The Effects Of S100A9 On Proliferation And Migration Of Human Squamous Cervical Carcinoma Cell Lines And Its Mechanisms

Backgrounds and objectivesS100 protein family is a class of calcium-binding protein with EF-hand. The physiological roles of S100 protein family include cell structure formation, energy metabolism, cell signal transduction and so on. There are 16 members(S100A9 included) located on chromosome 1q21, a region that is unstable and rearrangeable in tumors. Abnormal expression of some S100 proteins in tumor cells is associated with the staging and prognosis.S100A9(also known as calgranulin B) is a member of the S100 protein family. It was originally detected in myeloid cells, and express mainly in neutrophils and monocytes. Recently, over-expression of S100A9 has been observed in many tumor cells. Our previous studies have shown that S100A9 promoted the proliferation and invasion of hepatocellular carcinoma cells Hep G2, and also contributed to colorectal carcinoma cell survival and migration. All these results indicate that S100A9 is associated with tumor development. However, the precise roles of S100A9 in different tumors and potential molecular mechanisms have yet to be well investigated.Cervical cancer ranks as the third most commonly diagnosed cancer in females worldwide. The motility and invasiveness of cancer cells play a critical role in the mortality of cervical cancer patients, and provide valuable information for determining the approach for treatment. Therefore, clarifying the specific mechanism of migration of cervical cancer in patients is of great clinical value to lower mortality.In the previous study, it was confirmed that S100A9 expression is up-regulated in cervical cancer tissue samples compared with matching distal normal tissue samples by gene chip. Therefore, the expression of S100A9 is associated with cervical cancer progression.In the present study, we aimed to investigate the effects(proliferation and migration) of S100A9 on cervical cancer cells as well as the underlying molecular mechanisms.These findings can provide experimental evidence to clarify the mechanisms of cervical cancer, and also point new ways for diagnosis and treatment of cervical cancer.Methods1. Preparation tools for down and up regulation of S100A9 gene expression.1.1Establishment of the shuttle vector(p SES1), and then vector was cleaved with a restriction endonuclease to linearize it and transformed into E. coli strain BJ5183. Recombinants were selected with kanamycin and screened by restriction endonuclease digestion. Then the recombinant adenoviral construct was cleaved with Pac I to expose its inverted terminal repeats, the recombinant adenoviral vector DNA mixed with Lipo2000 was transfected into a packaging cell line(HEK293).1.2Adsi S100A9, Adsi Control and the recombinant adenoviruses carrying human S100A9 gene(Ad S100A9), the control recombinant adenoviruses carrying GFP gene(Ad GFP) were amplified in HEK293 cells.2. Detection of the endogenous expression of S100A9 in three cervical cancer cell lines(Siha, Caski and Hela); Hela cells with lowest expression of S100A9 were infected with Ad S100A9 for S100A9 over-expression; Siha cells with highest expression of S100A9 were infected with Adsi S100A9.The levels of m RNA and protein were confirmed by RT-PCR and Western Blot.3. The roles of S100A9 on proliferation and migration of Hela and Siha cells: the MTT assay, Wound healing assays and Transwell assay were used to detect proliferation and migration of Hela and Siha cells after infection with Ad S100A9 and Adsi S100A9 respectively.4. The effect of S100A9 on EMT(epithelial-mesenchymal transition): Western Blot and Immunofluorescence assays were used to detect EMT-relevant markers(E-cadherin, Vimentin) of Hela and Siha cells after infection with Ad S100A9 and Adsi S100A9 respectively.5. The effect of S100A9 on activity of Wnt/β-catenin signaling pathway: After Hela and Siha cells treated with Ad S100A9 and Adsi S100A9 respectively, Western blot was used to detect the β-catenin(total and nuclear β-catenin) which is a key effector of this signaling pathway. RT-PCR was used to detect transcriptional levels of c-myc, Snail and Twist, which are classic target genes of Wnt/β-catenin signaling pathway.6. The effect of the Wnt/β-catenin signaling pathway on S100A9-induced proliferation and migration of Hela cells: MTT assay and the Transwell assay were used to detect cell proliferation and migration in Hela cells treated with GST-h S100A9 and Adsiβ-catenin.7. The effect of the Wnt/β-catenin signaling pathway on S100A9-induced EMT in Hela cells: Western blot was used to detect the levels of E-cadherin and Vimentin in Hela cells treated with with GST-h S100A9 and Adsiβ-catenin.Results1. Establishment, generation and amplification of the recombinant adenovirus:1.1The shuttle vector was linearized and transformed into E. coli for recombination. Then the recombinant adenoviral vector DNA was transfected into the HEK293, and transfected cells were monitored by RFP expression. As RFP gradually enhanced, after 7–10 days, viruses were harvested; The Same methods to get the control adenovirus(Adsi Control).1.2 After infection with Adsi S100A9, Adsi Control and Ad S100A9, Ad GFP for 24, 36 and 48 h, the expression of red flurescent protein(RFP) and green flurescent protein(GFP) in HEK293 cells were gradually increased and then these viruses were harvested and then used in our study.2. The expression level of S100A9 in cervical cancer was examined. The results show that Hela cells have lowest expression of S100A9 and Siha cells have highest level expression among these cell lines. After Hela and Siha cells were treated with Ad S100A9 and Adsi S100A9 respectively, up- and down-regulation of S100A9 were confirmed, versus GFP(Ad GFP infection) group and si Control(Adsi Control infection) group.3. S100A9 promoted proliferation and migration of cervical cancer cells.3-1 S100A9 promoted proliferation of cervical cancer cells(MTT): 1) In Hela cells: The difference of the OD490nm(OD) between three groups was not obvious from 1d to 2d(P>0.05); Cell growth was highly enhanced by 0.51-fold in S100A9-overexpressing cells at 3d, versus GFP group(P<0.05);2) In Siha cells: The difference of the OD between three groups was not obvious from 1d to 2d(P>0.05); Cell growth was significantly suppressed in S100A9 down-regulated cells at 3d, versus si Control groups(P<0.05).3-2 S100A9 promoted proliferation of cervical cancer cells(Wound healing assays and Transwell assays):1) In Hela cells: S100A9-overexpressing cells migrated and almost filled the gap 24 h after wounding, however, the gap filling was significantly retarded in the Blank and GFP groups; In consistent with that, the numbers of migrated Hela cells in Ad S100A9 group(128.67±22.03) was, significantly higher than the blank group(74.67±14.05) and GFP group(66.00±14.42), P<0.05.2) In Siha cells: After 36 h, the healing ability of Adsi S100A9 group was significantly weaker than that of Blank group and si Control group; In consistent with that, the numbers of migrated Siha cells in Adsi S100A9 group(77.50±7.77)was, significantly lower than the Blank group(182.5±10.61) and the si Control group(155.50±4.94), P<0.01.4. S100A9 promoted EMT of cervical cancer cells(Western blot analysis and immunofluorescence assays): The result revealed that epithelial marker E-cadherin was partially rescued(P<0.05), mesenchymal factors Vimentin was significantly upregulated(P<0.01) in the Ad S100A9 group, versus GFP group in Hela cells; However, E-cadherin was significantly upregulated(P<0.01), Vimentin was significantly reduced(P<0.05) in the Adsi S100A9 group, versus si Control group in Siha cells.5. S100A9 enhanced the activity of Wnt/β-catenin signaling pathway. 1) The levels of total β-catenin and nuclear β-catenin were increased in Ad S100A9 group by 0.43-fold and 1.12-fold, versus GFP group(P<0.01) in Hela cells, but also promoted the transcription of its target genes c-myc, Snail and Twist(P<0.05,P<0.01,P<0.05); 2)The levels of total β-catenin and nuclear β-catenin were reduced in Adsi S100A9 group by 0.73-fold(P<0.001) and 0.50-fold(P<0.05), versus si Control group in Siha cells, but also reduced the transcription of the target genes c-myc, Snail and Twist(P<0.01, P<0.01, P<0.001).6. S100A9 induces cell viability and migration via the Wnt/β-catenin signaling pathway: The expreesion of β-catenin was significantly reduced by Adsiβ-catenin. Adsiβ-catenin-infected Hela cells were treated with the GST-h S100A9 and then cell viability and migration were measured. We found that down-regulation of β-catenin partly suppressed S100A9-induced viability and migration of Hela cells.7. S100A9 induces EMT via the Wnt/β-catenin signaling pathway. Adsiβ-catenin-infected Hela cells were treated with the GST-h S100A9 and then the levels of EMT markers were examined. We found that down-regulation of β-catenin partly suppressed S100A9-induced decreased E-cadherin expression and increased Vimentin expression in Hela cells.Conclusions1. S100A9 promotes proliferation and migration of cervical cancer cells.2. S100A9 promotes EMT of cervical cancer cells.3. S100A9 activates the Wnt/β-catenin signaling pathway.4. The activation of the Wnt/β-catenin signaling pathway by S100A9 is involved in the roles of S100A9 on cervical cancer cells, including promoting cell proliferation, migration and EMT.