expression of PTCH1.Each performed in triplicate.C-F：Knock-down of PTCH 1 promoted miR-132-dependent cell proliferation，but inhibited cell migration and invasion.C，D：Knock-down of PTCH 1 in H1299-pEGP-miR-132 cells increased cell growth rate compared with that of siRNA control in the presence of anti-miR-132（C），or in the absence of anti-miR-132（D）.Each experiment was performed in sextuplicate.E，F：Co-transfected anti-miR-132 and PTCH 1 siRNA into H1299-pEGP-miR-132，the cell ability of migration（E）and invasion（F）were suppressed by PTCH 1 siRNA.Transfection of PTCH 1 siRNA alone in H1299-pEGP-miR-132 cells inhibited the capacity of cells to migrate（E）or invade（F）.Each assay performed in triplicate.

In conclusion，we here described that miR-132 may have oncogenic properties in H1299 cells，and may have potential effect on Hh signaling pathway by mediating the expression of PTCH 1.Our study provides deep insights into the roles of miRNAs in an important signal network，which may provide an approach to therapeutic miRNA intervention in cancer.

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miR-l32调节非小细胞肺癌hedgehog信号通路受体基因PTCH l及细胞增殖、迁移和侵袭

杨秀方1▲褚凯丽1▲李 媛1李 瑶1黄 燕1△

miR-l32 regulates the hedgehog signaling pathway receptor PTCH l and cell proliferation，migration and invasion in non-small cell lung cancer cells

YANG Xiu-fang1▲，CHU Kai-li1▲，LI Yuan1，LI Yao1，HUANG Yan1△
（1State Key Laboratory of Genetic Engineering-Institute of Genetics，School of Life Sciences，Fudan University，Shanghai 200438，China）

ObjectiveTo study the role of miR-132 in non-small cell lung cancer（NSCLC）cells H1299 and its target gene.MethodsWe detected the expression of miR-132 in 7 cell lines by realtime PCR.We explored the cell growth rate of H1299 cells stably expressing miR-132（H1299-p EGP-miR-132）and H1299 cells stably expressing miR-null（H1299-p EGP-miR-null）using Cell Counting Kit-8.We carried out experiments to assess the effect of miR-132 on colony formation in H1299-p EGP-miR-132 cells compared with H1299-p EGP-miR-null cells.We studied the influence of miR-132 on cellmigration and cell invasion of H1299-pEGP-miR-132 compared with H1299-pEGP-miR-null using Transwell and Matrigel after 72 h since transfection.Besides，we also confirmed the results in H1299-pEGP-miR-132 cells transfected with anti-miR-132 or anti-miR-NC.We predicted the target gene of miR-132 in H1299 cells using biology information method and confirmed it by Dual luciferase assay，real-time PCRand Western blot.We investigated cell proliferation，cell migration and cell invasion of H1299-pEGP-miR-132 cells cotransfected with anti-miR-132 and PTCH1 siRNA.ResultsRelative expression level of miR-132 in NSCLC cell lines was significantly increased.miR-132 plays a role in NSCLC cell H1299 by targeting PTCH 1.Overexpressing miR-132 promoted cell proliferation（P＜0.01），cell migration（P＜0.05）and cell invasion（P＜0.05）in H1299 cells.Blocking PTCH1 expression by siRNA enhanced cell proliferation（P＜0.05），but suppressed cell migration（P＜0.001）and invasion（P＜0.001）in H1299 cells.Overexpressing miR-132 improved anchorage-independent growth up to 1.41-fold（P＜0.05）on day 15 in H1299 cells.Western blot revealed that the expression of PTCH1 protein was down-regulated to 0.68-fold by miR-132 and was up-regulated to 1.97-fold by anti-miR-132 in H1299 cells.ConclusionsmiR-132 may involve in NSCLC cell proliferation，migration and invasion，and its oncogenic activity was partially due to the regulation of the key negative regulatory protein PTCH1 of Hh signaling pathway.

miR-132； hedgehog signaling pathway； non-small cell lung cancer； PTCH1； cell migration and invasion

R 73 【文献标志码】 A doi：10.3969/j.issn.1672-8467.2015.03.002

上海市自然科学基金（14ZR1403700）

▲Co-first authors

△Corresponding author E-mail：huangyuyu@hotmail.com

2014-12-26；编辑：张秀峰）