Preliminary Study On The Role Of A Novel Gene PRR11in Lung Cancer

PRR11（proline rich11）is a novel cancer-associated gene，located inchromosome17q22. We had cloned the full-length of PRR11cDNA in ourprevious study. Bioinformatics analysis showed PRR11may be anevolutionarily conserved gene, and might play an important role in thegrowth and proliferation in eukaryotic cells. According to our previousstudy, PRR11were overexpressed in several types of tumors.To further investigate whether PRR11was involved in tumorigenesis,especially in lung cancer, we carried out the following studies.(1) Overexpression of PRR11in lung cancer.Cancer microarray database mining revealed that PRR11wasupregulated in several types of cancers including lung cancer. Real timeRT-PCR demonstrated that, PRR11was significantly upregulated at mRNAlevel in lung cancer samples compared with normal lung tissues. Notably,Overexpression of PRR11mRNA was significantly associated with tumorstages. Consistently, Immunohistochemistry (IHC) analysis furtherrevealed that PRR11was also significantly upregulated in lung cancersamples compared with normal adjunct tissues.(2)PRR11depletion inhibits proliferation and effects cell cycleprogression in lung cancer cells.To investigate the functional involvement of PRR11in lung cancer,the PRR11expression was depleted via siRNA-mediated silencing in threedifferent lung cancer cell lines H1299, A549and HCC827, and the cellular proliferation was ubsequently analyzed. Quantitative RT-PCR and Westernblot analysis demonstrated that the PRR11expression was significantlyinhibited at both mRNA and protein levels in all three cell lines. PRR11siRNA strongly caused a significant growth retardation of all three lungcancer cell lines studied whereas control siRNA did not affect theproliferation of the cells.Next, we analyzed the effect of PRR11depletion on cell cycleprogression in lung cancer cells using flow cytometry. Cell cycle analysisrevealed that silencing PRR11expression also resulted in a remarkableincreased percentage of cells in S phase in all three lung cancer cell linesand a modest increased percentage of cells in G2/M phase in H1299cells.Quantitative RT-PCR and Western blot analysis demonstrated that p21alsosignificantly increased in PRR11-depleted cells. Consistent with ourprevious observation in HeLa cells, these results further confirmed thatPRR11is a novel cell cycle gene, and regulates S phase and mitoticprogression.(3)PRR11depletion inhibits lung tumorigenesis in vitro and invivo.To further investigate the role of PRR11in tumorigenesis, we usedH1299to establish a PRR11knockdown stable cell line. QuantitativeRT-PCR and Western blot analysis confirmed that the PRR11expressionwas dramatically depleted at both mRNA and protein levels in the stablePRR11knockdown cells.Wound healing assay indicated that the stablePRR11knockdown cells showed a significantly reduced migration activity.Colony formation assay demonstrated that the stable PRR11knockdowncells showed a significantly reduced colony formation activity in bothnumber and size, as compared with the control cells. To examine whethersilencing of PRR11expression could inhibit the tumorigenicity in vivo, the stable PRR11knockdown and the control cells were injectedsubcutaneously into nude mice. Silencing of PRR11expression remarkablyinhibited the tumor growth in both weight and size in nude mice. At the endof the study (day45), tumor weight and tumor size of PRR11knockdowngroup (0.1±0.05g,90±59mm3) was only12%and11%of the controlgroup (0.8±0.5g,815±404mm3).In summary, our present study found that, PRR11was overexpressedin lung cancer, and PRR11depletion via RNAi caused significant lungcancer cell growth, S phase arrest, and reduced lung tumorigenesis both invitro and invio. Our results strongly indicated that PRR11had a criticalrole in both cell cycle progression and tumorigenesis, and might serve as apotential target in the diagnosis and/or treatment of human lung cancer.