Abstract:

Objective To discuss the effect of KH-type splicing regulatory protein（KHSRP） on the malignant biological behaviors of colorectal cancer （CRC） by activating the Janus kinase（JAK）/signal transducer and activator of transcription（STAT） signaling pathway， and to clarify its possible mechanism. Methods The CRC tissue and adjacent normal tissue from 64 CRC patients were selected. The human CRC cells （HT29， SW620， SW480， DLD-1， LOVO， and RKO） and normal human colorectal mucosal FHC cells were cultured in vitro. The total RNA from CRC tissue and cells were extracted， real-time fluorescence quantitative PCR （RT-qPCR） was used to detect the expression levels of KHSRP in the CRC tissue， adjacent normal tissue and all kinds of cells. The HT29 and SW620 cells were divided into sh-NC group （lentiviral plasmid inserted with non-targeting nucleotide sequence） and sh-KHSRP group （transfected with KHSRP knockdown lentivirus）. The SW480 and DLD-1 cells were divided into oe-NC group （lentiviral plasmid inserted with non-targeting nucleotide sequence） and oe-KHSRP group （transfected with KHSRP overexpression lentivirus）. Immunohistochemistry （IHC） staing in method was used to analyze the expressions of KHSRP in CRC tissue and adjacent normal tissue； CCK-8 method was used to detect the proliferation activities of the CRC cells in various groups； Transwell assay was used to detect the numbers of migration and invasion cells in various groups； Western blotting method was used to detect the expression levels of KHSRP， JAK1， phosphorylated JAK1（p-JAK1）， JAK2， phosphorylated JAK2（p-JAK2）， STAT1， STAT2， STAT3， and STAT5 proteins in the CRC cells in various groups. The subcutaneous xenograft tumor models in the nude mice were used to measure the tumor volumes and weights of the mice in various groups. Results Compared with adjacent normal tissue， the expression level of KHSRP in the CRC tissue was increased （P<0.05）. Compared with FHC cells， the expression levels of KHSRP in the CRC cells were increased （P<0.05）. Therefore， the HT29 and SW620 cells were selected for knockdown of KHSRP， while the SW480 and DLD-1 cells were selected for over-expression of KHSRP. The Western blotting results showed that the expression amounts of KHSRP protein in the CRC tissue and cells were higher than those in adjacent normal tissue and FHC cells. The IHC results showed that compared with adjacent normal tissue， the expression level of KHSRP protein in CRC tissue was increased （P<0.01）. The RT-qPCR results showed that compared with sh-NC group， the expression levels of KHSRP mRNA in the HT29 and SW620 cells in sh-KHSRP group were decreased （P<0.01）； compared with oe-NC group， the expression levels of KHSRP mRNA in the SW480 and DLD-1 cells in oe-KHSRP group were increased （P<0.01）， indicating successful transfection. The CCK-8 results showed that compared with sh-NC group， the proliferation activities of the HT29 and SW620 cells in sh-KHSRP group after knockdown of KHSRP were decreased （P<0.05 or P<0.01）； compared with oe-NC group， the proliferation activities of the SW480 and DLD-1 cells in oe-KHSRP group after over-expression of KHSRP were increased （P<0.05 or P<0.01）. Compared with sh-NC group， the numbers of migration and invasion cells in the HT29 and SW620 cells in sh-KHSRP group after knockdown of KHSRP were decreased （P<0.05）； compared with oe-NC group， the numbers of migration and invasion cells in the CRC cells in oe-KHSRP group after over-expression of KHSRP were increased （P<0.05）. After knockdown of KHSRP， the tumor volume and weight in sh-KHSRP group were smaller than those in sh-NC group （P<0.05 or P<0.01）， while the tumor volume andweight in oe-KHSRP group were larger than those in oe-NC group after over-expression of KHSRP （P<0.05 or P<0.01）. Compared with sh-NC group， the expression levels of JAK1， p-JAK1， and STAT3 proteins in the CRC cells in sh-KHSRP group were significantly decreased （P<0.05 or P<0.01）； compared with oe-NC group， the expression levels of JAK1， p-JAK1， and STAT3 proteins in the CRC cells in oe-KHSRP group were significantly increased （P<0.05 or P<0.01）. Conclusion High expression of KHSRP promotes the proliferation， migration， and invasion of the CRC cells and enhances the growth of subcutaneous xenograft tumors in the mice； its mechanism may be associated with its activation of the JAK1/STAT3 signaling pathway.

Key words: Colorectal neoplasms, KH-type splicing regulatory protein, Janus kinase, Signal transducer and activator of transcription, Malignant progression, Cell proliferation, Cell invasion, Cell migration