| Background/Objective:Cervical cancer is the third most common cancer in women worldwide,accounting for nearly 7.5% of cancer deaths in women in the world.According to reports,about 520,000 women in the world are newly diagnosed with cervical cancer every year,and about 260,000 of them die from the disease.In 2015,there were98,900 new cases of cervical cancer and 30,500 deaths in China.The treatment of cervical cancer is mainly surgery and radiotherapy,supplemented by chemotherapy.Surgery is the first choice for the treatment of early cervical cancer,and standardized comprehensive treatments such as postoperative radiotherapy and cis-platinum(CDDP)-based chemotherapy are recommended.Lymph node metastasis and distant metastasis are two of the reasons for the poor prognosis of patients with cervical adenocarcinoma.Evidence indicated that the 5-year survival rate of patients with early stage cervical cancer after treatment can reach 90%,while it is only about 20%for the patients with locally advanced or distant metastasis,especially for stage IV patients.Although CDDP-based concurrent chemoradiotherapy improves overall survival and progression-free survival stage of patients with locally advanced disease,approximately 50% of patients with locally advanced disease experience relapse within 2 years after the initial treatment.In addition,due to the emergence and development of tumor cell drug resistance,the efficacy of CDDP-based chemotherapy for advanced or metastatic cervical tumors is not satisfactory,often resulting in poor prognosis.Therefore,it is necessary to find new markers and effective targets for cervical cancer metastasis,and to reveal the relevant molecular mechanisms of cervical cancer resistance in order to find new and effective treatments for cervical cancer at an early date.Long non-coding RNA UCA1(long non-coding RNA UCA1,Lnc RNA UCA1)plays an important role in the metastasis,invasion and drug resistance of many tumors including cervical cancer,but the underlying molecular mechanisms have not been fully elucidated.In this study,we mainly explored whether UCA1 could promote cervical cancer proliferation,metastasis and invasion by targeting and inhibiting the expression of mi R-143.And we investigated if UCA1 can enhance the resistance of cells to CDDP.The important role of UCA1 in cervical cancer progression may become an effective target for cervical cancer treatment,providing theoretical and experimental references for finding new methods to overcome the problem of cervical cancer chemotherapy resistance.Research methods:1.QRT-PCR was used to detect the expression of UCA1 in cervical cancer tissues,human cervical epithelial cells H8,cervical cancer cell lines Hela and Si Ha.2.The expression of UCA1 and mi R-143 was up-regulated or down-regulated by lentiviral vector transfection in He La and Si Ha cells.3.Cell Counting Kit 8(CCK-8),clone formation experiment,scratch test,Transwell chamber experiment,flow cytometry and western blot were used to evaluate UCA1 and mi R-143 on cell proliferation activity,colony forming ability,apoptosis,metastasis,invasion and other tumor biological behaviors.The effects of UCA1/mi R-143 axis on tumor biological behavior of Hela and si Ha cells and on epithelial–mesenchymal transition(EMT)process were analyzed.4.The dual-luciferase reporter assay was used to confirm the axial relationship between UCA1 and mi R-143 in cervical cancer cells.5.An in vivo tumor-bearing mouse model was constructed to analyze the effect of UCA1/mi R-143 axis on the tumorigenic ability and intraperitoneal metastasis ability of cervical cancer cells6.CCK-8,scratch test,flow cytometry(PI staining)and mouse tumor-bearing experiments were used to evaluate CDDP treatment and / or UCA1,mi R-143,UCA1/mi R-143 regulation on cervical cancer cell proliferation activity,apoptosis,metastasis and the size of tumor.They were also used to evaluate the role of UCA1/mi R-143 in CDDP-mediated antitumor process.Result:1.The effect of Lnc RNA UCA1 on the activity of cervical cancer cells.1.1 The expression level of UCA1 in 45 primary cervical cancer tissues was significantly higher than that in adjacent normal tissues,p<0.001.UCA1 expression level in cervical cancer cell lines Hela and Si Ha was also significantly higher than that in human cervical epithelial cell H8,p(27)0.01 and p(27)0.001.1.2 After cervical cancer Hela and Si Ha cell lines were transfected with OE-UCA1,the expression of UCA1 in the cells was significantly higher than that in cells infected with OE-NC,p(27)0.001 and p(27)0.01;while the expression level of UCA1 in cells transfected with sh-UCA1-3 was significantly lower than that in sh-NC transfected cells,P<0.05.1.3 The proliferation activity of He La and Si Ha cells overexpressing UCA1 was significantly higher than that of the control group,P(27)0.5 and P(27)0.01,and the proliferation activity of cells with down-regulated UCA1 was significantly lower than that of the control group,P(27)0.05.The results of this study suggest that UCA1 overexpression can enhance cervical cancer cell proliferation.1.4 The clonogenic ability of He La and Si Ha cells overexpressing UCA1 was significantly higher than that of the control group,P<0.05,and the clonogenic ability of cells with down-regulated UCA1 was significantly lower than that of control group,P<0.05.The results of this study suggest that UCA1 overexpression can promote the clonogenic activity of cervical cancer cells.1.5 The cell migration distance of He La and Si Ha cells overexpressing UCA1 was significantly longer than that of the control cell group,P<0.05;the cell migration distance of the cells in sh-UCA1 group was significantly shorter than that of the control cell group,P<0.05.This result shows UCA1 overexpression can enhance the metastatic ability of cervical cancer cells.1.6 Compared with the control cell group,the invasive ability of He La and Si Ha X cells overexpressing UCA1 was significantly enhanced,P<0.05.Compared with the control cell group,the invasive ability of the cell group down-regulated UCA1 was significantly decreased,P<0.05.This suggests UCA1 overexpression enhances the invasive ability of cervical cancer cells.1.7 Flow cytometry result showed that the apoptosis level of He La and Si Ha cells overexpressing UCA1 was significantly lower than that of the control cell group,P<0.05,the apoptosis level of cells with down-regulated UCA1 was significantly higher than that of the control cell group,P<0.05.It shows UCA1 overexpression could inhibit the apoptosis of cervical cancer cells.2.The effect of mi R-143 on cervical cancer cells2.1 The results of the dual-luciferase reporter assay showed that compared with cells in the mimics-NC group,up-regulation of mi R-143 expression could significantly reduce the luciferase activity of Luc-UCA1-WT(Luc-UCA1 wild type)(P<0.05)..However,there was no significant effect on the luciferase activity of Luc-UCA1-MUT(Luc-UCA1 mutant).2.2 In vitro loss-of-function experiments showed that the expression level of mi R-143 in He La and Si Ha cells with down-regulated mi R-143(mi R-143-inhibitor)was significantly inhibited compared with that in inhibitor-NC cells,P<0.05.2.3 Compared with inhibitor-NC cells,He La and Si Ha cells that have down-regulated mi R-143 could significantly increase the proliferation activity,P<0.05.2.4 Compared with inhibitor-NC cells,the down-regulation of mi R-143 could significantly enhance the metastatic ability of He La and Si Ha cells,P<0.05.2.5 Compared with inhibitor-NC cells,low-level mi R-143 expression could significantly enhance the invasive ability of He La and Si Ha cells,P<0.05.2.6 Inhibition of mi R-143 expression activity can reduce the apoptosis level of He La and Si Ha cells,P<0.05).3.Long noncoding RNA UCA1 contributes to cervical cancer progression by targeting mi R-1433.1 The expression of mi R-143 in He La and Si Ha cells co-transfected with OE-UCA1(overexpressing UCA1)and mimics-NC(mi R-143-mimics negative control)compared with cervical cancer cells transfected with OE-NC(UCA1 negative control)was significantly decreased,P<0.05.He La and Si Ha cells co-transfected with OE-UCA1 and mi R-143-mimics(overexpressing mi R-143)had significantly higher expression levels of mi R-143 than the OE-UCA1+mimics-NC group,P<0.05.It was shown that overexpression of mi R-143(mi R-143-mimics)could significantly reverse the inhibitory effect of UCA1 on mi R-143.3.2 Compared with cervical cancer cells transfected with OE-NC,the viability of He La and Si Ha cells co-transfected with OE-UCA1 and mimics-NC was significantly enhanced,P<0.05.The viability of He La and Si Ha cells in the OE-UCA1+mi R-143-mimics group was significantly decreased than the OE-UCA1+mimics-NC group P<0.05.This result suggests UCA1 enhances cervical cancer cell activity by targeting mi R-143.3.3 Compared with cervical cancer cells transfected with OE-NC,the migration ability of He La and Si Ha cells transfected with OE-UCA1 and mimics-NC lentiviral vectors was significantly enhanced,P < 0.05;the migration ability of He La and Si Ha cells transfected with OE-UCA1 and mi R-143-mimics was significantly lower compared with the OE-UCA1+mimics-NC group,P<0.05.It showed that UCA1 enhanced the migration ability of cervical cancer cells by targeting mi R-143.3.4 Compared with cervical cancer cells transfected with OE-NC,the invasive ability of He La and Si Ha cells transfected with OE-UCA1 and mimics-NC lentiviral vectors was significantly enhanced,P<0.05.The invasive ability of He La and Si Ha cells coinfected OE-UCA1 and mi R-143-mimics was significantly lower compared with the OE-UCA1+mimics-NC group,P<0.05.It was shown that UCA1 enhanced the invasive ability of cervical cancer cells by targeting mi R-143.3.5 Compared with cervical cancer cells transfected with OE-NC,the ratio of XII apoptosis in He La and Si Ha cells transfected with OE-UCA1 and mimics-NC lentiviral vectors was significantly lower,P<0.05;The apoptosis rate of He La and Si Ha cells in OE-UCA1 and mi R-143-mimics group was significantly higher than that in OE-UCA1+mimics-NC group,P<0.05.It showed that UCA1 inhibits cervical cancer cell apoptosis by targeting mi R-143.3.6 Compared with cervical cancer cells transfected with OE-NC,the expression of E-cadherin protein was significantly decreased in He La and Si Ha cells co-transfected with OE-UCA1 and mimics-NC lentiviral vectors,while the expression levels of ICAM1 and Vimentin proteins were significantly increased,P<0.05.Overexpression of mi R-143 significantly inhibited this effect.This experiment showed that UCA1 induced EMT in cervical cancer cells by targeting mi R-143.3.7 The results of tumor-bearing experiments showed that compared with cervical cancer cells transfected with OE-NC,the volume and mass of tumors in mice injected He La and Si Ha cells transfected with OE-UCA1 lentiviral vectors were significantly increased,P<0.05.While overexpressing mi R-143 could reduce the volume and mass of tumors in mice significantly,P<0.05,indicating that UCA1 induce cervical cancer cell tumorigenesis by targeting mi R-143.3.8 Intraperitoneal tumor cell injection experiments showed He La cells overexpressing UCA1 increased the number of abdominal metastatic nodules,while overexpressing mi R-143 could reduce the number of abdominal metastatic nodules,and the difference was statistically significant P<0.05.It showed that UCA1 enhanced the peritoneal metastasis ability of cervical cancer cells by targeting mi R-143.4.Lnc RNA UCA1 enhances the cisplatin resistance of cervical cancer cells by targeting mi R-1434.1 The expression of UCA1 in He La and Si Ha cells treated with CDDP was significantly higher than that in untreated cells,P<0.05.4.2 The IC50 values for CDDP were 3.52 μM and 7.398 μM for He La cells in the OE-NC group and OE-UCA1 group,respectively,and 4.265 μM and 10.48 μM for Si Ha cells.Compared with the OE-NC group,the OE-UCA1 group could significantly increase the IC50 of cervical cancer cells.4.3 Compared with the OE-NC group,the viability of He La and Si Ha cells was significantly inhibited after CDDP treatment.After cells overexpressing UCA1 were treated with CDDP,the viability of cancer cells was significantly increased compared with the control group treated with CDDP,P<0.05.4.4 Compared with the OE-NC group,the migration ability of He La and Si Ha cells was significantly inhibited after CDDP treatment,P<0.05;and the effect of CDDP was significantly inhibited after UCA1 overexpression.4.5 After CDDP treatment,the apoptosis level of He La and Si Ha cells in the OE-NC group was significantly higher than that in the OE-NC group,P < 0.05 while the effect of CDDP-induced apoptosis was significantly inhibited after UCA1 overexpression,P < 0.05.4.6 Compared with the control group,inhibiting the expression of mi R-143 can significantly increase the IC50 of cervical cancer cells,while overexpression of mi R-143 can significantly reduce the IC50 of cervical cancer cells to CDDP.4.7 Compared with the inhibitor-NC group,the viability of He La and Si Ha cells after CDDP treatment in the mi R-143 overexpression group were significantly inhibited,P < 0.05,the effect of CDDP on inhibiting cell activity was limited after down-regulating mi R-143,while the cell viability of the mi R-143 overexpression group was significantly decreased compared with the control group,.4.8 Compared with the inhibitor-NC group,the apoptosis ratio of He La and Si Ha cells was significantly increased after CDDP treatment,P < 0.05.After inhibiting the expression of mi R-143,the effect of CDDP-induced apoptosis was significantly inhibited,while the effect of CDDP-induced apoptosis was enhanced in the mi R-143 overexpression group.4.9 Compared with OE-NC group,the IC50 value of CDDP in He La and Si Ha cells in OE-UCA1 group was significantly increased,while the effect was significantly inhibited by mi R-143 overexpression.4.10 Compared with control group,the cell viability of He La and Si Ha cells in OE-UCA1 group was significantly increased after CDDP treatment,P<0.05.While the activity of cancer cells in mi R-143 overexpression group after CDDP treatment was significantly lower than that in OE-UCA1 group.4.11 Compared with control group,the apoptotic cells of He La and Si Ha cells overexpressing UCA1 were significantly decreased after CDDP treatment,P < 0.05;however,the number of apoptotic cells in the two cancer cells in the mi R-143 overexpression group increased significantly after CDDP treatment,P < 0.05.4.12 In vivo mouse tumor-bearing experiments showed that compared with the control group,the tumorigenic ability of He La and Si Ha cells was significantly enhanced after UCA1 overexpression,and this effect was significantly inhibited after up-regulation of mi R-143.Conclusion:1.This study shows that UCA1 has the biological effect of enhancing the proliferation,metastasis and invasion of cervical cancer cells,and promotes the occurrence and progression of cervical cancer;while mi R-143 inhibits this tumorigenesis process and has tumor suppressor effect.2.The results revealed that UCA1 may regulate the development of cervical cancer by targeting micro RNA-143;the target molecular regultaion of UCA1/mi R-143 may have potential clinical value in the diagnosis and treatment of cervical cancer progression.3.The experimental results also showed that UCA1 could enhance the cisplatin resistance of cervical cancer cells by targeting mi R-143,suggesting that the UCA1/mi R-143 axis may become an effective target to overcome the drug resistance of cervical cancer. |
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