Study On The Role And Mechanism Of LRP11 In The Development Of Cervical Cancer

BackgroundCervical cancer is the most common gynecological malignancy.According to China Cancer Statistics Report published in 2015,the incidence and death rate of cervical cancer increased year by year from 2000 to 2011,and the incidence rate of cervical cancer was the second highest among female malignant tumors in the age group of 34-44 years old.Although the diagnosis and treatment technology of cervical cancer has improved significantly in recent years,according to the 2018 I RAC global statistics report,there were still 569,847 new cases of cervical cancer and 311,365 deaths worldwide in 2018.The proportion of new cases of cervical cancer(13.1%)and the proportion of deaths(6.9%)are the fourth highest among female tumors.Therefore,cervical cancer is still a serious threat to women s health worldwide.Cervical cancer is usually caused by abnormal cell growth caused by persistent infection with high-risk human papillomavirus(HPV).In recent decades,the incidence of cervical cancer has been declining in most countries with the spread of cancer screening and the introduction of the HPV vaccine.Unfortunately,the clinical treatment strategy for cervical cancer progresses slowly,and surgery,chemotherapy and radiotherapy are still the main treatment methods for cervical cancer patients.Although concurrent chemoradiotherapy can achieve certain clinical efficacy for locally advanced cervical cancer,the prognosis improvement for patients with metastatic and recurrent advanced cervical cancer is limited,so it is urgent to find a low-toxicity and effective treatment,and the development of molecular targeted therapy provides a new direction for it.However,currently,the targeted therapeutic drugs with a definite effect on cervical cancer have limited effects.Exploring new molecular markers for prognosis and looking for more effective molecular targets are of great significance for the early diagnosis and survival improvement of cervical cancer.Lipids play an important role in cell survival,proliferation and apoptosis by participating in the formation of cell signals,cell membranes and intercellular connections,and are closely related to the transformation,progression and metastasis of cancer,suggesting that bioactive lipids are mediators of carcinogenic processes.It has been reported that family members of low-density lipoprotein receptor-related protein 1(LRP1)and low-density lipoprotein receptor-related protein 1B(LRP1B),as prognostic markers of colon cancer,breast cancer,thyroid cancer,urothelial carcinoma,and clear-cell renal cell carcinoma,play an important role in tumor development.Low density lipoprotein receptor related protein 11(LRP11),as a newly described member of the LRP family,was found highly expressed in cervical cancer patients and was associated with poor prognosis according to our bioinformation analysis.However,the mechanism is unclear.In this project,we intend to detect the expression level of LRP11 in cervical cancer and its biological functional significance,and use P16 as a common detection index to explore the possibility of LRP11 as a target for diagnosis and treatment of cervical cancer and a prognostic factor.The study is divided into the following three parts:Part I:Correlation study of LRP11 expression and clinical prognosis in cervical cancerObjectives:To detect the expression level of LRP11 in patients with cervical cancer and to explore the correlation between LRP11 and the clinicopathological features and survival prognosis of cervical cancer.Methods:1.LRP11 bioinformation analysis were performed from the GEO database,Oncomine database,GEPIA database.In the GEO dataset,we need to download the original data of LRP11 in the form of GEO profile,which can be opened by Excel software.The mRNA expression level of LRP11 is relatively quantified.Therefore,we use GraphPad software directly for student's t-test and mapping.In the Oncomine database and the GEPIA database,we can directly input LRP11 and other related molecular names,then we can obtain corresponding expression quantitative graphs,expression related graphs and survival prognosis graphs.2.The expression of LRP11 in cervical cancer tissues and normal cervical tissues was detected by Western blot.The expression differences of LRP11 and P16 in normal cervical tissues,high-grade squamous intraepithelial lesions and cervical carcinoma were detected by immunohistochemical staining IHC).3.Pearson's chi-square test was used to evaluate the relationship between LRP11 and P16 expression and the clinicopathological characteristics,and their correlation with overall survival rates in cervical cancer patients.Kaplan-meier method was used to plot the survival curve,and the survival and prognosis value of LRP11 and P16 in patients with cervical cancer was analyzed.Results:1.LRP11 is highly expressed in cervical cancer and positively correlated with P16 expression:Westen blot results confirmed that the relative protein content of LRP11/?-actin in cervical cancer tissues was significantly increased compared with that in normal cervical tissues(0.74±0.25 vs.0.30±0,13,p<0.05).Immunohistochemical results showed that the expression level of LRP11 in the cervical high-grade squamous intraepithelial lesions(HSIL)and cervical cancer tissues was significantly increased compared with the normal cervical tissues(p<0.0001),and the expression level of P16 was also significantly correlated with the changes in pathological grade(p<0.001).Pearson r test results suggested that LRP11 expression was positively correlated with P16 expression(r= 0.5407,p<0.001).2.High expression of LRP11 is associated with low differentiation of cervical cancer:According to the median value of immunohistochemical score of tissue samples collected in this study(LRP11=126.02 vs.P16=142.04),50 cervical cancer tissue samples were divided into the group with high expression of LRP11(28 cases,56%),the group with low expression of LRP11(22 cases,44%),the group with high expression of P16(34 cases,68%)and the group with low expression of P16(16 cases,32%).The results showed that:the expression of LRP11 was correlated with the degree of differentiation of cervical cancer(p=0.0266),but not with age,histology,clinical stage,tumor size,lymph node metastasis or involvement of lymph vessel space.There was no significant correlation between the expression level of P16 and the above clinicopathological variables of cervical cancer.3.High expression of LRP11 indicates poor prognosis:Survival analysis results of kaplan-meier method and log-rank test showed that,compared with patients with low expression of LRP11 with cervical cancer,Overall survival(OS)of patients with high expression of LRP11 was significantly shortened(log rank p=0.0210).However,there was no correlation between P16 expression and OS of patients with cervical cancer(log rank p= 0.7941).Conlusions:1.LRP11 is highly expressed in cervical cancer,positively correlated with the expression level of P16,and significantly correlated with the degree of tumor differentiation.2.The high expression of LRP11 significantly shortens the OS of cervical cancer patients,which is expected to be an effective predictor of the clinical prognosis of cervical cancer patients.Part ?:Effect of LRP11 on proliferation,apoptosis and invasion of cervical cancer cells in vitroObjectives:To explore the effects of LRP11 on the biological behaviors of cervical cancer cell proliferation,apoptosis,migration and invasion,and to analyze the relevant molecular mechanisms.Methods:1.sh-LRP11 plasmid down-regulated the expression of LRP11 was constructed,and the empty plasmid NC group was used as the control group.After lentivirus vector transfection and purinomycin screening,cell lines with stable down-regulated LRP11 expression level were obtained.Western blot was used to detect the expression of LRP11 protein in the sh-LRP11 group and NC group corresponding to SiHa and CaSki of two cervical cancer cell lines,to determine the low expression of LRP11 in the transfected cervical cancer cells,and to detect the expression of P16 in the two cervical cancer cell lines sh-lrp11 group and NC group,and to analyze the effect of LRP11 on P16 expression.2.Changes in proliferation,apoptosis and invasion ability of cervical cancer cells after down-regulation of LRP11 expression were detected by CCK8 assay,flow cytometry and Transwell invasion assay,and the expression of the cell cycle regulatory proteins(CDK 2/4,cyclin D1/E1),apoptosis related proteins(cleaved PARP,BcL-xL,Bax)and invasion related proteins(MMP-2,MMP-9,VEGF)was further detected.Results:1.Down-regulated expression of LRP11 can reduce the expression of P16 in cervical cancer cells:Western blot results showed that after transfection of sh-LRP11 with SiHa cells and CaSki cells,both the LRP11 and P16 protein expression were decreased compared with the NC group.The optical density values of the LRP11 and P16 and the ?-actin internal reference protein were calculated,respectively.The results showed that,compared with the NC group,the expression of LRP11 in cervical cancer cells was down-regulated,resulting in a decrease in the expression of P16.2.Down-regulation of LRP11 expression can inhibit the proliferation activity of cervical cancer cell lines:CCK8 results showed that the cell viability of SiHa sh-LRP11 group was lower than that of NC group at 12 h and 24h,and that of CaSki sh-LRP11 group was lower than that of NC group at 24h and 48 h.After transfection for 12h,the cell viability of the sh-LRP11 group and the NC group were 16.1%and 18.3%,respectively(p<0.05).Cell viability was 19.8%and 21.3%(p<0.01)at 24h after transfection.When CaSki cells were transfected at 24h and 48h,the growth vitality of sh-LRPp11 group and NC group was 21.6%vs.22.3%(p<0.05)and 39.5%vs.42.3%(p<0.001),respectively.3.Down-regulation of LRP11 expression can block the expression of SiHa and CaSki cells in the GO/G1 phase of cervical cancer,and inhibit the expression of CDK 2/4 and cyclinDl/E1:Compared with NC group,SiHa cells and CaSki cells sh-LRP11 were inhibited in G0/G1 phase.After transfection of SiHa cells with sh-LRP11,the proportion of G0/G1 phase cells was 59.8%and 54.1%,respectively,compared with the NC group.The proportion of S+G2/M cells was 40.2%and 45.9%(p<0.001),respectively.After CaSki cells were transfected with sh-LRP11,the proportion of G0/G1 phase cells was 37.8%and 24.6%,respectively.The proportion of S+G2/M cells was 62.2%and 75.4%(p<0.001).Western blot results showed that CDK 2,CDK 4,Cyclin D1,Cyclin E1 and other cell cycle regulation proteins were affected by the expression of LRP11.Compared with the NC group,down-regulation of LRP11 in cervical cancer could inhibit the expressions of CDK 2,CDK 4,Cyclin D1 and Cyclin E1.4.Down-regulation of LRP11 could induce early apoptosis of SiHa cells,but had no effect on apoptosis of CaSki cells:The results of flow cytometry apoptosis detection confirmed that LRP11 could induce early apoptosis of SiHa cells,but had no effect on the apoptosis of CaSki cells.After down-regulation of LRP11,the early apoptosis rates(PI-/Annexin+)of SiHa cells in the sh-LRP11 group and the NC group were 2.33%and 1.94%,respectively(p<0.05).However,there was no significant difference between the two groups in the rate of late apoptosis(5.89%and 5.40%).The early apoptosis rate and late apoptosis rate of CaSki sh-LRP11 group were 4.08%and 1.03%,respectively,which were not significantly different from the NC group(4.12%and 0.98%).Further Western blotting showed that the expression of cleaved-PARP and anti-apoptotic protein bcl-xl was decreased and the expression of pro-apoptotic protein Bax was increased after the down-regulation of LRP11,but there was no statistical difference.5.Down-regulation of LRP11 can significantly inhibit the invasion ability of cervical cancer cells and reduce the expression of MMP-2,MMP-9,VEGF:Compared with the NC group,the number of cell migration and invasion through the Transwell cell in the sh-LRP11 group of two cervical cancer cells was significantly decreased.After down-regulation of LRP11,the number of cells in the NC group and sh-LRP11 group crossing the Tranwell compartment was 212.3±13.0 and 170.2±14.2(p<0.01).CaSki cells:147.6±14.5 and 76.8±12.0(p<0.001).Compared with the negative control group,down-regulated LRP11 could significantly inhibit the invasive ability of cervical cancer cells.Western blot results showed that the down-regulation of LRP11 could reduce the expression of MMP-2,MMP-9,VEGF.However,after the down-regulation of LRP11 in SiHa cells,MMP-2 protein expression was decreased,which was not statistically different from that in the NC group.The relative protein expressions of MMP-9/?-actin in the LRP11 group and the NC group were 0.63±0.11 and 1.36±0.12,respectively(p<0.05).After CaSki cells down-regulated LRP11,VEGF protein(0.90±0.13 vs.1.48±0.10,p<0.05)and MMP-9 protein(0.58±0.09 vs.1.07±0.11,p<0.01)were significantly decreased compared with the NC group.Conclusion:1.Down-regulating the expression of LRP11 in cervical cancer cells can reduce the proliferation and invasion ability of tumor cells.2.LRP11 had no significant effect on the expression of apoptosis-related proteins and the apoptosis rate of cervical cancer cells.3.LRP11 plays an important role in the biological progress of cervical cancer.Part ?:Effect of LRP11 on the growth of cervical cancer cells in vivo and its related mechanismObjectives:1.Establish the nude mouse model of SiHa cervical cancer transplantation.2.The effect of LRP11 expression on the growth of transplanted tumor in siha-bearing nude mice was detected.3.To investigate the effect of LRP11 expression on the expression levels of LRP11 and P16 in xenograft tumors of siha-bearing nude mice.Methods:1.SiHa cells of cervical cancer(sh-LRP11 group and NC group)were cultured and inoculated under the skin of nude mice with approximately the same body weight.The number of tumor cells inoculated in each nude mouse was 5×106.2.The volume of transplanted tumor was measured every 3 days after tumor seeding 5 days,tumor volume=(width)2 × length/2.Mice were sacrificed after 41 days of tumor seeding,the transplanted tumors were stripped off the back,weighed and recorded.3.The exfoliated transplanted tumor was taken to make paraffin sections in equal amounts,and the expressions of LRP11 and P16 in the transplanted tumor tissues were detected by immunohistochemistry.Results:1.Down-regulating the expression of LRP11 can decrease the growth capacity of SiHa cells in vivo:Lentivirus down-regulated LRP11 expression was transfected into cervical cancer SiHa cells and inoculated subcutaneously in nude mice.NC group was used as the control group.The results showed that the tumor growth rate of sh-LRP11 group was significantly lower than that of NC group within 41 days after inoculation(p<0.05).The average volume and weight of sh-LRP11 group were smaller than NC group(p<0.05).The results suggested that after down-regulated expression of LRP11,the growth ability of SiHa cells in cervical cancer decreased,which was consistent with the results of in vitro experiments.2.The down-regulation of LRP11 can reduce the expression of P16 in cervical cancer cells:The expression of LRP11 and P16 in SiHa cells transfected with sh-LRP11 was detected by immunohistochemical method.The results showed that compared with the NC group,the expression of LRP11 in the sh-LRP11 group was decreased(23.6±4.5 vs.44.8±7.1,p<0.05),and the expression of P16 was also significantly decreased(18.4±3.9 vs.77.7±9.2,p<0.05).Conclusion:1.Down-regulating the expression of LRP11 in cervical cancer can inhibit the growth capacity of cervical cancer cells in vivo.2.The down-regulation of LRP11 can reduce the expression of P16 in cervical cancer cells in vivo.The innovation of this study1.Innovation of LRP11 in the field of cervical cancer diagnosis and prognosis:this study first studied the expression level of LRP11 in cervical cancer and its relationship with the clinical survival and prognosis of cervical cancer patients.2.Innovative research on the role of LRP11 in the biological progress of cervical cancer:this study is the first to explore the effect of LRP11 on the biological functions of cervical cancer cell proliferation,apoptosis,invasion,etc Currently,LRP family plays an important role in the occurrence and development of a variety of malignant tumors,but there are few relevant studies on cervical cancer.