Research About The Functional Study Of RRAGD In The Progress Of Cervical Cancer

Background:Cervical cancer(CC),one of the most common cancers in women,is the fourth most common malignancy and the second incidence in female malignant tumors.According to statistics,about 530000 new cases are diagnosed every year in the world,and about 275000 patients die of the disease.The incidence of cervical cancer in developing countries accounts for 85percentof the world’s total women,and is also the main cause of cancer deaths in these areas.According to the survey,the incidence of cervical cancer is 7.5/100 thousandin China,and nearly half of them die,and the death rate is as high as 3.4/10 thousand.Thus,the cervical cancer has become one of the malignant tumors that seriously threaten the life and health of women.At present,treatments of cervical cancer include radiotherapy,chemotherapy and surgery.The surgical treatment is limited to women with early diseases and women who have no requirements for birth.The combination of radiotherapy and platinum based chemotherapy is the most common method for the treatment of cervical cancer and can be effective in the treatment of cervical cancer.However,either radiotherapy or chemotherapy may damage normal cells in the body as well as the tumor.In recent years,many studies have been done on the causes of cervical cancer,and many risk factors and tumor related signaling pathways including human papillomavirus have been found.A study has shown that the mutation of upstream regulatory gene or loss of function of the mTOR has been proved to be closely related to the occurrence of a variety of clinical tumors.More importantly,it has been found in many kinds of human malignant tumors for the excessive activation of PI3K and Akt,or loss or mutation of a negative regulator of inhibitors of phosphatase and tensin homologue or PI3K signaling(phosphatase and tensin homologue,PTEN).The excessive activation of mTOR pathway plays a very important role in the occurrence and development of cancer.Therefore,targeting the mTOR pathway component may be an effective cancer treatment strategy.Ras related GTP binding protein D(Ras-related GTP-binding protein D,RRAGD Rag),a GTP binding protein family,is a single amino acid guanine nucleotide binding protein.Guanine nucleotide protein construct set two dimers to heterologous amino acids that induced mTORCl reset to lysosomes,followed by activation of RHE GTPase that is the key step TOR amino acid signaling cascade,and the amino acid mediated activation of mTOR signaling pathway plays a central role.There have been many reports on the mechanism of RRAGD mediated amino acid activation of the mTOR signaling pathway.However,the mechanism of how RRAGD is regulated is not completely clear.Studies have confirmed that NF-kappa B can control the microphthalmia associated transcription factor(MITF)expression in cervical cancer cells.In another study,microarray data for melanoma metastasis patients(cancer genome map dataset)and melanoma cell line(gene expression comprehensive database)showed that there was a significant correlation between MITF and RagD gene expression level.Moreover,publicly available MITF chromatin Chromatin Immunoprecipitation(ChIP)dataset shows that MITF occupies the RRAGD proximal promoter in COL0829 melanoma cells,which is consistent with our previous results.Therefore,MITF may be positively regulating RRAGD.However,it is unknown whether MITF can regulate RRAGD through mTOR signaling pathway to regulate the development of cervical cancer.Therefore,the expressions of RRAGD and MITF mRNA were tested in 15 strains of human cervical cancer cell linesin this experiment,and ChIP-seq and si-RNA transfection were used to analyse the correlation between RRAGD and MITF,and we hypothesize that the MITF may be involved in the regulation of RRAGD.Then Luciferase reporter vector PGL4.21 carrying RRAGD gene promoter(Luc2P/Puro)-promoter recombinant plasmid vector RRAGD and corresponding promoter mutation were constructed,and experiments were divided into fifteen groups.A luciferase reporter array was used to verify the hypothesis.We construct human cervical carcinoma cell RRAGD low expression system by the lentivirus infection and CRISPR Cas9 gene knockout technology,while establish human cervical cancer cells MITF expression system.The protein level and molecular level of related genes RRAGD,MITF,S6K and 4EBP1 were detected by different treatments.The contents were mainly divided into three parts:Part One The expression of RRAGD and MITF in cervical cancer cell line.Purpose:The purpose of our study is to investigate the mRNA expression and correlation of RRAGD and MITF in cervical cancer cell line.After all,we further investigate the physiological role of RRAGD and MITF in the progress of cervical cancer,in order to provide evidence.Materials and Methods:1.To create the melatonin cell line.Using human melatonin cell as a reference,examine the RRAGD and MITF mRNA expression of 15 human cervical cancer cell line using real time PCR(all the specimen came from Fu Dan University).2.Examine the relationship of RRAGD and MITF in human primary melanocytes using ChIP-seq.3.Measure the MITF and RRAGD mRNA expression using RT-PCR in siRNA-Control,siRNA-MITF-1 and siRNA-MITF-3 transfected HeLa,HeLa S3,HeLa229 and Hep2 cells.4.Statistical analysis was done using Graph Pad Prism 7(Graph Pad Software 7,Inc.,La Jolla,CA,USA).Data are presented as Mean ± SD.Comparison was done using Student’s t-test or Mann-Whitney’s,α = 0.05.Results:1.Human melatonin cell line was successfully created.2.RRAGD and MITF mRNA expression in cervical cancer cell line.Real time PCR results showed that,RRAGD mRNA expression was higher in HR5,X1/5,HeLa,C-4 Ⅱ,HeLa S3,HeLa229,Hep2(HeLa derivative),HeLa B,Bu25 TK-,HeLa Ohio cell line;in contrast,RRAGD mRNA expression was lower in HeLa DH,HtTA-1,HR5-CL11,C-4 I,Ca Ski cell line.MITF mRNA expression was higher in HeLa,HeLa S3,HeLa229 cell line but lower in HeLa DH,HtTA-1 HR5,X1/5,HR5-CL11,C-4 Ⅰ,C-4 Ⅱ,Ca Ski(small bowel metastasis),Hep2(HeLa derivative),HeLa B,Bu25 TK-,HeLa Ohio cell line.3.The correlation of RRAGD and MITF in human primary melanocytes.(1)MITF ChIP-seq showed that MITF is occupies the RRAGD promoter in human primary melanocytes.(2)H3K27ac ChIP-seq showed that MITF knockdown strikingly decreases H3K27ac levels at the RRAGD promoter region in melanocytes.(3)So,MITF and H3K27ac ChIP-seqs suggests that MITF binds to RRAGD proximal promoter and positively regulates its transcription in melanocytes.4.MITF and RRAGD condition after transfection of siRNA-MITF in human cervical cancer cells.(1)Compare to negative control,MITF mRNA expression of siRNA-MITF-1 and siRNA-MITF-3 transfected Hela,HeLa S3,HeLa229 and Hep2 are relatively lower(P<0.001).(2)Compare to negative control,RRAGD mRNA expression of siRNA-MITF-3 transfected Hela,HeLa S3,HeLa229 and Hep2 are relatively lower(P<0.001).RRAGD mRNA expression of siRNA-MITF-1 transfected Hela,HeLa S3and HeLa229 are relatively lower(P<0.001).(3)siRNA-MITF-3 has higher transfection efficiency than siRNA-MTF-1,RRAGD mRNA level was significantly reduced when siRNA-MTF transfection efficiency reached 80%.Conclusion:RRAGD and MITF has higher expression in cervical cancer cells line HeLa,HeLa S3,HeLa229,MITF could play a role in regulating RRAGD.Part 2 Research on the regulation of RRAGD promoter activity by MITFPurpose:In the current study,we aim to investigate if MITF can regulate RRAGD,the following contains evidenes of RRAGD participates in regulating the progress in cervical cancer.Materials and Methods:1.Using Gibson method,melatonin cell DNA as reference,propagate RRAGD using PCR(-1869 bp—213 bp),Construction of luciferase reporter vector PGL4.21(Luc2P/Puro)-RRAGDpromoter recombinant plasmid carrying the promoter of RRAGD gene,using gene sequencing technology to identify recombinant plasmids(All sequencing was completed at the Harvard Medical School/Massachusetts General Hospital Center For Computational&Integrative Biology DNA Core).2.Making PGL4.21(Luc2P/Puro)-RRAGD-mut(E-box)plamid,using gene sequencing technology to identify recombinant plasmids(All sequencing was completed at the Harvard Medical School/Massachusetts General Hospital Center For Computational&Integrative Biology DNA Core).3.Using Gibson methods,to make pcDNA3,1(+)-HA-hMITF-M plamid.4.Luciferase reporter array assay was used to test promoter activity:The experimental grouping is as followsTransfection control group:Renilla-PGL4-pLKO.1(shRNANegative transfection control group)Renilla-PGL4-shMITF-2(shRNATransfection control group)Renilla-PGL4-shMITF-5(shRNATransfection control group)Renilla-PGL4-pCW45(Overexpression negative transfection controlgroup)Renilla-PGL4-pcDNA3.1(+)-HA-hMITF-M(Overexpression transfection controlgroup)Transfection experimental group:Renilla-PGL4-RRAGD promoter-pLKO.1(shRNANegative transfection experiment group)Renilla-PGL4-RRAGD promoter-shMITF-2(shRNA transfection experiment group)Renilla-PGL4-RRAGD promoter-shMITF-5(shRNA transfection experiment group)Renilla-PGL4-RRAGD promoter-pCW45(Overexpression negative transfection experimental group)Renilla-PGL4-RRAGD promoter-pcDNA3.1(+)-HA-hMITF-M(Overexpression transfection experimental group)Mutation test group:Renilla-PGL4-RRAGD mut-pLKO.1(Negative transfection mutation test group)Renilla-PGL4-RRAGD mut-shMITF-2(shRNA transfection mutation experiment group)Renilla-PGL4-RRAGD mut-shMITF-5(shRNA transfection mutation experiment group)Renilla-PGL4-RRAGD mut-pCW45(Overexpression negative transfection mutation test group)Renilla-PGL4-RRAGD mut-pcDNA3.1(+)-HA-hMITF-M(Overexpression transfection mutation test group)Experimental method:Transfection of HeL and HeLa229 cells simultaneously by liposome method according to the above subgroups.After 72 hours of transfection,the activities of firefly luciferase and Renilla luciferase in cell lysates were measured using dual luciferase monitoring system,to calculate the ratio,the ratio of the two indirectly reflects the activity of the promoter.5.Data analysis is same as above.Results:1.Successfully construct PGL4.21(Luc2P/Puro)-RRAGD promoter,PGL4.21(Luc2P/Puro)-RRAGD-mut(E-box),pcDNA3.1(+)-HA-hMITF-M plasmid.2.Luciferase reporter array showed:(1)The overall activity of the transfection experiment group was significantly higher than that of the transfection control group,difference was significant(P<0.01).(2)The overall activity of the mutant experimental group was lower than that of the transfected control group,difference was significant(P<0.05).(3)Compared with shRNA transfection control group,the activity of shRNA transfection control group decreased,difference was significant(P<0.01);Compared with the shRNA negative transfection experimental group,the activity of the shRNA transfection experiment group decreased,difference was significant(P<0.01);Overexpression of negative transfection control group overexpression Transfection control group activity increased,difference was significant(P<0.01);Overexpression of negative transfection experimental group overexpression transfection increased activity of the experimental group,difference was significant(P<0.01).(4)The overall activity level of transfection in the five experimental groups was not significantly different.difference was significant(P<0.01).Conclusion:MITF is positively associated with RRAGD transcriptional activity and regulates RRAGD at the transcriptional level.Part 3 Functional study of RRAGD in cervical cancer progressionPurpose:In the current study,we aim to investigate the function of RRAGD in cervical cancer and examine RRAGD regulate the progression of cervical cancer via mTOR pathway.Materials and Methods:1.Transfection with lentivirus infectionshluc(Negative control),using shRRAGD-l,shRRAGD-2 knockout cervical cancer cell Hela,to promotes stable expression of HeLa-shluc,HeLa-shRRAGD-1,HeLa-shRRAGD-2,this was further validated by Real time PCR.2.Using CRISPR Cas9 method to knockout RRAGD in cervical cancer Hela to make Hela CRISPR control,HeLa-CRISPR-RRAGD-1,HeLa-CRISPR-RRAGD-2,this was further validated using western blot.3.Use western blot to detect the expression of RRAGD,MITF,S6K,and 4EBP1 protein in HeLa cells with low expression of RRAGD induced by Lentivirus infection.4.Use RT-PCR to measure RRAGD,MITF,S6K,and 4EBP1 after transfected Hela cells with pLJM.l-EGFP5 pcDNA3.1(+)-HA-hMITF-M.5.Data analysis is same as Part One.Results:1.Succesfully construct Hela cells with low RRAGD expression.Stably transfection of shluc,shRRAGD-1 shRRAGD-2 in Hela cells was validated by RT-PCR,Compare to negative control,the mRNA level of RRAGD in the experimental group are relatively lower(P<0.05);Compared with HeLa-CRISPR-control group,RRAGD protein levels in HeLa-CRISPR-RRAGD-1 and HeLa-CRISPR-RRAGD-2 groups were significantly lower(P<0.05);Compared with HeLa-CRISPR-control group,RRAGD protein levels in HeLa-CRISPR-RRAGD-1 and HeLa-CRISPR-RRAGD-2 groups were significantly lower(P<0.05).2.HeLa-shRRAGD-1 and HeLa-shRRAGD-2 has significantly lower RRAGD,S6K,4EBP1 protein expression than Hela-Shluc cells(P<0.05).3.HeLa-CRISPR-RRAGD-1,HeLa-CRISPR-RRAGD-2 cells has significantly lower RRAGD,S6K,4EBP1 protein expression than HeLa-CRISPR-control cells(P<0.05).4.Expression of HeLa cell-associated factor protein expression in RRAGD constructed by stimulation of lentivirus-infected cells without amino acid mediaThe protein levels of RRAGD,S6K,and 4EBP1 in the HeLa cells with low expression of RRAGD constructed by Lentivirus infection were significantly increased after stimulation with amino acid-free medium for 72 hours,and the difference was statistically significant(P<0.05).5.Expression of related factors in the MITF high expression HeLa cell systemHeLa-pLJM.1-EGFP has higher RRAGD,MITF,S6K,4EBP1 mRNA expression when compare to negative control(P<0.05)Conclusion:Amino acid deficiency upregulated RRAGD and the activation of mTORl,overexpression of MITF led to the upregulation of RRAGD and the activation of mTOR1,RRAGD plays an important role in cervical cancer progression.