Biological Effects Of MiR-494 By Targeting SOCS6 In Cervical Cancer

BackgroundCervical cancer(CC)is one of the most common cancers in women worldwide.Despite of the improvement of medical technology including the Pap smear,CC remains the second commonest cancers in women after breast cancer while the mortality rate ranked fourth universally.There are about 460,000 new cases and 270,000 deaths annually,approximately 90%of cases occur in developing countries.According to the data announced by National Health Commission of the People's Republic of China,one-third new cases of the world are found in China with 80,000 deaths annually.Human Papillomavirus(HPV)has been diagnosed in about 99%of the cervical cancers,which is infected by skin or sex.HPV infection is very common in women due to open sex concept,shortage of qualified health care personnel and environmental pollution.Thus,the age of patients suffering from CC was trending younger.More than 100 types of HPV have been identified,whereas most of them are not related with CC.14 types of HPV contribute to CC,and the most common type in CC deals with HPV-16 type followed by HPV 18 causing more than 70%CC cases.Based on that the HPV infection is the main causative agent of CC,the Advisory Committee on Immunization Practices(ACIP)has recommended a quadrivalent HPV vaccine to prevent HPV-associated CC.However,the current HPV vaccines do not cover all 14 HPV types causing CC.For example,the 2-valent HPV vaccine contains HPV-16 type and HPV-18 type,while the 9-valent HPV vaccine contains HPV-6,1 1,16,18,31,33,45,52 and 58 types.In addition,the knowledge and awareness of HPV vaccines are still clear.Therefore,effective cancer treatments and biomarkers for CC are urgently needed to explore.Currently the CC screening and diagnosis depend on Papanikolaou test detecting the precancerous and cancerous cell lesions and HPV test detecting HPV DNA due to the mechanism under which HPV induces CC has not been uncovered.However,these two tests are utilized wildly in developing countries because of inadequate healthcare services and limited funds.Moreover,there is not yet a reliable method and diagnostic marker in clinics for fast screening or early diagnosis of CC.Surgery combined with chemoradiotherapy is still the preference for CC.Unfortunately,the prognosis of CC patients is poor.Five-year survival rate is less than 40%while the recurrence rate of patients in the terminal stage more than 30%.Thus,novel molecular therapies are needed to explore.Growing evidence has indicated that HPV induces CC through regulating miRNAs.miRNAs are endogenous,noncoding RNA molecules of 22 nt in length,modulating gene expression by binding to complementary sequences in the 3' untranslated region(UTR)of target messenger RNAs(mRNAs)posttranscriptionally.miRNAs play key roles in cancer formation,progression,metastasis and prognosis functioned as oncogenes or cancer suppressor.Previous studies have shown that miRNAs are involved in every stage of CC progression.For instance,HPV infection promotes CC cells proliferation and migration through inhibiting miR-3156-3p expression.In addition,HPV-E7 increases miR-182 expression through TGF-?/Smad4 pathway to induce CC.Moreover,miR-944 overexpression is a biomarker for poor prognosis of CC.Numerous studies have demonstrated that miR-494 is a tumor suppresser.For example,miR-494 inhibits cell proliferation in oral cancer through repressing homeobox protein A10(HOXA10)expression.In addition,miR-494 suppresses the progression of breast cancer by targeting C-X-C motif chemokine receptor 4(CXCR4)via the Wnt/beta-catenin signaling pathway.Moreover,Cinobufacin suppresses gastric cancer cell proliferation via regulating miR-494 expression.Recent studies have indicated that miR-494 inhibits epithelial ovarian cancer growth by targeting c-Myc and fibroblast growth factor receptor 2(FGFR2)to induce cancer cell apoptosis.However,the role of miR-494 in CC has not been identified,though a retracted paper reported that miR-494 suppresses CC cell migration through down-regulating pituitary tumor-transforming 1(PTTG1).Suppressor of cytokine signaling 6(SOCS6)is a putative target of miR-494.Previous studies have revealed that SOCS6 is also a tumor suppresser.In colorectal cancer,miR-301a promotes cancer cell growth and invasion by targeting SOCS6.Similarly,miR-21 and MiR-155 promote non-small cell lung cancer progression by decreasing SOCS6 expression.Furthermore,SOCS6 can inhibit gastric cancer cell proliferation.However,the function of SOCS6 in CC is still unclear.Objective1.To identify the expression of miR-494 in the serum both of health and CC patients,and expression levels of miR-494 and SOCS6 in or around CC tumor tissue.2.To demonstrate the relationship between the expression of miR-494 in the serum of CC patients and clinical pathological changes,including the five-year survival rate.3.To reveal the targeted regulatory relationship between miR-494 and SOCS6.4.To investigate the roles of miR-494 and SOCS6 in CC cell growth and migration in vitro.5.To further identify the roles of miR-494 and SOCS6 in CC formation in vivo.Method1.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was utilized to detect the expression of miR-494 in the serum both of health and CC patients,the expression of miR-494 in the serum of CC patients both before and after surgery,and the level of miR-494 in or around CC tumor tissue.2.Correlation analysis was applied to indicate the relationship between the expression of miR-494 in the serum of CC patients and clinical pathological changes including five-year survival rate.3.qRT-PCR and Western Blotting(WB)were used to detect the expression of SOCS6 in or around CC tumor tissue.4.qRT-PCR and WB were utilized to identify the expression levels of miR-494 and SOCS6 in CC cell lines.5.qRT-PCR was used to detect the expression levels of miR-494 and SOCS6 in Hela cells after transfection of miR-494 mimic and inhibitor or SOCS6 siRNAs.6.shRNA vector was applied to construct SOCS6 expression vector and identified by DNA sequencing;qRT-PCR was used to test the level of SOCS6 mRNA in Hela cells after transfection of SOCS6 expression vector.7.psiCHECKTM-2 vector was applied to create vector contained wild type SOCS6 3'UTR or mutated SOCS6 3' UTR and identified by DNA sequencing.8.The dual luciferase reporter system was performed to identify the targeted regulatory relationship between miR-494 and SOCS6.9.MTS assay and cell clone experiment were performed to verify roles of miR-494 and SOCS6 in CC cell growth.10.Flow cytometry was utilized to effects of miR-494 and SOCS6 on CC cell cycle and apoptosis.11.Transwell chamber experiment was performed to reveal roles of miR-494 and SOCS6 in CC cell migration and invasion.12.The tumor formation of nude mice experiment was applied to determine effects of miR-494 and SOCS6 on CC formation.ResultsI.Compared with the health,the expression of miR-494 in the serum of CC patients was dramatically decreased;the expression of miR-494 in the serum of 90%CC patients after surgery was obviously increased than that before treatment;Moreover,the level of miR-494 in CC tumor tissue was lower than that in paracancerous tissue.2.The serum miR-494 level was associated with the tumor size,tumor differentiation and prognosis of CC patients.3.The level of SOCS6 was significantly lower in CC tumor tissue than that in para-cancerous tissue.4.Compared with normal human endometrial epithelial cells,the expression of miR-494 was dramatically decreased in CC cell lines,the level of SOCS6 was decreased.5.miR-494 mimic.miR-494 inhibitor and SOCS6 siRNAs could regulate expression level of miR-494 and SOCS6 in Hela cells.6.The SOCS6 expression vector was constructed correctly.7.The vector contained wild type SOCS6 3' UTR or mutated SOCS6 3' UTR was created successfully.8.Results of luciferase activities indicated that SOCS6 was the target of miR-494.9.Results of MTS assay and cell clone experiment revealed that miR-494 suppressed CC cell growth,whereas SOCS6 also inhibited CC cell growth.10.Results of flow cytometry showed that miR-494 overexpression and SOCS6 overexpression suppressed the entrance of cell cycle from G1 phase to S phase and induced cell apoptosis,while miR-494 silence and SOCS6 siRNAaccelerated the entrance of cell cycle from G1 phase to S phase and inhibited cell apoptosis.:11.Results of flow cytometry showed that miR-494 overexpression and SOCS6 overexpression suppressed the entrance of cell cycle from G1 phase to S phase and induced cell apoptosis,while miR-494 silence and SOCS6 siRNAaccelerated the entrance of cell cycle from G1 phase to S phase and inhibited cell apoptosis.12.Results of the tumor formation of nude mice experiment revealed that miR-494 overexpression and SOCS6 overexpression inhibited CC formation,while iR-494 silence and SOCS6 overexpression promoted CC formation.Conclusion1.The expression of miR-494 in the serum of CC patients in CC tumor tissue was lower,while the expression of miR-494 in the serum of 90%CC patients after surgery was obviously increased;the level of SOCS6 was significantly lower in CC tumor tissue.2.The serum miR-494 level was closely associated with the tumor size,tumor differentiation and prognosis of CC patients.3.For the first time,the present study identified the targeted regulatory relationship between miR-494 and SOCS6.4.Results identified that miR-494 regulated CC cell growth,apoptosis,cell cycle,migration,invasion and CC formation through targeting SOCS6 both in vitro and in vivo.5.The mechanism under which miR-494 and SOCS6 regulated CC formation and progression provided new biomarkers for CC early diagnosis and novel targets for following CC treatment.