SMC1A Knockdown Induces Growth Suppression Of Human Lung Adenocarcinoma A549and H1299Cells

Lung cancer is the most common malignancy and the leading cause ofcancer-related mortality worldwide. Despite significant progress in surgicaltechniques and other conventional therapeutic modalities like chemotherapy andradiotherapy, most of the patients diagnosed with lung cancer succumb to the diseasein a short period. Therefore, understanding of the molecule mechanisms underlyingtumorogenesis of lung cancer is crucially important for the development of moreeffective therapy of lung cancer.The recent discovery of cohesin complex in yeast helps us further understand themolecular basis underlying genome instability, which has been recognized as ahallmark of human carcinomas. Cohesin complex, evolutionarily conserved fromyeast to humans, comprises four subunits: a pair of SMC （structural maintenance ofchromosomes） proteins, namely SMC1A and SMC3, two non-SMC proteins,RAD21/SCC1and STAG/SCC3/SA. SMC1A and SMC3are constituted by twocoiled domains and interact with each other by their hinge domain to form anantiparallel heterodimer. Their head domains interact with RAD21, creating aring-like structure. By trapping DNA within the ring-like structure, cohesin isassociated with chromosomes, holding pairs of sister chromatids from the time ofreplication in S phase until their separation in anaphase to ensure faithful chromosomesegregation during mitosis. It has been shown that cohesin complex participate inmany aspects of DNA repair, cell cycle, gene expression regulation and genomicimprinting, making key contribution to genome stability. Additionally, studies havedemonstrated that dysfunction of cohesin and cohesin regulatory genes makes themstrong candidates for promoting genome instability and cancer development.The RNAi technique, a powerful tool for carrying out loss-of-function assay,represents a novel alternative to gene inhibition and provides a new approach forstudying cancer gene therapy. Applications of RNAi for mammalian cells haveemerged. In this study, we adopted a lentiviral vector-mediated RNAi system toachieve highly stable silence of SMC1A. The safety of lentiviral vectors has been quite recognized in the scientific community.Here, we constructed SMC1A-specific small interfering RNA （siRNA）-lentiviralvector that is capable of effectively inhibiting the expression of SMC1A gene inhuman lung adenocarcinoma A549and H1299cells, and investigated systemically theimpacts of SMC1A silence on the growth and invasive ability of the cancer cells invitro. Furthermore, we determined the effects of SMC1A knockdown on the cell cycledistribution and apoptosis of A549and H1299cells. As result, we found that SMC1Ais a novel regulator, which modulates the proliferation and migration capabilities oflung cancer cells via arrested G1/S phase cell cycle and apoptosis.Materials and methods1Construction of SMC1A shRNA expressing lentivirusTo overcome drawbacks of short-time action and lack of regenerating ability ofsiRNA and permit robust inducible RNAi mediated SMC1A silencing, the encodingshort hairpin RNA （shRNA） lentiviral vector was constructed. The RNAi wasdesigned based on a19nt SMC1A （NM₀06306）-targeting sequence （5’-TAGGAGGTTCTTCTGAGTACA-3’） of oligonucleotides and negative control sequence（5’-TTCTCCGAACGTGTCACGT-3’）. The sequences were annealed, and ligatedinto the Nhe I/Pac I （NEB, Ipswich, MA, USA）-linearized pFH1UGW vector（Shanghai Hollybio Co. LTD., Shanghai, China）. The lentiviral-based shRNA-expressing vectors were confirmed by DNA sequencing.2Lentivirus infectionRecombinant lentiviral vectors and packaging vectors were then cotransfectedinto293T cells using Lipofectamine2000（Invitrogen, Carlsbad, CA, USA）,according to the instructions of manufacture for the generation of recombinantlentiviruses （SMC1A shRNA （Lv-shSMC1A） and negative control shRNA（Lv-shCon））. Supernatants containing lentiviruses expressing Lv-shSMC1A andLv-shCon were harvested72h after transfection, respectively. Lentiviruses werepurified using ultracentrifugation. A549and H1299cells were infected with thelentiviruses at multiplicity of infection （MOI）=30. The uninfected A549and H1299cells were used as controls.Quantitative Real-time PCRIn brief, total RNA was extracted from A549and H1299cells96h after infectionusing the RNeasy Midi Kit （Promega, Madison, Wis, USA）. cDNA was synthesized with SuperScriptII reverse transcriptase （Invitrogen, Carlsbad, CA, USA）. The primersequences for PCR amplification of SMC1A gene were5′-AAGTGAGGAGGAGGAGGAG-3′and5′-ACTTTCTTCAGGGTCTTGTTC-3′. β-actin was applied as aninternal control.Western blotIn brief, A549and H1299cells were collected and lysed by precooled lysisbuffer after96h of infection. Total protein was extracted from cells and determinedby BCA method. The resulting membrane was probed with goat anti-SMC1A （1:1000dilution; Sigma, Cat#SAB4300451） and mouse anti-GAPDH （1:6,000; Santa Cruz,CA） overnight at4℃. The protein level of GAPDH was used as control and detectedby an anti-GAPDH antibody. The membrane was washed three times withTris-buffered saline tween-20（TBST）, followed by incubation for2h withanti-mouse IgG at a1:5,000dilution （Santa Cruz, CA）. The membrane was developedusing enhanced chemiluminescence （Amersham, UK）. Bands on the developed filmswere quantified with an ImageQuant densitometric scanner （Molecular Dynamics）.3MTT assayBriefly, exponentially growing cells were inoculated into96-well plates with2×103A549cells or6×10⁴H1299cells per well. After incubation for24,48,72,96and120h,10μl of sterile MTT （5mg/ml） was added into each well. Followingincubation at37℃for4h, the reaction was stopped by adding100μl of dimethylsulfoxide. The formazan production was detected by measurement of thespectrometric absorbance at595nm. The values obtained are proportional to theamount of viable cells.4Colony formation assayIn brief, A549and H1299cells infected with Lv-shSMC1A or-shCon anduninfected cells （Con） were seeded in six-well plates （2×102cells/well of A549,5×10⁴cells/well of H1299） and cultured at37℃with5%CO2for8days. The cellcolonies were washed twice with PBS, fixed in4%paraformaldehyde for15min andstained with Giemsa for30min. Individual colonies with more than50cells werecounted under a fluorescence microscope.5Cell migration assayIn brief, A549and H1299cells infected with Lv-shSMC1A or–shCon for96hand uninfected cells （Con） were harvested and their ability of migration in vitro was determined using a Transwell chamber （Corning, NY, USA）. Cells were seeded intothe upper chamber （3.0×10⁴cells/well of A549,8.0×10⁴cells/well of H1299） in100μl of serum-free medium. Amount of1ml medium containing20%FBS was added tothe lower chamber as a chemo-attractant was performed. After incubation for24h at37℃in5%CO2, cells that invaded to the lower surface of the filter were fixed by4%paraformaldehyde and stained with cristal purple. Cell numbers were counted in fiverandom fields （×100） per filter and detected by the spectrometric absorbance at570nm.6Fluorescence-activated cell sorting analysisIn brief, A549and H1299cells were seeded in six-well plates （1.5×106cells/wellof A549,2×106cells/well of H1299）. After48h, cells were collected, washed withPBS, and fixed with75%cold ethanol. The cells were then incubated for more than24h at4℃. After washing the cells with PBS, propidium iodide （PI） was added to thecell suspension and the analysis of cell cycle distribution was performed by FACS.ResultEfficacy of lentivirus-mediated RNAi targeting SMC1AReal-time PCR analysis showed that the expression level of SMC1A mRNA ofthe Lv-shSMC1A infected cells was significantly lower than that of the parent cells（Con） and Lv-shCon infected cells. Moreover, western blot assay further showed that,SMC1A protein levels decreased significantly in Lv-shSMC1A infected cellscompared with that of Lv-shCON infected cells. Therefore, this indicated the highefficacy of lentivirus mediated SMC1A shRNA on SMC1A expression in lung cancercells.Impact of downregulation of SMC1A expression on cell growth in vitroThe growth dynamics of parent or Lv-shCon and Lv-shSMC1A infected A549and H1299cells was determined by MTT assay and colony formation assay,respectively. MTT assay showed that, during the120h incubation period, the growthof Lv-shCon infected cells did not differ from uninfected parent cells and showedstrong proliferation, whereas the growth of Lv-shSMC1A infected cells wasprominently slower than parent or Lv-shCon infected cells at the time points of48h,72h,96h and120h. Quantitative analysis of colonies showed that after incubationfor8days, the number of colonies in Lv-shSMC1A infected cells was lower than thatin the parent and Lv-shCon infected cells （P<0.01）. Therefore, the low viability and colony-forming efficiency of Lv-shSMC1A infected A549and H1299cellsdemonstrated that downregulation of SMC1A expression inhibits the growth of lungcancer cells in vitro.Impact of downregulation of SMC1A expression on cell invasionTranswell chamber assay demonstrated that the invasive ability of Lv-shConinfected cells did not significantly differ from its parent cells and showed stronginvasiveness. However, invasive ability of Lv-shSMC1A infected cells wasremarkably lower than that of the parent and Lv-shCon infected cells. Therefore, thisindicated that downregulation of SMC1A expression mitigates the invasion of lungcancer cells in vitro.Impact of downregulated SMC1A expression on cell-cycle distribution in vitroThe cell cycling patterns of parent or Lv-shCon and Lv-shSMC1A infectedcancer cells were determined by FACS Flow Cytometric analysis96hours afterinfection. There was no obvious difference in the frequency at G2stage of each groupof cells. Neither did the frequency of Lv-shCon infected cells at G1stage nor at Sstage significantly differ from its parent cells. At G1stage, the frequency ofLv-shSMC1A infected cells was significantly higher than that of its parent orLv-shCon infected cells. In contrast, at S stage, the frequency of Lv-shSMC1Ainfected cells was lower than that of the controls （p<0.01）. These results suggestedthat downregulation of SMC1A expression resulted in cell cycle arrest at G1/Stransition in A549and H1299cells, which contributed to the inhibition of SMC1Acell growth.Impact of downregulated SMC1A expression on apoptosis in vitroSub-G1cells are usually considered as the result of apoptotic DNAfragmentation: during apoptosis, the DNA is degraded by cellular endonucleases.Therefore, nuclei of apoptotic cells contain less DNA than nuclei of healthy G0/G1cells, resulting in a sub-G1peak in the fluorescent histogram that can be used todetermine the relative amount of apoptotic cells. As shown in Fig.5c and6c, therewas no obvious difference in the cell population at sub-G1phase between parent andLv-shCon infected cells, whereas Lv-shSMC1A infected cells exhibited significantlyhigher frequency in sub-G1phase than that of parent or Lv-shCon infected cells. Thissuggested that downregulation of SMC1A expression might trigger apoptosis in lungcancer cells, which contributed to the suppression of SMC1A cell growth. Lung cancer is well established as a highly heterogeneous disease, withmultitude of cellular components and patterns of gene expression that affect tumordevelopment. In-depth understanding of the molecular mechanisms underlying cancerproliferation is critical for the development of optimal therapeutic modalities.Moreover, there is evidence to suggest that therapeutic drugs specifically targeting attumor-related molecules are expected to be highly specific to malignant cells and haveminimal adverse reactions due to their actions through well-defined mechanisms. Thehallmarks of cancer involve several critical biological capabilities acquired in cellproliferation and invasion-metastasis cascade of malignant tumors. Genome instabilityhas been found to foster these multiple hallmarks function via generating the geneticdiversity that expedites their acquisition. Cohesin defects represent novel notionamongst molecular mechanisms underlying genome stability that involve defects inDNA repair, cell cycle checkpoints, and epigenetic processes et al. Research studiesreveal that apart from its role in sister chromatid cohesion, cohesin also acts as a keyplayer in various respects of DNA damage response, cell cycle and gene expressionregulation. SMC1A, an indispensible component of the versatile cohesin complex, isimplicated as an important molecular target of malignancies. Our observation isconsistent with the previous report and emphasized that SMC1A facilitates importantregulatory roles in lung cancer cell proliferation and invasiveness.In conclusion, our findings strongly suggest the significance of cohesin geneSMC1A in modulating the growth and invasiveness of lung cancer, and indicate thatdownregulation of SMC1A expression induces growth suppression of humanpulmonary adenocarcinoma A549and H1299cells via arrested G1/S phase cell cycleand apoptosis pathways. Hence, this study extends our knowledge about theoncogenesis of lung cancer, and indicates that SMC1A may serve as a new moleculartarget.