Study On The Role And Molecular Mechanism Of CircIQCH In Breast Cancer Progression

[Background]Breast cancer(BC)is the most common malignant tumors in women.According to the latest research,the number of new cases of breast cancer worldwide reached 2.26 million in 2020(accounting for 11.7%of all malignant tumors),surpassing lung cancer for the first time to become the No.1 malignant tumor worldwide.In recent years,with the continuous improvement in the level of breast cancer diagnosis and comprehensive treatment,the five-year survival rate of early-stage breast cancer patients has been significantly improved,but the mortality rate of middle and late stage breast cancer is still high,which is closely related to distant metastasis.The most common site of metastasis for breast cancer is bone,followed by lung,liver and brain.Breast cancer patients with lung metastases have a poor prognosis,with a median survival time of approximately 21 months.Therefore,it is particularly important to explore the molecular mechanisms of breast cancer metastasis,to find novel and accurate biomarkers and to develop new therapeutic targets.Circular RNAs(circRNAs)are a new class of non-coding RNAs with a closed loop structure formed by back-splicing.Most of circRNAs are mainly located in the cytoplasm and have attracted attention because of their biological characteristics such as tissue specificity,temporal sequence specificity,disease specificity and high stability,and are expected to become a novel molecular marker for tumors.However,the role of circRNAs in lung metastasis of breast cancer and the exact molecular mechanisms remain to be revealed and elucidated.[Purpose and significance]The aim of this study is to investigate the expression and biological function of circIQCH in breast cancer and its molecular mechanisms regulating breast cancer progression.This study will provide potential molecular markers and therapeutic targets for breast cancer.[Methods](1)The circRNA microarray results of breast cancer primary tissues and lung metastasis tissues from our previous study were analyzed,and top 5 circRNAs with the most significant differences were selected for qRT-PCR validation;the circIQCH(hsa_circ₀104345)with the most significantly up-regulated expression was selected as the study target,and the expression of circIQCH was detected in normal breast epithelial cells MCF-10A,multiple breast cancer cell lines,breast cancer tissues and paracancerous normal tissues by qRT-PCR;The source and basic information of hsa_circ₀104345 was determined by searching the circBase and circBANK online database;RNase R assay to identify the circular RNA characteristics of circIQCH;Actinomycin D assay to detect the stability of circIQCH and IQCH mRNA.(2)The si-circIQCH silencing vector was constructed and transfected into breast cancer cells SKBR3 and BT474.qRT-PCR was used to verify the transfection efficiency of circIQCH siRNA.The effects of circIQCH silencing on proliferation,migration and invasion of breast cancer cells were detected by CCK-8,wound healing assay and transwell assay,respectively.Breast cancer cell lines with transfection of si-circIQCH and control vector were inoculated subcutaneously into nude mice to construct a nude mouse transplantation tumor model and tail vein injection to construct a breast cancer lung metastasis model.The changes in tumorigenic and lung metastatic abilities of breast cancer cells after circIQCH silencing were examined respectively,and the expression of Ki-67 in tumor tissues was detected by immunohistochemistry.(3)Cellular localization of circIQCH was performed using nucleoplasmic isolation assay;miRNAs bound to circIQCH were predicted using the online Circular RNA Interactome database(https://circinteractome.nia.nih.gov);qRT-PCR was performed to detect miR-145 in breast cancer cell lines;luciferase reporter gene assay and RIP assay to detect whether circIQCH binds to miR-145.(4)Targetscan online database was used to predict the target gene of miR-145;qRT-PCR to detect the expression of DNMT3A in breast cancer cell lines;qRT-PCR to detect the expression of DNMT3A after overexpression or inhibition of miR-145;luciferase reporter gene assay to detect whether miR-145 binds to DNMT3A;RIP assay was used to detect whether circIQCH,miR-145 and DNMT3A bind to each other.(5)Knockdown of circIQCH or inhibiting miR-145 alone or knockdown of circIQCH and inhibiting miR-145 at the same time,Western blot detected the expression of DNMT3A and oncogenes PTEN and BRCA1;knocking down circIQCH and inhibiting miR-145 or overexpressing DNMT3A at the same time,CCK-8 and transwell assay detected the proliferation and migration ability of breast cancer.[Results]1.CircIQCH expression was upregulated in breast cancer cells,primary breast cancer tissues and lung metastases The qRT-PCR results showed that hsa_circ₀104345 was most significantly up-regulated in breast cancer lung metastasis tissues.The online database revealed that hsa_circ₀104345 originated from the gene IQCH located on chromosome 15p23(chr15:67636400-67665771).Therefore,hsa_circ₀104345 was named circIQCH in this study.qRT-PCR assay revealed that the expression level of circIQCH was upregulated in breast cancer cell lines compared with MCF-10A,especially the highest expression was found in SKBR3 and BT474 cells(P<0.05).Similarly,the expression level of circIQCH was significantly higher in breast cancer tissues than in normal tissues adjacent to the cancer(P<0.05).the results of RNase R assay confirmed that circIQCH has a stable circular RNA profile resistant to RNAase digestion.In the actinomycin D assay,circIQCH was more stable than linear IQCH mRNA in SKBR3 cells.2.Down-regulation of circIQCH inhibits the proliferation of breast cancer cells in vitro and in vivo To investigate the biological function of circIQCH,two circIQCH siRNA interference sequences were designed to silence the expression of circIQCH in SKBR3 and BT474 cells.The success of circIQCH interference was verified by qRT-PCR.The results showed that the expression of circIQCH was significantly reduced after si-circIQCH transfection into SKBR3 and BT474 cells,while its linear IQCH mRNA expression did not change.CCK-8 assay analysis showed that the cell activity in the circIQCH siRNA group was significantly lower than that in the control group.Subcutaneous injection of nude mice into tumorigenic assays also yielded consistent results that knockdown of circIQCH expression could significantly inhibited the tumor growth.3.Down-regulation of circIQCH inhibits the ability of breast cancer cells to migrate,invade and metastasize in vitro and in vivo In the scratch assay,when circIQCH expression was down-regulated,the percentage of wound healing was significantly lower in SKBR3 and BT474 cells compared to the control group.Transwell assay showed that the number of tumor cells crossing transwell compartments was significantly reduced after transfection with circIQCH siRNA,suggesting that silencing circIQCH expression significantly inhibited the invasive ability of breast cancer cells.The results of the tail vein injection lung metastasis assay in nude mice suggested that the number of lung metastases was significantly reduced after knocking down the expression of circIQCH compared with the control group.4.CircIQCH acts as a molecular sponge for miR-145 The Circular RNA Interactome database predicts several miRNAs that potentially interact with circIQCH.Among them,miR-145 has binding sites that interact with circIQCH.qRT-PCR results showed that miR-145 is lowly expressed in breast cancer cell lines.Nucleoplasmic segregation experiments revealed that circIQCH was mainly present in the cytoplasm,suggesting that it may function by acting as a sponge for miRNA.Luciferase reporter gene experiments showed that luciferase activity was reduced after transfection with circIQCH wild-type reporter gene and miR-145 mimics.RNA immunoprecipitation(RIP)results indicated that miR-145 was mainly enriched in the MS2bs-circIQCH group.These results suggest that circIQCH functions as a molecular sponge for miR-145.5.CircIQCH regulates DNMT3A expression through miR-145 sponge TargetScan online software predicted the presence of a miR-145 binding site in the DNMT3A 3'-UTR sequence.Luciferase reporter gene experiments showed that luciferase activity was reduced after cotransfection with miR-145 mimics and wild-type 3'-UTR-DNMT3A reporter gene in SKBR3 and BT474 cells.qRT-PCR experiments demonstrated that overexpression of miR-145 reduced DNMT3A expression levels,and miR-145 inhibitors upregulated DNMT3A expression levels,indicating that DNMT3A was regulated by miR-145.In addition,RIP analysis associated with Ago2 showed that circIQCH,DNMT3A and miR-145 were enriched to Ago2 in both SKBR3 and BT474 cells.circIQCH downregulation significantly increased DNMT3A enrichment to Ago2.Western blot experiments showed that knockdown of circIQCH decreased DNMT3A expression,and this effect could be reversed by inhibition of miR-145.6.CircIQCH/miR-145/DNMT3A axis regulates the progression of breast cancer Silencing of circIQCH expression was followed by upregulation of the expression of two classical tumor suppressor oncogenes,PTEN and BRCA1,and this effect could be partially reversed by inhibition of miR-145 overexpression.In addition,knockdown of circIQCH along with overexpression of DNMT3 A or inhibition of miR-145 partially reversed the cell proliferation and migration inhibitory effects mediated by knockdown of circIQCH.[Conclusions]1.CircIQCH was highly expressed in breast cancer cells and tissues,with higher expression in lung metastatic tissues.2.Knockdown of circIQCH could inhibit proliferation,migration,invasion and metastasis of breast cancer cells.3.CircIQCH was mainly localized in the cytoplasm and could indirectly promote the expression of target gene DNMT3A by sponging miR-145 with oncogenic effects and inhibit PTEN and BRAC1 expression.