The Cell Function Study Of Long Non-coding RNA-PVT1 And Sorafenib In Papillary Thyroid Cancer Cell Lines

Objective:The incidence of thyroid cancer has been raised dramatically,though the rise of incident rate didn’t accompany with a significant rise of the death rate,the occurrence of lymph node and distance metastasis is the major cause of the decrease of survival and even death.In order to practice precise diagnosis and therapy,lots of researchers focus on the mechanism of thyroid cancer’s occurrence and biomarkers.Long Noncoding RNA PVT1 has proved to be highly expressed in many cancer types and cancer cell lines,sorafenib has already used in Iodine refractory differentiated thyroid cancer,our study aim to knock down Long Noncoding RNA PVT1,and combined with molecular target drug sorafenib,to evaluate the impact of the proliferation,colony formation,invasion,migration,apoptosis,the cell cycle of the Papillary Thyroid Cancer cell lines.Methods:(1)A retrospective analysis of 38 papillary thyroid carcinoma patients who were treated in the First Hospital of Shanxi Medical University and confirmed by surgery and pathology.During the operation,tumor nodules were collected and placed in a cryopreserved tube immediately until RNA extraction.The expression level of PVT1 in tissue was detected by RT-qPCR.Analyze ultrasound images,surgery record to explore the relationship between C-TIRADS point and the expression level of LncRNA-PVT1,lymph node metastasis,invasion of capsule.(2)To explore the time-dose curve and IC50 of the sorafenib in B-CPAP、TPC-1、IHH-4、K1 cell lines.(3)To test the level of Long Noncoding RNA-PVT1 through quantity PCR within the B-CPAP、TPC-1、IHH-4、K1 cell lines.(4)According to the expression level of LncRNA-PVT1,we use the B-CPAP,TPC-1cell lines to knock down LncRNA-PVT1.Experimental groups divided into 6 groups,namely blank,knockdown negative control,knockdown LncRNA-PVT1,sorafenib,sorafenib combined knockdown negative control,sorafenib combined knockdown LncRNA-PVT1.(5)Conduct cell proliferation,colony formation,Transwell migration and invasion,cell apoptosis,cell cycle,to verify the function of LncRNA-PVT1 and sorafenib.(6)Statistics method is to use t test or One-Way Anova,use t’ test or Kruskal-Wallis H test if the data is the heterogeneity of variance.Results:(1)The relative expression of PVT1 in PTC nodule tissues of C-TIRADS 4 group was higher than that of C-TIRADS 3 groups(P<0.05).There was a difference between groups in cervical lymph node metastasis variables,C-TIRADS 3 group were higher than C-TIRADS 2 groups.(2)In TPC-1,B-CPAP,K1,IHH-4 cell line,the cell survival rate is lower when the concentration of sorafenib is higher and with prolonged applied time.(3)The expression level of LncRNA-PVT1 from high to low is TPC-1,BCPAP,K1,IHH4 cell lines,we use TPC-1,BCPAP to knock down LncRNA-PVT1.(4)The proliferation of knockdown LncRNA-PVT1 in BCPAP,TPC-1 lower than the negative control group and blank group using cck-8 kit(P<0.05).Using 5μM sorafenib combined with knockdown LncRNA-PVT1 in the TPC-1 and B-CPAP cell line reduces the live cells numbers(P<0.05).(5)Compared with the blank group and negative control group,knockdown LncRNA-PVT1 group,negative control combined sorafenib group,knockdown LncRNA-PVT1 combined sorafenib group’s clone formation numbers have been decreased,the clone formation numbers in knockdown LncRNA-PVT1 combined with sorafenib group is lower than negative control combined with sorafenib group in B-CPAP cell line(P<0.05).Clone formation numbers is lower in knockdown LncRNA-PVT1combined with sorafenib than the blank and negative control group in TPC-1 cell line(P<0.05).(6)In the Transwell migration and invasion test,The migration and invasion numbers are lower in knockdown LncRNA-PVT1 combined with sorafenib group compared with negative control and blank group(P<0.05),the migration numbers of knockdown LncRNA-PVT1 group is lower in the blank group in BCPAP and TPC-1 cell line(P<0.05).(7)In cell line IHH-4,The proportion of apoptotic is higher in sorafenib group than blank and DMSO group(P<0.05).In cell line B-CPAP,The proportion of earlier apoptotic cell is higher in sorafenib group,negative control combined with sorafenib group,knockdown LncRNA-PVT1 combined sorafenib group than blank group in B-CPAP cell line(P<0.05).The total proportion of apoptotic cell is higher in the knockdown LncRNA-PVT1 combined sorafenib group than the knockdown LncRNA-PVT1 group,sorafenib group,negative control combined sorafenib group(P<0.05).The proportion of total proportion of apoptotic is higher in the sorafenib group,negative control combined sorafenib group,knockdown LncRNA-PVT1 combined sorafenib group than blank TPC-1 cell line(P<0.05).(8)The G1 proportion of B-CPAP,IHH-4 cell line is relatively higher compared with blank and DMSO group(P<0.05).Conclusion:(1)There were significant differences between the expression level of PVT1 and C-TIRADS grades,the lymph node metastases rate in C-TIRADS 3 point group is higher than C-TIRADS 2 point group.(2)Sorafenib can inhibit papillary thyroid carcinoma TPC-1,BCPAP,IHH4,K1 cell line,cell survival rate decrease accompanied with time prolong and the higher concentration of the sorafenib applied.(3)The expression level of LncRNA-PVT1 from high to low is TPC-1,BCPAP,K1,IHH4.(4)Inhibit the expression of LncRNA-PVT1 can reduce the proliferation,form fewer clone cells,inhibit migration,and promote cell apoptosis.(5)Inhibit the expression of LncRNA-PVT1 combined with 5μM sorafenib have a synergistic effect on inhibit cell survive.B-CPAP at 9μM,TPC-1 at 13μM concentration combined with knockdown LncRNA-PVT1 can form fewer clone cells,inhibit migration and invasion,and promote cell apoptosis.(6)Sorafenib can arrest the cell cycle in the G1 stage in B-CPAP,IHH-4 cell line.