Objective

2

To investigate the potential mechanism of the

Jianpi Yishen Huatan Formula

（健脾益肾化痰方，JPYSHF） in treating squamous cell lung cancer through the LncRNA ALAL-1/USP4/HDAC2 signaling pathway.

Methods

2

Forty Sprague-Dawley （SD） rats were randomly divided into a control group and high-， medium-， and low-dose JPYSHF group with 10 rats in each group. Rats in the JPYSHF groups were administered JPYSHF concentrated liquid at doses of 45， 30， and 15 g/（kg·d） via intragastric gavage， respectively， while the control group received 10 ml/（kg·d） of normal saline， once daily for 10 consecutive days before preparation of drug containing serum. Human lung squamous carcinoma SK-MES-1 cells were divided into a control group and low-， medium-， and high-dose JPYSHF-medicated serum groups. The control group was cultured with 10% saline-containing serum， while the JPYSHF groups were cultured with 10% low-， medium-， or high-dose medicated serum. After 48 hours of incubation， flow cytometry was used to detect apoptosis rates， and a cell scratch assay was performed to evaluate migration areas at 0 h and 24 h to calculate migration rate. Additional SK-MES-1 cells were divided into control serum， JPYSHF-medicated serum （low-， medium-， high-） do

se， LncRNA-silenced group （transfected with ALAL-1 siRNA）， USP4-inhibited group （treated with 35 μmol/L PR-619， a deubiquitinase inhibitor）， and HDAC2-inhibited group （treated with 60 μmol/L Vorinostat）. After 24 and 48 hours of culture， cell viability was assessed using the CCK-8 assay；

LncRNA ALAL

-

1

，

USP4

， and

HDAC2

mRNA levels were quantified by qPCR after 24 hours； USP4 and HDAC2 protein levels were measured by Western Blot after 48 hours.

Results

2

Compared with the control serum group， the total apoptosis rate of cells in middle- and high-JPYSHF-medicated serum group significantly increased， and the cell migration rate of cells in the low-， middle- and high-JPYSHF-medicated serum group significantly decreased （

P

＜0.05 or

P

＜0.01）. The cell migration rate of the low-， medium- and high-JPYSHF-medicated serum groups decreased with the increase of concentration in a concentration-dependent manner （

P

＜0.05 or

P

＜0.01）. Compared with the control serum group at the same time， the cell viability at 24 h and 48 h significantly decreased in all groups （

P

＜0.05 or

P

＜0.01）. Compared with the low-JPYSHF-medicated serum group at the same time， the cell viability at 24 h and 48 h also decreased in the high-JPYSHF-medicated serum group and the LncRNA silencing group （

P

＜0.05）. Compared with the control serum group， the expression of

USP4

and

HDAC2

mRNA reduced in the low- and medium-dose JPYSHF-medicated serum groups and the USP4 inhibitor group， and the expression of

LncRNA ALAL

-

1

，

USP4

and

HDAC2

mRNA reduced in the high-dose JPYSHF-medicated serum group and LncRNA-silencing group， and

HDAC2

mRNA expression reduced in the HDAC2 inhibitor group. USP4 and HDAC2 protein levels were reduced in cells of

all groups except for USP4 protein level in HDAC2 inhibitor group （

P

＜0.05 or

P

＜0.01）.

Conclusion

2

JPYSHF-medicated serum inhibits proliferation and promotes apoptosis of human lung squamous carcinoma cells， and its mechanism of action may be related to its inhibition of the LncRNA ALAL-1/USP4/HDAC2 pathway， with best effect at a high concentration.