The Expression Of Lysophosphatidic Acid Receptor 6 (LPAR6) And Its Related Molecules In Breast Cancer, Its Effect On Prognosis And Its Functional Mechanism

Objective: As a universal bioactive lipid,lysophosphatidic acid(LPA)functions in many physical and pathological processes.LPA receptor 6(LPAR6)has been documented to play multiple roles in different cancers,which were even contradictory.This study was initiated to uncover its biological effects and regulating mechanisms in breast cancer which are not fully known.Methods:1.Different open databases were used to identify the expression of LPAR6 between normal mammary tissues(including para-cancer tissues)and breast cancer tissues and the relationship between LPAR6 expression and clinicopathological characteristics.Western blot was used to verify the LPAR6 protein expression between para-cancer tissues and breast cancer tissues.2.The Breast Cancer Gene-Expression Miner v4.5(bc-GenExMiner v4.5)was used to analyze the prognostic value of LPAR6 for patients with breast cancer.3.The biological function of LPAR6 on breast cancer was evaluated via CCK-8 assay,plate colony formation assay,flow cytometry analysis,and wound healing assay.Meanwhile,gain-and loss-of-function assays were utilized to certify the results.4.Bioinformatics analyses were used to analyze the potential pathways which LPAR6 may affect to inhibit breast cancer growth,thus postulating the underlying mechanisms of the antitumor effect of LPAR6.5.By exploring the web tools and in vitro experiments,possible microRNAs which regulate the expression of LPAR6 were supposed to be validated.6.The SWISS-MODEL and I-TASSER methods were used to establish and analyze the advanced structure model of human LPAR6.7.By constructing full-length and truncated LPAR6 plasmids,the key domain was identified which is essential to subcellular location via the laser confocal microscopy.Results:1.LPAR6 expression was significantly decreased in breast cancer tissues compared with normal mammary tissues or para-cancer tissues,which suggested that LPAR6 acted as a tumor suppressor in breast cancer.Western blot assay also supported that LPAR6 expression was downregulated in breast cancer in protein level.And the decreased expression of LPAR6 in cancer tissues was significantly correlated with clinicopathological characteristics,including pathological subtypes,grades,and clinical stages.2.Bioinformatics analyses showed that decreased LPAR6 expression was significantly correlated with poor prognosis.Regardless of all patients with breast cancer or patients belonging to hormone receptor-positive(HR+)or lymph node metastasis-positive(LN+)subgroups,the LPAR6 expression level was suitable to predict prognosis with regard to overall survival(OS),disease-free survival(DFS),and distant metastasis-free survival(DMFS).3.Overexpression of LPAR6 significantly inhibited cell proliferation and plate colony formation ability and increased cell early apoptosis in the SK-BR3 cell line.And vice versa,LPAR6 knockdown significantly increased cell proliferation and colony formation ability in the MCF-7 cell line.Meanwhile,ectopic expression of LPAR6 significantly impaired the migration ability in the SK-BR3 cell line,and LPAR6 knockdown,in turn,increased migration ability in the MCF-7 cell line.4.Bioinformatics analyses suggested that LPAR6 inhibited breast cancer progression mainly via inactivating hallmark pro-cancerous pathways,i.e.,“E2F_targets”,“MTORC1_signaling”,“G2M_checkpoint”and “Myc-targets”.5.After exploring the web tools of miRWalk and Target Scan,we identified and validated that miR-27a-3p acted as a positive regulator for LPAR6 expression by RT-PCR and in vitro proliferation assays.We verified the value of the miR-27a-3p-LPAR6 axis in antitumor effects against breast cancer.6.The advanced structure model of human LPAR6 was established using the I-TASSER method and the model displayed more stable structure binding with target ligands than zebrafish Lpar6 a.7.The subcellular location of truncated LPAR6(the first-half of CDS deleted)was different from that of full-length LPAR6 in HEK-293 T cells,which hinted that the first-half domain is essential to the subcellular location of LPAR6.Conclusion:1.LPAR6 expression was decreased in breast cancer tissues and correlated with clinicopathological characteristics,including pathological subtypes,grades,and clinical stages.The decreased LPAR6 expression predicted a poor prognosis.2.LPAR6 inhibited breast cancer progression partly regulated by miR-27a-3p,which indicated that LPAR6 acted as a tumor suppressor in breast cancer.3.The LPAR6 advanced structure model analysis demonstrated that human LPAR6 showed a more stable structure than zebrafish lpar6 a.Further research revealed that the first-half CDS of LPAR6 was vital to protein subcellular location.Objective: Breast cancer is the leading diagnosed cancer in women,with its high mortality just inferior to lung cancer.Homeobox A4(HOXA4)was documented to have crucial roles in lung cancer,ovarian cancer,and leukemia.However,the study about HOXA4 in breast cancer has not been fully understood.Methods:1.Bioinformatics methods were used to analyze the differential expression of HOXA4 and the differential methylation level in HOXA4 promoter between normal mammary tissues and breast cancer tissues.In addition,the correlation analyses and the prognostic analyses of HOXA4 expression on patients with breast cancer were also included.2.RT-PCR assay,western blot assay,IP assay,and mass spectrometry were used to measure the expression of associated molecules or their interaction.3.CCK-8 assay,plate colony formation assay,flow cytometry,scratch assay,and transwell assay were used to evaluate the biological functions of HOXA4 in breast cancer,such as proliferation ability,apoptosis,and migration ability.4.The transcriptome analysis was used to determine the underlying mechanism of HOXA4 in breast cancer.Results:1.HOXA4 expression was significantly decreased in breast cancer tissues compared with normal mammary tissues and decreased HOXA4 expression was associated with poor prognosis with regard to OS,DFS,and DMFS.2.HOXA4 promoter was hypermethylated in breast cancer tissues than normal mammary tissues,thus downregulating HOXA4 expression.Meanwhile,treatment with 5-AZA rescued HOXA4 expression in breast cancer cell lines.3.HOXA4 inhibited cell proliferation ability in breast cancer.Overexpression of HOXA4 impaired the proliferative activity and colony formation ability in the MDA-MB231 cell line,and knockdown of HOXA4 enhanced the proliferative activity and colony formation ability in the MCF-7 cell line.4.HOXA4 increased cell apoptosis in breast cancer.Overexpression of HOXA4 increased cell apoptosis and the protein level of cleaved caspase3 in MDA-MB231 and SK-BR3 cell lines.In addition,the knockdown of HOXA4 reduced the protein level of cleaved caspase 3 in the MCF-7 cell line.5.HOXA4 inhibited cell migration ability in breast cancer.Overexpression of HOXA4 impaired cell migration ability and reduced the protein level of integrin β1 in the MDA-MB231 cell line.6.The transcriptome analysis demomstrated that the epithelial-mesenchymal transition pathway and KRAS signal pathway were significantly activated.7.HOXA4 regulated the mRNA and protein levels of integrin β1 in breast cancer cell lines.8.HOXA4 bound with Salvador family WW domain containing protein 1(SAV1)and also regulated the mRNA and protein levels of SAV1 in breast cancer cell lines.Conclusion:1.HOXA4 expression was significantly decreased in breast cancer tissues.Decreased HOXA4 expression was partly due to the hypermethylation of HOXA4 promoter and associated with poor prognosis.2.HOXA4 acted as a tumor suppressor in breast cancer,which could inhibit cell proliferation and migration and promote cell apoptosis.3.HOXA4 inhibited breast cancer progression probably by regulating the integrin β1/SAV1-Hippo pathway.