Anwen Xiong

Lung cancer is a highly heterogeneous disease. Cancer cells and cells within the tumor microenvironment together determine disease progression, as well as response to or escape from treatment. To map the cell type-specific transcriptome landscape of cancer cells and their tumor microenvironment in advanced non-small cell lung cancer (NSCLC), we analyze 42 tissue biopsy samples from stage III/IV NSCLC patients by single cell RNA sequencing and present the large scale, single cell resolution profiles of advanced NSCLCs. In addition to cell types described in previous single cell studies of early stage lung cancer, we are able to identify rare cell types in tumors such as follicular dendritic cells and T helper 17 cells. Tumors from different patients display large heterogeneity in cellular composition, chromosomal structure, developmental trajectory, intercellular signaling network and phenotype dominance. Our study also reveals a correlation of tumor heterogeneity with tumor associated neutrophils, which might help to shed light on their function in NSCLC.

Vertebrate muscle differentiation is coordinated by an intricate network of transcription factors requiring proliferating myogenic precursors to withdraw irreversibly from the cell cycle. Recent studies have implicated a large number of microRNAs exerting another layer of control in many aspects of muscle differentiation. By annealing to short recognition sequences in the 3′-untranslated region, microRNAs attenuate target gene expression through translation repression or mRNA degradation. Here, we show that miR-214 promotes myogenic differentiation in mouse C2C12 myoblasts at a step preceding the induction of p21 and myogenin. Blocking miR-214 function with a 2′-O-methylated double-stranded inhibitor maintained C2C12 cells in the active cell cycle, thereby inhibiting the myogenic differentiation. By global gene expression profiling, we identified the proto-oncogene N-ras as one of miR-214 targets. Furthermore, manipulating the N-Ras level with small interfering RNA or adenovirus-mediated forced expression either augmented or attenuated the effect of miR-214, respectively. Thus, our data uncovered a novel microRNA-mediated mechanism that controls myogenic differentiation. Vertebrate muscle differentiation is coordinated by an intricate network of transcription factors requiring proliferating myogenic precursors to withdraw irreversibly from the cell cycle. Recent studies have implicated a large number of microRNAs exerting another layer of control in many aspects of muscle differentiation. By annealing to short recognition sequences in the 3′-untranslated region, microRNAs attenuate target gene expression through translation repression or mRNA degradation. Here, we show that miR-214 promotes myogenic differentiation in mouse C2C12 myoblasts at a step preceding the induction of p21 and myogenin. Blocking miR-214 function with a 2′-O-methylated double-stranded inhibitor maintained C2C12 cells in the active cell cycle, thereby inhibiting the myogenic differentiation. By global gene expression profiling, we identified the proto-oncogene N-ras as one of miR-214 targets. Furthermore, manipulating the N-Ras level with small interfering RNA or adenovirus-mediated forced expression either augmented or attenuated the effect of miR-214, respectively. Thus, our data uncovered a novel microRNA-mediated mechanism that controls myogenic differentiation. IntroductionThe vertebrate skeletal muscle is developed from mesodermal stem cells that are committed to a muscle fate within paraxial somites, giving rise to immature myoblasts (1Rudnicki M.A. Le Grand F. McKinnell I. Kuang S. Cold Spring Harbor Symp. Quant. Biol. 2008; 73: 323-331Crossref PubMed Scopus (188) Google Scholar, 2Arnold H.H. Winter B. Curr. Opin. Genet. Dev. 1998; 8: 539-544Crossref PubMed Scopus (244) Google Scholar, 3Buckingham M. Bajard L. Chang T. Daubas P. Hadchouel J. Meilhac S. Montarras D. Rocancourt D. Relaix F. J. Anat. 2003; 202: 59-68Crossref PubMed Scopus (630) Google Scholar, 4Buckingham M. Relaix F. Annu. Rev. Cell Dev. Biol. 2007; 23: 645-673Crossref PubMed Scopus (348) Google Scholar). Committed myoblasts are proliferating progenitors (5Buckingham M. Montarras D. Curr. Opin. Genet. Dev. 2008; 18: 330-336Crossref PubMed Scopus (89) Google Scholar) that must exit from the cell cycle before myogenic regulatory factors such as MyoD and Myf5 activate myocyte enhancer factors and other downstream muscle-specific genes to drive the formation of multinucleated myotubes and eventually the contractile muscle fibers (6Black B.L. Olson E.N. Annu. Rev. Cell Dev. Biol. 1998; 14: 167-196Crossref PubMed Scopus (843) Google Scholar, 7Brand-Saberi B. Ann. Anat. 2005; 187: 199-207Crossref PubMed Scopus (77) Google Scholar, 8Tapscott S.J. Development. 2005; 132: 2685-2695Crossref PubMed Scopus (546) Google Scholar, 9Walsh K. Perlman H. Curr. Opin. Genet. Dev. 1997; 7: 597-602Crossref PubMed Scopus (264) Google Scholar). This process can be recapitulated in vitro with cell culture systems, the most widely used of which is the mouse C2C12 cell (10Andrés V. Walsh K. J. Cell Biol. 1996; 132: 657-666Crossref PubMed Scopus (502) Google Scholar, 11Yaffe D. Saxel O. Nature. 1977; 270: 725-727Crossref PubMed Scopus (1542) Google Scholar). In proliferating myoblasts, the MyoD targets of myogenic genes are repressed through chromosome remodeling and epigenetic histone modifications (12McKinsey T.A. Zhang C.L. Olson E.N. Curr. Opin. Genet. Dev. 2001; 11: 497-504Crossref PubMed Scopus (348) Google Scholar, 13Pownall M.E. Gustafsson M.K. Emerson Jr., C.P. Annu. Rev. Cell Dev. Biol. 2002; 18: 747-783Crossref PubMed Scopus (464) Google Scholar). Under the influence of extracellular signals in developing muscle tissues or upon serum depletion in culture systems, MyoD orchestrates an orderly exit from the cell cycle, involving concerted interplays between myogenic regulatory factors and the cell cycle machinery (7Brand-Saberi B. Ann. Anat. 2005; 187: 199-207Crossref PubMed Scopus (77) Google Scholar, 9Walsh K. Perlman H. Curr. Opin. Genet. Dev. 1997; 7: 597-602Crossref PubMed Scopus (264) Google Scholar, 13Pownall M.E. Gustafsson M.K. Emerson Jr., C.P. Annu. Rev. Cell Dev. Biol. 2002; 18: 747-783Crossref PubMed Scopus (464) Google Scholar, 14Polesskaya A. Rudnicki M.A. Dev. Cell. 2002; 3: 757-758Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The retinoblastoma gene product Rb and its homologue p107 (15Zacksenhaus E. Jiang Z. Chung D. Marth J.D. Phillips R.A. Gallie B.L. Genes Dev. 1996; 10: 3051-3064Crossref PubMed Scopus (264) Google Scholar, 16Schneider J.W. Gu W. Zhu L. Mahdavi V. Nadal-Ginard B. Science. 1994; 264: 1467-1471Crossref PubMed Scopus (338) Google Scholar, 17Gu W. Schneider J.W. Condorelli G. Kaushal S. Mahdavi V. Nadal-Ginard B. Cell. 1993; 72: 309-324Abstract Full Text PDF PubMed Scopus (641) Google Scholar) are essential in initiating an irreversible withdrawal from the cell cycle by up-regulating cyclin-dependent kinase inhibitor p21 (18Halevy O. Novitch B.G. Spicer D.B. Skapek S.X. Rhee J. Hannon G.J. Beach D. Lassar A.B. Science. 1995; 267: 1018-1021Crossref PubMed Scopus (1089) Google Scholar, 19Guo K. Wang J. Andrés V. Smith R.C. Walsh K. Mol. Cell. Biol. 1995; 15: 3823-3829Crossref PubMed Scopus (361) Google Scholar). Certain oncogene products including H- and N-Ras exhibit potent inhibition on myogenic differentiation by blocking Rb function (20Olson E.N. Spizz G. Tainsky M.A. Mol. Cell. Biol. 1987; 7: 2104-2111Crossref PubMed Scopus (144) Google Scholar).In addition to the complex network of protein-encoding genes, recent molecular and genetic studies have uncovered a myriad of microRNAs (miRNAs) 2The abbreviations used are: miRNAmicroRNAUTRuntranslated regionRTreverse transcriptionMHCmyosin heavy chainBrdUrdbromodeoxyuridinePBSphosphate-buffered saline214mimiR-214 mimic214inmiR-214 inhibitorqPCRquantitative PCRFACSfluorescence-activated cell sorterMAPmitogen-activated protein. that exert a different layer of control over myogenic differentiation (21Callis T.E. Deng Z. Chen J.F. Wang D.Z. Exp. Biol. Med. 2008; 233: 131-138Crossref PubMed Scopus (105) Google Scholar, 22van Rooij E. Liu N. Olson E.N. Trends Genet. 2008; 24: 159-166Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 23Brennecke J. Stark A. Cohen S.M. Genes Dev. 2005; 19: 2261-2264Crossref PubMed Scopus (29) Google Scholar). miRNAs are a class of small noncoding RNAs that are synthesized as pri-miRNAs from either dedicated transcription units or introns of protein-encoding genes (24Ambros V. Nature. 2004; 431: 350-355Crossref PubMed Scopus (8921) Google Scholar, 25Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29004) Google Scholar, 26Tomari Y. Zamore P.D. Genes Dev. 2005; 19: 517-529Crossref PubMed Scopus (735) Google Scholar). Pri-miRNAs are processed in the nucleus by the RNase Drosha, yielding precursor miRNAs, which have a characteristic “stem-loop” structure and are ∼70 nucleotides in length. Precursor miRNAs are then exported by Exportin 5 to the cytoplasm to be further processed by the RNase Dicer into mature miRNAs of ∼22 nucleotides (25Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29004) Google Scholar, 26Tomari Y. Zamore P.D. Genes Dev. 2005; 19: 517-529Crossref PubMed Scopus (735) Google Scholar). Mature miRNAs are incorporated into the RNA-induced silencing complex, where they anneal to their recognition sequences in the 3′-UTR of mRNA genes to attenuate gene expression through translation repression or mRNA degradation (27Carmell M.A. Hannon G.J. Nat. Struct. Mol. Biol. 2004; 11: 214-218Crossref PubMed Scopus (311) Google Scholar). Of many miRNAs that are involved in the regulation of muscle biology, miR-1 (28Zhao Y. Samal E. Srivastava D. Nature. 2005; 436: 214-220Crossref PubMed Scopus (1347) Google Scholar), miR-133 (29Chen J.F. Mandel E.M. Thomson J.M. Wu Q. Callis T.E. Hammond S.M. Conlon F.L. Wang D.Z. Nat. Genet. 2006; 38: 228-233Crossref PubMed Scopus (2192) Google Scholar), and miR-206 (30Kim H.K. Lee Y.S. Sivaprasad U. Malhotra A. Dutta A. J. Cell Biol. 2006; 174: 677-687Crossref PubMed Scopus (636) Google Scholar) are specifically expressed in the muscles where they superimpose on the intricate network of transcription factors and other regulatory proteins to control growth and myogenesis. Another muscle-specific microRNA, miR-208, is involved in myosin heavy chain production (31van Rooij E. Sutherland L.B. Qi X. Richardson J.A. Hill J. Olson E.N. Science. 2007; 316: 575-579Crossref PubMed Scopus (1370) Google Scholar).Here we investigate the mechanism of mouse miR-214, which was first shown to play an essential role in specifying slow muscle development in zebrafish (32Flynt A.S. Li N. Thatcher E.J. Solnica-Krezel L. Patton J.G. Nat. Genet. 2007; 39: 259-263Crossref PubMed Scopus (273) Google Scholar). In a mouse model of cardiac hypertrophy, miR-214 was found to be expressed at an elevated level, but forced expression of miR-214 did not lead to increased muscle growth (33Watanabe T. Sato T. Amano T. Kawamura Y. Kawamura N. Kawaguchi H. Yamashita N. Kurihara H. Nakaoka T. Dev. Dyn. 2008; 237: 3738-3748Crossref PubMed Scopus (118) Google Scholar). Using the in vitro differentiation of mouse C2C12 cells as a model, we report here that miR-214 promotes myogenic differentiation by facilitating the exit from the cell cycle, and we identified N-ras as a target of this function through microarray analysis.DISCUSSIONIn the current study, we investigated the mechanism by which miR-214 promotes myogenic differentiation and uncovered one aspect of miR-214 functions in facilitating the cells exiting from the cell cycle, a prerequisite to differentiation of all cell lineages. However, we were puzzled by the strong inhibition of miR-214 on BMP-induced osteogenic differentiation (Fig. 2F), which suggested that the role of miR-214 was not simply limited to promoting differentiation per se; rather it showed a specific preference for generating myocytes. This dichotomy was finally resolved by our global gene expression profiling studies, which identified N-ras as one of the functional targets of miR-214. We subsequently confirmed this. As a proto-oncogene, N-ras is not only a strong cell growth promoter, it also has a proven role in inhibiting muscle differentiation (20Olson E.N. Spizz G. Tainsky M.A. Mol. Cell. Biol. 1987; 7: 2104-2111Crossref PubMed Scopus (144) Google Scholar, 38Takahashi C. Contreras B. Bronson R.T. Loda M. Ewen M.E. Mol. Cell. Biol. 2004; 24: 10406-10415Crossref PubMed Scopus (33) Google Scholar). So, by targeting N-ras, miR-214 forced the cells out of the cell cycle, but in a way that shepherded them down the pathway toward muscle formation.Irreversible withdrawal from the cell cycle is mediated by the retinoblastoma protein, pRB (15Zacksenhaus E. Jiang Z. Chung D. Marth J.D. Phillips R.A. Gallie B.L. Genes Dev. 1996; 10: 3051-3064Crossref PubMed Scopus (264) Google Scholar, 16Schneider J.W. Gu W. Zhu L. Mahdavi V. Nadal-Ginard B. Science. 1994; 264: 1467-1471Crossref PubMed Scopus (338) Google Scholar). In proliferating myoblasts, pRB is progressively inactivated by phosphorylation in the G1 phase by concerted actions of multiple cyclin-dependent kinase complexes to allow for proper progression through the cell cycle until late in the mitosis when it is degraded (39De Falco G. Comes F. Simone C. Oncogene. 2006; 25: 5244-5249Crossref PubMed Scopus (70) Google Scholar). Mitogenic signals through the intracellular MAP kinase pathways initiated from one of the three Ras small GTPases maintain pRB as an inactive hyperphosphorylated protein. At the onset of cell differentiation triggered by serum withdrawal in the case of C2C12 model system, the RAS-MAP kinase pathways are silent, allowing hypophosphorylated pRB to block cell growth by repressing the key cell cycle transcription factor, E2F, through binding. Of the three ras genes in the mammalian genomes, N-ras was shown previously to possess a muscle specific function (38Takahashi C. Contreras B. Bronson R.T. Loda M. Ewen M.E. Mol. Cell. Biol. 2004; 24: 10406-10415Crossref PubMed Scopus (33) Google Scholar). In pRb-deficient mouse embryos, thoracic skeletal muscles showed a dramatic reduction in fiber density and the length of myotubes; however, removal of both N-Ras and pRb simultaneously restored normal muscle development (37Betel D. Wilson M. Gabow A. Marks D.S. Sander C. Nucleic Acids Res. 2008; 36: D149-D153Crossref PubMed Scopus (2012) Google Scholar). Our results were consistent with this earlier observation in that down-regulation of N-ras by miR-214 led to an increase in the myotube formation while reducing the BMP-mediated osteogenic differentiation (Fig. 2). Thus, by controlling the level of N-Ras, miR-214 exerts an additional layer of control of myogenic differentiation in this complex regulatory network.Promoting cell cycle exit as a way to regulate myogenic differentiation was demonstrated previously with miR-206 (30Kim H.K. Lee Y.S. Sivaprasad U. Malhotra A. Dutta A. J. Cell Biol. 2006; 174: 677-687Crossref PubMed Scopus (636) Google Scholar), one of the three microRNAs that are specifically expressed in the muscle. Also through global expression profiling analysis, several miR-206 targets were identified, including DNA polymerase α, and three other genes. However, the exact roles of miR-214 and miR-206 were likely to be different, because miR-206 was absent in proliferating C2C12 cells, and its expression did not commence until 2 days after induction of differentiation. The late appearance of miR-206 argues that its role probably lies in preventing the reversal of the cell cycle exit, which would result in a catastrophic consequence. In contrast, mi-214 was already expressed in proliferating C2C12 cells, albeit at a level lower than that in the differentiated cells (Fig. 2E). This suggests that miR-214 likely plays a permissive rather than an inductive role in allowing the cells to exit from the cell cycle. Moreover, the fact that forced expression of both 214in and 214mi exerted an effect on the myogenic differentiation argues that the cells are sensitive to fluctuations of the miR-214 level. So, it is possible that miR-214 constitutes a part of a measuring mechanism that senses the extent of the myogenic differentiation in a developing tissue environment and allocating an appropriate number of proliferating muscle precursors accordingly.Recently, miR-214 was found to target the polycomb group protein, Ezh2, in an independent study (40Juan A.H. Kumar R.M. Marx J.G. Young R.A. Sartorelli V. Mol. Cell. 2009; 36: 61-74Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). The MyoD locus in undifferentiated embryonic stem cells and several MyoD target genes in proliferating myoblasts were known to be occupied by Ezh2, which represses the transcription from these genes via chromatin remodeling. This study identified MyoD and myogenin as the transcription factors that control the pri-miR-214 transcription, thus suggesting a positive feedback loop, in which activation of MyoD and myogenin turns on the expression of miR-214, which in turn reinforces MyoD and myogenin expression by negatively feeding back to Ezh2. These and our current data demonstrate that miR-214 can function through multiple to the a that was proven to be for all IntroductionThe vertebrate skeletal muscle is developed from mesodermal stem cells that are committed to a muscle fate within paraxial somites, giving rise to immature myoblasts (1Rudnicki M.A. Le Grand F. McKinnell I. Kuang S. Cold Spring Harbor Symp. Quant. Biol. 2008; 73: 323-331Crossref PubMed Scopus (188) Google Scholar, 2Arnold H.H. Winter B. Curr. Opin. Genet. Dev. 1998; 8: 539-544Crossref PubMed Scopus (244) Google Scholar, 3Buckingham M. Bajard L. Chang T. Daubas P. Hadchouel J. Meilhac S. Montarras D. Rocancourt D. Relaix F. J. Anat. 2003; 202: 59-68Crossref PubMed Scopus (630) Google Scholar, 4Buckingham M. Relaix F. Annu. Rev. Cell Dev. Biol. 2007; 23: 645-673Crossref PubMed Scopus (348) Google Scholar). Committed myoblasts are proliferating progenitors (5Buckingham M. Montarras D. Curr. Opin. Genet. Dev. 2008; 18: 330-336Crossref PubMed Scopus (89) Google Scholar) that must exit from the cell cycle before myogenic regulatory factors such as MyoD and Myf5 activate myocyte enhancer factors and other downstream muscle-specific genes to drive the formation of multinucleated myotubes and eventually the contractile muscle fibers (6Black B.L. Olson E.N. Annu. Rev. Cell Dev. Biol. 1998; 14: 167-196Crossref PubMed Scopus (843) Google Scholar, 7Brand-Saberi B. Ann. Anat. 2005; 187: 199-207Crossref PubMed Scopus (77) Google Scholar, 8Tapscott S.J. Development. 2005; 132: 2685-2695Crossref PubMed Scopus (546) Google Scholar, 9Walsh K. Perlman H. Curr. Opin. Genet. Dev. 1997; 7: 597-602Crossref PubMed Scopus (264) Google Scholar). This process can be recapitulated in vitro with cell culture systems, the most widely used of which is the mouse C2C12 cell (10Andrés V. Walsh K. J. Cell Biol. 1996; 132: 657-666Crossref PubMed Scopus (502) Google Scholar, 11Yaffe D. Saxel O. Nature. 1977; 270: 725-727Crossref PubMed Scopus (1542) Google Scholar). In proliferating myoblasts, the MyoD targets of myogenic genes are repressed through chromosome remodeling and epigenetic histone modifications (12McKinsey T.A. Zhang C.L. Olson E.N. Curr. Opin. Genet. Dev. 2001; 11: 497-504Crossref PubMed Scopus (348) Google Scholar, 13Pownall M.E. Gustafsson M.K. Emerson Jr., C.P. Annu. Rev. Cell Dev. Biol. 2002; 18: 747-783Crossref PubMed Scopus (464) Google Scholar). Under the influence of extracellular signals in developing muscle tissues or upon serum depletion in culture systems, MyoD orchestrates an orderly exit from the cell cycle, involving concerted interplays between myogenic regulatory factors and the cell cycle machinery (7Brand-Saberi B. Ann. Anat. 2005; 187: 199-207Crossref PubMed Scopus (77) Google Scholar, 9Walsh K. Perlman H. Curr. Opin. Genet. Dev. 1997; 7: 597-602Crossref PubMed Scopus (264) Google Scholar, 13Pownall M.E. Gustafsson M.K. Emerson Jr., C.P. Annu. Rev. Cell Dev. Biol. 2002; 18: 747-783Crossref PubMed Scopus (464) Google Scholar, 14Polesskaya A. Rudnicki M.A. Dev. Cell. 2002; 3: 757-758Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The retinoblastoma gene product Rb and its homologue p107 (15Zacksenhaus E. Jiang Z. Chung D. Marth J.D. Phillips R.A. Gallie B.L. Genes Dev. 1996; 10: 3051-3064Crossref PubMed Scopus (264) Google Scholar, 16Schneider J.W. Gu W. Zhu L. Mahdavi V. Nadal-Ginard B. Science. 1994; 264: 1467-1471Crossref PubMed Scopus (338) Google Scholar, 17Gu W. Schneider J.W. Condorelli G. Kaushal S. Mahdavi V. Nadal-Ginard B. Cell. 1993; 72: 309-324Abstract Full Text PDF PubMed Scopus (641) Google Scholar) are essential in initiating an irreversible withdrawal from the cell cycle by up-regulating cyclin-dependent kinase inhibitor p21 (18Halevy O. Novitch B.G. Spicer D.B. Skapek S.X. Rhee J. Hannon G.J. Beach D. Lassar A.B. Science. 1995; 267: 1018-1021Crossref PubMed Scopus (1089) Google Scholar, 19Guo K. Wang J. Andrés V. Smith R.C. Walsh K. Mol. Cell. Biol. 1995; 15: 3823-3829Crossref PubMed Scopus (361) Google Scholar). Certain oncogene products including H- and N-Ras exhibit potent inhibition on myogenic differentiation by blocking Rb function (20Olson E.N. Spizz G. Tainsky M.A. Mol. Cell. Biol. 1987; 7: 2104-2111Crossref PubMed Scopus (144) Google Scholar).In addition to the complex network of protein-encoding genes, recent molecular and genetic studies have uncovered a myriad of microRNAs (miRNAs) 2The abbreviations used are: miRNAmicroRNAUTRuntranslated regionRTreverse transcriptionMHCmyosin heavy chainBrdUrdbromodeoxyuridinePBSphosphate-buffered saline214mimiR-214 mimic214inmiR-214 inhibitorqPCRquantitative PCRFACSfluorescence-activated cell sorterMAPmitogen-activated protein. that exert a different layer of control over myogenic differentiation (21Callis T.E. Deng Z. Chen J.F. Wang D.Z. Exp. Biol. Med. 2008; 233: 131-138Crossref PubMed Scopus (105) Google Scholar, 22van Rooij E. Liu N. Olson E.N. Trends Genet. 2008; 24: 159-166Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 23Brennecke J. Stark A. Cohen S.M. Genes Dev. 2005; 19: 2261-2264Crossref PubMed Scopus (29) Google Scholar). miRNAs are a class of small noncoding RNAs that are synthesized as pri-miRNAs from either dedicated transcription units or introns of protein-encoding genes (24Ambros V. Nature. 2004; 431: 350-355Crossref PubMed Scopus (8921) Google Scholar, 25Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29004) Google Scholar, 26Tomari Y. Zamore P.D. Genes Dev. 2005; 19: 517-529Crossref PubMed Scopus (735) Google Scholar). Pri-miRNAs are processed in the nucleus by the RNase Drosha, yielding precursor miRNAs, which have a characteristic “stem-loop” structure and are ∼70 nucleotides in length. Precursor miRNAs are then exported by Exportin 5 to the cytoplasm to be further processed by the RNase Dicer into mature miRNAs of ∼22 nucleotides (25Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29004) Google Scholar, 26Tomari Y. Zamore P.D. Genes Dev. 2005; 19: 517-529Crossref PubMed Scopus (735) Google Scholar). Mature miRNAs are incorporated into the RNA-induced silencing complex, where they anneal to their recognition sequences in the 3′-UTR of mRNA genes to attenuate gene expression through translation repression or mRNA degradation (27Carmell M.A. Hannon G.J. Nat. Struct. Mol. Biol. 2004; 11: 214-218Crossref PubMed Scopus (311) Google Scholar). Of many miRNAs that are involved in the regulation of muscle biology, miR-1 (28Zhao Y. Samal E. Srivastava D. Nature. 2005; 436: 214-220Crossref PubMed Scopus (1347) Google Scholar), miR-133 (29Chen J.F. Mandel E.M. Thomson J.M. Wu Q. Callis T.E. Hammond S.M. Conlon F.L. Wang D.Z. Nat. Genet. 2006; 38: 228-233Crossref PubMed Scopus (2192) Google Scholar), and miR-206 (30Kim H.K. Lee Y.S. Sivaprasad U. Malhotra A. Dutta A. J. Cell Biol. 2006; 174: 677-687Crossref PubMed Scopus (636) Google Scholar) are specifically expressed in the muscles where they superimpose on the intricate network of transcription factors and other regulatory proteins to control growth and myogenesis. Another muscle-specific microRNA, miR-208, is involved in myosin heavy chain production (31van Rooij E. Sutherland L.B. Qi X. Richardson J.A. Hill J. Olson E.N. Science. 2007; 316: 575-579Crossref PubMed Scopus (1370) Google Scholar).Here we investigate the mechanism of mouse miR-214, which was first shown to play an essential role in specifying slow muscle development in zebrafish (32Flynt A.S. Li N. Thatcher E.J. Solnica-Krezel L. Patton J.G. Nat. Genet. 2007; 39: 259-263Crossref PubMed Scopus (273) Google Scholar). In a mouse model of cardiac hypertrophy, miR-214 was found to be expressed at an elevated level, but forced expression of miR-214 did not lead to increased muscle growth (33Watanabe T. Sato T. Amano T. Kawamura Y. Kawamura N. Kawaguchi H. Yamashita N. Kurihara H. Nakaoka T. Dev. Dyn. 2008; 237: 3738-3748Crossref PubMed Scopus (118) Google Scholar). Using the in vitro differentiation of mouse C2C12 cells as a model, we report here that miR-214 promotes myogenic differentiation by facilitating the exit from the cell cycle, and we identified N-ras as a target of this function through microarray

BACKGROUND: This trial aimed to analyse the safety, effectiveness and transcriptomic characteristics of neoadjuvant toripalimab plus chemotherapy in II-III non-small-cell lung cancer (NSCLC). METHODS: Patient eligibility mainly involved treatment-naive, clinical stage II-III and wild-type EGFR/ALK NSCLC. The patients received 2-4 cycles of toripalimab (240 mg q3w) plus carboplatin-based chemotherapy. After the second treatment cycle, all patients were re-evaluated by a multidisciplinary team. Candidates eligible for surgery underwent surgery; otherwise, patients received the remaining treatment cycles. The primary endpoints were safety and major pathological response (MPR). Secondary endpoints were R0 resection rate, progression-free survival (PFS) and overall survival (OS). RNA sequencing of baseline and post-treatment samples was conducted to explore the transcriptomic characteristics of the therapeutic response. RESULTS: In total, 50 eligible patients were enrolled, including 12 (24.0%) with resectable disease (RD) and 38 (76.0%) with potentially resectable disease (PRD). Treatment-related adverse events (TRAEs) were recorded in 48 cases (96.0%). Severe TRAEs occurred in 3 (6.0%) cases, including myelosuppression, drug-induced liver injury and death related to haemoptysis. The objective response rate (ORR) was 76.0%, with 8 (16.0%) patients having a complete response (CR), 30 (60.0%) partial response (PR), 10 (20.0%) stable disease (SD) and 2 (4.0%) progressive disease (PD). Surgery could be achieved in 12 (100%) patients with RD and 25 (65.8%) with PRD; 1 (2.0%) with PRD refused surgery. Therefore, R0 resection was performed for all 36 (100%) patients who underwent surgery; 20 (55.6%) achieved MPR, including 10 (27.8%) with a complete pathological response (pCR). The CHI3L1 (chitinase-3-like protein 1) immunohistochemistry (IHC) expression of baseline tumour samples could predict the therapeutic response (AUC=0.732), OS (P=0.017) and PFS (P=0.001). Increased PD-1 expression, T cell abundance and immune-related pathway enrichment were observed in post-treatment samples compared to baseline in the response group (CR+PR) but not in the non-response group (SD+PD). CONCLUSIONS: Neoadjuvant toripalimab plus chemotherapy was safe and effective, with a high MPR and manageable TRAEs for II-III NSCLC, even converting initially PRD to RD. Disparate transcriptomic characteristics of therapeutic efficiency were observed, and CHI3L1 expression predicted therapeutic response and survival. TRIAL REGISTRATION: ChiCTR1900024014, June 22, 2019.

BACKGROUND: Immunotherapy for malignant tumors has made great progress, but many patients do not benefit from it. The complex intratumoral heterogeneity (ITH) hindered the in-depth exploration of immunotherapy. Conventional bulk sequencing has masked intratumor complexity, preventing a more detailed discovery of the impact of ITH on treatment efficacy. Hence, we initiated this study to explore ITH at the multi-omics spatial level and to seek prognostic biomarkers of immunotherapy efficacy considering the presence of ITH. METHODS: Using the segmentation strategy of digital spatial profiling (DSP), we obtained differential information on tumor and stromal regions at the proteomic and transcriptomic levels. Based on the consideration of ITH, signatures constructed by candidate proteins in different regions were used to predict the efficacy of immunotherapy. RESULTS: Eighteen patients treated with a bispecific antibody (bsAb)-KN046 were enrolled in this study. The tumor and stromal areas of the same samples exhibited distinct features. Signatures consisting of 11 and 18 differentially expressed DSP markers from the tumor and stromal areas, respectively, were associated with treatment response. Furthermore, the spatially resolved signature identified from the stromal areas showed greater predictive power for bsAb immunotherapy response (area under the curve=0.838). Subsequently, our stromal signature was validated in an independent cohort of patients with non-small cell lung cancer undergoing immunotherapy. CONCLUSION: We deciphered ITH at the spatial level and demonstrated for the first time that genetic information in the stromal region can better predict the efficacy of bsAb treatment. TRIAL REGISTRATION NUMBER: NCT03838848.