SPARC is a highly conserved, multifunctional matricellular protein that regulates matrix remodeling, cell adhesion and migration, cell cycle regulation and angiogenesis. Although there is growing evidence for an important role for SPARC in a variety of cancers, there is no unifying model, which explains all aspects of its function
[9, 11]. For example, higher levels of SPARC expression have been reported in breast cancer
[12, 13], melanoma
[14, 15], hepatocellular carcinoma
[16, 17], prostate cancer
 and colorectal cancer
[19, 20]. However, the opposite effect has also been demonstrated, suggesting that SPARC may be able to inhibit tumorigenesis or tumor progression in breast cancer
[21, 22], ovarian carcinoma
[23, 24], hepatocellular carcinoma
, prostate cancer
, and colorectal cancer
[27, 28]. In cervical cancer, recent researches reveal that increased expression of SPARC is related to the progression of cervical cancer and often accompanied with aberrant methylation
[29, 30]. In our study, we found that SPARC was highly expressed in the high invasive subclones HeLa-1 and SiHa-1, compared to the low invasive subclones HeLa-25 and SiHa-23. These results revealed that SPARC was related to the invasive phenotype of cervical cancer cells. Further researches showed that lentivirus-mediated knockdown of SPARC expression significantly suppressed cervical cancer cell proliferation, induced cell apoptosis, and inhibited cell invasion and metastasis.
In this study, using MTT assay, soft agar colony formation assay and flow cytometer, we concluded that knockdown of SPARC suppressed cervical cancer cell proliferation. Flow cytometer showed that knockdown of SPARC reduced the number of cells in S-phase, while increased the number of cells in G1/G0-phase, indicating G1/G0 arrest. To understand the mechanism of SPARC in cervical cancer cell proliferation, we detected the different expressions of Cyclin D1, PCNA, P53 and P21 between SPARC shRNA infected cells and control shRNA infected cells. Cyclin D1, a member of G1 cyclins, and PCNA (proliferating cell nuclear antigen) are proliferation biomarkers
[31, 32]. P53, a tumor suppressor gene, and P21 (cyclin-dependent kinase inhibitor), a key mediator of p53, can induce cell-cycle arrest in the G1-S checkpoint
[33, 34]. In this study, we found that knockdown of SPARC expression could decrease the expressions of Cyclin D1 and PCNA and increase the expressions of P53 and P21, which suggested that depletion of SPARC could inhibit cervical cancer cell proliferation through activation of a p53⁄p21Cip1⁄Waf1 pathway dependent on G1-S checkpoint. In melanoma, SPARC also could regulate cell cycle progression and proliferation through the p53/p21 (Cip1/Waf1) pathway
. In brief, SPARC could play an important role in cervical cancer cell proliferation by controlling cell cycle progression, but how depletion of SPARC can lead to activation of the p53/p21 (Cip1/Waf1) signaling pathway needs further study.
Further researches showed that knockdown of SPARC induced cervical cancer cell apoptosis. Apoptosis is modulated partially by Bcl-2 family including apoptosis-inhibiting genes (Bcl-2, Bcl-xL, Mcl-1, A1 and Bcl-w) and apoptosis-accelerating genes (Bax, Bak, Bcl-xS and Bim)
. In this study, we found that knockdown of SPARC could decrease the expression of apoptosis-inhibiting gene Bcl-2 and increase the expression of apoptosis-accelerating gene Bax, indicating the down-regulated ratio of Bcl-2/Bax. Our result revealed that SPARC could play an effect on apoptosis by changing the ratio of Bcl-2/Bax. Similar results were found in human melanoma; suppression of SPARC in several human melanoma cells triggered apoptotic cell death dependent on p53 and induction of Bax
. Apoptosis, programmed cell death, is vital for normal development and tissue homeostasis. Our data suggested that SPARC as an antistress factor could promote cervical cancer cell survival through suppression of apoptotic pathways.
Using Boyden chambers and xenografts in nude mice, we concluded that knockdown of SPARC expression inhibited cervical cancer cell invasion and metastasis. To clarify the mechanism of SPARC in cervical cancer cell invasion and metastasis, after viral infection, we detected the expression of the cellular adhesion molecule E-cadherin and integrins, which mediated cell-cell adhesion and cell-extracellular matrix adhesion, and the expression of proteolytic enzymes such as plasminogen activator/plasmin system (uPA-uPAR) and matrix metalloproteinases (MMPs), which degraded the extracellular matrix (ECM). These results revealed that knockdown of SPARC up-regulated the expression of E-cadherin and had no effect on the expressions of integrin β1 and integrin β3. SPARC can break down cell-cell connections to improve tumor invasion by changing E-cadherin expression. Similar results were also found in melanoma
[38, 39]; SPARC can down-regulate E-cadherin and stimulate an invasive melanoma phenotype. Next, knockdown of SPARC decreased the expressions of MMP2 and MMP9, but no significant differences were found in the expressions of u-PA, uPAR, PAI-1, TIMP1 and TIMP2 between SPARC shRNA infected cells and control shRNA infected cells. SPARC can make the extracellular matrix degradation by MMP2 and MMP9. Similar results were also found in glioma
[40, 41]. Our data suggested that depletion of SPARC could promote the homophilic cell-cell adhesion by up-regulating E-cadherin and restrained extracellular matrix degradation by down-regulating MMPs expressions to inhibit cervical cancer cell invasion and metastasis.
In conclusion, SPARC is associated with cervical cancer cell growth and metastasis. Knockdown of SPARC expression significantly suppresses cervical cancer cell proliferation, induces cell apoptosis and inhibits cell invasion and metastasis. All of these informations contribute to a better understanding that SPARC, as a promoter, improves cervical cancer cell proliferation, invasion and metastasis.