• 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • br CDCA contributes to the growth of breast


    3.2. CDCA8 contributes to the growth of breast cancer cells
    CDCA8 shRNA and control shRNA were transfected into the MCF7 and T47D cells to decipher the biological functions of CDCA8 on E2-stimulated breast cancer cells. Western blot analysis demonstrated that the two shRNA were well designed to significantly down-regulate the expression of CDCA8 in both MCF7 and T47D cells (Fig. 2A). It was further testified that the CDCA8 knockdown could inhibit E2 stimulated MCF7 and T47D cells proliferation (Fig. 2B and C, p < 0.01) and colony formation (Fig. 2D and E, p < 0.01), while CDCA8 knockdown did not show any significant effect on cells proliferation or colony formation in MCF7 and T47D cells without E2 stimulation (Fig. 2B–E). Such results suggested that CDCA8 mediated the growth of breast cancer cells induced by E2.
    3.3. CDCA8 promotes the survival of breast cancer cells
    CDCA8 knockdown could promote the apoptosis of E2-stimulated MCF7 (Fig. 3A) and T47D cells (Fig. 3B) (p < 0.01), and there was no such effect happened without E2 stimulation. Cell cycle analysis showed that about 70% tumor cells were arrested at G1 stage without
    Fig. 1. CDCA8 is upregulated in estrogen-stimulated breast cancer cells. MCF7 and T47D cells were grown in 6-well plates (phenol red-free media, 5% char-coal-stripped FBS. with or without E2 (10 nmol/L)) (A) RT-PCR analysis of CDCA8 mRNA in MCF7 and T47D cells. GAPDH served as loading control. (B) Western blot analysis of CDCA8 levels in MCF7 and T47D breast cancer cells. β-Actin served as loading controls. Data were shown as mean ± S.D. of three independent experiments. **p < 0.01.
    E2 stimulation (Fig. 3C and D), and such distribution could be altered after E2 stimulation (about 40% at G1 stage and above 20% at S stage). CDCA8 knockdown could restore the Minocycline HCl distribution on E2 sti-mulated tumor cells, while such treatment did not show any effect on the cells without E2-stimulation (Fig. 3C and D).
    Next, the survival relevant mechanism involved was investigated. It was demonstrated that CDCA8 knockdown could up-regulate the ex-pression of P21 and P27, and down-regulate the expression of CCND1 and BCL2 both at the mRNA (Fig. 4A, p < 0.05) and protein levels (Fig. 4B and C).
    3.4. Up-regulated CDCA8 could predict poor prognosis in breast carcinoma patients
    Immunohistochemistry analysis showed up-regulated CDCA8 ex-pression in tumor samples of breast carcinoma patients (Fig. 5A) and such increase could be also observed in mRNA level (Fig. 5B, p < 0.01). Kaplan-Meier's analysis of the correlation between CDCA8 expression and overall survival of breast carcinoma patients indicated that higher CDCA8 expression could predict the poor prognosis of breast carcinoma with a probability lower than 0.4 at the five-year interval (Fig. 5C, p = 0.035). Association of CDCA8 expression with clinicopathological parameters of patients in breast cancer was supplied in Supplementary Table 2, which showed that the expression of CDCA8 correlates with the tumor size of the breast cancer patient.
    Fig. 2. CDCA8 knockdown inhibits breast cancer cell growth. (A) Western blot assay for CDCA8 in MCF7 and T47D cells stably expressing shCtrl or shCDCA8. β-Actin served as loading control. Total cell number assays. (B) MCF7 and (C) T47D cells number was determined at the indicated days with or without E2 (10 nmol/L). (D) MCF7 and (E) T47D cells were subjected to cell colony formation assay. Data were shown as mean ± S.D. of three independent experiments. *p < 0.05; **p < 0.01.
    Fig. 3. Knockdown of CDCA8 induces cell apoptosis in breast cancer cells. Flow cytometry analysis of (A) MCF7 and (B) T47D cells apoptosis. Cell cycle analysis of (C) MCF7 and (D) T47D cells. Data were mean ± S.D. of three independent experiments and each measured in triplicate, **p < 0.01.
    Fig. 4. Cellular expression of marker genes is analyzed in breast cancer cells. (A) mRNA levels of GREB1, TFF1, ESR1, MYC, MYB, SRC, P21, P27, CCND1 and BCL2 were detected in MCF7 and T47D cells by RT-PCR. GAPDH was detected as a control. (B) (C) Western blot analysis of marker protein ex-pression. β-Actin served as an internal control. Data were mean ± S.D. of three independent experiments and each measured in triplicate (*p < 0.05).
    4. Discussion
    In the present investigation, up-regulated CDCA8 expression can be observed in E2-stimulated MCF7 cells, T47D cells, and human breast cancer samples, which can predict the poor prognosis of breast carci-noma patients. It is further demonstrated that CDCA8 can promote the E2 induced growth and survival of both MCF7 cells and T47D cells by down-regulated P21 and P27, while up-regulated CCND1 and BCL2. It has been reported that the interrelationships among 14-3-3ζ, 
    forkhead box protein M1, and mitosis associated CPC gene signature could contribute to the endocrine resistance in breast cancer (Anna et al., 2011), and the up-regulated CDCA8 expression could dramati-cally reduce survival ratio of breast cancer patients (Phan et al., 2018). In fact, for the first time, CDCA8 has been demonstrated to be indis-pensable for the E2-stimulated MCF7 and T47D cells growth and sur-vival, which can be significantly inhibited by shRNA against CDCA8. Such results are consistent with the previous findings that CDCA8 knockdown could inhibit cutaneous melanoma cell proliferation,