1. MiR-19b/20a/92a regulates the self- renewal and proliferation of gastric cancer stem cells Journal of Cell Science (IF=5.325) 报告人：黄美玲 2014-11-27

2. 传统观念：肿瘤是由体细胞突变而成, 每个肿瘤细胞都可以无限制 地生长。 肿瘤干细胞 (CSCs) 理论 : 肿瘤的生长是肿瘤中极少量肿瘤干细胞 增殖的结果，只有消灭这些细胞才可能彻底治愈癌症。 肿瘤干细胞的概念于 2001 年被正式提出 Cancer stem cell theory 1

3. Finding and Development of CSCs 只有少数表型为 CD34+CD38– 的癌细胞 可成功在 NOD/SCID 小鼠体内形成肿瘤 。 Hewitt 将小鼠白血病细胞移植到同种 小鼠，发现仅有 1%-4% 细胞能在小 鼠脾脏内形成克隆集落。 此后脑部、前列腺、胰腺、结肠、直肠等部 位的 CSC 被陆续分离出来。。。 Al Hajj 等提取 CD44+CD24– 乳腺癌细胞，植 入小鼠体内，约 12 周出现明显肿瘤，首次分 离出实体瘤干细胞。 2

4. CSCs and traditional stem cells 相同点： ① 具有无限增殖的能力。 ② 有共同的调节机制及信号转导通路。 ③ 具有异质性。 ④ 具有端粒酶活性。 ⑤ 具有比较类似的表面标志物 ⑥ 通过两种方式分裂 不同点： 不能控制细胞的数量，恶性增殖。 3

5. CSCs in tumorigenesis and metastasis Niche: the specific microenvironment = blood vessels (red) + myofibroblasts (orange) + extracellular matrix components Purple: CSCs Blue: nontumorigenic cells 2: tumorigenic CSCs 4

6. Cancer therapy targeting CSCs Targeting CSCs is effective for cancer treatment 5

7. Signal pathways Microenvironment to CSCs 6

8. The isolation and identification of CSCs 无血清培养基自然筛选法：最广泛，最简单易行。 体内移植 鉴定肿瘤形成 体外培养鉴定集落形成 特殊无血清培养基培养 2 肿瘤组织分离单个细胞 或（ FCM 分选） 1 非干细胞营养缺陷死亡 干细胞正常生长 3 鉴 定鉴 定 分 离分 离 7

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10. Design GCSCs isolation Identification of proliferation and differentiation Verify the function of miR-19b/20a/92a to tumor growth and the self-renewal in vivo and in vitro (overexpression) miR-19b/20a/92a was reduced during differentiation miR-92a is an independent prognostic factor in gastric cancer miR-19b/20a/92a target E2F1 and HIPK1 and activate the b-catenin signaling pathway 9

11. 1 Isolation GCSCs by EpCAM+/CD44+ Gastric cancer stem cells (GCSCs) has been identified in primary tissues by the cell surface markers EpCAM+ and CD44. SGC7901, 80 % expressed EpCAM and CD44. MKN28 cells, only 0.2% EpCAM+/CD44+ cells Cells were isolated by FACs cultured in suspension in low adherence flasks with non-serum medium. 15 days later, 85–95% of SGC7901 cells formed tumorspheres VS 15–20% of MKN 28 cells. 10

12. Cells containing 10 4 EpCAM+/CD44+ cells generated tumors in BALB/C nude mice (IVIS 100 Imaging System) 2 Identification (non-serum medium) 11

13. CFSE: 能穿透细胞膜，与胞内蛋白共价结合，水解后释放绿色荧光。在增殖过程中荧 光强度会随细胞分裂而逐级递减，标记荧光可平均分配至两个子代细胞中, 可被用于 检测细胞增殖。另外， CFSE 标记的细胞用于体内观察可以长达数周之久。 GCSCs proliferate well Identification ( differentiation in serum-containing medium) 12

14. Cells develop into elongated cells with highly expressing CK14 and CK18 13

15. Successful isolation of GCSC by EpCAM+/CD44+ Containing more cells Good tumorigenetic ability Growing rapidly and differentiating well when attached Because miRNAs can regulate the self-renewal and differentiation of stem cells. So we compare miRNA expression in GCSCs and the differentiated cells. 14

16. 3 miR-19b/20a/92a reduced during differentiation Lane1: freshly dissociated tumorspheric cells; lane 2-5: 8 h,12 h, 1 d, 8d after resuspended in normal culture conditions. miRNA array RT-PCR 15

17. GCSCs from human gastric cancer tissue (undifferentiation) high expressed miR-19b/20a/92a 16

18. 4 Overexpression of miR-19b/20a/92a is required to maintain GCSCs tumorspheres Successful overexpression of miRNA by RT-PCR Methods: infected EpCAM - /CD44 - (separated from SGC7901 cells) and MKN28 cells with lentiviruses containing precursor microRNAs: pre-miR-19b, pre-miR-20a and pre-miR-92a. 17

19. synergistic effect Sphere and cell numbers increased after overexpression 18

20. miR-19b/20a/92a inhibitors markedly inhibited tumorspheres formation not only sustains the self-renewal of GCSCs but also maintains resistance to chemotherapeutic drugs 5-FU 19

21. 5 Overexpression of miR-19b/20a/92a upregulates the selfrenewal of GCSCs in NOD-SCID mice NOD-SCID mouse was injected with2×103 miR-19b/20a/92a-infected cells or NC-infected cells. 28th day 20

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23. 6 miR-19b/20a/92a miRNAs promote gastric cancer cell growth and progression. MTT Clony formation 22

24. luciferase-labeled lenti-miR-17-92-infected SGC7901-Luc cell 23

25. 7 The miR-17-92 cluster members target E2F1 and HIPK1 and activate the b-catenin signaling pathway 24 3’-UTR of human E2F1 contains two conserved putative target sites for miR-20a 3’UTR of human HIPK1 contains one conserved site for miR-19b and one for miR-92a 3’UTRs of human E2F1 and HIPK1 were inserted in downstream of the luciferase gene in the pGL3- Control vector, providing sense (Luc-1S, 2S and S) and antisense (Luc-1AnS, 2AnS and AS) constructs HEK-293

26. upregulation of β-catenin proteins in miR- 19b/20a/92a- transfected cells and GCSCs from human GC tissues 25

27. 8 miR-92a can be used as an independent prognostic factor in gastric cancer detect each member of the miR-17-92 cluster in RNA samples from the stomach tissues of 97 gastric cancer patients miR-92a as an independent factor that was associated with overall survival 26

28. Conclusion miR-19b, miR-20a and miR-92a might play important roles in the development of gastric cancer stem cells miR-92a has the potential to be used as a predictive prognostic marker in gastric cancer 27