The Role Of WHSC1 In The Advancement Of Cervical Cancer And Its Related Mechanisms

Aim:Cervical cancer,the fourth most common malignant tumor in women,poses a serious threat to the lives and health of women around the world.Different stages of cervical cancer directly affect the survival rate of patients.Patients with early cervical cancer and locally advanced cervical cancer have the opportunity to receive conventional treatment with a good prognosis.However,patients with metastatic cervical cancer show heterogeneity and low survival rate.Currently,there is no standard treatment for metastatic cervical cancer.In order to reduce the mortality rate of cervical cancer,it is necessary to find an effective therapeutic target for metastatic cervical cancer,which has become an important direction for our research group to study cervical cancer.Over-expressed Wolf-Hirschhom syndrome candiadate(WHSC1),one of the histone lysine methyltransferases,is involved in epigenetic regulation during the development of various solid tumors.This study examined the expression of WHSC1 in cervical cancer tissues and cells.And in vitro,We observed the effects of WHSC1 on cell growth,proliferation,invasion,migration,and tube formation experiments of HUVECs by knocking down theexpression of WHSC1 in C-33 A cells.The study was designed to explore the possible role of WHSC1 in the progression of cervical cancer and related mechanisms.Methods:(1)Tissue microarray(TMA)was collected from 96 patients,and 72 cases of cervical cancer,6 cases of cervical intraepithelial neoplasia,and 18 cases of chronic cervicitis were detected by immunohistochemistry.The protein expression of WHSC1 in the specimen and the relationship between the abnormal expression of WHSC1 and clinicopathological features were analyzed.(2)The expression of WHSC1 mRNA in 72 cases of cervical cancer,6cases of CINIII,and 18 cases of chronic cervicitis were detected by PCR.(3)Western Blot was used to detect the protein expression of WHSC1 in Siha cells,C-33 A cells,HeLa cells and HEK cells.(4)qPCR was used to detect the expression of WHSC1 mRNA in Siha cells,C-33 A cells,HeLa cells and HEK cells.(5)Design and chemically synthesize three pairs of recombinant plasmids containing the target WHSC1 gene.And double enzyme digestion assay and DNA sequencing were used to identify the recombinant plasmid.(6)Sequencing the correct target gene plasmid was transfected into293 T cells,and the lentivirus packaging cells were constructed.The virus was transfected into C-33 A cells,and the plasmid with the best inhibitory effect was screened.(7)Transfection and selection of stably transfected cells.qPCR and WB were used to detect the expression of WHS1 mRNA and protein in the cells of the transfected group and the negative control group,respectively,to identify the inhibitory effect of WHS1 gene expression.(8)CCK-8 was used to detect the effect of transfection on the growth of C33 A cells.(9)Transwells was used to detect changes in invasion and migration ability of C-33 A cells after interference with WHSC1-shRNA.(10)Observe the effect on angiogenesis of HUVECs cells in matrigel by C-33 A cells after interference with WHSC1-shRNA.(11)WB was used to detect the expression of AKT,VEGF and eNOS proteins in the pathway involved in angiogenesis after interference with WHSC1-shRNA.Results:(1)The positive rate of WHSC1 protein expression was positively correlated with the severity of the lesion(P<0.0001).The expression of WHSC1 mRNA in cervical cancer tissues was significantly higher than that of chronic cervicitis and cervical intraepithelial neoplasia(P< 0.05),but there was no significant difference in the expression of CIN3 compared with chronic cervicitis group(P>0.05).(2)WHSC1 was highly expressed in advanced cervical cancer(≥III phase)(P=0.0040)and poorly differentiated patients(P=0.0156),and it was statistically significant.There was no statistical difference in the expressionof WHSC1 between tumor size(P=0.4999),lymph node metastasis(P=0.5131),and patient age(P=0.5929).(3)The survival rate of cervical cancer patients with high expression of WHSC1 protein was significantly lower than that of patients with low expression of cervical cancer(P<0.05).(4)The expression of WHSC1 in cervical cancer cell lines C-33 A,HELA and Siha cells was higher than that in normal epithelial cells HEK,and the expression level of WHSC1 protein was the highest in C-33 A cells.(5)The expression of WHSC1 mRNA in C-33 A and Siha cells was higher than that in normal epithelial cells,while the expression level of WHSC1 mRNA in HELA cells was lower than that in HEK cells.(6)Successfully designed and synthesized three WHS1-specific shRNAs.Double enzyme digestion experiments and DNA sequencing reports showed that the gene recombination technology successfully constructed the WHSC1 gene RNA interference lentiviral vector.(7)Lentiviral-mediated WHSC1-shRNA1#,WHSC1-shRNA2#,WHSC1-shRNA3# and negative control successfully infected C-33 A cells,and qPCR was used to verify the inhibitory effect of WHSC1.Among them,WHSC1-shRNA1# had the best inhibitory effect.(8)WHSC1-shRNA1# transfected C-33 A cells to obtain stable cells(WHSC1-KD-1#),qPCR and WB detected the expression of WHSC1 mRNA and protein in transfected cells and negative control cells,respectively,and the results showed that both mRNA and protein expression levels of the target genes were significantly inhibited.(9)In the CCD-8 experiment,the proliferation of cells in the WHSC1-KD-1# group was inhibited in the late stage compared with the NC group,and was more obvious on the 5th and 6th day.(10)Transwell detection of WHSC1-KD-1# cells compared with NC group,their invasive ability and migration ability were weakened.(11)In the co-culture,the cell culture supernatant of WHSC1-KD-1#group was co-cultured with HUVEC,the tube formation effect was significantly inhibited compared with the NC group.(12)After knocking down the expression of WHSC1 gene,AKT and VEGF were slightly decreased,but the decrease was not obvious,and the expression of eNOS was significantly increased.Conclusions:(1)The mRNA and protein levels of WHSC1 are highly expressed in tissues of cervical cancer patients,and the high expression of WHSC1 is associated with poorly differentiated,late-stage cervical cancer,and high expression of WHSC1 protein and low survival rate in cervical cancer.In addition,in a variety of cervical cancer cell lines,the protein expression of WHSC1 was higher than that of normal squamous cells HEK cells,and the expression of WHSC1 in C-33 A cells was the most obvious.These results suggest that WHSC1 may play a role in the development of cervical cancer,but the relevant mechanism is still to be further explored.C-33 A cells were selected as target cells for further study.(2)The WHSC1 gene-specific recombinant plasmid with the best inhibition was successfully constructed and screened.Successfully transfected with lentiviral vector and screened for stable transfected cervical cancer C-33 A cell line(WHSC1-KD-1#).The experimental basis was laid to further study the biological behavior and molecular mechanism of WHSC1 in the development of cervical cancer.(3)After silencing of WHSC1 gene,the proliferation,invasion and migration ability of C-33 A cells were significantly inhibited.In co-culture,WHS1 gene silencing significantly inhibited the tube formation of HUVEC cells.After knocking down the WHS1 gene,the expression of eNOS protein in the cells was significantly increased.These results suggest that WHSC1 as an important genetic epigenetic regulator has an important role in promoting malignant behaviors such as cervical cancer proliferation,invasion,metastasis,and angiogenesis;high expression of WHSC1 may cause abnormalities in AKT-VEGF-eNOS signaling pathway.Therefore,WHSC1 is expected to become a new therapeutic target for cervical cancer for further scientific research.