MiR-21 Promotes The Proliferation And Migration Of Cervical Cancer Cells Via The Inhibition Of PTEN

BackgroundCervical cancer remains the second leading malignancy for women worldwide (1,2),although the incidence of this type of cancer is on the decrease in developed countries due to early diagnosis. Nevertheless, cervical cancer remains a serious health issue for women in developing countries, such as China, where diagnostic programs are not well established. Moreover, local recurrence remains challenging for those patients with cervical cancers(particularly at the advanced stage)(3),even though such therapies as surgery, chemotherapy and/or radiotherapy have been utilized.Persistent cervical infection with high-risk human papillomavirus (HPV),particularly with HPV type 16 or 18,contributes to the development of cervical cancer(4-6).The early oncoproteins of HPVs E5,E6 and E7 are known to contribute to tumor progression, such as the proliferation, migration and invasion of cervical cancer cells(7).Accumulating evidence has identified molecular mechanisms involved in cervical cancer. Molecules such as vascular endothelial growth factor-C (VEGF-C)(8), Src homology-2 domain containing protein, tyrosine phosphatase-2 (SHP-2)(9),or CD 147 isoform-4(CD147-4)(10) are known to promote the proliferation, migration or invasion of cervical cancers by activating focal adhesion kinases (8),through inhibition of interferon-? production(9),or with an upregulated expression of the cancerous inhibitors PP2A(CIP2A),polo-like kinase (PLK) and cyclin D1 but a downregulated p27 expression(10).By contrast, tumor-suppressive molecules, such as Beclinl(11)and histone deacetylase(HDAC)10(12) have been confirmed to inhibit the invasion and migration of cervical cancer cells by decreasing the expression of VEGF and matrix metalloproteinase (MMP)-9 proteins(11),or through the inhibition of MMP-2 and -9 expression(12).A type of non-coding RNA with ?22 nucleotides, known as microRNAs (miRNAs)(13),has been found to be important in the development and progression of cervical cancers (14-16).Dysregulated miRNAs, such as miR-135a(17),miR-10a(18),or miR-205(19) promote cell growth, migration and invasion in human cervical cancer cells, by regulating ?-catenin(17),by targeting CHL1(18),or by downregulating CYR61 and CTGF(19).Some tumor-suppressive microRNAs, such as miR-218(20),miR-372 (21),or miR-214(22) are also deregulated in cervical cancers and contribute to cancer progression. In addition, the oncogenic miR-21 has been widely recognized to play a role in non-small cell lung cancers (NSCLCs)(23,24),as well as colorectal(25),ovarian (26),breast(27) and esophageal cancers (28). Accumulating evidence shows that the promoting role of miR-21 in NSCLCs or in colorectal cancers occurs through the modulation of the phosphatase and the tensin homolog (PTEN) signaling pathway(24,25).Moreover, miR-21 has been demonstrated to be deregulated in cervical cancers, with a marked association with the worsening clinical diagnosis of cervical cancers (29).In the present study, we examined the expression of miR-21 and PTEN in cervical cancer specimens, and investigated the regulation of miR-21 and PTEN on the proliferation and migration of the cervical cancer CaSki and HeLa cells. We also determined the regulation of miR-21 on the PTEN expression. The present study demonstrated the regulation of miR-21 on the progression of the cervical cancer cells.Chapter 1. MiR-21 highly express in cervical cancer, and negatively correlat with PTEN expression.Objective:To detect the expression of miR-21 in cervical cancer specimens and normal cervical specimens, and analyze the differences between the them. Then to clarify the correlation between the expression of miR-21 and cervical cancer. And further study the correlation between the expression of miR-21 and PTEN in cervical cancer specimens described above, and the correlation between the expression of miR-21 and HPV-positive/negative.To lay the foundation for exploring miR-21's role in cervical cancer.Methods:We collected 36 cases of cervical cancer tumor tissue samples as the experimental group,21 cases of normal cervical tissue sample as a control group. Cellular mRNA was extracted from specimens using TRIzol reagent. cDNA from each sample was synthesized with the Superscript First-Strand Synthesis System for the RT-PCR kit with a random Uni-12 primer. The PTEN mRNA was quantified by RT-qPCR performed using a TaqManAssay. The PTEN mRNA level in each sample was normalized to ?-actin. miRNA extraction was performed using the mirVana miRNA Isolation kit. Quantification of the miR-21 level in the cervical cancer specimens and cell samples was conducted using the mirVana RT-qPCR miRNA Detection kit, with the U6 small nuclear RNA used as the internal control. The results obtained were statistically analyzed to study the correlation of miR-21 and PTEN in cervical cancer specimens. And because there are 36 HPV-positive cases and 8 HPV-negative cases in these 36 cases of cervical cancer specimens, statistical analyze the correlation between the expression of miR-21 and HPV-positive/negative.Results:The mean value of miR-21 was 2.14±0.19 in the 36 samples from patients with cervical cancer and 1.00±0.09 in the healthy controls (P<0.001). there was a significant reduction of PTEN mRNA in the invasive cervical cancer specimens (P<0.01).Downregulation of PTEN correlated with the miR-21 upregulation in the specimens (R2=0.2713, P=0.0011).No significant difference was identified in the miR-21 level between the HPV-positive and -negative samples.Conclusions:miR-21 is overexpressed in invasive cervical cancer specimens, in association with a reduced PTEN expression. No significant difference was identified in the miR-21 level between the HPV-positive and -negative samples.Chapter 2. The impact on biological behavior in vitro by overexpressing miR-21.Objective:To identify the regulation of miR-21 on the proliferation, migration and invasion of the cervical cancer cells, we manipulated the miR-21 level in the CaSki and HeLa cells by transfecting the cells with miR-21 mimics, miR-21 inhibitor or miRNA control. Thereby clarify the role of miR-21 in the development of cervical cancer.Methods:The CaSki and HeLa cervical cancer cell lines grow in RPMI-1640 medium or EMEM supplemented with 10% fetal bovine serum respectively.The two cell types were incubated at 37?,with 5% CO2. miR-21 mimics, miR-21 inhibitor or miRNA control were transfected with Lipofectamine 2000 into the CaSki or HeLa cells. Then conduct proliferation, colony formation, migration and invasion detection.Firstly, to detect influence of expression of miR-21 levels on proliferation, CaSki or HeLa cells transfected with miR-21 mimics, miR-21 inhibitor or miR-21 control were incubated in CCK-8. Absorbance at 450 nm of the treated cells was detected following incubation at 37?,with 5% CO2,for 24h. Additional, to detect changs of proliferation over time by expression of miR-21 changed, absorbance at 450 nm of the treated cells was detected following incubation at 37?,with 5% CO2,for 24,36 or 48h.Secondly, For the cell colony-forming assay,300 CaSki or HeLa cells were incubated in 12-well plates at 37?,5% CO2,and were then transfected with or without 50 nM miR-21 mimics, miR-21 inhibitor or miRNA control. Following 48h post-incubation, the cells were stained with crystal violet (0.005%) for 20 min and the colony numbers were recorded using Image J software. To detect the influence of expression of miR-21 levels on the ability of colony formation.Thirdly, the cells were seeded in 12-well plates and cultivated to a confluence of 85%. The 85% confluent HeLa cells were then transfectedwith miR-21 mimics,miR-21 inhibitor or miR-21 control. Cell growth was observed at 0,24 and 48h. To detect the influence of expression of miR-21 levels on the ability of migration.Lastly, Cell invasion was examined by the Matrigel-coated Transwell assay. Briefly, the cells were seeded at a density of 1×105 cells in serum-free medium on the upper chamber with the a non-coated membrane. The lower chamber contained medium with 20% FBS as a chemoattractant. The cells in the upper chamber were discarded using cotton wool after 24h and the migratory cells in the lower chamber were counted under an optical microscope. To detect the influence of expression of miR-21 levels on the ability of invasion.Results:Transfecting miR-21 mimics in CaSki and HeLa cells lead to expression levels of miR-21 increased significantly, and transfected with miR-21 inhibitor can lead to a significant decrease in the expression level of miR-21. Compared with the control, the ability of proliferation, the number of colony formation, the speed of wound healing and the number of cells through the Matrigel of CaSki and HeLa cells transfected miR-21 mimics increase significantly. In contrast, the ability of proliferation, the number of colony formation, the speed of wound healing and the number of cells through the Matrigel of CaSki and HeLa cells transfected miR-21 mimics increase significantly.Conclusions:The ability of proliferation, colony formation, migration and invasion increase significantly after overexpressed miR-21. This shows that miR-21 is a oncogene of cervical cancer. High expression of miR-21 significantly promotes occurrence and development of cervical cancer.Chapter 3. miR-21 regulate proliferation of cervical cancer cell by inhibiting PTEN expressionObjective:Preliminary work found that miR-21 express highly and PTEN express lowly expression in cervical cancer tumor tissue, their expression levels were significantly negatively correlated, in order to clarify the relationship between miR-21 and PTEN and study the molecular mechanism of miR-21, we conducted a study of this section.Methods:To determine whether PTEN is in the downstream of miR-21,CaSki or HeLa cells transfected with miR-21 mimics, miR-21 inhibitor or miR-21 control were detected changes in PTEN by Real-Time PCR and Western blot, respectively at the mRNA and protein levels. PTEN-pcDNA3.1 was constructed by molecular cloning means and it was stably transfected into CaSki cervical cancer cells to make it overexpress PTEN. To determine whether overexpressed PTEN successfully, detect changes in PTEN by Real-Time PCR and Western blot, respectively at the mRNA and protein levels. Then we detect the influence of overexpression of PTEN on proliferation of cell by CCK-8 assay, and on ability of colony formation by colony formation assay. Finally, determine whether miR-21 affects cell proliferation through PTEN.Results:Compared with the control group, CaSki or HeLa cells transfected with miR-21 mimics, the expression level of PTEN reduce significantly at the mRNA and protein levels.PTEN-pcDNA3.1 can success in overexpressing PTEN in CaSki cells. The ability of proliferation and colony formation reduce significantly after overexpressed PTEN.Conclusions:Transfected miR-21 mimics can significantly reduce the expression level of PTEN in Cervical cancer cell. PTEN is a downstream gene of miR-21.And miR-21 influences cell proliferation by inhibiting PTEN.