The Role Of POU4F3 In Proliferation,Migration And Apoptosis Of LUAD And The Related Mechanisms

Background:Lung cancer ranks the first in the incidence of malignancies,with about 781 thousand new cases in China each year.Lung cancer is the most common malignancy in men and the second in women.Lung adenocarcinoma(LUAD)is the most common type of lung cancer,and its pathogenesis is not completely clear.POU domain class Ⅳ transcription factor Ⅲ(POU4F3),also known as brain-specific homeobox protein 3C(Brn-3C),is located in chromosome 5q31-p33.Mounting studies have shown that POU4F3 plays a vital role in the occurrence of many malignancies,including Merckle cell carcinoma,astrocytoma,cervical intraepithelial neoplasia,endometrial carcinoma,ovarian cancer,and acute myeloid leukemia.POU4F3 is closely related to the occurrence of a variety of tumors,but the relationship between POU4F3 and LUAD is still unknown.In this study,human LUAD tissues,LUAD cell lines,and animals were used as experimental subjects to explore the role of POU4F3 in LUAD and its related mechanisms,which is expected to provide new ideas for the diagnosis,prognosis,and treatment of LUAD.Methods:1.Immunohistochemical staining was used to detect the expression level of POU4F3 protein in carcinoma(n=122)and paracancerous tissues(n=118)of human LUAD tissue microarray and to analyze the relationship between POU4F3 expression and the clinicopathological characteristics of LUAD patients.2.We constructed LUAD cell lines with stable overexpression or knockdown of POU4F3.POU4F3 overexpression lentivirus(Lv-POU4F3)and its negative control lentivirus(Lv-control)were produced by Genomeditech.POU4F3 knockdown lentivirus(shPOU4F3)and its nontargeting control lentivirus(control shRNA)were constructed by GeneChem.They were stably transfected into LUAD cell lines,respectively.All stably transfected cell lines were screened by puromycin,and the transfection efficiency was detected by Western Blotting.3.CCK-8 assay and cell clone formation assay were used to investigate the effect of POU4F3 on cell proliferation in vitro.Subcutaneous tumor experiments were conducted to detect the impact of POU4F3 on cell proliferation in vivo.4.The effect of POU4F3 on the cell cycle was detected by flow cytometry.5.The effect of POU4F3 on apoptosis was detected by flow cytometry.Western Blotting was conducted to detect the expression of Caspase3 and Cleaved caspase3.6.Wound scratch and Transwell migration assays were carried out to detect cell migration.A Phalloidine fluorescence experiment was applied to determine the effect of POU4F3 on cell microfilaments.7.Western Blotting was used to detect the expression of GRP78,a key molecule in the endoplasmic reticulum stress(ERS)signaling pathway,and to detect the expression of critical molecules of two ERS sub-pathways PERK/eIF2a/ATF4/CHOP and IRE1α/XBP-1s in LUAD cells with stable overexpression or inhibition of POU4F3.Results:1.POU4F3 was low expressed in LUAD tissues,and patients with higher POU4F3 expression had a longer overall survival.POU4F3 positive rate of LUAD tissue(n=122)and para-carcinoma tissue(n=118)was significantly different(26.23%vs.92.37%,χ2=105.6,P<0.0001).There were no statistically significant differences in age,gender,tumor size,tumor location,TNM stage,pathological differentiation,and lymph node metastasis between the high POU4F3 expression group and the low POU4F3 expression group(P>0.05).The log-rank in 92 LUAD patients showed that the median overall survival of LUAD patients with higher POU4F3 expression is longer than that with lower POU4F3 expression(83 vs.34 months,χ2=7.758,P=0.0053).2.POU4F3 inhibited the proliferation of LUAD cells in vitro.CCK-8 cell proliferation curve showed that overexpression of POU4F3 significantly inhibited the proliferation rate of LUAD cells(P24 h<0.05;P48 h<0.05;P72 h<0.01).Cell clone formation showed that the number of clones of LUAD cells was reduced considerably by POU4F3 overexpression(P<0.0001).Under the same experimental conditions,POU4F3 knockdown significantly promoted the proliferation rate of LUAD cells(P24 h<0.05;P48 h<0.05;P72 h<0.01)and increased LUAD cell clones’ number(P<0.01).3.POU4F3 inhibited the proliferation of LUAD cells in vivo.Animal experiments showed that POU4F3 overexpression reduced tumor weight(P<0.01)and volume(P<0.01)significantly compared with their control group.POU4F3 knockdown increased tumor weight(P<0.001)and volume(P<0.01)significantly compared with their control group.4.POU4F3 had no significant effect on LUAD cells cycle.Cell cycle showed that there was no statistical difference in the proportion of G0/G1,S and G2/M phases between SPCA1-Lv-control group and SPCA1-Lv-POU4F3 group(P>0.05).Under the same experimental conditions,there was no statistical difference in the proportion of G0/G1,S and G2/M phases between SPCA1-control-shRNA group and SPCA1-shPOU4F3 group(P>0.05).5.POU4F3 promoted apoptosis of LUAD cells.Cell apoptosis assay showed that POU4F3 overexpression significantly increased the apoptosis rate of LUAD cells(P<0.001),and POU4F3 knockdown reduced the apoptosis rate of LUAD cells(P<0.05).Western Blotting showed that the relative expression of Cleaved caspase3 in LUAD cells was significantly increased by POU4F3 overexpression(P<0.05)and was reduced by POU4F3 knockdown(P<0.01).The relative expression level of Caspase3 was not changed apparently by POU4F3 overexpression or knockdown(P>0.05).6.POU4F3 inhibited the migration of LUAD cells.The scratch healing showed that overexpressed POU4F3 inhibited the scratch healing rate(P24h<0.01;P48h<0.01).Transwell sho wed that POU4F3 overexpression reduced the number of transmembrane cells(P<0.01).Under the same experimental conditions,POU4F3 knockdown increased the scratch healing rate(P24h<0.01;P48h<0.001)and increased the number of transmembrane cells(P<0.0001).7.POU4F3 changed the morphology of cell microfilaments and decreased the content of cell microfilaments.The microfilaments of SPCA1 cells in the overexpressed POU4F3 group were thinner and the fluorescence content was lower than that in the control group(P=0.0016).Under the same experimental conditions,the microfilaments of SPCA1 cells in knockdown POU4F3 group were thicker and more complete,and the mean fluorescence intensity of cellswas higher than that in control group(P=0.0017).8.POU4F3 activated the ERS sub-pathways PERK/eIF2α/ATF4/CHOP and IRE1α/XBP-1s.Western Blotting showed that overexpressed POU4F3 upregulated the relative expression levels of GRP78(P<0.05),P-PERK(P<0.01),p-eIF2α(P<0.05),ATF4(P<0.01),and CHOP(P<0.05),while the relative expression levels of PERK and eIF2α had no significant changes(P>0.05).POU4F3 knockdown downregulated the relative expression levels of GRP78(P<0.01),P-PERK(P<0.01),p-eIF2α(P<0.001),ATF4(P<0.01),and CHOP(P<0.01),while the relative expression levels of PERK and eIF2α had no significant changes(P>0.05).These results indicate that POU4F3 can activate the endoplasmic reticulum stress sub-pathway PERK/eIF2α/ATF4/CHOP.Besides,overexpressed POU4F3 upregulated the relative expression levels of p-IRE1α(P<0.05)and XBP-1 s(P<0.05),while the relative expression levels of IRE1α had no significant changes(P>0.05).POU4F3 knockdown downregulated the relative expression levels of p-IRE1α(P<0.01)and XBP-1s(P<0.01),while the relative expression levels of IRE1α had no significant changes(P>0.05).These results indicate that POU4F3 can activate the endoplasmic reticulum stress sub-pathway IRE1α/XBP-1s.Conclusion:1.POU4F3 expression in LUAD tissues is lower than that in paracancerous tissues,and patients with higher POU4F3 expression has more prolonged overall survival.2.POU4F3 can significantly inhibit the proliferation and migration of LUAD cells and promote cell apoptosis.3.POU4F3 activates the sub-pathways of ERS signaling pathway PERK/eIF2α/ATF4/CHOP and IRE1α/XBP-1s.This study indicates that the expression of POU4F3 is significantly lower in human LUAD tissues than in para-cancerous tissues.LUAD patients with high expression of POU4F3 have a better prognosis.POU4F3 could inhibit the proliferation and migration of LUAD cells and promote cell apoptosis.Our study suggests that POU4F3 may be a novel tumor suppressor of LUAD and a new target for the diagnosis and treatment of LUAD.