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

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

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

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

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

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

Signal pathways Microenvironment to CSCs 6

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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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

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

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

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

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

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

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

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

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

synergistic effect Sphere and cell numbers increased after overexpression 18

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

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

luciferase-labeled lenti-miR infected SGC7901-Luc cell 23

7 The miR 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

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

8 miR-92a can be used as an independent prognostic factor in gastric cancer detect each member of the miR 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

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