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B7-H3 silencing by RNAi inhibits tumor progression and enhances chemosensitivity in U937 cells.

Zhang W, Wang J, Wang Y, Dong F, Zhu M, Wan W, Li H, Wu F, Yan X, Ke X - Onco Targets Ther (2015)

Bottom Line: Downregulation of B7-H3 significantly decreased U937 cell growth and colony-forming ability.The cell migration rate of B7-H3 knockdown cells was reduced more than fivefold, and invasion capacity decreased by 86.7%.B7-H3 RNAi profoundly increased the antitumor effect of chemotherapy in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Lymphoma Research Center, Peking University Third Hospital, Beijing, People's Republic of China.

ABSTRACT

Background: The role of B7-H3 in acute monocytic leukemia U937 cells has not been thoroughly investigated.

Materials and methods: B7-H3 knockdown in the U937 cell line was performed using small hairpin (sh)RNA lentivirus transduction. The effects on cell proliferation, cycle, migration, and invasion were investigated by Cell Counting Kit-8 assay, methyl cellulose colony-forming assay, propidium iodide staining, and Transwell assays in vitro. Changes in cell growth inhibition and apoptosis, when combined with chemotherapy drugs, were determined using the Cell Counting Kit-8 and Annexin V-FITC/PI assays. U937 xenograft models were used to assess the effects of B7-H3 on tumorigenicity and the therapeutic effect of B7-H3 knockdown in combination with chemotherapy drugs in vivo.

Results: Downregulation of B7-H3 significantly decreased U937 cell growth and colony-forming ability. The mean inhibition rate of tumor growth with B7-H3 knockdown was 59.4%, and the expression of both Ki-67 and PCNA in xenografts was significantly reduced. After B7-H3 silencing, the U937 cell cycle was arrested at the G0/G1 phase. The cell migration rate of B7-H3 knockdown cells was reduced more than fivefold, and invasion capacity decreased by 86.7%. B7-H3 RNAi profoundly increased the antitumor effect of chemotherapy in vitro and in vivo. On day 19, inhibition rates of tumor growth in B7-H3 shRNA combined with idarubicin, cytarabine, and idarubicin plus cytarabine were 70.5%, 80.0%, and 90.0%, respectively (P=0.006, P=0.004, and P=0.016, respectively).

Conclusion: B7-H3 may promote U937 cell progression, and shRNA targeting B7-H3 significantly enhances sensitivity to chemotherapeutic drugs. These results may provide new insight into the function of B7-H3 and a promising therapeutic approach targeting B7-H3 in acute monocytic leukemia.

No MeSH data available.


Related in: MedlinePlus

B7-H3 knockdown inhibits tumor proliferation in vitro and in vivo.Notes: (A) B7-H3 silencing inhibited cell growth analyzed by CCK-8 assay. (B and C) B7-H3 knockdown inhibited the colony formation detected by methyl cellulose colony-forming assay (×100). The data are from at least three separate experiments. (D) Silencing of B7-H3 significantly inhibited tumor growth in vivo compared with the NC. (E) Tumor weight of the B7-H3 KD group was decreased compared to the NC group on day 19. (F) Excised xenograft tumors on day 19. (G) Immunohistochemical staining of Ki-67 and PCNA for proliferative cells in a B7-H3 knockdown xenograft was reduced compared to the NC group on day 19 (×400). *Indicates that the results are statistically significant compared to the NC group; P<0.05.Abbreviations: CON, control group of nontransfected cells; NC, transfected group with negative nontargeted control sequence; KD, transfected group with B7-H3 small hairpin RNA; h, hours; d, days; CCK-8, Cell Counting Kit-8; PCNA, proliferating cell nuclear antigen; CFA, colony-forming ability.
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f2-ott-8-1721: B7-H3 knockdown inhibits tumor proliferation in vitro and in vivo.Notes: (A) B7-H3 silencing inhibited cell growth analyzed by CCK-8 assay. (B and C) B7-H3 knockdown inhibited the colony formation detected by methyl cellulose colony-forming assay (×100). The data are from at least three separate experiments. (D) Silencing of B7-H3 significantly inhibited tumor growth in vivo compared with the NC. (E) Tumor weight of the B7-H3 KD group was decreased compared to the NC group on day 19. (F) Excised xenograft tumors on day 19. (G) Immunohistochemical staining of Ki-67 and PCNA for proliferative cells in a B7-H3 knockdown xenograft was reduced compared to the NC group on day 19 (×400). *Indicates that the results are statistically significant compared to the NC group; P<0.05.Abbreviations: CON, control group of nontransfected cells; NC, transfected group with negative nontargeted control sequence; KD, transfected group with B7-H3 small hairpin RNA; h, hours; d, days; CCK-8, Cell Counting Kit-8; PCNA, proliferating cell nuclear antigen; CFA, colony-forming ability.

Mentions: We used CCK-8 and colony-forming assays to evaluate the effects of B7-H3 knockdown on tumor cell proliferation in vitro. The stable knockdown of B7-H3 in U937 cells significantly reduced cell growth. Compared to the NC group, the growth of KD cells was decreased by 32.8% after 72 hours of incubation (P=0.017) (Figure 2A). The colony formation assay further confirmed that B7-H3 silencing inhibits U937 cell proliferation. After 14 days of incubation, the average CFA of KD cells was 22.5% compared with 92.8% in the NC group (P=0.000) (Figure 2B and C).


B7-H3 silencing by RNAi inhibits tumor progression and enhances chemosensitivity in U937 cells.

Zhang W, Wang J, Wang Y, Dong F, Zhu M, Wan W, Li H, Wu F, Yan X, Ke X - Onco Targets Ther (2015)

B7-H3 knockdown inhibits tumor proliferation in vitro and in vivo.Notes: (A) B7-H3 silencing inhibited cell growth analyzed by CCK-8 assay. (B and C) B7-H3 knockdown inhibited the colony formation detected by methyl cellulose colony-forming assay (×100). The data are from at least three separate experiments. (D) Silencing of B7-H3 significantly inhibited tumor growth in vivo compared with the NC. (E) Tumor weight of the B7-H3 KD group was decreased compared to the NC group on day 19. (F) Excised xenograft tumors on day 19. (G) Immunohistochemical staining of Ki-67 and PCNA for proliferative cells in a B7-H3 knockdown xenograft was reduced compared to the NC group on day 19 (×400). *Indicates that the results are statistically significant compared to the NC group; P<0.05.Abbreviations: CON, control group of nontransfected cells; NC, transfected group with negative nontargeted control sequence; KD, transfected group with B7-H3 small hairpin RNA; h, hours; d, days; CCK-8, Cell Counting Kit-8; PCNA, proliferating cell nuclear antigen; CFA, colony-forming ability.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4508088&req=5

f2-ott-8-1721: B7-H3 knockdown inhibits tumor proliferation in vitro and in vivo.Notes: (A) B7-H3 silencing inhibited cell growth analyzed by CCK-8 assay. (B and C) B7-H3 knockdown inhibited the colony formation detected by methyl cellulose colony-forming assay (×100). The data are from at least three separate experiments. (D) Silencing of B7-H3 significantly inhibited tumor growth in vivo compared with the NC. (E) Tumor weight of the B7-H3 KD group was decreased compared to the NC group on day 19. (F) Excised xenograft tumors on day 19. (G) Immunohistochemical staining of Ki-67 and PCNA for proliferative cells in a B7-H3 knockdown xenograft was reduced compared to the NC group on day 19 (×400). *Indicates that the results are statistically significant compared to the NC group; P<0.05.Abbreviations: CON, control group of nontransfected cells; NC, transfected group with negative nontargeted control sequence; KD, transfected group with B7-H3 small hairpin RNA; h, hours; d, days; CCK-8, Cell Counting Kit-8; PCNA, proliferating cell nuclear antigen; CFA, colony-forming ability.
Mentions: We used CCK-8 and colony-forming assays to evaluate the effects of B7-H3 knockdown on tumor cell proliferation in vitro. The stable knockdown of B7-H3 in U937 cells significantly reduced cell growth. Compared to the NC group, the growth of KD cells was decreased by 32.8% after 72 hours of incubation (P=0.017) (Figure 2A). The colony formation assay further confirmed that B7-H3 silencing inhibits U937 cell proliferation. After 14 days of incubation, the average CFA of KD cells was 22.5% compared with 92.8% in the NC group (P=0.000) (Figure 2B and C).

Bottom Line: Downregulation of B7-H3 significantly decreased U937 cell growth and colony-forming ability.The cell migration rate of B7-H3 knockdown cells was reduced more than fivefold, and invasion capacity decreased by 86.7%.B7-H3 RNAi profoundly increased the antitumor effect of chemotherapy in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Lymphoma Research Center, Peking University Third Hospital, Beijing, People's Republic of China.

ABSTRACT

Background: The role of B7-H3 in acute monocytic leukemia U937 cells has not been thoroughly investigated.

Materials and methods: B7-H3 knockdown in the U937 cell line was performed using small hairpin (sh)RNA lentivirus transduction. The effects on cell proliferation, cycle, migration, and invasion were investigated by Cell Counting Kit-8 assay, methyl cellulose colony-forming assay, propidium iodide staining, and Transwell assays in vitro. Changes in cell growth inhibition and apoptosis, when combined with chemotherapy drugs, were determined using the Cell Counting Kit-8 and Annexin V-FITC/PI assays. U937 xenograft models were used to assess the effects of B7-H3 on tumorigenicity and the therapeutic effect of B7-H3 knockdown in combination with chemotherapy drugs in vivo.

Results: Downregulation of B7-H3 significantly decreased U937 cell growth and colony-forming ability. The mean inhibition rate of tumor growth with B7-H3 knockdown was 59.4%, and the expression of both Ki-67 and PCNA in xenografts was significantly reduced. After B7-H3 silencing, the U937 cell cycle was arrested at the G0/G1 phase. The cell migration rate of B7-H3 knockdown cells was reduced more than fivefold, and invasion capacity decreased by 86.7%. B7-H3 RNAi profoundly increased the antitumor effect of chemotherapy in vitro and in vivo. On day 19, inhibition rates of tumor growth in B7-H3 shRNA combined with idarubicin, cytarabine, and idarubicin plus cytarabine were 70.5%, 80.0%, and 90.0%, respectively (P=0.006, P=0.004, and P=0.016, respectively).

Conclusion: B7-H3 may promote U937 cell progression, and shRNA targeting B7-H3 significantly enhances sensitivity to chemotherapeutic drugs. These results may provide new insight into the function of B7-H3 and a promising therapeutic approach targeting B7-H3 in acute monocytic leukemia.

No MeSH data available.


Related in: MedlinePlus