The Role And Mechanism Of LncRNA-HOST2 Targeting MiR-let-7b In HPV-Positive Cervical Cancer

Cervical cancer(CC)is one of the more common gynecological malignancies with a high incidence,ranking fourth among female malignancies,and being the leading cause of cancer-related deaths in women.The main therapeutic factor of cervical cancer is the persistent infection of high-risk human papillomavirus(HPV).However,the pathogenesis of cervical cancer caused by HPV infection is unknown.Therefore,it is very important to study the pathogenesis of HPV-positive cervical cancer and find clinical markers and therapeutic targets for early diagnosis.lncRNA is a class of long non-coding RNA with a length of about 200 nucleotides.They can degrade miRNAs after competitive binding,thus affecting the expression of target genes and exerting biological regulatory functions.lncRNA can also play the role of molecular sponge and adsorb miRNA,thus inhibiting the degradation of miRNA to target genes.Human ovarian cancer specific transcript 2(HOST2)is a member of the lncRNA family.Its effects on the development of cervical cancer and its molecular mechanism remain unclear.It is predicted on Lnc Tar’s website that lncRNA-HOST2 may target miR-let-7b.In this study,the expression of HOST2 in HPV-negative and HPV-positive cervical cancer tissues was first detected,and the correlation between the expression level of HOST2 and the clinicopathological characteristics of patients with cervical cancer was analyzed.Secondly,the influence of HOST2 on the malignant biological behavior of HPV-positive cervical cancer cell lines in vitro was explored.Finally,the targeted binding of HOST2 and miR-let-7b was verified to explore the molecular mechanism of HOST2’s influence on the malignant biological behavior of HPV-positive cervical cancer cells.Part One Study on the expression of lncRNA-HOST2 and miR-let-7b in cervical cancer tissues and their relationship with patholo-gical features of clinical patientsObjective: The expression of lncRNA-HOST2 and miR-let-7b in cervical cancer tissues were analyzed,and the relationship between the expression levels and the clinicopathological characteristics of patients with cervical cancer was respectively discussed,so as to provide evidence for the discovery of new prediction targets of cervical cancer and the study of molecular mechanism.Methods: From June 2013 to June 2017,cervical cancer tissues of 45 patients with HPV16/18 positive cervical cancer and 22 patients with HPV-negative cervical cancer were collected in Hebei General Hospital.Meanwhile,normal cervical tissues of 20 patients with hysteromyoma surgery were collected as normal control.Quantitative real-time fluorescence PCR(q RT-PCR)was used to detect the relative expression levels of lncRNAHOST2 and miR-let-7b in the above cervical cancer tissues and normal cervical tissues,and the relationship between the expression levels and the clinicopathological characteristics of patients was analyzed respectively.The expression levels of lncRNA-HOST2 and miR-let-7b in cervical cancer tissue of HPV16 were analyzed compared with those of cervical cancer tissue of HPV18.Results:1.The expression level of lncRNA-HOST2 in cervical cancer tissues was significantly higher than that in normal cervical tissues,and the expression level of lncRNA-Host2 in HPV-positive cervical tissues was higher than that in HPV-negative cervical tissues(P<0.05).2.The expression level of miR-let-7b in cervical cancer tissues was significantly lower than that in normal cervical tissues,and the expression level of miR-let-7b in HPV positive cervical cancer tissues was lower than that in HPV-negative cervical cancer tissues(P<0.05).3.The expression levels of lncRNA-HOST2 and miR-let-7b in tissues of patients with cervical cancer had significant a correlation with tumor size,FIGO stage,and HPV positive(P<0.05),but had no significant correlation with age,lymph node metastasis,and vascular infiltration(P>0.05).4.The expressions of lncRNA-HOST2 and miR-let-7b in HPV16 and 18 positive cervical cancer patients were not significantly changed(P>0.05).Conclusions:1.Compared with normal cervical tissues,the relative expression level of lncRNA-HOST2 in cervical cancer tissues was increased,while the relative expression level of miR-let-7b was significantly decreased,and the changes were more significant in HPV-positive group.2.The expression levels of lncRNA-HOST2 and miR-let-7b in cervical cancer tissues were not related to age,lymph node metastasis,and vascular invasion,but were related to tumor size,FIGO stage,and HPV positive.3.The expressions of lncRNA-HOST2 and miR-let-7b in HPV16 and 18 positive cervical cancer were not significantly different.Part Two Effects of lncRNA-HOST2 on biological function of cervical cancer cellsObjective: To explore the expression of lncRNA-HOST2 in cervical cancer cell lines and its effect on malignant biological behavior of cervical cancer cell lines.Methods:1.Using normal cervical epithelial cell lines(Ha Ca T)as control,the expression of lncRNA-HOST2 was detected in HPV16-positive cervical cancer cell lines(Ca Ski and Si Ha),HPV18-positive cervical cancer cell lines(He La)and HPV-negative cervical cancer cell lines(C33A)by q RT-PCR.2.NC(negative control),pc DNA3.0-HOST2 and si HOST2 were transfected into Ca Ski and Hela cell lines,respectively,and the expression of HOST2 was detected by q RT-PCR to verify the transfection effect.3.The above transfected cells were inoculated into the cell culture plate,cell proliferation was detected by MTT,cell apoptosis was detected by flow cytometry,cell migration was detected by wound healing assay,and cell invasion was detected by transwell.Results:1.Compared with normal cervical epithelial cell lines(Ha Ca T),HOST2 expression was significantly increased in cervical cancer cell lines(Ca Ski,Si Ha,He La and C33A).The expression of HOST2 was the highest in HPV16-positive cervical cancer cell line(Ca Ski)and HPV18-positive CC cell line(He La)(P<0.05).2.Compared with the NC group,the expression of HOST2 in Ca Ski and Hela cell lines transfected with pc DNA3.0-HOST2 was significantly increased,while the expression of HOST2 in si HOST2 group was significantly decreased(P<0.05).3.Compared with NC group,OD value,cell mobility,and cell invasion number of Ca Ski and Hela cell lines transfected with pc DNA3.0-HOST2 were significantly increased,while apoptosis rate was significantly decreased.In Ca Ski and Hela cell lines transfected with si HOST2,OD value,cell mobility and cell invasion number were significantly decreased,while apoptosis rate was significantly increased.Conclusions:1.lncRNA-HOST2 expression was elevated in cervical cancer cell lines and was most pronounced in hpv-positive cervical cancer cell lines(Ca Ski and Hela).2.Overexpression of HOST2 promoted the proliferation,migration,and invasion of HPV positive cervical cancer cell lines in vitro,and inhibited cell apoptosis;At the same time,knockdown HOST2 inhibited the proliferation,migration and invasion of HPV-positive cervical cancer cell lines in vitro,and promoted cell apoptosis.Part Three lncRNA-HOST2 affects the proliferation,apoptosis,migration and invasion of HPV-positive cervical cancer cells through targeted regulation of miR-let-7bObjective: To explore the molecular mechanism of lncRNA-HOST2 affecting the malignant biological behavior of HPV-positive cervical cancer cell lines.Methods:1.Using normal cervical epithelial cell lines(Ha Ca T)as control,the expression of miR-let-7b was detected in HPV16-positive cervical cancer cell lines(Ca Ski and Si Ha),HPV18-positive cervical cancer cell lines(He La)and HPV-negative cervical cancer cell lines(C33A)by q RT-PCR.2.NC,pc DNA3.0-HOST2,and si HOST2 were transfected into Ca Ski and Hela cell lines,respectively,and the expression of miR-let-7b was detected by q RT-PCR.3.The binding site of HOST2 and miR-let-7b was predicted on Lnc Tar.In Ca Ski and Hela cell lines,the targeted binding relationship between HOST2 and miR-let-7b was detected by double luciferase reporting assay.4.Ca Ski and Hela cells were transfected into NC group,miR-let-7b mimics group,and pc DNA3.0-HOST2+miR-let-7b mimics group.The expression of miR-let-7b was detected by q RT-PCR.5.The above transfected cells were inoculated into the cell culture plate,cell proliferation was detected by MTT,cell apoptosis was detected by flow cytometry,cell migration was detected by wound healing assay,and cell invasion was detected by transwell.Results:1.Compared with normal cervical epithelial cell lines(Ha Ca T),the expression level of miR-let-7b in cervical cancer cell lines(Ca Ski,Si Ha,He La,and C33A)was significantly decreased.The expression of miR-let-7b was lowest in HPV16-positive cervical cancer cell line(Ca Ski)and HPV18-positive CC cell line(He La)(P<0.05).2.Compared with the NC group,the expression level of miR-let-7b in Ca Ski and Hela cell lines transfected with pc DNA3.0-HOST2 was significantly decreased,while the expression level of miR-let-7b in si HOST2 group was significantly increased(P<0.05).3.The results of the double luciferase reporting experiment showed that the luciferase activity in Ca Ski and Hela cell lines co-transfected with miR-let-7b mimics and p GL3-HOST2-wt decreased significantly compared with the NC and p GL3-HOST2-wt cells(P<0.05).4.Compared with the NC group,the expression level of miR-let-7b in Ca Ski and Hela cell lines transfected with miR-let-7b mimics was significantly increased,while the expression level of miR-let-7b in the co-transfected pc DNA3.0-HOST2 and miR-let-7b mimics group was reduced to the level close to that of the NC group.5.Compared with NC group,OD value,cell mobility,and cell invasion number of Ca Ski and Hela cell lines transfected with miR-let-7b mimics were significantly decreased,while the apoptosis rate was significantly increased.OD value,cell mobility,and cell invasion number in the co-transfected pc DNA3.0-HOST2 and miR-let-7b mimics groups increased to the level close to the NC group,and cell apoptosis rate also decreased to the level close to the NC group.Conclusions:1.In HPV-positive cervical cancer cell lines(Ca Ski and Hela),HOST2 can be targeted to miR-let-7b and degrade it.2.Overexpression of HOST2 promoted the proliferation,migration,and invasion of HPV-positive cervical cancer cell lines in vitro and inhibited cell apoptosis through targeted binding to miR-let-7b.