Experimental Study On The Expression And Effects Of Pim-1in Human Primary Non-small Cell Lung Cancer

Objective: Pim-1gene is localized to the conserved region of humanchromosome6q21, and the protein it encodes belongs to the serine/threoninekinase family. Pim-1and AKT are thought to be two major kinases thatregulate cellular survival and metabolism. As a proto-oncogene, Pim-1aloneor in collaboration with other oncogenes （such as c-myc, N-myc and etc.）,could induce malignant transformation of cells, promote genomic instability,promote cell proliferation and suppress programmed cell death. Accordingly,it is involved in the process of tumorigenesis and progression. Pim-1is amajor downstream target of many cytokine signaling pathways, sustainedPim-1expression could be induced by a variety of cytokines, mitogen andhormone. By now, the related studies about Pim-1oncogene were mainly seenin hematological malignancies. More importantly, the anti-tumor effects ofPim-1kinase inhibitor have reached preclinical testing program. In contrast,deregulated expression of Pim-1kinase and its role upon solid tumorprogression was extensively studied in prostate cancer. However, it is yet to bedetermined how Pim-1works in human primary non-small cell lung cancer（NSCLC）. Therefore, in the present study, Pim-1expression and its biologicalfunctions involved in NSCLC were studied systematically.Methods:1The expression of Pim-1in human NSCLC:Pim-1expression at protein level was detected from27cases of humanprimary NSCLCs and matched normal lung tissue, as well as NSCLC celllines （H460, A549, H1299, H157, SK and H358） using Western Blot method.Then, Pim-1expression at mRNA level was further analyzed from24available cases of human primary NSCLCs and matched normal lung tissueusing Real-time PCR method. Immunohistochemical staining was used to detecte the localization of Pim-1protein in tumor tissues from NSCLC.2The biological function of Pim-1in NSCLC:The effects of Pim-1were evaluated in A549and H1299cells aftertransfected with specific Pim-1siRNA in vitro firstly. Cell proliferation wasaccessed by MTT and Colony formation assay. Cell cycle distribution wasexamined by Flow Cytometry, and the effects on cell migration ability werealso evaluated by Wound healing and Boyden chamber assay. Then, in vivotests were further performed in tumors from transplanted nude mice afterintratumoral injection of pis-Hi-pim-1or pis-Hi-vector to observe the role ofPim-1down-regulation on tumor proliferation.3The expression of Pim-1under different tumor microenvironment:Pim-1expression at protein level was detected in A549and H1299cellsafter treated with EGF, CoCl2or Chemotherapeutic drugs （Docetaxel orCisplatin） using Western Blot assay. Furthermore, the effects of Pim-1siRNAcombined with Docetaxel or Cisplatin treatment on cell proliferation wereobserved by MTT assay.Results:1The expression of Pim-1in human primary non-small cell lung cancerWestern bolt results showed that Pim-1protein is over-expressed in24out of27cases of NSCLC tumor tissue compared to that in paired normal lungtissues. At mRNA level, Pim-1was over-expressed only in37.5%tumortissues （9out of24cases） when normalized to the paired normal lung tissuesby Real-time PCR assay. Further analysis showed that there is no significantdifferences in the average level of Pim-1mRNA between the24tumor tissuesand the matched normal lung tissues （p<0.05）. Immunohistochemistry resultsshowed that the positive Pim-1protein expression was located in nucleus andcytoplasm of tumor cells. Furthermore, Pim-1protein expression wasexamined in six NSCLC cell lines H460, A549, H1299, H157, SK-MES-1andH358. Except for H460, all the other cells indicated relatively higher level ofPim-1protein expression.2The biological function of Pim-1in non-small cell lung cancer 2.1The effects of Pim-1in NSCLC in vitroTo further investigate the biological effects of Pim-1inNSCLC, we usedRNA interference （RNAi） to knockdown the Pim-1expression in A549andH1299cells to observe the role of Pim-1on cell proliferation, cell cycledistribution, and cell motility.2.1.1The efficiency of siRNA interferenceThe expressions of Pim-1at mRNA and protein level were detected48hafter transfected with Pim-1siRNA or negative control siRNA in A549cellsand H1299cells by Real-time PCR and Western Blot method, respectively. Incomparison with control group, Pim-1mRNA expression was decreased by80%and86%in A549cells and H1299cells, respectively. Consistant withReal-time PCR results, Pim-1protein was inhibited obviously by Pim-1siRNA compared to the control group in the two cell lines, suggesting that thePim-1siRNA sequences could inhibit Pim-1expression effectively.2.1.2Effects of Pim-1siRNA transfection on cell proliferationCell growth was significantly inhibited in A549and H1299cells withPim-1siRNA transfection compared with the control group both at72h and96h after transfection （p<0.05） by MTT assay. We further observed the effectsof Pim-1siRNA transfection on cell proliferation by colony formation assay inA549cells. The results indicated that the numbers of the colonies werereduced significantly in cells transfected with Pim-1siRNA （p<0.05）. Theabove results revealed that Pim-1siRNA transfection could inhibit theproliferation of A549cells and H1299cells.2.1.3Effects of Pim-1siRNA transfection on cell cycle distributionCompared to the control group, the proportion of cells in G0/G1phase wasincreased significantly and the proportion of cells in S phase was significantlydecreased in Pim-1siRNA transfected group （p<0.05） by FCM assay. Theresults were confirmed both in A549and H1299cells, indicating that Pim-1siRNA can lead cells to be arrested at G0/G1phase.2.1.4Effects of Pim-1siRNA transfection on cell migrationMonolayer wound healing assay was performed to observe the role of Pim-1siRNA transfection in A549and H1299cells. The results showed thatthe speed which cells migrated towards the scratch was lower in Pim-1siRNAtransfected cells when compared with the control ones. In addition, Boydenchamber assay was used to observe the effect of Pim-1on A549cellsmigration. Cells which crossed the membrane were counted and photographedat24hs. In Pim-1siRNA group, cells in lower chamber were much less thanthat in control group （P<0.05）.2.2The effects of Pim-1on tumor proliferation in vitro2.2.1Pim-1shRNA plasmid construction and efficiency testPim-1shRNA plasmids were constructed by Guangzhou GenecopoeiaCorporation. The knockdown efficiency was evaluated in H1299cells of aftertransfection with the Pim-1shRNA or empty vector. Real-time PCR resultsshowed that HSH013152-1-CH1-1plasmid showed the most significantinterference on Pim-1mRNA expression （⁶0%）. Therefore,HSH013152-3-CH1（psi-H1-Pim-1） plasmid was used for further research.2.2.2Preparation Nude mice with translating tumor1×10⁷A549cells per nude mouse were injected subcutaneously into theright flank, and the mice were randomly divided into two groups when thevolumes of the tumor reached to100mm3three weeks after implantation. Then,mice were treated by intratumoral injection of100μg/100μl pis-Hi-pim-1（shPim-1group） or pis-Hi-vector （empty plasmid group） once every4days for16days. The effects on tumor proliferation were accessed using the parameterof Relative Tumor Volume （RTV）.2.2.3Knowdown Pim-1on tumor cell proliferation in vivoIt showed that no obvious difference on tumor proliferation betweenshPim-1group and empty plasmid group within the10days after intratumoralinjection by RTV calculation. However, the tumor proliferation was decreasedsignificantly in shPim-1group begining at13days after treatment （p<0.05）。H&E staining of tumor tissues was performed18days after intratumoralinjection. The results confirmed that the histological type of the tumors fromtransplanted nude mice were all adenocarcinoma, as the same as A549cells types. Meanwhile, the total RNA and protein were extracted from tumor tissueto detect Pim-1expression in the two groups. Real-time PCR results showedthat, compared with the control group, the average level of Pim-1mRNA inshPim-1group was reduced by30%. Western Bolt and immunohistochemicalstaining results indicated that Pim-1at protein level in the shPim-1plasmidinjection group was significantly lower than the empty plasmid group. All theabove data confirmed that Pim-1expression can be inhibited to a certaindegree through intratumoral injection with shPim-1plasmid, and knockdownPim-1gene can inhibit tumor cell proliferation activity subsequently in vivo.3Pim-1expression under different tumor microenvironment3.1The effect of Pim-1expression with EGF treatmentCells were starved in0.5%serum medium for24h followed by50ng/μlEGF stimulation for2h. Whole cell extracts were then prepared by lyses andanalyzed by Western Blot. The results showed that Pim-1level wassignificantly increased in A549and H1299cells cells with EGF stimulationthan the control group, indicating that EGF can induce Pim-1expression.3.2The effect of Pim-1expression with CoCl2treatmentA549and H1299cells were treated with200μmol/L CoCl₂for24h toimitate hypoxia environment in vitro. Western Blot results showed that theexpression of Pim-1in the cells with CoCl2treatment was significantly higherthan the control group, suggested that hypoxia could induce Pim-1expressionin A549and H1299cells.3.3The effect of Pim-1expression with chemotherapeutic drugs （Doctaxel orCisplatin） treatmentAfter A549cells were treated with10nM Doctaxel or2μg/ml Cisplatinfor48h, Pim-1protein expression was increased compared to the controlgroup. In H1299cells, the Pim-1protein level was also increased after100nMDoctaxel or2μg/ml Cisplatin treatment for24h, Pim-1protein expression wassignificantly increased.3.4The effect of Pim-1siRNA combined with Doctaxel or Cisplatin treatmenton cell proliferation MTT assay results showed that downregulation of endogenous Pim-1expression by Pim-1siRNA led to decreased A549cell proliferation at72hafter docetaxel application （p<0.05） or at48h and72h after Cisplatin treatment（p<0.05） relative to that in NC siRNA group. In addition, inhibiton Pim-1expression by Pim-1siRNA was able to suppress survival of H1299cells, asreflected by the MTT assay, at72h after docetaxel exposure （p<0.05） or at48hafter Cisplatin exposure （p<0.05）. The above results suggested that Pim-1siRNA can increase the tumor cells to the sensitivity of the chemotherConclusion:1Pim1was freque ntly overexpression in human primary NSCLC.2Pim-1siRNA transfection can inhibit A549and H1299cell proliferation,induce cell cycle G0/G1phase arrest, and reduce the ability of cellmigration.3Intratumoral injection with shPim-1plasmid can reduce Pim-1expressionin tumors from transplanted nude mice, and inhibit tumor cell proliferationin vivo.4Pim-1expression in A549and H1299cells could be induced by EGF,CoCl₂, Docetaxel or Cisplatin treatment.5Pim-1siRNA transfection can increase the sensitivity of the tumor cellsagainst drugs, therefore, Pim-1specific inhibitors is expected to becomethe new target for NSCLC.