PLOD2 Promotes The Transformation Of Cervical Cancer-associated Fibroblasts

Background and ObjectiveThe PLOD2 gene encodes lysine hydroxylase 2(LH2),which mainly affects the morphology,structure,and molecular cross-linking of collagen fibers in the extracellular matrix.It is highly expressed in cervical cancer,breast cancer,lung cancer and many other cancer types,resulting in cells The increased hardness of the outer matrix is closely related to tumor invasion and metastasis.Studies have shown that the invasion and metastasis of malignant tumors are closely related to the tumor microenvironment.Extracellular matrix remodeling is a key event in the process of tumor invasion and metastasis.Tumor-related fibroblasts(CAFs)are the main source of collagen fibers in the extracellular matrix In this process played an important role.Current research suggests that CAFs can be derived from normal fibroblasts(NFs)and other cells in the tumor matrix,and environmental stimulation such as stiff matrix can induce the conversion of normal fibroblasts into CAFs.But so far the source of cervical cancer-related fibroblasts is still unclear.The previous research of this research group showed that PLOD2 is highly expressed in cervical cancer,and vascular tumor thrombus,deep muscle invasion,pelvic lymph node metastasis,and poor prognosis of patients are positively correlated.Those with high PLOD2 expression have stronger invasive ability,but the specific mechanism of action clear.This study intends to preliminary investigate whether the PLOD2 gene in cervical cancer cells promotes the conversion of NFs to CAFs to create a good tumor microenvironment,which leads to the enhancement of tumor invasion and metastasis.The integrity of the environment,weakening its invasion and metastasis ability provides new theoretical basis and therapeutic targets.Methods1.Lentiviral transfection technology was used to infect HeLa cells(adenocarcinoma)and SiHa cells(squamous cell carcinoma)with lentivirus overexpressing PLOD2 and negative control lentivirus,respectively.The efficiency of lentivirus transfection was detected by fluorescence;qRT-PCR and Western blot were used to detect the expression of PLOD2.Therefor,cervical cancer cell line stably overexpressing PLOD2 gene was constructed.2.Transwell chamber were used to co-culture the cervical cancer cell lines HeLa or SiHa with normal fibroblasts(HFF-1 cells):HeLa cells or SiHa cells are inoculated in the Transwell chamber(upper chamber),and fibroblasts are inoculated in the cell culture plate(lower chamber).The experimental grouping is:inoculation of fibroblasts in the cell culture plate,and its corresponding Transwell chamber is not inoculated with cervical cancer cells,which is the BLANK group;cervical cancer cells transfected with negative control virus and fibroblasts co-culture were HeLa-LV-NC group and SiHa-LV-NC group respectively;cervical cancer cells overexpressing PLOD2 and fibroblasts co-culture were HeLa-LV-PLOD2 group and SiHa-LV-PLOD2 group.3.After co-culturing the two kinds of cells for 48 hours,examined the morphological status of the fibroblasts.Collected the fibroblasts in the lower chamber and then perform wound healing assay to analyze their migration ability.And gene expression of α-SMA,FAP-α,FSP1 and PLOD2 in fibroblasts were validated by qRT-PCR and Western blot.In addition,wound healing assay and transwell assay were carried out to evaluate the migration and invasion ability of HeLa and SiHa cells after co-cultured.The secretion of VEGF and MMP9 in the culture medium were detected by ELISA.Results1.After transfection with lentivirus,We could see clear green fluorescence in SiHa and HeLa cells under inverted fluorescence microscope.The results of qRT-PCR and Western blot confirmed that the expression of PLOD2 was significant increase after transfection with overexpressed PLOD2 lentivirus.And there was no significant difference in the expression of PLOD2 in SiHa and HeLa cells transfected with the negative control virus and in the control group.That was,cervical cancer cell lines that stably overexpressed PLOD2 was successfully constructed.2.The shape and size of fibroblasts in the BLANK group remained basically the same,showing a long spindle shape and irregular alignment after co-cultivation.However,The size and shape of fibroblasts in the LV-NC group and LV-PLOD2 group were significantly different,with more protrusions at the edges,severe overlapping growth between cells,and the morphological characteristics of CAFs.Wound healing assay showed enhanced migration ability of co-cultured fibroblasts.The results showed that PLOD2 promoted cervical cancer cells to induce fibroblasts to acquire the morphological characteristics and migration characteristics of CAFs.3.The expression levels of α-SMA,FAP-α,and FSP1,which are the markers of CAFs,were significantly higher than those in the BLANK group.At the same time,the expression levels that in the LV-PLOD2 group were higher than those in the LV-NC group;The expression of PLOD2 in the LV-NC group was not significantly different from that in the BLANK group,but in the LV-PLOD2 group it was higher than that in the LV-NC group.The result showed that PLOD2 promoted cervical cancer cells to induce fibroblasts to express CAFs markers,that is,transform into CAFs.And the expression of fibroblasts PLOD2 is increased.4.After co-cultivation,the contents of MMP9 and VEGF in the culture medium of LV-NC group and LV-PLOD2 group were significantly higher than that of BLANK group,and that in LV-PLOD2 group was significantly higher than that of LV-NC group.The result showed that PLOD2 promoted the transformation of cervical cancer cells into fibroblasts to CAFs,which increased the secretion of metastasis-related cytokines.5.Wound healing assay of cervical cancer cells after co-cultivation showed that the healing rate of the scratch area in LV-PLOD2 group was significantly increased than that in LV-NC group,suggesting that the migration ability of cervical cancer cell was enhanced.The result showed that fibroblasts transform to CAFs and then act on cervical cancer cells,which enhanced their migration ability.6.Transwell assay of cervical cancer cells after co-culture showed that the number of cancer cells in the LV-PLOD2 group passing through the membrane from the upper compartment to the lower compartment was significantly higher than that in the LV-NC group,and the same results were observed in the matrigel transwell assay.The result showed that fibroblasts transform to CAFs and then act on cervical cancer cells,which enhance the invasion ability of cervical Cancer cell.Conclusion1.PLOD2 promotes cervical cancer cells to induce normal fibroblasts(NFs)to transform into cancer-associated fibroblasts(CAFs).2.PLOD2 promotes cervical cancer cells to induce normal fibroblasts to transform into CAFs,which can increase the secretion of metastasis-related cytokines.3.After the fibroblasts transform into CAFs,they can act on cervical cancer cells to enhance their migration and invasion ability.4.Our research suggests that PLOD2 can promote the transformation of cervical cancer-related fibroblasts to create a good tumor microenvironment in order to accelerate the invasion and metastasis of cervical cancer.